Advanced pharmacognosy notes


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This notes preparation is based on M.G.R Medical university Syllabus


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INTRODUCTION Living plants considered as biosynthetic laboratory p r i m a r y as well as secondary metabolite. i Different biosynthetic pathway: - Shikmic acid pathway Mevalonic acid pathway Acetate pathway ii Various intermediate and steps are involved in biosynthetic pathway in plants can be investigated by means of following techniques: - Tracer technique Use of isolated organ Grafting methods Use of mutant strain

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• Definition: - It can be defined as technique which utilizes a labelled compound to find out or to trace the different intermediates and various steps in biosynthetic pathways in plants at a given rate time. OR • In this technique different isotope mainly the radioactive isotopes which are incorporated into presumed precursor of plant metabolites and are used as marker in biogenic experiments.

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The labelled compound can be prepared by use of two types of isotopes. » Radioactive isotopes. » Stable isotopes. Radioactive isotopes: - e.g. 1 H 14 C 24 Na 42 K 35 S 35 P 131 I decay with emission of radiation – For biological investigation – carbon hydrogen. – For metabolic studies – S P and alkali and alkaline earth metals are used. – For studies on protein alkaloids and amino acid – labelled nitrogen atom give more specific information. – 3 H compound is commercially available. vii Stable isotopes: - e.g. 2 H 13 C 15 N 18 O – Used for labelling compounds as possible intermediates in biosynthetic pathways. – Usual method of detection are: – MASS spectroscopy 15 N 18 O – NMR spectroscopy 2 H 13 C

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SIGNIFICANCE OF TRACER TECHNIQUE • Tracing of Biosynthetic Pathway: - e.g. By incorporation of radioactive isotope of 14 C into phenylalanine the biosynthetic cyanogenetic glycoside prunasin can be detected. • Location Quantity of compound containing tracer: - 14 C labelled glucose is used for determination of glucose in biological system • Different tracers for different studies: - For studies on nitrogen and amino acid. Labelled nitrogen give specific information than carbon • Convenient and suitable technique CRITERIA FOR TRACER TECHIQUE • The starting concentration of tracer must be sufficient withstand resistance with dilution in course of metabolism. • Proper Labelling: - for proper labelling physical chemical nature of compound must be known. • Labelled compound should involve in the synthesis reaction. • Labelled should not damage the system to which it is used.

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ADVANTAGES High sensitivity. Applicable o all living organism. Wide ranges of isotopes are available. More reliable easily administration isolation procedure. Gives accurate result if proper metabolic time technique applied. LIMITATION Kinetic effect Chemical effect Radiation effect Radiochemical purity High concentration distorting the result.

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REQUIREMENT FOR TRACER TECHNIQUE – Preparation of labelled compound. – Introduction of labelled compound into a biological system. – Separation determination of labelled compound in various biochemical fractions at later time. I. Preparation of Labelled Compound: -  The labelled compound produce by growing chlorella in atmosphere of  14 CO 2  . All carbon compounds 14 C labelled.  The  3 H tritium labelled compound are commercially available. Tritium labelling is effected by catalytic exchange in aqueous media by hydrogenation of unsaturated compound with tritium gas. Tritium is pure β – emitter of low intensity its radiation energy is lower than 14 C.  By the use of organic synthesis: - CH3MgBr + 14 CO 2 CH 3 14 COOHMgBr+H 2 O CH3 14 COOH + MgOHBr

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II. Introduction of labelled compound: - PRECAUTION: - •The precursor should react at necessary site of synthesis in plant. •Plant at the experiment time should synthesize the compound under investigation •The dose given is for short period. 1. Root feeding 2. Stem feeding 3. Direct injection 4. Infiltration 5. Floating method 6. Spray technique III. Separation and detection of compound: - a Geiger – Muller counter. b Liquid Scintillation counter. c Gas ionization chamber. d Bernstein – Bellentine counter. e Mass spectroscopy. f NMR eletrodemeter. g Autoradiography.

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METHODS IN TRACER TECHNIQUE 1. PRECURSOR PRODUCT SEQUENCE: - In this technique the presumed precursor of the constituent under investigation on a labelled form is fed into the plant and after a suitable time the constituent is isolated purified and radioactivity is determined. Disadvantage: - The radioactivity of isolated compound alone is not usually sufficient evidence that the particular compound fed is direct precursor because substance may enter the general metabolic pathway and from there may become randomly distributed through a whole range of product. Application: - •Stopping of hordenine production in barley seedling after 15 – 20 days of germination. •Restricted synthesis of hyoscine distinct from hyoscyamine in Datura stramonium. •This method is applied to the biogenesis of morphine ergot alkaloids

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2. DOUBLE MULTIPLE LABELLING: - This method give the evidence for nature of biochemical incorporation of precursor arises double triple labelling. In this method specifically labelled precursor and their subsequent degradation of recover product are more employed. Application: -  This method is extensively applied to study the biogenesis of plant secondary  metabolite.  Used for study of morphine alkaloid. E.g. Leete use Doubly labeled lysine used to determine which hydrogen of lysine molecule was involved in formation of piperidine ring of anabasine in Nicotina glauca. N. glauca N H N N H H 2 N 2 - Anabasine COO Lysine - 2 - 14 C ε − 15 Ν N. glauca N H 2 N H 2 N N H COOH Anabasine Lysine - 2 - 14 C α − 15 Ν

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3. COMPETITIVE FEEDING: - If incorporation is obtained it is necessary to consider whether this infact the normal route of synthesis in plant not the subsidiary pathway. Competitive feeding can distinguish whether B B‟ is normal intermediate in the formation of C from A. B OR A C A C A B C Application: B - A B C  This method is used for elucidation of biogenesis of propane alkaloids.  Biosynthesis of hemlock alkaloids conline conhydrine etc e.g. biosynthesis of alkaloids of Conium maculactum hemlock using 14 C labelled compounds.

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4. ISOTOPE INCORPORATION: - This method provides information about the position of bond cleavage their formation during reaction. E.g. Glucose – 1- phosphatase cleavage as catalyzed by alkaline phosphatase this reaction occur with cleavage of either C – O bond or P – O bond. CH 2 OH CH 2 OH O O 18 OH + H 2 O OH OH + H 2 PO 4 OPO 3 H OH OH OH OH

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5. SEQUENTIAL ANALYSIS: - The principle of this method of investigation is to grow plant in atmosphere of 14 CO 2 then analyze the plant at given time interval to obtain the sequence in which various correlated compound become labelled. Application: -  14 CO 2  sequential analysis has been very successfully used  in elucidation of carbon in photosynthesis.  Determination of sequential formation of opium hemlock and  tobacco alkaloids.  Exposure as less as 5 min.  14 CO 2 is used in detecting  biosynthetic sequence as –  Piperitone --------- - Menthone ------ ---- - Menthol in Mentha piperita.

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APPLICATION OF TRACER TECHNIQUE 1. Study of squalene cyclization by use of 14 C 3 H labelled mevalonic acid. 2. Interrelationship among 4 – methyl sterols 4 4 dimethyl sterols by use of 14 C acetate. 3. Terpenoid biosynthesis by chloroplast isolated in organic solvent by use of 2- 14 C mevalonate. 4. Study the formation of cinnamic acid in pathway of coumarin from labelled coumarin. 5. Origin of carbon nitrogen atoms of purine ring system by use of 14 C or 15 N labelled precursor. 6. Study of formation of scopoletin by use of labelled phenylalanine. 7. By use of 45 Ca as tracer - found that the uptake of calcium by plants from the soil. CaO CaCO 2 . 8. By adding ammonium phosphate labelled with 32 P of known specific activity the uptake of phosphorus is followed by measuring the radioactivity as label reaches first in lower part of plant than the upper part i.e. branches leaves etc.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY PLANT TISSUE CULTURE TECHNIC 1. Introduction Plant tissue culture can be defined as the in vitro manipulation of plant cells and tissues and is a keystone in the foundation of plant biotechnology. It is useful for plant propagation and in the study of plant growth regulators. It is generally required to manipulate and regenerate transgenic plants. Whole plants can be regenerated under in vitro conditions using plant organs tissues or single cells by inoculating them in an appropriate nutrient medium under sterile environment. Plant tissue culture relies on the fact that many plant cells have the capacity to regenerate into a whole plant–a phenomena known as totipotency. Plant cells cells without cell walls protoplasts leaves or roots can be used to generate a new plant on culture media containing the necessary nutrients and plant growth regulators. Plant tissue culture was first attempted by Haberlandt 1902. He grew palisade cells from leaves of various plants but they did not divide. In 1934 White generated continuously growing cultures of meristematic cells of tomato on medium containing salts yeast extract and sucrose and vitamin B pyridoxine thiamine and nicotinic acid and established the importance of additives. In 1953 Miller and Skoog University of Wisconsin – Madison discovered Kinetin a cytokine that plays an active role in organogenesis. Plant cell cultures are an attractive alternative source to whole plants for the production of high-value secondary metabolites. 2. Advantages of plant tissue culture over conventional agricultural production The most important advantage of in vitro grown plants is that it is independent of geographical variations seasonal variations and also environmental factors. It offers a defined production system continuous supply of products with uniform quality and yield. Novel compounds which are not generally found in the parent plants can be produced in the in vitro grown plants through plant tissue culture. In addition stereo- and region- specific biotransformation of the plant cells can be performed for the production of bioactive compounds from economical precursors. It is also independent of any political interference. Efficient downstream recovery of products and rapidity of production are its added advantages Figure 31.1.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Figure 31.1: Steps involved in the production of secondary metabolites from plant cell 3. Plant secondary metabolites Plant products can be classified into primary plant metabolites and secondary metabolites. Primary plant metabolites are essential for the survival of the plant. It consists of sugars amino acids and nucleotides synthesized by plants and are used to produce essential polymers. Typically primary metabolites are found in all species within broad phylogenetic groupings and are produced using the same metabolic pathway. Secondary metabolites are the chemicals which are not directly involved in the normal growth and development or reproduction of an organism. Secondary metabolites are not indispensable for the plants but play a significant role in plant defense mechanisms. Primary metabolites essentially provide the basis for normal growth and reproduction while secondary metabolites for adaptation and interaction with the environment. The economic importance of secondary metabolites lies in the fact that they can be used as sources of industrially important natural products like colours insecticides antimicrobials fragrances and therapeutics. Therefore plant tissue culture is being potentially used as an alternative for plant secondary metabolite production. Majority of the plant secondary metabolites of interest to humankind fit into categories which categorize secondary metabolites based on their biosynthetic origin. Secondary metabolism in plants is activated only in particular stages of growth and development or during periods of stress limitation of nutrients or attack by micro-organisms. Plants produce several bioactive compounds that are of importance in the healthcare food flavor and cosmetics industries. Many pharmaceuticals are produced from the plant secondary metabolites. Currently many natural products are produced solely from massive quantities of whole plant parts. The source plants are cultured in tropical subtropical geographically remote areas which are subject to drought disease and changing land use patterns and other environmental factors. Secondary metabolites can be derived from primary metabolites through modifications like methylation hydroxylation and glycosylation. Secondary metabolites are naturally more complex than primary metabolites and are classified on the basis of chemical structure e.g. aromatic rings sugar composition

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY containing nitrogen or not their solubility in various solvents or the pathway by which they are synthesized Table 31.1. They have been classified into terpenes composed entirely of carbon and hydrogen phenolics composed of simple sugars benzene rings hydrogen and oxygen and nitrogen and or sulphur containing compounds Figure 31.2. It has been observed that each plant family genus and species produces a characteristic mix of these bioactive compounds. All plants produce secondary metabolites which are specific to an individual species genus and are produced during specific environmental conditions which makes their extraction and purification difficult. As a result commercially available secondary metabolites for example pharmaceuticals flavours fragrances and pesticides etc. are generally considered high value products as compared to primary metabolites and they are considered to be fine chemicals. Table 31.1: Classification of secondary metabolites Figure 31.2: The production of secondary metabolites is tightly associated with the pathways of primary/central metabolism such as glycolysis shikimate and production of aliphatic amino acids. 4. Strategies for enhanced production of secondary metabolites in plant cell cultures

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY 4.1. Proper selection of cell lines The heterogeneity within the cell population can be screened by selecting cell lines capable of accumulating higher level of metabolites. 4.2. Manipulation of medium The constituents of culture medium like nutrients phytohormones and also the culture conditions like temperature light etc. influence the production of secondary metabolites. For e.g. if sucrose concentration is increased from 3 to 5 production of rosamarinic acid is increased by five times. In case of shikonin production IAA enhances the yield whereas 24-D and NAA are inhibitory. 4.3. Addition of Elicitors Elicitors are the compounds which induce the production and accumulation of secondary metabolites in plant cells. Elicitors produced within the plant cells include cell wall derived polysaccharides like pectin pectic acid cellulose etc. Product accumulation also occurs under stress conditions caused by physical or chemical agents like UV low or high temperature antibiotics salts of heavy metals high salt concentrations which are grouped under abiotic elicitors. Addition of these elicitors to the medium in low concentration enhances the production of secondary metabolites. 4.4. Addition of precursors Precursors are the compounds whether exogenous or endogenous that can be converted by living system into useful compounds or secondary metabolites. It has been possible to enhance the biosynthesis of specific secondary metabolites by feeding precursors to cell cultures. For example amino acids have been added to suspension culture media for production of tropane alkaloids indole alkaloids. The amount of precursors is usually lower in callus and cell cultures than in differentiated tissues. Phenylalanine acts as a precursor of rosmarinic acid addition of phenylalanine to Salvia officinalis suspension cultures stimulated the production of rosmarinic acid and decreased the production time as well. Phenylalanine also acts as precursor of the N- benzoylphenylisoserine side chain of taxol supplementation of Taxus cuspidata cultures with phenylalanine resulted in increased yields of taxol. The timing of precursor addition is critical for an optimum effect. The effects of feedback inhibition must surely be considered when adding products of a metabolic pathway to cultured cells. 4.5. Permeabilisation Secondary metabolites produced in cells are often blocked in the vacuole. By manipulating the permeability of cell membrane they can be secreted out to the media. Permeabilisation can be achieved by electric pulse UV pressure sonication heat etc. Even charcoal can be added to medium to absorb secondary metabolites. 4.6. Immobilisation Cell cultures encapsulated in agarose and calcium alginate gels or entrapped in membranes are called immobilised plant cell cultures. Immobilization of plant cells allows better cell to cell contact and the cells are also protected from high shear stresses. These immobilized systems can effectively increase the productivity of secondary metabolites in a number of species. Elicitors can also be added to these systems to stimulate secondary metabolism.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY 4.7. Limitations • Production cost is often very high. • Lack of information of the biosynthetic pathways of many compounds is a major drawback in the improvement of their production. • Trained technical manpower is required to operate bioreactors. 5. Advantages of cell tissue and organ cultures as sources of secondary metabolites 5.1. Plant cell cultures Once interesting bioactive compounds have been were identified from plant extracts the first part of the work consisted in collecting the largest genetic pool of plant individuals that produce the corresponding bioactive substances. However a major characteristic of secondary compounds is that their synthesis is highly inducible therefore it is not certain if a given extract is a good indicator of the plant potential for producing the compounds. The ability of plant cell cultures to produce secondary metabolites came quite late in the history of in vitro techniques. For a long time it was believed that undifferentiated cells such as callus or cell suspension cultures were not able to produce secondary compounds unlike differentiated cells or specialized organs. 5.2. Callus culture Callus is a mass of undifferentiated cells derived from plant tissues for use in biological research and biotechnology. In plant biology callus cells are those cells that cover a plant wound. To induce callus development plant tissues are surface sterilized and then plated onto in vitro tissue culture medium. Different plant growth regulators such as auxins cytokinins and gibberellins are supplemented into the medium to initiate callus formation. It is well known that callus can undergo somaclonal variations usually during several subculture cycles. This is a critical period where due to in vitro variations production of secondary metabolite often varies from one subculture cycle to another. When genetic stability is reached it is necessary to screen the different cell callus lines according to their aptitudes to provide an efficient secondary metabolite production. Hence each callus must be assessed separately for its growth rate as well as intracellular and extracellular metabolite concentrations. This allows an evaluation of the productivity of each cell line so that only the best ones will be taken for further studies for example for production of the desired compound in suspensions cultures. 5.3. Cell suspension cultures Cell suspension cultures represent a good biological material for studying biosynthetic pathways. They allow the recovery of a large amount of cells from which enzymes can be easily separated. Compared to cell growth kinetics which is usually an exponential curve most secondary metabolites are often produced during the stationary phase. This lack of production of compounds during the early stages can be explained by carbon allocation mainly distributed for primary metabolism when growth is very active. On the other hand when growth stops carbon is no longer required in large quantities for primary metabolism and secondary compounds are more actively synthesized. However some of the secondary plant products are known to be growth-associated with undifferentiated cells such as betalains and carotenoids. 5.4. Organ cultures

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Plant organs are alternative to cell cultures for the production of plant secondary metabolites. Two types of organs are generally considered for this objective: hairy roots and shoot cultures. A schematic representation of various organized cultures induced under in vitro conditions is given in Figure 31.3. 5.4.1. Shoot cultures Shoots exhibit some comparable properties to hairy roots genetic stability and good capacities for secondary metabolite production. They also provide the possibility of gaining a link between growth and the production of secondary compounds. 5.4.2. Hairy root cultures Hairy roots are obtained after the successful transformation of a plant with Agrobacterium rhizogenes. They have received considerable attention of plant biotechnologists for the production of secondary compounds. They can be subcultured and indefinitely propagated on a synthetic medium without phytohormones and usually display interesting growth capacities owing to the profusion of lateral roots. This growth can be assimilated to an exponential model when the number of generations of lateral roots becomes large. Cell Suspension culture

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY  Tissues and cells cultured in a agitated liquid medium produce a suspension of single cells and cells clumps of few to may cell these are called suspension cultures. PROTOPLAST CULTURES  Isolated protoplasts have been described as "naked" cells because the cell wall has been removed by either a mechanical or an enzymatic process.  Protoplasts can be induced to reform a cell and divide if placed in a suitable nutrient medium than form callus.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Ovary/ovule culture Ovary or ovule culture involves development of haploid from unfertilized cells of embryosac present in ovary.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY CELLULAR TOTIPOTENCY In the preceding units of this course you have read that innumerable cells which constitute the body of a higher plant or animal and containing identical genetic material can be traced to a single cell-the zygote. During development cells undergo diverse structural and functional specialisation depending upon their position in the body. Leaf cells bear chloroplasts and act as the site of photosynthesis. The colourless root hairs perform the function of absorbing nutrients and water from the soil and some other cells become part of the colourful petals. Normally fully differentiated cells do not revert back to a meristematic: state which suggests that the cells have undergone a permanent change. In earlier sections of this unit you have read that the regenerative capacity is retained by all living cells of a plant. Several horticultural plants regenerate whole plant from root leafiand stem cuttings. Highly differentiated and mature cells such as those of pith and cortex and highly specialised cells as those of microspores and endospermretain full potential to give rise to full plants under suitable culture conditions. G. Haberlandt was the first to test this idea experimentally. This endowment called "cellular totipotency" is unique to plants. Animal cells possibly because of their higher degree of specialisation do not exhibit totipotency. Whole plant regeneration from cultured cells may occur in one of the two pathways: shoot bud differentiation organogenesis and ii embryo formation Embryogenesis. The Embryos are bipolar structures with no organic connection with the parent tissue and can germinate directly into a complete plant. On the other hand shoots are monopolar. They need to be removed from the parent tissue and rooted to establish a plantlet. Often the same tissue can be induced to form shoots or embryos by manipulating the components of the culture conditions. In the following sub sections we will discuss organogenesis and embryogenesis in detail. Organogenesis Organogenesis refers to the differentiation of organs such as roots shoots or flowers. Shoot bud differentiation may occur directly from the explant or from the callus. The stimulus for organogenesis may come from the medium from the endogenous compounds produced by the cultured tissue or substances carried over from the original explant. Organogenesis is chemically controlled by growth regulators. Skoog while working with tobacco pith callus observed that the addition of an auxin Indole Acetic Acid IAA enhanced formation of roots and suppressed shoot differentiation. He further observed that adenine sulphate Cytokinin reversed the inhibition of auxin and promoted the formation of shoots. You should know that:

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY 1 Organogenesis is contolled by a balance between cytokinin and auxin concentration i.e. it is their relative rather than the absolute concentration that determines the nature of differentiation. 2. A relatively high auxin: Cytokinin ration induces root formation whereas a high cytokinin: auxin ratio favours shoot bud differentiation. 3. Differential response to exogenously applied growth regulators may be due to differences in the endogenous levels of the hormones within the tissue. Organogenesis is a complex process. Whereas in the cultured tissues of many species organogeiiesis can be demonstrated in this pattern some plants notably the monocots are exceptions. Somatic Embryogenesis The process of embryo development is called embryogenesis. It is not the monopoly of the egg to form an embryo. Any cell of the female gametophyte Embryo sac or even of the sporophytic tissues around the embryo sac may give rise to an embryo. Thus we can say that The phenomenon of embryogenesis is not necessarily confined to the reproductive cycle". In this subsection we will discuss -- some examples of "embeos formed in culture" also referred to as "somatic - embryos". The first observation of somatic embryos were made m Dacus Carota. Other plants in which the phenomenon has been studied in some detail are Ranunculus scleratus citrus and coflea spp. In Rarrunculus scleratus somatic as well as various floral tissues including anthers proliferated to form callus which after limited unorganised growth differentiated several embryos. These embryos germinated in situ and a fresh crop of embryos appeared on the surface of the seedling. The embryos were derived from individual epidernal cells of the hypocotyl Citrus is commonly cited as an example of natural polyembryony Figure 31.3: Guidelines for the production of secondary metabolites from plant organ cultures. 1. Laboratory Design and Development

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY The size of tissue culture lab and the amount and type of equipment used depend upon the nature of the work to be undertaken and the funds available. A standard tissue culture laboratory should provide facilities for: • washing and storage of glassware plasticware • preparation sterilization and storage of nutrient media • aseptic manipulation of plant material • maintenance of cultures under controlled temperature light and humidity • observation of cultures data collection and photographic facility • acclimatization of in vitro developed plants. The overall design must focus on maintaining aseptic conditions. At least three separate rooms should be available one for washing up storage and media preparation the media preparation room a second room containing laminar-air-flow or clean air cabinets for dissection of plant tissues and subculturing dissection room or sterilization room and the third room to incubate cultures culture room. This culture room should contain a culture observation table provided with binoculars or stereozoom microscope and an adequate light source. Additionally a green house facility is required for hardening-off in vitro plantlets. For a commercial set-up a more elaborate set-up is required. 1.1. Media preparation room The washing area in the media room should be provided with brushes of various sizes and shapes a large sink preferably lead-lined to resist acids and alkalis and running hot and cold water. It should also have large plastic buckets to soak the labware to be washed in detergent hot-air oven to dry washed labware and a dust-proof cupboard to store them. If the preparation of the medium and washing of the labware are done in the same room a temporary partition can be constructed between the two areas to guard any interference in the two activities. A continuous supply of water is essential for media preparation and washing of labware. A water distillation unit of around 2 litre/h a Milli-Q water purification systems needs to be installed.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Figure 2.1: A floor plan for plant tissue culture laboratory 1.2. Culture room The room for maintaining cultures should be maintained at temperature 25 ±2°C controlled by air conditioners and heaters attached to a temperature controller are used. For higher or lower temperature treatments special incubators with built-in fluorescent light can be used outside the culture room. Cultures are generally grown in diffuse light from cool white fluorescent tubes. Lights can be controlled with automatic time clocks. Generally a 16-hour day and 8-hour nights are used. The culture room requires specially designed shelving to store cultures. Some laboratories have shelves along the walls others have them fitted onto angle-iron frames placed in a convenient position. Shelves can be made of rigid wire mesh wood or any building material that can be kept clean and dust-free. Insulation between the shelf lights and the shelf above will ensure an even temperature around the cultures. While flasks jars and petridishes can be placed directly on the shelf or trays of suitable sizes culture tubes require some sort of support. Metallic wire racks or polypropylene racks each with a holding capacity of 18-24 tubes are suitable for the purpose. 1.3. Dissection room or sterilization room This area should have restricted entry which is needed to ensure the sterile conditions required for the transfer operations. For sterile transfer operations the laminar-air-flow cabinets are used. Temperature control is essential in this room as the heat is produced continuously from the flames of burners in the hoods. The room should be constructed in a way to minimize the dust particles and for easy cleaning. Several precautions can be taken including the removal of shoes before entering the area. The laminar horizontal flow sterile transfer cabinets are available in various sizes from many commercial sources. They should be designed with horizontal air flow from the back to the front and equipped with gas cocks if gas burners are to be used. Electrical outlets are needed for use of electric sterilizers and microscopes and if weighing is to be done in the hoods. A stainless steel working platform is most durable easy to keep clean and to prevent the unwanted damage due to accidental fire. Sometimes it is fitted with

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Ultraviolet light to maintain sterility inside the cabinet. UV light is a source of ozone which can be mutagenic therefore utmost care is to be taken while using this. Although UV light is not necessary a short exposure time of 3-5 min to cabinet is fine sometimes. Work can be started after 10-15 min of switching on the air flow and one can work uninterrupted for long hours. A Laminar-air-flow cabinet has small motor to blow air which first passes through a coarse filter where it loses large particles and subsequently through a fine filter known as „high efficiency particulate air HEPA. The HEPA filters remove particles larger than 0.3 µm and the ultraclean air flows through the working area. The velocity of the ultra clean air is about 27 ± 3 m min -1 which is adequate for preventing the contamination of the working area as long as the flow is on. The flow of the air does not in any way hamper the use of a spirit lamp or a Bunsen burner. 1.4. Greenhouse The greenhouse facility is required to grow parent pants and to acclimatize in vitro raised plantlets. The size and facility inside the green house vary with the requirement and depends on the funds available with the laboratory. However minimum facilities for maintaining humidity by fogging misting or a fan and pad system reduced light cooling system for summers and heating system for winters must be provided. It would be desirable to have a potting room adjacent to this facility. 1.5. Equipments and apparatus 1.4.1. Media preparation area • benches at a height suitable to work while standing • pH meter is used to determine the pH of various media used for tissue culture. pH indicator paper can also be used for the purpose but it is less accurate. The standard media pH is maintained at 5.8. • hot-plate-cum-magnetic stirrer for dissolving chemicals and during media preparation • an autoclave or domestic pressure cooker is crucial instrument for a tissue culture laboratory. High pressure heat is needed to sterilize media water labware forceps needles etc. Certain spores from fungi and bacteria can only be killed at a temperature of 121°C and 15 pounds per square inch psi for 15-20 min. A caution should be taken while opening the door of autoclave and it should be open when the pressure drops to zero. Opening the door immediately can lead to a rapid change in the temperature resulting in breakage of glassware and steam burning of operator. • plastic carboys for storing distilled water required for media preparation and final washing of labware. • balances near dry corner of the media room. High quality microbalance are required to weigh smallest of the quantities. Additionally a top pan balance is required for less sensitive quantities. • hot-air oven to keep autoclaved medium warm before pouring into vessels. It is also used for the dry heat sterilization of clean glassware like Petridishes culture tubes pipettes etc. Typical sterilizing conditions are 160-170 °C/1hr. • Dish washer for cleaning glass pipettes in running water

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY 1.4.2. Storage area • a deep freezer -20°C to -80°C / refrigerator for storage of enzyme solutions stock solutions plant materials and all temperature-sensitive chemicals. • microwave oven to melt agar solidified media • Upright and inverted light microscope with camera attachment for recording the morphogenic responses from various explants calli cells and protoplasts. Inverted microscope gives the clear views of cultures settled at the bottom of Petridishes. 1.4.3. Dissection room • laminar-air-flow cabinet within which tissue culture work can be carried out under sterilized environment • glass bead sterilizer where temperature of beads is raised to 250°C in 15-20 min with 15 s cut off. Here the sterilization of instruments is effecting by pushing them into the beads for 5-7 s. This is much safer compare to the Bunsen burner heating of instruments like forceps needles scalpels etc. • binocular microscope to observe surface details and morphogenic responses of cultures and their possible contamination. • low speed table-top centrifuge to sediment cells or protoplasts 1.4.4. Culture room • air or heating / cooling system to maintain 25±2 °C temperature • racks for holding test-tubes • lights to provide diffuse light and to maintain photoperiod • shakers with various sized clamps for different sized flasks to grow cells in liquid medium • thermostat and time clock for lights • wall cabinets for dark incubation of cultures 1.4.5. Other apparatus • beakers 100 mL 250 mL 1 L 5 L • measuring cylinders 5 mL 10 mL 25 mL 50 mL 100 mL 500 mL 1L 2 L 5 L • graduated pipettes and teats • reagent bottles for storing liquid chemicals and stock solutions glass or plastic • culture tubes and flasks glass or polypropylene or disposable

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY • plastic baskets • filter membrane preferably nylon of sizes 0.22 µm and 0.45 µm holders and hypodermic syringes for solutions requiring filter sterilization • large forceps blunt and fine points and scalpels for dissecting and subculturing plant material. • Scalpel handles no. 3 and blades no. 11 • Chemicals and reagents for preparing culture media • Disposable gloves and masks. • Micropipettes of maximum volume size 5000 µL 1000 µL 500 µL 250 µL 100 µL A Syringe with filter assembly fitted on conical flask B Disassembled filter assembly Forceps and scalpels for dissection Micropipettes . Tissue culture media 1. Preparation and handling The simplest method of preparing media is to use commercially available dry powdered media containing mineral elements and growth regulators. By following the procedure written on the packets dissolve the powder in distilled or demineralized water 10 less than the final volume of the medium. After adding sugar and other desired supplements like plant growth regulators make up the final volume with distilled water adjust the pH add agar and then autoclave the medium. An alternative method of media preparation is to prepare a series of concentrated stock solutions which can be combined later as required. For preparing stock solutions and media use glass-distilled or demineralized water and chemicals of high purity analytical reagent AR grade. 1.1. Composition of widely used tissue culture media Both the media listed in the below tables 2 3 can be prepared from stock solutions of: i. Macronutrients: As its name suggests in plant tissue culture media these components provide the elements which are required in large amounts concentrations greater than 0.5 mmole l -1 by cultured plant cells. Macronutrients are usually considered to be carbon nitrogen phosphorous magnesium potassium calcium and sulphur. ii. Micronutrients: It provides the elements that are required in trace amounts concentrations less than 0.5 mmole l -1 for plant growth and development. These include manganese copper cobalt boron iron molybdenum zinc and iodine.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY iii. Iron source: It is considered the most important constituent and required for the formation of several chlorophyll precursors and is a component of ferredoxins proteins containing iron which are important oxidation : reduction reagents. iv. Organic supplements vitamins: Like animals in plants too vitamins provide nutrition for healthy growth and development. Although plants synthesize many vitamins under natural conditions and therefore under in vitro conditions they are supplied from outside to maintain biosynthetic capacity of plant cells in vitro. There are no firm rules as to what vitamins are essential for plant tissues and cell cultures. The only two vitamins that are considered to be essential are myo-inositol and thiamine. Myo-inositol is considered to be vitamin B and has many diverse roles in cellular metabolism and physiology. It is also involved in the biosynthesis of vitamin C. v. Carbon source: This is supplied in the form of carbohydrate. Plant cells and tissues in the culture medium are heterotrophic and are dependent on external source of carbon. Sucrose is the preferred carbon source as it is economical readily available relatively stable to autoclaving and readily assimilated by plant cells. During sterilization by autoclaving of medium sucrose gets hydrolyzed to glucose and fructose. Plant cells in culture first utilize glucose and then fructose. Besides sucrose other carbohydrates such as lactose maltose galactose are also used in culture media but with a very limited success. Table 3.1: The media elements and their functions The steps involved in preparing a medium are summarized below: Add appropriate quantities of various stock solutions including growth regulators and other special supplements. Make up the final volume of the medium with distilled water. Add and dissolve sucrose. After mixing well adjust the pH of the medium in the range of 5.5-5.8 using 0.1 N NaOH or 0.1 N HCl above 6.0 pH gives a fairly hard medium and pH below 5.0 does not allow satisfactory gelling of the agar. Add agar stir and heat to dissolve. Alternatively heat in the autoclave at low pressure or in a microwave oven. Once the agar is dissolved pour the medium into culture vessels cap and autoclave at 121°C for 15 to 20 min at 15 pounds per square inch psi. If using pre-sterilized non-autoclavable plastic culture vessels the medium may be autoclaved in flasks or media bottles. After autoclaving allow the medium to cool to around 60°C before pouring under aseptic conditions.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Allow the medium to cool to room temperature. Store in dust-free areas or refrigerate at 7°C temperature lower than 7°C alter the gel structure of the agar. 1.2. Gelling agents The media listed above are only for liquids often in plant cell culture a „semi-solid medium is used. To make a semi-solid medium a gelling agent is added to the liquid medium before autoclaving. Gelling agents are usually polymers that set on cooling after autoclaving. i. Agar: Agar is obtained from red algae- Gelidium amansii . It is a mixture of polysaccharides. It is used as a gelling agent due to the reasons: a It does not react with the media constituents b It is not digested by plant enzymes and is stable at culture temperature. ii. Agarose: It is obtained by purifying agar to remove the agaropectins. This is required where high gel strength is needed such as in single cell or protoplast cultures. iii. Gelrite: It is produced by bacterium Pseudomonas elodea . It can be readily prepared in cold solution at room temperature. It sets as a clear gel which assists easy observation of cultures and their possible contamination. Unlike agar the gel strength of gelrite is unaffected over a wide range of pH. However few plants show hyperhydricity on gelrite due to freely available water. iv. Gelatin: It is used at a high concentration 10 with a limited success. This is mainly because gelatin melts at low temperature 25°C and as a result the gelling property is lost.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY 1.3. Plant growth regulators In addition to nutrients four broad classes of growth regulators such as auxins cytokinins gibberellins and abscisic acid are important in tissue culture. In contrast with animal hormones the synthesis of a plant growth regulator is often not localized in a specific tissue but may occur in many different tissues. They may be transported and act in distant tissues and often have their action at the site of synthesis. Another property of plant growth regulators is their lack of specificity- each of them influences a wide range of processes. The growth differentiation organogenesis and embryogenesis of tissues become feasible only on the addition of one or more of these classes of growth regulators to a medium. In tissue culture two classes of plant growth regulators cytokinins and auxins are of major importance. Others in particular gibberellins ethylene and abscisic acid have been used occasionally. Auxins are found to influence cell elongation cell division induction of primary vascular tissue adventitious root formation callus formation and fruit growth. The cytokinins promote cell division and axillary shoot proliferation while auxins inhibit the outgrowth of axillary buds. The auxin favours DNA duplication and cytokinins enable the separation of chromosome. Besides cytokinin in tissue culture media promote adventitious shoot formation in callus cultures or directly from the explants and occasionally inhibition of excessive root formation and are therefore left out from rooting media. The ratio of plant growth regulators required for root or shoot induction varies considerably with the tissue and is directly related to the amount of growth regulators present at endogenous levels within the explants. In general shoots are formed at high cytokinin and low auxin concentrations in the medium roots at low cytokinin and high auxin concentrations and callus at intermediate concentrations of both plant growth regulators. Commonly used plant growth regulators are listed in Table 4. Stock solutions of growth regulators

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY 1 molar the molecular weight in g/l 1 mM the molecular weight in mg/l ppm parts per million mg/l 2. Establishing aseptic cultures Plant tissue culture media contain sugar and so support the growth of many microorganisms bacteria and fungi. When these microorganisms reach a medium they generally grow much faster than the cultured plant materials. Their growth and toxic metabolites will affect and may even kill the tissue cultures. It is therefore essential to maintain a completely aseptic environment inside the culture vessels. There are several possible sources of contamination of the medium: • the culture vessel • the medium itself • the explant plant tissue • the environment of the transfer area • the instruments used to handle plant material during establishment and subculture • the environment of the culture room. Autoclaving media will eliminate contamination from the culture vessel or the medium. In some cases substances such as gibberellic acid abscisic acid ABA urea and certain vitamins are thermolabile and break down upon autoclaving. These chemicals can be sterilized by membrane filtration using microfilters of pore size 0.22-0.45 µm which is suitable enough to exclude pathogens. Later the filter sterilized compound can be added to autoclaved medium cooled to around 40°C. To prevent the environment of the culture room from being the source of contamination keep the culture room as dust- free as possible and remove contaminated cultures from the area as soon as they are detected. Ideally the culture room should be clean filtered air which has passed through high efficiency particulate air HEPA filters. The transfer area in most laboratories is within a laminar air-flow cabinet. A laminar air-flow cabinet has a small fan which blows air through a coarse filter to remove large dust particles and then through a fine HEPA filter to remove microbes their spores and other particles larger than 0.3 µm. The velocity of the air coming out of the fine filter is about 27 ± 3 m/min which keeps airborne microorganisms out of the working area. The working area is swabbed with 70 alcohol or equivalent and instruments dipped in 70 alcohol flamed and cooled before use. Caution : Prolonged contact with alcohol can cause skin irritation and other health problems can result from the inhalation of fumes. Use ethanol rather than methanol and surgical gloves when handling. Take care

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY with ultraviolet light as it can permanently damage eyes and promote skin cancer. Laminar flow cabinets equipped with ultraviolet light for surface sterilization should be fitted with safety doors which can be closed when ultraviolet light is used. Plant surfaces carry a wide range of microorganisms. The tissue must be thoroughly surface-sterilized before being placed on the nutrient medium. Discard cultures with fungal or bacterial contamination. Solutions of sodium or calcium hypochlorite are usually effective in disinfecting plant tissues. Placing tissues in a 0.5 to 1 solution of sodium hypochlorite for 10 to 15 minutes will disinfect most tissues. Surface sterilants are toxic to plant tissues. Choose the concentration of the sterilizing agent and the length of time to minimize tissue damage which shows up as white bleached areas. Other techniques for surface sterilisation include dipping plant material for a few seconds in 90 ethanol or placing in running water for 30 minutes and 2 hours before disinfection. Caution : Take care with powdered calcium hypochlorite as it is a powerful reducing agent. If calcium hypochlorite is stored moist and the container opened later it can explode. Store calcium hypochlorite in a sealed container in a dry place. A summary of the six steps commonly involved in establishing and maintaining aseptic plant tissue culture follows. i. Collect pieces of plant material ex-plants in a screw-cap bottle. Immerse them in a dilute solution of the disinfectant containing a wetting agent. Replace the lid and store the bottle in the laminar air flow cabinet. Shake the bottle two or three times during the sterilization period. ii. Remove the lid and drain carefully. Thoroughly rinse the plant material in sterilized distilled water and replace the lid. After shaking a few minutes discard the water. Rinse two or three times more. iii. Transfer the material to a pre-sterilized Petri-dishes or test-tubes. iv. Sterilize the required instruments by dipping them in 70 ethanol and flamed them. Allow to cool. Sterilize the instruments after each time they are used to handle tissue. v. Prepare suitable explants from the surface sterilized material using sterilized instruments scalpels needles forceps etc.. vi. Quickly remove the lid of the culture vessel transfer the explants on to the medium flame the neck of the vessel only if glass and replace the lid. If handling aseptic plant materials during routine subculture omit the first two steps. Plant tissue culture techniques 1. Introduction Plant tissue culture has become popular among horticulturists plant breeders and industrialists because of its varied practical applications. It is also being applied to study basic aspects of plant growth and development. The discovery of the first cytokinin kinetin is based on plant tissue culture research.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY The earliest application of plant tissue culture was to rescue hybrid embryos Laibach 1925 1929 and the technique became a routine aid with plant breeders to raise rare hybrids which normally failed due to post- zygotic sexual incompatibility. Currently the most popular commercial application of plant tissue culture is in clonal propagation of disease-free plants. In vitro clonal propagation popularly called micropropagation offers many advantages over the conventional methods of vegetative propagation: 1 many species e.g. palms papaya which are not amenable to in vivo vegetative propagation are being multiplied in tissue cultures 2 the rate of multiplication in vitro is extremely rapid and can continue round the year independent of the season. Thus over a million plants can be produced in a year starting from a small piece of tissue. The enhanced rate of multiplication can considerably reduce the period between the selection of plus trees and raising enough planting material for field trials. In tissue culture propagation occurs under pathogen and pest-free conditions. An important contribution made through tissue culture is the revelation of the unique property of plant cells called “cellular totipotency”. The totipotency of plant cells was predicted in 1902 by Haberlandt and the first true plant tissue culture on agar was established. Since then plant tissue culture techniques have greatly evolved. The technique has developed around the concept that a cell has the capacity and ability to develop into a whole organism irrespective of their nature of differentiation and ploidy level. Therefore it forms the backbone of the modern approach to crop improvement by genetic engineering. The principles involved in plant tissue culture are very simple and primarily an attempt whereby an explant can be to some extent freed from inter-organ inter-tissue and inter-cellular interactions and subjected to direct experimental control. Regeneration of plants from cultured cells has many other applications. Plant regeneration from cultured cells is proving to be a rich source of genetic variability called “somaclonal variation”. Several somaclones have been processed into new cultivars. Regeneration of plants from microspore/pollen provides the most reliable and rapid method to produce haploids which are extremely valuable in plant breeding and genetics. With haploids homozygosity can be achieved in a single step cutting down the breeding period to almost half. This is particularly important for highly heterozygous long-generation tree species. Pollen raised plants also provide a unique opportunity to screen gametic variation at sporophytic level. This approach has enabled selection of several gametoclones which could be developed into new cultivars. Even the triploid cells of endosperm are totipotent which provides a direct and easy approach to regenerate triploid plants difficult to raise in vivo. The entire plant tissue culture techniques can be largely divided into two categories based on to establish a particular objective in the plant species: I. Quantitative Improvement  Micropropagation Adventitious shoot proliferation leaves roots bulbs corm seedling- explants etc. Nodal segment culture Meristem/Shoot-tip culture Somatic embryogenesis Callus culture

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY II. Qualitative Improvement  Anther/ Microspore culture Ovary/ Ovule culture Endosperm culture Cell culture Protoplast culture The above techniques are discussed in detail in subsequent chapters. 2. Micropropagation Growing any part of the plant explants like cells tissues and organs in an artificial medium under controlled conditions aseptic conditions for obtaining large scale plant propagation is called micropropagation. The basic concept of micropropagation is the plasticity totipotency differentiation dedifferentiation and redifferentiation which provide the better understanding of the plant cell culture and regeneration. Plants due to their long life span have the ability to withhold the extremes of conditions unlike animals. The plasticity allows plants to alter their metabolism growth and development to best suit their environment. When plant cells and tissues are cultured in vitro they generally exhibit a very high degree of plasticity which allows one type of tissue or organ to be initiated from another type. Hence whole plants can be subsequently regenerated and this regenerated whole plant has the capability to express the total genetic potential of the parent plant. This is unique feature of plant cells and is not seen in animals. Unlike animals where differentiation is generally irreversible in plants even highly mature and differentiated cells retain the ability to regress to a meristematic state as long as they have an intact membrane system and a viable nucleus. However sieve tube elements and xylem elements do not divide any more where the nuclei have started to disintegrate According to Gautheret 1966 the degree of regression a cell can undergo would depend on the cytological and physiological state of the cell. The meristematic tissues are differentiated into simple or complex tissues called differentiation. Reversion of mature tissues into meristematic state leading to the formation of callus is called dedifferentiation. The ability of callus to develop into shoots or roots or embryoid is called redifferentiation. The inherent potentiality of a plant cell to give rise to entire plant and its capacity is often retained even after the cell has undergone final differentiation in the plant system is described as cellular totipotency. 2.1. Micropropagation vs. conventional method of propagation All living plant cells irrespective of their nature of specialization and ploidy level have been shown to regenerate plants via organogenesis or embryogenesis. The latter involves a highly specialized mode of development that normally occurs only inside the seed under the cover of several layers of parental tissues. Consequently the observation of developing embryos and their isolation in intact and living conditions for experimental studies have been extremely difficult. In vitro production of embryos from somatic and gametic cells has opened up the possibility of obtaining large numbers of embryos of different stages enabling investigations on cellular genetic and physiological control of embryogenesis induction pattern formation organ differentiation and maturation. In vitroexpression of cellular totipotency and other techniques of plant tissue culture have also facilitated and/ or accelerated the traditional methods of plant improvement propagation and conservation.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY 2.2. Micropropagation vs. vegetative propagation The vegetative propagation has been conventionally used to raise genetically uniform large scale plants for thousands of years. However this technique is applicable to only limited number of species. In contrast to this micropropagation has several advantages which are summarized here: i. The rapid multiplication of species difficult to multiply by conventional vegetative means. The technique permits the production of elite clones of selected plants. ii. The technique is independent of seasonal and geographical constraints. iii. It enable large numbers of plants to be brought to the market place in lesser time which results in faster return on the investment that went into the breeding work. iv. To generate disease-free particularly virus-free parental plant stock. v. To raise pure breeding lines by in vitro haploid and triploid plant development in lesser time. vi. It can be utilized to raise new varieties and preservation of germplasm vii. It offers constant production of secondary medicinal metabolites. 2.3. Cell differentiation During in-vitro and in vivo cytodifferentiation cell differentiation the main emphasis has been on vascular differentiation especially tracheary elements TEs. These can be easily observed by staining and can be scored in macerated preparations of the tissues. Tissue differentiation goes on in a fixed manner and is the characteristic of the species and the organs 2.4. Factors affecting vascular tissue differentiation Vascular differentiation is majorly affected qualitatively and quantitatively by two factors auxin and sucrose. Cytokinins and gibberellins also play an important role in the process of xylogenesis. Depending upon the characteristics of different species concentration of phytohormones sucrose and other salt level varies and accordingly it leads to the vascular tissue differentiation. 3. Micropropagation techniques 3.1. Strategies for propagation in vitro Typical micropropagation system can be broadly divided into five distinct stages Figure 4.1:  The stage zero is the selection of mother plant and preparation of explant. The first stage is the initiation of a sterile culture of the explant in a particular enriched medium for specific species. The second stage includes initiation of cell division from almost any part of the plant system to initiate regeneration or multiplication of shoots or other propagules from the explant. Adventitious shoot proliferation is the most frequently used multiplication technique in micropropagation systems. The culture media and growth conditions used in second stage need to be optimized for maximum rate of multiplication.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY The third stage is the development of roots on the shoots to produce plantlets. Specialized media may or may not be required to induce roots depending upon the species. The final or the fourth stage is to produce self-sufficient plants. This stage usually involves a hardening-off process and acclimatization of plants in soil under green-house conditions for later transplanting to the field. Mode of differentiation Regenerants may differentiate either directly from the explants or indirectly via callusing. Dedifferentiation favours unorganized cell growth and the resultant developed callus has meristems randomly distributed in the callus. Most of these meristems if provided appropriate invitro conditions would differentiate shoot- buds roots or embryos. Figure 4.1: Micropropagation stages 4. Trouble shooting  • Few explants exude dark colored compounds like phenols pigments etc which leach into the medium from the cut ends of the explant. It results in the browning of tissues and the medium as well. The browning of medium is associated with poor culture establishment and low regeneration capacity of the explants. This can be overcome by: i. minimizing the wounding of explants during isolation and surface disinfection to reduce this browning response. ii. washing or incubation of explants for 3-5 hrs in sterile distilled water to remove phenolics responsible for browning of medium or explants. iii. frequent subculture of explants with excision to fresh medium at regular intervals.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY iv. initial establishment of cultures in liquid medium and later transfer to the semi-solid medium. vi. culture of explants on porous substrate or paper bridges. vi. addition of activated charcoal AC or polyvinylpyrrolidone PVP for adsorbtion of phenolics. vii. antioxidants like ascorbic acid citric acid etc. can also be used to prevent browning of tissues in culture. • Appearance of vitrified tissues hyperhydricity a physiological disorder occurring in the in vitro cultures due to which the tissues look transparent and fluffy resulting from excessive intake of water. Hyperhydricity can be caused by a high concentration of cytokinin or low concentration of gelling agent or high water retention capacity of explants if the container is tightly closed. • Loss of regeneration ability in long-term cultures due to epigenetic variations temporary variations and culture aging including transition from juvenile to mature stage. Epigenetic variation are phenotypic temporary variations which disappear as soon as the culture conditions are removed. • Genotypic variations are also seen in the cultures therefore cytological biochemical and molecular analyses are required to confirm clonal fidelity of in vitro regenerants. Besides morphological and physiological testing is also required to remove undesired genetic variability. Plant tissue culture Plant tissue culture is a collection of techniques used to maintain or grow plant cells tissues or organs under sterile conditions on a nutrient culture medium of known composition. Plant tissue culture is widely used to produce clones of a plant in a method known as micropropagation. Different techniques in plant tissue culture may offer certain advantages over traditional methods of propagation including:  The production of exact copies of plants that produce particularly good flowers fruits or have other desirable traits.  To quickly produce mature plants.  The production of multiples of plants in the absence of seeds or necessary pollinators to produce seeds.  The regeneration of whole plants from plant cells that have been genetically modified.  The production of plants in sterile containers that allows them to be moved with greatly reduced chances of transmitting diseases pests and pathogens.  The production of plants from seeds that otherwise have very low chances of germinating and growing i.e.: orchids and Nepenthes.  To clean particular plants of viral and other infections and to quickly multiply these plants as cleaned stock for horticulture and agriculture. Plant tissue culture relies on the fact that many plant cells have the ability to regenerate a whole plant totipotency. Single cells plant cells without cell walls protoplasts pieces of leaves stems or roots can often be used to generate a new plant on culture media given the required nutrients and plant hormones. Techniques Modern plant tissue culture is performed under aseptic conditions under HEPA filtered air provided by a laminar flow cabinet. Living plant materials from the environment are naturally contaminated on their surfaces and sometimes interiors with microorganisms so surface sterilization of starting material explants in chemical solutions usually alcohol and sodium or calcium hypochlorite or mercuric chloride 1 is required. Mercuric chloride is seldom used as a plant sterilant today unless other sterilizing

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY agents are found to be ineffective as it is dangerous to use and is difficult to dispose of. Explants are then usually placed on the surface of a solid culture medium but are sometimes placed directly into a liquid medium particularly when cell suspension cultures are desired. Solid and liquid media are generally composed of inorganic salts plus a few organic nutrients vitamins and plant hormones. Solid media are prepared from liquid media with the addition of a gelling agent usually purified agar. The composition of the medium particularly the plant hormones and the nitrogen source nitrate versus ammonium salts or amino acids have profound effects on the morphology of the tissues that grow from the initial explant. For example an excess of auxin will often result in a proliferation of roots while an excess of cytokinin may yield shoots. A balance of both auxin and cytokinin will often produce an unorganised growth of cells or callus but the morphology of the outgrowth will depend on the plant species as well as the medium composition. As cultures grow pieces are typically sliced off and transferred to new media subcultured to allow for growth or to alter the morphology of the culture. The skill and experience of the tissue culturist are important in judging which pieces to culture and which to discard. As shoots emerge from a culture they may be sliced off and rooted with auxin to produce plantlets which when mature can be transferred to potting soil for further growth in the greenhouse as normal plants. Choice of explant The tissue obtained from a plant to be cultured is called an explant based on work with certain model systems particularly tobacco it has often been claimed that a totipotent explant can be grown from any part of the plant and may include portions of shoots leaves stems flowers roots and single undifferentiated cells. citation needed however this has not been true for all plants. 3 In many species explants of various organs vary in their rates of growth and regeneration while some do not grow at all. The choice of explant material also determines if the plantlets developed via tissue culture are haploid or diploid. Also the risk of microbial contamination is increased with inappropriate explants. The specific differences in the regeneration potential of different organs and explants have various explanations. The significant factors include differences in the stage of the cells in the cell cycle the availability of or ability to transport endogenous growth regulators and the metabolic capabilities of the cells. The most commonly used tissue explants are the meristematic ends of the plants like the stem tip auxiliary bud tip and root tip. These tissues have high rates of cell division and either concentrate or produce required growth regulating substances including auxins and cytokinins. The pathways through which whole plants are regenerated from cells and tissues or explants such as meristems broadly fall into three types: 1. The method in which explants that include a meristem viz. the shoot tips or nodes are grown on appropriate media supplemented with plant growth regulators to induce proliferation of multiple shoots followed by rooting of the excised shoots to regenerate whole plants 2. The method in which totipotency of cells is realized in the form of de novo organogenesis either directly in the form of induction of shoot meristems on the explants or indirectly via a callus unorganised mass of cells resulting from proliferation of cells of the explant and plants are regenerated through induction of roots on the resultant shoots 3. Somatic embryogenesis in which asexual adventive embryos comparable to zygotic embryos in their structure and development are induced directly on explants or indirectly through a callus phase. The first method involving the meristems and induction of multiple shoots is the preferred method for the micropropagation industry since the risks of somaclonal variation genetic variation induced in tissue culture are minimal when compared to the other two methods. Somatic embryogenesis is a method that has

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY the potential to be several times higher in multiplication rates and is amenable to handling in liquid culture systems like bioreactors. Some explants like the root tip are hard to isolate and are contaminated with soil microflora that become problematic during the tissue culture process. Certain soil microflora can form tight associations with the root systems or even grow within the root. Soil particles bound to roots are difficult to remove without injury to the roots that then allows microbial attack. These associated microflora will generally overgrow the tissue culture medium before there is significant growth of plant tissue. Aerial above soil explants are also rich in undesirable microflora. However they are more easily removed from the explant by gentle rinsing and the remainder usually can be killed by surface sterilization. Most of the surface microflora do not form tight associations with the plant tissue. Such associations can usually be found by visual inspection as a mosaic de-colorization or localized necrosis on the surface of the explant. An alternative for obtaining uncontaminated explants is to take explants from seedlings which are aseptically grown from surface-sterilized seeds. The hard surface of the seed is less permeable to penetration of harsh surface sterilizing agents such as hypochlorite so the acceptable conditions of sterilization used for seeds can be much more stringent than for vegetative tissues. Tissue cultured plants are clones. If the original mother plant used to produce the first explants is susceptible to a pathogen or environmental condition the entire crop would be susceptible to the same problem. Conversely any positive traits would remain within the line also. Applications Plant tissue culture is used widely in the plant sciences forestry and in horticulture. Applications include:  The commercial production of plants used as potting landscape and florist subjects which uses meristem and shoot culture to produce large numbers of identical individuals.  To conserve rare or endangered plant species. 4  A plant breeder may use tissue culture to screen cells rather than plants for advantageous characters e.g. herbicide resistance/tolerance.  Large-scale growth of plant cells in liquid culture in bioreactors for production of valuable compounds like plant-derived secondary metabolites and recombinant proteins used as biopharmaceuticals. 5  To cross distantly related species by protoplast fusion and regeneration of the novel hybrid.  To cross-pollinate distantly related species and then tissue culture the resulting embryo which would otherwise normally die Embryo Rescue.  For production of doubled monoploid dihaploid plants from haploid cultures to achieve homozygous lines more rapidly in breeding programmes usually by treatment with colchicine which causes doubling of the chromosome number.  As a tissue for transformation followed by either short-term testing of genetic constructs or regeneration of transgenic plants.  Certain techniques such as meristem tip culture can be used to produce clean plant material from virused stock such as potatoes and many species of soft fruit.  Production of identical sterile hybrid species can be obtained.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Callus Culture: When the cells divide into an undifferentiated mass it is called as callus. Any part of a plant can be used to produce the calli. It may be a stem leaf meristem or any other part. It is used to produce variations among the plantlets. Callus formation is induced from plant tissues after surface sterilization and plating onto in vitro tissue culture medium. Plant growth regulators such as auxins cytokinins andgibberellins are supplemented into the medium to initiate callus formation or somatic embryogenesis. Plant callus is usually derived from somatic tissues. The tissues used to initiate callus formation depends on plant species and which tissues are available for explant culture. The cells that give rise to callus and somatic embryos usually undergo rapid division or are partially undifferentiated such as meristematic tissue. In alfalfaMedicago truncatula however callus and somatic embryos are derived from mesophyll cells that undergo dedifferentiation. 17 Plant hormones are used to initiate callus growth. Specific auxin to cytokinin ratios in plant tissue culture medium give rise to an unorganized growing and dividing mass of callus cells. Callus cultures are often broadly classified as being either compact or friable. Friable calluses fall apart easily and can be used to generate cell suspension cultures. Callus can directly undergo direct organogenesis and/or embryogenesis where the cells will form an entirely new plant. Callus induction and tissue culture A callus cell culture is usually sustained on gel medium. Callus induction medium consists of agar and a mixture of macronutrients and micronutrients for the given cell type. There are several types of basal salt mixtures used in plant tissue culture but most notably modified Murashige and Skoog medium 13 Whites medium 14 and woody plant medium. 15 Vitamins are also provided to enhance growth such as Gamborg B5 vitamins. 16 For plant cells enrichment with nitrogen phosphorus andpotassium is especially important. Callus cells deaths Callus can brown and die during culture but the causes for callus browning are not well understood. In Jatropha curcas callus cells small organized callus cells became disorganized and varied in size after

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY browning occurred. 18 Browning has also been associated with oxidation and phenolic compounds in both explant tissues and explant secretions. Suspension culture: The callus produced from the explants are grown on nutrient solutions that are semi solid for a period of time and they are induced to produce plants with new traits. A callus crumbles into smaller clumps and single cells in liqudmedium by gentle agitation 100-120rPM on a shaker. Shaking the cultures also helps to aerate the cells. Such suspension cultures however rarely comprise single cells alone because cells tend to aggregate in clusters of 2-100. Suspension cultures can be maintained indefinitely by inoculations of known aliquot5 of cells to a fresh medium. This process is termed as "batch cultures". Alternatively the medium is replenished at regu lar intervals. This process is termed as "continuous culture". In the continuous culture process at the time of replenishing the medium cells are also harvested open continuous system or the biomass is allowed tincrease close continuous system. Suspension cultures are useful in studying problems related to cell biology including cell cycle and production of secondary metabolites like alkaloids steroids glycosides napthaquinones flavones etc. which find medicinal and industrial application. Pharmaceutical industries use large bioreactors for suspension cultures to obtain valuable bioorganic compounds. A bioreactor is a vessel of glass or steel in which cells are cultured aseptically and culture conditions are closely monitored. This results in higher yield of metabolites. In a bioreactor there is provision for adding fresh medium for harvesting cells for the aeration of products for mixing and sampling for controlling pH 02 content and temperature Plant cells are immobilised in alginate agarose polyacrylamide beads. Immobilisation of cells enables i re-use of biomass by rotation of cells ii separation of cells from the medium and iii leaching of metabolites in it . Immobilised cells are cultured in column reactors. Column reactors are of different types with different agitation and flow systems. Such reactors may be i stirred tank type ii air lift type iii bubble column type and iv rotating drum type. 11.3.2 Single Cell Culture This is an important invitro technique which enables the cloning of selected cells. Single cells can be obtained directly from plant organs by treatment with enzymes that dissolve middle lamellae. The separate cells can sieve into liquid medium to start a suspension culture. The most widely used technique for single cell culture is the Bergmanns method of Cell Plating and. Microchamber technique. Bergmanns Method of Cell Plating: In this method free cells are suspended in a liquid medium at a density twice the Plant Tissue And Organ finally desired plating density. Melted agarcontaining medium of otherwise the Culture same composition as the liquid medium is maintained at 35Oc in water bath. Equal volumes of the two media are mixed and rapidly spread out in petri dishes in such a manner that the cells are evenly distributed and fixed in a thin layer about 1 mm thick of the medium after it has cooled and solidified. The dishes are sealed with parafilm. The cells to be followed are marked on the outside of the plate and before the colonies derived from individual cells grow large enough to merge with each other. They are transferred to.separate plates. Fig. 11.3. Another popular method for single cell culture is the microchamber technique developed by Jones et al. 1960. In this method mechanically isolated single cells are cultured in separate droplets of liquid medium. While Jones et al. used sterile microslides and three coverglasses to make microchamber it is now possible to buy pre-sterilised plastic plates with several microwells Cuprak dishes. Individual cells are cultured in separate wells each containing 0.25 ml of the liquid medium. The culture requirement of single cells increases with decrease in the plating cell density and the cell cultured in complete isolation require a very complex culture medium. A simple medium conditioned by growing cell suspension for some time rlso fulfils the requirements of single cell culture at low density

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Clonal Propagation Most cultivars of ornamental and fruit species and forest trees are highly heterozygous. Consequently their seed progeny is not true-to-type. To preserve the unique characters of selected cultivars of horticultural plants nurserymen practise vegetative propagation using stem leaf or root cuttings or propagules such as tubers corms bulbs or bulbils. For plants which do not set seeds such as edible bananasgrapes citrus petunia rose and chrysanthemum vegetative propagation is the only means of multiplication. A population of plants derived from a single individual by vegetative propagation is genetically uniform and is called a clone. The conventional methods of clonal propagation are slow and often not applicable. For example the only in-vivo method for clonal multiplication of cultivated orchids which are complex hybridsis "back- bulb" propagation. It involves separating the oldest pseudobulbil to force the development of dormant buds. This process allows at best doubling the plant number every year. Moreover Diagrammatic summary of steps involved in aseptic multiplication of plants. Shoot multiplication is achieved through enhanced axillary branching adventitious budding from explants directly or after callusing The shoots are rooted individually in a me- dium containing an auxin. The plantlets so obtained are transferred to well drained potting mix. After maintaining them under high humidity for3-4 weeks the plants are transferred to ordinary glasshouse or field conditions Plant multiplication involving a callus phase may occur via shoot bud differentiation or somatic embryogenesis. In the latter case the rooting step is eliminated as the embryos possess a pre-formed root primordiurn. monopodial orchids do not form pseudobulbils and therefore cannot be clonally multiplied. In 1960 a French scientist G.More1 described an in-vitro method for rapid clonal multiplication of orchids. This revolutionised the orchid industry and today tissue culture is the only economically feasible method for clonal multiplication of orchids and is being widely used. In-vitro clonal propagation popularly called Micropropagation has been extended to a large number of species other than orchids and is being practised on commercial scale for numerous ornamental and fruit bearing plant and some forest trees. After the initiation of aseptic cultures micropropagation generally involves three steps: Shoot multiplication rooting and transplantation. Shoot Multiplication: This is the most important step with respect to the rate of propagation and genetic uniformity of the product. The most reliable and therefore themost popular method of shoot multiplication is forced proliferation of axillary shoots. For this cultures are initiated from apical or nodal cuttings carrying one or more vegetative buds. In the presence of a cytokinin alone or in combination with a low concentration of an auxin such as IAA or NAA the pre-existing buds grow and produce 4-6 shoots sometimes up to 30-40 shoots within 3-4 weeks. By periodic removal of individual shoots and planting them on fresh medium of the original composition the shoot multiplication cycle can be repeated almost indefinitely and a stock of large number of shoots built up in a short period of time. Treatments with PGRs as described above can also help in a rapid build up of shoots by inducing adventitious buds by the explant directly or after callusing. Somatic embryogenesis which generally occurs after callusing of the explant is another method of micro propagation. Somatic embryogenesis is not only fast but may also allow partial automation of micropropagation and the propagules so produced somatic embryos bear both shoot and root meristems. However adventitive differentiation of shoots or somatic embryos especially from callus tissue has the risk of genetic variability in the progeny. Such variation that develops in tissue culture called "somaclonal variation" is not desirable for micropropagation but is being exploited as a novel source of useful variations for crop improvement. Rooting:

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Shoots produced through axillary branching or adventitious differentiation are rooted in-vitro on a medium containing a suitable auxin such as IAA NAA or IBA. Alternatively where possible the shoots are treated with auxin and directly planted in potting mixture for in-vivo rooting. Transplantation: The shoots or plantlets multiplied on a medium containing organic nutrients show poor photosynthetic capability. Moreover in these plants mechanisms to prevent loss of water from leaves are poorly developed. Therefore they require gradual acclimatization to the field conditions. In practice the plants are maintained under high humidity 80-90 for 10-15 days after they are removed from culture vessels. During the next few weeks the huidity around the plants is gradually lowered before they are transferred to naturtil conditions. The special merits of micropropagation are: i it considerably increases the rate of multiplication 2 high rate of multiplication can be maintained throughout the year 3 the multiplied plants are maintained in disease-free conditions 4 being free from microbes and insects valuable genotypes of exotic plants can be multiplied for export purpose and 5 small size of the propagules and their ability to proliferate in a soil-less environment facilitates their convenient storage handling and rapid transfer by air across international quarantine baniers. Uses of plant tissue culture Plant tissue culture now has direct commercial applications as well as value in basic research into cell biology genetics and biochemistry. The techniques include culture of cells anthers ovules and embryos on experimental to industrial scales protoplast isolation and fusion cell selection and meristem and bud culture. Applications include:  micropropagation using meristem and shoot culture to produce large numbers of identical individuals  screening programmes of cells rather than plants for advantageous characters  large-scale growth of plant cells in liquid culture as a source of secondary products  crossing distantly related species by protoplast fusion and regeneration of the novel hybrid  production of dihaploid plants from haploid cultures to achieve homozygous lines more rapidly in breeding programmes  as a tissue for transformation followed by either short-term testing of genetic constructs or regeneration of transgenic plants  removal of viruses by propagation from meristematic tissues  IMPORTANCE AND HISTORICAL VIEW OF PLANT TISSUE CULTURE  Objective  To begin with one should know the importance of plant tissue culture in theimprovement of useful crop plants and also the ways in which it has helped mankind. Planttissue culture forms an integral part of any plant biotechnology activity. It offers an alternativeto conventional vegetative propagation. But tissue culture requires attention-to-detail andunless practiced as art and science the entire process is ratherunforgiving. The various objectives achievable or achieved by plant tissue culture may besummarized as under:  a. Crop Improvement  As you all understand that for any crop improvement conventional breeding methodsare employed which involve six to seven generations of selfing and crossing- over to obtain apure line. With plant tissue culture techniques production of haploids through distant crossesor using pollen anther or ovary culture followed by chromosome doubling reduces this timeto two generations.  b. Micropropagation

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY  Plant tissue culture techniques have also helped in large- scale production of plantsthrough micropropagation or clonal propagation of plant species. Small amounts of tissue canbe used to raise hundreds or thousands of plants in a continuous process. This is beingutilized by industries in India for commercial production of mainly ornamental plants likeorchids and fruit trees e.g. banana. Using this method millions of genetically identical plantscan be obtained from a single bud. This method has therefore become an alternative tovegetative propagation. Shoot tip propagation is exploited intensively in horticulture and thenurseries for rapid clonal propagation of many dicots monocots and gymnosperms.  c. Genetic Transformation  Tissue culture in combination with genetic engineering is very useful in gene transfers.For example the transfer of a useful bacterial gene say cry crystal protein gene from  Bacillus thuringiensis  into a plant cell and ultimately regeneration of whole plants containing andexpressing this gene transgenic plants can be achieved.  d. Production of Pathogen-free Plants  Eradication of virus has been an outstanding contribution of tissue culture technology.It was found that even in infected plants the cells of shoot tips are either free of virus or carry anegligible amount of the pathogen. Such shoot tips are culturedin a suitable culture medium to obtain virus- free plants. This technique is economical andused very frequently in horticulture production of virus- free ornamentals etc.  e. Production of Secondary Metabolites  Cultured plant cells are also known to produce biochemicals secondary metaboliteslike alkaloids terpenoids phenyl propanoids etc. of interest. The technology is now availableto the  industry. The commercial production of „shikonin‟a naphthoquinone from cell cultures  of Lithospermum erythrorhizon has been particularly encouraging Applications of immobilized enzymes The first industrial use of an immobilized enzyme is amino acid acylase by Tanabe Seiyaku Company Japan for the resolution of recemic mixtures of chemically synthesized amino acids. Amino acid acylase catalyses the deacetylation of the L form of the N-acetyl amino acids leaving unaltered the N-acetyl-d amino acid that can be easily separated racemized and recycled. Some of the immobilized preparations used for this purpose include enzyme immobilized by ionic binding to DEAE-sephadex and the enzyme entrapped as microdroplets of its aqueous solution into fibres of cellulose triacetate by means of fibre wet spinning developed by Snam Progetti. Rohm GmbH have immobilized this enzyme on macroporous beads made of flexiglass-like material By far the most important application of immobilized enzymes in industry is for the conversion of glucose syrups to high fructose syrups by the enzyme glucose isomerase 95 . Some of the commercial preparations have been listed. It is evident that most of the commercial preparations use either the adsorption or the cross-linking technique. Application of glucose isomerase technology has gained considerable importance especially in nontropical countries that have abundant starch raw material. Unlike these countries in tropical countries like India where sugarcane cultivation is abundant the high fructose syrups can be obtained by a simpler process of hydrolysis of sucrose using invertase. Compared to sucrose invert sugar has a higher humectancy higher solubility and osmotic pressure. Historically invertase is perhaps the first reported enzyme in an immobilized form 96 . A large number of immobilized invertase systems have been patented 97 . The possible use of whole cells of yeast as a source of invertase was demonstrated by D‟Souza and Nadkarni as early as 1978. A systematic study has been carried out in our laboratory for the preparation of invert sugar using immobilized invertase or the whole cells of yeast. These comprehensive studies carried

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY out on various aspects in our laboratory of utilizing immobilized whole-yeast have resulted in an industrial process for the production of invert sugar. L-aspartic acid is widely used in medicines and as a food additive. The enzyme aspartase catalyses a one- step stereospecific addition of ammonia to the double bond of fumaric acid. The enzymes have been immobilized using the whole cells of Escherichia coli. This is considered as the first industrial application of an immobilized microbial cell. The initial process made use of polyacrylamide entrapment which was later substituted with the carragenan treated with glutaraldehyde and hexamethylenediamine. Kyowa Hakko Kogyo Co. uses Duolite A7 a phenolformaldehyde resin for adsorbing aspartase used in their continuous process 99 . Other firms include Mitsubishi Petrochemical Co. 100 and Purification Engineering Inc 101 . Some of the firms specially in Japan like Tanabe Seiyaku and Kyowa Hakko have used the immobilized fumarase for the production of malic acid for pharmaceutical use 94 . These processes make use of immobilized nonviable cells of Brevibacterium ammoniagenes or B. flavus as a source of fumarase. Malic acid is becoming of greater market interest as food acidulant in competition with citric acid. Studies from our laboratory have shown the possibility of using immobilized mitochondria as a source of fumarase 6 . One of the major applications of immobilized biocatalysts in dairy industry is in the preparation of lactose- hydrolysed milk and whey using b -galactosidase. A large population of lactose intolerants can consume lactose-hydrolysed milk. This is of great significance in a country like India where lactose intolerance is quite prevalent 102 . Lactose hydrolysis also enhances the sweetness and solubility of the sugars and can find future potentials in preparation of a variety of dairy products. Lactose-hydrolysed whey may be used as a component of whey-based beverages leavening agents feed stuffs or may be fermented to produce ethanol and yeast thus converting an inexpensive byproduct into a highly nutritious good quality food ingredient 99 . The first company to commercially hydrolyse lactose in milk by immobilized lactase was Centrale del Latte of Milan Italy utilizing the Snamprogetti technology. The process makes use of a neutral lactase from yeast entrapped in synthetic fibres 103 . Specialist Dairy Ingredients a joint venture between the Milk Marketing Board of England and Wales and Corning had set up an immobilized b -galctosidase plant in North Wales for the production of lactose-hydrolysed whey. Unlike the milk the acidic b -galactosidase of fungal origin has been used for this purpose 31 . Some of the commercial b -galactosidase systems have been summarized in Table 3. An immobilized preparation obtained by cross-linking b -galactosidase in hen egg white lyophilized dry powder has been used in our laboratory for the hydrolysis of lactose 47 . A major problem in the large-scale continuous processing of milk using immobilized enzyme is the microbial contamination which has necessitated the introduction of intermittent sanitation steps. A co-immobilizate obtained by binding of glucose oxidase on the microbial cell wall using Con A has been used to minimize the bacterial contamination during the continuous hydrolysis of lactose by the initiation of the natural lacto-peroxidase system in milk 88 . A novel technique for the removal of lactose by heterogeneous fermentation of the milk using immobilized viable cells of K. fragilis has also been developed 10 . One of the major applications of immobilized enzymes in pharmaceutical industry is the production of 6- aminopenicillanic acid 6-APA by the deacylation of the side chain in either penicillin G or V using penicillin acylase penicillin amidase 104 . More than 50 of 6-APA produced today is enzymatically using the immobilized route. One of the major reasons for its success is in obtaining a purer product thereby minimizing the purification costs. The first setting up of industrial process for the production of 6-APA was in 1970s simultaneously by Squibb USA Astra Sweden and Riga Biochemical Plant USSR. Currently most of the pharmaceutical giants make use of this technology. A number of immobilized systems have been patented or commercially produced for penicillin acylase which make use of a variety of techniques either using the isolated enzyme or the whole cells 100105106 . This is also one of the major applications of the immobilized enzyme technology in India. Similar approach has also been used for the production of 7- aminodeacetoxy-cephalosporanic acid an intermediate in the production of semisynthetic cephalosporins.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Immobilized oxidoreductases are gaining considerable importance in biotechnology to carry out synthetic transformations. Of particular significance in this regard are oxidoreductase-mediated asymmetric synthesis of amino acids steroids and other pharmaceuticals and a host of speciality chemicals. They play a major role in clinical diagnosis and other analytical applications like the biosensors. Future applications for oxidoreductases can be in areas as diverse as polymer synthesis pollution control and oxygenation of hydrocarbons 107 . Immobilized glucose oxidase can find application in the production of gluconic acid removal of oxygen from beverages and in the removal of glucose from eggs prior to dehydration in order to prevent Maillard reaction. Studies carried out in this direction in our laboratory have shown that glucose can be removed from egg using glucose oxidase and catalase which are co-immobilized either on polycationic cotton cloth 57 or in hen egg white foam matrix 50 . Alternatively glucose can also be removed by rapid heterogeneous fermentation of egg melange using immobilized yeast 108 . Immobilized D-amino acid oxidase has been investigated for the production of keto acid analogues of the amino acids which find application in the management of chronic uremia. Keto acids can be obtained using either L- or D-amino acid oxidases. The use of D-amino acid oxidase has the advantage of simultaneous separation of natural L-isomer from DL-recemates along with the conversion of D-isomer to the corresponding keto acid which can then be transamina-ted in the body to give the L-amino acid. Of the several microorganisms screened the triangular yeast T.variabilis was found to be the most potent source of D-amino acid oxidase with the ability to deaminate most of the D-amino acids 109 . The permeabilized cells entrapped either in radiation polymerized acrylamide 24 Ca-alginate 23 or gelatin 25 have shown promise in the preparation of a -keto acids. Another interesting enzyme that can be used profitably in immobilized form is catalase for the destruction of hydrogen peroxide employed in the cold sterilization of milk. A few reports are available on its immobilization using yeast cells 1122 . Lipase catalyses a series of different reactions. Although they were designed by nature to cleave the ester bonds of triacylglycerols hydrolysis lipase are also able to catalyse the reverse reaction under microaqueous conditions viz. formation of ester bonds between alcohol and carboxylic acid moieties. These two basic processes can be combined in a sequential fashion to give rise to a set of reactions generally termed as interesterification. Immobilized lipases have been investigated for both these processes. Lipases possess a variety of industrial potentials starting from use in detergents leather treatment controlled hydrolysis of milk fat for acceleration of cheese ripening hydrolysis glycerolysis and alcoholysis of bulk fats and oils production of optically pure compounds flavours etc. Lipases are spontaneously soluble in aqueous phase but their natural substrates lipids are not. Although use of proper organic solvents as an emulsifier helps in overcoming the problem of intimate contact between the substrate and enzyme the practical use of lipases in such psuedohomogeneous reactions poses technological difficulties. Varieties of approaches to solve these using immobilized lipases have recently been reviewed 110 . Significant research has also been carried out on the immobilization and use of glucoamylase. This is an example of an immobilized enzyme that probably is not competitive with the free enzyme and hence has not found large-scale industrial application 111 . This is mainly because soluble enzyme is cheap and has been used for over two decades in a very optimized process without technical problems. Immobilization has also not found to significantly enhance the thermostability of amylase 111 . Immobilized renin or other proteases might allow for the continuous coagulation of milk for cheese manufacture 112 . One of the major limitations in the use of enzymes which act on macromolecular substrates or particulate or colloidal substrates like starch or cellulose pectin or proteins has been the low retention of their realistic activities with natural substrates due to the steric hindrance. Efforts have been made to minimize these problems by attaching enzymes through spacer arms 113 . In this direction application of tris hydroxymethyl phosphine as a coupling agent 114 may have future potentials for the immobilization of enzymes which act on macromolecular substrates. Other problem when particulate materials are used as the substrates for an enzyme is difficulty in the separation of the immobilized enzyme from the final mixture. Efforts have been

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY made in this direction to magnetize the bicatalyst either by directly binding the enzyme on magnetic materials magnetite or stainless steel powder or by co-entrapping magnetic material so that they can be recovered using an external magnet 98115 . Magnetized biocatalysts also help in the fabrication of magnetofluidized bed reactor 116 . A variety of biologically active peptides are gaining importance in various fields including in pharmaceuti- cal industries and in food industries as sweeteners flavourings antioxidants and nutritional supplements. Proteases have emerged over the last two decades as powerful catalysts for the synthesis and modification of peptides. The field of immobilized proteases may have a future role in this area 117118 . One of the important large scale applications will be in the synthesis of peptide sweetener using immobilized enzymes like the thermolysin 119 . Proteolytic enzymes such as subtilisin a-chymotrypsin papain ficin or bromelain which have been immobilized by covalent binding adsorption or cross-linking to polymeric supports are used Bayer AG to resolve A N-acyl-DL-phenylglycine ester racemate yielding N-acyl-D-esters or N-acyl-D- amides and N-acyl-L-acids 100 . Immobilized aminopeptidases have been used to separate DL- phenylgycinamide racemates 100 . SNAM-Progetti SpA-UK have used the immobilized hydropyrimidine hydrolase to prepare D-carmamyl amino acids and the corresponding D-amino acids from various substituted hydantoins 100 . Application of Immobilized Cells Immobilization of plant cells is considered to be of importance in research and development in plant cell cultures because of the potential benefits that could be provided 24 92: a The extended viability of cells in the stationary and producing stage enabling maintenance of biomass over a prolonged time period b Simplified downstream processing if products are secreted c The putative promotion of differentiation linked with enhanced secondary metabolism d Higher cell density enabling a reduced bioreactor size thereby reducing costs and the risk of contamination e Reduced shear sensitivity especially with entrapped cells f Promotion of secondary metabolite secretion in some cases g Flow-through reactors can be used enabling greater flow rates h Minimization of fluid viscosity increase which in cell suspension causes mixing and aeration problems. An immobilization system which could maintain viable cells over an extended period of time and release the bulk of the product into the extracellular medium in a stable form could dramatically reduce the costs of phytochemicals production in plant cell culture 1. However an immobilized system also has the problems described below: a Immobilization is normally limited to cases where production is decoupled from cell growth b The initial biomass must be grown in suspension

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY c Secretion of product into the extracellularly medium is imperative d Where secretion occurs there may be problems of extracellular degradation of the products e When gel entrapment is used the gel matrix introduces an additional diffusion barrier. Due to these problems a system with commercial potential has not yet been developed in plant tissue cultures. However various immobilization methods have been developed ie. entrapment adsorption and covalent coupling. Some preliminary results have been obtained with immobilized cells. Early work with C. roseus showed that agar agarose and carageenan were all suitable immobilization matrices suitable for maintenance of cell viability but alginate was superior in terms of ajmalicine production 93. The accumulation of serpentine by C. roseus and anthraquinones by Morinda citrifolia were both enhanced in the immobilized state when compared with freely suspended cells. It should be noted however that the possibility that alginate acts as an elicitor of secondary metabolism cannot be ruled out 94. Agar has been shown to stimulate shikonin accumulation in L. erythrorhizon cultures 95. Lambe and Rosevear 96 have successfully immobilized C. roseus cells in polyacrylamide with alginate and observed prolonged viability and increased productivity. Adsorption immobilization has been successfully used with a number of plant species. Capsicum frutescens cells immobilized on polyurethane foam produced 50 times as much capsaicin as suspension cells 97. Similarly Solanum nigrum cells accumulated glycoalkaloids to levels exceeding those found in suspensions. Datura innoxia cells accumulated tropane alkaloids with a profile similar to that of the intact plant whilst in free suspensions productivity was markedly suppressed 98. In general it appears that mild immobilization either through gel entrapment or surface adsorption enhances productivity and prolongs the viability of cultured cells. As described in the section on Biotransformation immobilized cells can also be used as biocatalysts for biotransformations. Such a system compares favourably with the use of freely suspended cells since in the case of immobilization the catalyst is theoretically reusable and the product is easily separated from the biomass. The most appropriate example is that of the 12-hydroxylation of ß-methyldigitoxin to ß- methyldigoxin with alginate-entrapped Digitalis lanata cells 99. The enzyme activity was maintained by the immobilized cultures for a period of 61 days. Furthermore the product was located in the extracellular medium. Mild permeabilization of the cells may enable biotransformation rates to be increased. Polyurethane-immobilized C. frutescens cells fed capsaicin precursors produced this metabolite at levels of up to 10 times those of non-fed cultures 98. DiCosmo et al. 48 found that glass fibres can be used as a carrier of plant cells to produce useful plant metabolites. Papaver somniforum cells were immobilized on fabric of loosely woven polyester fibres arranged in a spiral configuration on stainless steel support frame by Kurz et al. 100 to produce sanguinarine an antibiotic in oral hygiene. The yield was 3.6 mg/g-fw. by immobilized cells and was more than twice as much as by suspension cells. INDUSTRIAL PRODUCTION OF PHYTOCONSTITUTIONS: Process for the preparation of sennosides A and B: A. Extraction evaporation and washing

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY 16 kg of senna pods are digested for 3 days in a mixture of 10 liters of methanol and 10 liters of tap water. The solution is circulated with a pump in order to make the extraction more effective. The temperature is maintained at 20° to 30° C. After the specified time the solution is drained and the pods washed with 4 l of a 50 methanol solution. The obtained methanol-water-solution is distilled under vacuum at 40° C. The distillation is stopped when the density of the distillation residue is 1.25 kg/l. The distillation residue is extracted with 8 l of n-butanol. The extraction is carrried out in a suitable reaction vessel by mixing the solution for 1.5 hours. The stirring is discontinued and the layers are left to separate over the night. After separation the aqueous phase is poured into the reduction vessel. B. Reduction To the raw aqueous sennoside solution in the reduction vessel at a temperature of 25° C. a 50 sodium hydroxide solution is added until the pH is 8.3. 120 to 150 g of lye is consumed. To the solution 500 g of sodium dithionite is added and the mixture is stirred for two hours. 3 l of water is added whereafter the pH is adjusted with sulphuric acid to a value of 4.7. l g of rheinanthrone-8-glucoside crystals are added and adjusted with sulphuric acid to a pH of 2.9. The mixture is cooled to 10° C. where it is kept for two hours. The crystallized rheinanthrone-8-glucoside is filtered onto a filter and washed with 1500 ml of hot water. The precipitate is dried by sucking nitrogen through the filter under vacuum whereby appr. 560 g of rheinanthrone-8-glucoside is obtained which contains about 20 moisture. C. Oxidation of rheinanthrone-8-glucoside 560 g of rheinanthrone-8-glucoside containing 15-20 moisture is slurried in 6000 ml of 80 vol./vol.isopropanol at +5° to +10° C. The rheinanthrone-8glucoside is made to dissolve by adding triethylamine to a pH of appr. 8. The pH may not rise above 8.5. Appr. 200 ml of triethylamine is consumed. Thereafter 50 g of OH- active carbon is added and the introduction of pressurized air into the mixture is started by bubbling through a sinter at a rate of appr. 3 liters in a minute. Air is bubbled for appr. 2 1/2hours the temperature of the reaction mixture being 5° to 10° C. When the reaction is complete the mixture is filtered through filter cardboard and adjusted with concentrated hydrochloric acid about 200 ml to a pH-value of 1.5 to 2.0. The mixture is left to crystallize over night at room temperature while stirring. The obtained precipitate is filtered through cardboard washed with 500 ml of isopropanol and dried in a vacuum chamber at a temperature of not more than appr. 40° C. The yield is appr. 310 g appr. 62.2 calculated from the rheinanthrone-8-glucoside. D. Preparation of the calcium salt 300 g of sennoside A + B acid is slurried in 1800 ml of water and dissolved by adding a calcium hydroxide- water slurry 30 g CaOH 2 +150 ml of water. The addition is continued to a pH value of 8±0.5 and appr. 110 ml of lime slurry is consumed to dissolve the acid. Thereafter the pH is adjusted with weak hydrochloric acid 40 ml 1:10 dilution to a pH-value of 6.7 in the course of one hour while making sure that the pH stays in the range of 6.7 to 6.9. Within 1/2hour 1000 ml of a 90 methanol solution and thereafter during appr. 2 hours 4400 ml pure 100 methanol are added. The mixture is stirred for another hour and filtered through cardboard. The precipitate is washed with a small amount of methanol. The precipitate is dried at a temperature of at the most 40° C. over night and weighed. The yield is appr. 317 g 100 as air-dry and 285 g appr. 91 as vacuum dry calculated from the sennoside A + B acid of which the sennoside content is appr. 82.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Vinca Alkaloids The dimeric indole alkaloids vinblastine and vincristine have become highly valued drugs in cancer chemotherapy due to their potent antitumor activity against various leukemias Hodgkins disease and solid tumors. They are currently produced commercially by extraction from Catharanthus roseus Apocyanaceae plants but the process is not efficient because of very low concentrations of the alkaloids in the plant. It was reported that the concentration of both vinblastine and vincristine was only 0.0005 as a dry weight basis. In order to produce these useful anticancer drugs much more efficiently many scientists have tried to apply plant tissue culture technology. In fact a large number of papers related to this approach have been presented since the first research carried out by Carew et al. in 1966 196. However production of both alkaloids by de novo synthesis using the callus or the suspension cultured cell of C. roseus is so far not promising because the productivity of the cultured cells reported was so far very low. Misawa and his colleagues of Allelix Inc. in Canada 197-199 studied on production of vinblastine by an alternative way in collaboration with Kurz of the National Research Council of Canada and Kutney of University of British Columbia and established an economically feasible process consisting of production of catharanthine by plant cell fermentation and a simple chemical or an enzymatic coupling. The vinblastine molecule is derived from two monomeric alkaloids catharanthine and vindoline as shown in Fig. 8. The concentration of vindoline in the intact C. roseus plant is approximately 0.2 as a dry weight basis which is much a higher level than catharanthine and the cost of vindoline is less expensive compared to catharanthine and vinblastine. The Allelix group therefore investigated the production of catharanthine by a cell suspension culture process with a selected C. roseus cell line induced from anthers on Gamborgs B5 medium containing 2 sucrose 1 mg/L 24-D and 0.1 mg/L kinetin. The cells were grown in 250 ml flasks containing 60 ml of MS liquid medium supplemented with 3 sucrose 1 mg/L NAA and 0.1 mg/L kinetin under continuous diffuse light on a rotary shaker 250 r.p.m. at 25° C. In experiments for optimization of catharanthine production they transferred 7 day old cells to a test medium and subcultured for 3 passages. In the 4th passage 60 ml cultures were harvested in triplicate after 2 or 3 weeks growth and the cell mass and alkaloid content were determined. Figure 8: Chemical Structures of Catharanthine Vindoline Vinblastine and Vincristine The results showed that the MS medium was the most favourable for catharanthine production but the optimal levels of phytohormones for the growth and the production were varied in different cell lines. For

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY example one line required no phytohormones but another line required 0.1 mg/L NAA and 0.1 mg/L kinetin. Addition of various chemically defined compounds to the medium as "inducers" was found to stimulate the production efficiently. Among them effects of vanadyl sulphate abscisic acid and NaCl on the production of catharanthine were significant 200. Based on the conditions optimized by using flasks Smart et al 201 scaled up the cultures to 10 30 and 100 L-air lift fermentors. When abscisic acid was added to the culture as an elicitor on the 7th day of cultivation the final titer of catharanthine was raised to 85 mg/L in a 30 L fermentor. The second stage in this project the Allelixs group tried to couple enzymatically or chemically catharanthine produced by the cell culture process with commercially available vindoline. As an enzyme source for the coupling a crude preparation obtained by 70 ammonium sulphate precipitation from the cultured cells of C. roseus was used. The reaction mixture containing both monomeric alkaloids Tris buffer pH 7.0 and the enzyme preparation was incubated at 30° C and for 3 hours. It was determined that the enzyme reaction gave various dimeric alkaloids including vinamidine 3-R-hydroxyvinamidine and 34- anhydrovinblastine. Leurosine and catharine oxidized derivatives of anhydrovinblastine were also detected in the early stages of the incubation. They found that MnCl 2 and either FAD or FMN stimulated the coupling. Although neither vinblastine nor vincristine was detected in the mixture it was recognized that a substantial amount of anhydrovinblastine was formed as a major coupling product when an excess amount of sodium borohydride was added to the mixture after incubation. In order to investigate properties of the coupling enzymes it was partially purified with gel filtration and isoelectric focusing and five isozymes were obtained by Endo et al. 202. One of them had MW 15000 and the other four had the same MW 37000. All of these isozymes were shown to have peroxidase activity. Using the partially purified enzymes anhydrovinblastine was formed with a conversion yield of about 50. Formation of vinblastine from vincristine as detected by Goodbody et al. 203 using a crude enzyme preparation obtained from suspension cultured cells of C. roseus. The highest yield of conversion obtained was 13 from 0.13 mg anhydrovinblastine in 1 ml of the reaction mixture after 3 hours incubation at 30° C Ph 7.0. During the course of these studies on coupling mechanisms they found that ferric ion catalyzed the coupling reaction significantly in the absence of the enzyme. It is of interest that the products of the chemical coupling were not only anhydrovinblastine but also vinblastine. The yields of both alkaloids were 52.8 and 12.3 respectively after 3 hours incubation at 30° C pH 7.0. These products including catharanthine were analyzed by high resolution mass spectrometry as further confirmation of their identification. Circular dichroism confirmed that a-coupling exists between the 2 monomeric units of both vinblastine and vincristine produced either enzymatically or chemically. This is a novel and an efficient process to produce an antitumor drug vinblastine and is likely to be applied commercially. The technology was transferred from the Canadian company to a Japanese company Mitsui Petrochemicals Industry for further development. Hara et al. 204 of Mitsui Petrochemical could increase the yield of catharanthine up to 150 mg/L in the MS medium supplemented with 1 mg/L NAA and 0.1 mg/L kinetin using the best producing cell line isolated from Allelixs cell line. The stimulating activity of NaCl and KCl on alkaloid production was also confirmed. Furthermore the scientists of Mitsui employed high-cell density cultures and reported yields of catharanthine of 230 mg/L/week 205.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY The yield of vinblastine by the chemical coupling reaction was also improved by the same group addition of ferric chloride oxalate maleate and sodium borohydrate stimulated the yield of vinblastine from anhydrovinblastine up to 50 206. Bede et al. 206 also investigated the production of anhydrovinblastine. They employed a two-enzyme system containing horseradish peroxidase and glucose oxidase to catalyze the formation of anhydrovinblastine from catharanthine and vindoline. Although peroxidase requires hydrogen peroxide for the coupling reaction its presence in excess in the reaction mixture may inhibit the reaction. But addition of glucose oxidase was used to allow the controlled continuous production of hydrogen peroxide at low levels minimizing oxidative reactions. Both enzymes were immobilized on Euperight C beads an oxirane matrix and the system was shown in catalyze the coupling reaction. Podophyllotoxin Podophyllum pelatatum May apple which is a common herb in eastern North America contains an antitumor lignan podophyllotoxin. It is active to KB cells and is used against certain virus diseases and skin cancer 190. A semi-synthetic derivative of podophyllotoxin etoposide V-16 was found to be active against brain tumor lymphosarcoma and Hodgkins disease and was approved by the FDA in the U.S. Bristol-Myers Squibb is one of the largest manufacturers of the drug. Production of podophyllotoxin by P. pelatum cell cultures was first attempted by Kadkade 191 and he found that a combination of 24-D and kinetin in the medium supported the highest amount of its production. Red light stimulated the production. Sakata et al. of Nippon Oil 192 induced embryogenic roots from a callus of the plant in a liquid MS medium supplemented 1 mg/L NAA 0.2 mg/L kinetin and 500 mg/L casein hydrolysates. The roots were then transferred to the medium without growth regulators. They detected 1.6 of podophyllotoxin in the dried tissues which was 6 times higher level than that in a mother plant. To increase the yield of podophyllotoxin Woerdenberg et al. in the Netherlands 193 added a complex of a precursor coniferyl alcohol and ß-cyclodextrin to Podophyllum hexandrum cell suspension cultures. Addition of 3 mM coniferyl alcohol complex gave 0.013 podophyllotoxin of the cells on a dry weight basis but the cultures without the precursor produced only 0.003. ß-D-glucoside of coniferyl alcohol coniferin was a more potent precursor in terms of the yield of the anticancer compound 0.055 but unfortunately this compound is not commercially available. The same authors reported that cell suspension cultures of Callitris drummondii conifer also accumulated podophyllotoxin-ß-D-glucose. In the dark the cells produced approximately 0.02 podophyllotoxin of the dry cell mass and 85-90 of the lignans were the ß-D-glucoside form while in the light the yield of podophyllotoxin-ß-D-glucose increased to 0.11. Smooly et al. 194 reported that callus tissues and suspension culture cells of Lilium album produced podophyllotoxin. One of the cell lines produced 0.3 podophyllotoxin of dried cells together with small amounts of 5-methylpodophyllotoxin lariciresinol and pinoresinol after 3 weeks of cultivation. The callus tissue induced from P. hexandrum was reported by Heyenga 195 to produce podophyllotoxin 4- demethyl-podophyllotoxin and podophyllotoxin-4-0-glucoside when the callus was incubated in B5 medium containing 24-Dichlorophenoxyacetic acid gibberellic acid and 6-benzylaminopurine. The levels of podophyllotoxin and its derivatives were similar to those in the mother plant. QUINOLINES.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY The Skraup synthesis is a chemical reaction used to synthesize quinolines. It is named after the Czech chemist Zdenko Hans Skraup 1850-1910. In the archetypal Skraup reaction aniline is heated with sulfuric acid glycerol and an oxidizing agent such as nitrobenzene to yield quinoline. 1234 In this example nitrobenzene serves as both the solvent and the oxidizing agent. The reaction which otherwise has a reputation for being violent "the Chemical Inquisition" is typically conducted in the presence of ferrous sulfate. 5 Arsenic acid may be used instead of nitrobenzene and the former is better since the reaction is less violent. 6 8-aminoquinolines• Drugs in this group have amino group at position 8 of quinoline ring• Important members of this family include 1- Pamaquine 2- Primaquine etc.  2. • Such drugs have OCH3 group at position 6• This molecule has antimalarial activity but when side chain is introduced at amino group antimalarial activity is intensified e.g pamaquine• It causes hemolysis of RBCs Diethyl amino pentyl side chain  3. • It contains tertiary amino group and when it is converted into primary amino group the compound is called primaquine which is – Less toxic – Well tolerated – It is the most commonly used agent in this group in the treatment of malaria  4. • OCH3 is not necessary for antimalarial activity but when replaced by OC2H5 the compound became – less active – Toxic in nature• OCH3 when replaced by CH3 the compound become inactive• Introduction of halogens increases toxicity• Presence of quinoline ring is necessary for antimalarial activity. When pyridine ring is converted to piperidine saturated the compound became inactive  5. • Pentyl side chain gives maximum activity increase or decrease of chain result is reduction of activity.• The branched side chain when converted into straight chain pentaquine is obtained• It has less antimalarial activity as compared to both pamaquine and primaquine  6. Chemical synthesis pamaquine• Glycerol undergoes dehydration to produce propene aldehyde• Dehydrating agent is sulphuric acid  7. • Addition reaction of propene aldehyde and 4 methoxy 2-nitro aniline to form 4 methoxy 2- nitro propene aldehyde  8. • Tautomerization: 4 methoxy 2-nitro propene aldehyde keto form converted in enol form  9. • Enol form undergoes cyclization to form 8 nitro 6 methoxy dihydroquinoline which then oxidized to form 8 nitro 6 methoxy quinoline  10. • 6 methoxy 8 nitro quinoline undergoes reduction to form 8 amino 6 methoxy quinoline  11. • 8 amino 6 methoxy quinoline reacts with 2 chloro diethyl amino pentane to form pamaquine

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY  12. Therapeutic uses• Active against hepatic stage of plasmodium• Provide radical cure hepatic stage of P. vivax and P. ovale• It also acts at gametocytes hence used as prophylactic drugs• Used in combination with chloroquine for complete eradication of malaria• Side effect: hemolysis in G6 phosphate dehydrogenase deficient people Aplication of Quinoline: Quinoline is used in the manufacture of dyes.Quinoline and quinoline derivative can be usedin manufacturing a wide range of Food Colors Lake Colors Salt Free Dyes etc. which areextensively used as Dyes in various industries such as Food Pharmaceutical and Cosmetic. Quinoline is used in the preparation of hydroxyquinoline sulfate and niacin. It has also used asa solvent for resins and terpenes. Quinoline is mainly used as a feedstock in the production of other specialty chemicals. Itsprincipal use is as a precursor to 8-hydroxyquinoline which is a versatile chelating agent andprecursor to pesticides. Its 2- and 4-methyl derivatives are precursors to cyanine dyes. Oxidationof quinoline affords quinolinic acid pyridine-23-dicarboxylic acid a precursor to the herbicidesold under the name "Assert". Non-Cancer: Quinoline is an irritant of the eye and respiratory tract. Acute inhalationoverexposure to quinoline vapors in humans may cause signs and symptoms such as headachesdizziness and nausea and coma. Quinoline overexposure has also been reported to cause injuryto the cornea retina and optic nerve. MENTHOL MANUFACTURING PROCESS TECHNOLOGY: The leaves of the "Mentha Arvenisis" are subjected to steam distillation the distillation products are condensed and separated into peppermint oil and water. The crude mint oil then obtained is refined by vacuum filteration and then chilled to about 5-10 degree C to obtain Menthol Crystals. The crystals thus formed are centrifuged and obtain about 45 yield of menthol. The spent oil is treated with sodium hydroxide and Boric Acid while crystalline borate esters which are formed are separated and decomposed by steam. The Menthol thus released is recovered by crystallisation under reduced temperatures and centrifuging. The mother liquor is distilled to obtain dementholised peppermint oil. The overall yield of menthol is about 50 and an equal amount of dementhonised oil is obtained as co-product. INDUSTRIAL PRODUCTION OF CITRIC ACID  Microorganism: Aspergillus niger mainly Candida yeast from carbohydrates or n-alkanes  Citric acid production is mixed growth associated mainly take place under nitrogen and phosphate limitation after growth has ceased.  Medium requirements for high production: - Carbon source: molasses or sugar solution. - Na-ferrocyanide is added to reduce Iron 1.3 ppm and manganese 0.1ppm. - High dissolved oxygen concentration - High sugar concentration - pH2 - Temperature: 30 o C  Bioreactor: batch or fed-batch 100m 3 - 5-25×10 6 A. niger spores/L may be introduced to the fermentor.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY - Aeration is provided to the fermenter by air sparging 0.1-0.4 vvm - Temperature is controlled by cooling coil. - Agitation: 50-100rpm to avoid shear damage on molds. - Fed-batch is used to reduce substrate inhibition and prolong the production phase one or two days after growth cessation. - Volumetric yield: 130 kg/m 3 Separation: - The biomass is separated by filtration - The liquid is transferred to recovery process: - Separation of citric acid from the liquid: precipitation calcium hydroxide is added to obtain calcium citrate tetrahydrate → wash the precipitate→ dissolve it with dilute sulfuric acid yield citric acid and calcium sulfate precipitate → bleach and crystallization → anhydrous or monohydrate citric acid. - Microorganism: S. cerevisae for hexose Candida sp. for lactose or pentose Genetically modified E. coli - Ethanol production is growth-associated with S. cerevisae. - Medium requirements for high production - Carbon source: sugar cane starch materials e.g. corn wheat cellulosic materials . yield: 0.51 g ethanol/g glucose. - N P minerals. - Anaerobic - 100g/L glucose are inhibitory for yeast. - 5 v/v of ethanol are inhibitory for yeast. - pH:4-6 for 30-35 o C. Bioreactor: batch continuous or with cell recycle 95 conversion of sugars with a residence time of 40 h in batch reactor 21 h in continuous reactor without cell recycle 1.6 h in continuous reactor with cell cycle

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY By-products: glycerol acetic acid succinic acid. Separation: - Distillation to obtaining 95 w/w of ethanol-water mixture followed by - Molecular sieves to removing water from the mixture to get anhydrous ethanol. Purification of Citric acid • A typical method used for purification of citric acid from a fermentation broth involves two major purification techniques: precipitation and filtration. • The following schematic displays a generic citric acid purification scheme:

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY To crack the calcium citrate precipitate sulfuric acid is needed. The temperature of this reaction should stay below 60ºC. The reaction will produce free citric acid and a new precipitate calcium sulfate which will need to be removed later. The stoichiometric coefficients for this reaction are all one. In this filter the calcium sulfate is washed away from the citric acid and the leftover biomass is removed. Again the contaminants that were present in the fermentation broth can be removed by additional filtration means such as microfiltration or ultra filtration. Applications : Food • Used as flavoring and preservative in food and beverages. • Can be added to e.g. ice cream as an emulsifying agent to keep fats from separating to caramel to prevent sucrose crystallization or to recipes in place of fresh lemon juice. • Citric acid is used with sodium bicarbonate in a wide range of effervescentformulae both for ingestion e.g. powders and tablets and for personal care e.g. bath salts bath bombs and cleaning of grease. • Citric acid is also often used in cleaning products and sodas or fizzy drinks. Cleaning and Chelating agent • Used to remove scale from boilers and evaporators. • Can be used to soften water which makes it useful in soaps and laundry detergents. • In industry it is used to dissolve rust from steel. • Can be used in shampoo to wash out wax and coloring from the hair. Cosmetics and pharmaceuticals

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY • Citric acid is widely used as a pH adjusting agent in creams and gels of all kinds. • Citric acid is commonly used as a buffer to increase the solubility of brown heroin. • Citric acid is used as one of the active ingredients in the production of antiviral tissues. Dyeing • Citric acid can be used in food coloring to balance the pH level of a normally basic dye. • It is used as an odorless alternative to white vinegar for home dyeing with acid dyes. Photography • Citric acid can be used as a lower-odor stop bath as part of the process for developing photographic film. INDUSTRIAL PRODUCTION OF DIOSGENIN: DIOSGENIN TUBERS COLLECTED---------- WASHED ------- DRIED----------- EXTRACTED WITH HOT WATER OR 90 ETHANOL FOR 6 HRS……………………… ALCOHOLIC EXTRACT CONCENTRATED UNDER VACUUM………….. FILTER IT ……….. FILTERATE + SOLVENT ETHER OR LEAD ACETATE SOLUTION…………HYDROLYSIS BY ACID …………………….. EXTRACTED WITH PET. ETHER……….EVAPORATE SOLVENT …………………… DIOSGENIN COLLECTED DRIED AND PACKED . INDUSTRIAL PRODUCTION OF SOLSODINE SOLSODINE BY TWO METHODS METHOD 1 B. METHOD 2 METHOD. 1 Dried berries is powdered-------- Oil is removed------------- Defatted is extracted with ethanol-- ------------------- Resultant is filtered Concentrated Treat with HCl Reflux ---------------- Extract is made alkaline by ammonia…………. Reflux for 1 hr……………. Filter it…………………Dry and wash Residue ……………. Mix in chloroform …………. Evaporate solvent……….. Solasodine solid residue is obtained. METHOD. 2 Powdered drug + ethanol-------- Soxhlation 6 hrs.------------- Solvent distilled off…………… Concentrated to syrupy mass ---------Add 5 ml HCl Boil …….. Reflux for 2 hr……………. Cool it Filter………… Residue + Boil water………. Adjust pH-9 by NH 3 10 …………. Boil under reflux for 2 hrs ……… Cool Filter…..Dry Ppt ……….. Solasodine solid residue is obtained. Atropine The final problem in the synthesis the combination of tropine and tropic acid was overcome by a Fischer-Speier esterification 13. The acid and alcohol were heated together in the presence of HCl to yield atropine

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY The biosynthesis of atropine starting from L-Phenylalanine first undergoes a transamination forming phenylpyruvic acid which is then reduced to phenyl-lactic acid. 14 Coenzyme A then couples phenyl-lactic acid with tropine forming littorine which then undergoes a radical rearrangement initiated with aP450 enzyme forming hyoscyamine aldehyde. 14 A dehydrogenase then reduces the aldehyde to a primary alcohol making --hyoscamine which upon racemization forms atropine. Atropine is a naturally occurring tropane alkaloid extracted from deadly nightshade Atropa belladonna Jimson weed Datura stramoniummandrake Mandragora officinarum and other plants of the family Solanaceae. It is a secondary metabolite of these plants and serves as a drugwith a wide variety of effects.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY In general atropine counters the "rest and digest" activity of glands regulated by the parasympathetic nervous system. This occurs because atropine is a competitive antagonist of the muscarinic acetylcholine receptors acetylcholine being the main neurotransmitter used by the parasympathetic nervous system. Atropine dilates the pupils increases heart rate and reduces salivation and other secretions. Chemistryedit Ergometrine 1-hydroxymethylethylamide lysergic acid is synthesized by esterification of D-lysergic acid using 2-aminopropanol indimethylformamide and direct treatment of the reaction mixture with phosgene. 5

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Diosgenin is steroidal sapogenin obtained from the tubers of Dioscorea species of the family Dioscoreacece. Chemically is a steroidal sapogenin. Sole source for steroidal contraceptives topical hormones estrogens progestogen androgen and sex hormone . Chemical nature Diosgenin is hydrolytic product of saponin Dioscin . Saponins – plant constituent which bring about frothing in an aqueous solution. Historically used for their detergent properties. Properties: Frothing property : hydrophobic large molecules C 27 - 30 glycone hydrophilic makes the molecule capable of lowering surface tension in water. Hemolytic property : destroys RBC by hemolysis toxic to cold-blooded animals - fish poison Methods of isolation: Methods of isolation 1 Alcoholic extraction method : Dioscorea tubers are cut into small pieces dried under sun ↓ Dried tubers are powdered extracted with ethanol / methanol twice for 6-8 hrs ↓ filter filterate is concentrated to a syrupy liquid ↓ the concentrated liq. Is then hydrolysed using an acid HCl or H2SO4 for 2 -12 hrs ↓ 85 of diosgenin is ppted ↓ Ppts are filtered washed with water ↓ purification with alcohol 2 Acid hydrolysis method Dried rhizomes are powdered 20 and first subjected to hydrolysis by refluxing with 5 HCl for 2 hours. ↓ The hydrolyzed mass is filtered washed twice with water and then twice with 5 sodium bicarbonate solution. ↓ It is finaly washed with water till the washing are neutral. The residue thus obtained is dried and futher extracted with toluene for 8 hours. ↓ The toluene extract concentrated during which diosgenin gets precipitated. ↓ Diosgenin filtered washed with little hexane and dried40-60 o c to yield about 95 pure product. Identification tests Thin Layer Chromatography: Identification tests Thin Layer Chromatography Stationary phase : silica gel G Solvent system : Toluene :Ethyl acetate 7:3 Spraying reagent : Anisaldehyde in sulfuric acid Standard solution : Dissolve Std .diosgenin 1mg in 1 ml chloroform. Test solution : Dissolve residue obtained through isolation in chloroform Rf for diosgenin : 0.62 14 Chemical Tests: Chemical Tests 1.Libermann-Burchard test : Treat the extract with few drops of acetic anhydride boil and cool. Then add conc .sulphuric acid from the sides of test tube brown ring is at the junction of two layers upper layer turns green steroids and formation of deep red colour triterpenoids

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY 2. Libermann „s reaction : mix 3 ml extract + 3 ml acetic anhydride heat cool add few drops of conc H2SO4 Blue color obtained 15 Nikita modi of pharmacy 3.Salkowski test : 2ml extract + 2ml CHCl3 +2ml conc H2SO4 CHCl3 layer appears red and acid layer shows greenish yellow florescence 4.Sulfur powder test : Add small amount of sulfur powder to the test solution it sinks at the bottom. Steroid present. PRODUCTION AND UTILIZATION OF podophyllotoxin It is obtained from the dried rhizomes and root of Podophyllum hexandrum Family: Berberidaceae ISOLATION OF PODOPHYLLOTOXIN: ISOLATION OF PODOPHYLLOTOXIN DRIED RHIZOME POWDER------------- EXTRACT WITH ETHANOL----------SOXHLATION----------------- DISTILLATION------------------ CONCENTRATE TO SYRUPY MASS ---------------------- ADD HCL + H 2 O--------------- COOL AT 5 0 C--------------- ALLOWED TO STAND FOR 2 HRS--------------- FILTER UNDER VACUUM--------------------WASH RESIDUE WITH ACIDIFIED WATER------------ COOL BELOW 5 0 C------------- RESIDUE + HOT ALCOHOL 90-------------- FILTER EVAPORATE------------------ DRY RESIDUE TO CONSTANT WEIGHT AT 80 0 C. STRUCTURE OF SOLASODINE Solasidine is obtained from the whole plant . Solanum xanthocarpum And dried full growth berries of Solanum khasianum Family:Solanaceae

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY ISOLATION OF SOLSODINE BY TWO METHODS METHOD 1 B. METHOD 2 METHOD. 1 Dried berries is powdered-------- Oil is removed------------- Defatted is extracted with ethanol-- ------------------- Resultant is filtered Concentrated Treat with HCl Reflux ---------------- Extract is made alkaline by ammonia…………. Reflux for 1 hr……………. Filter it…………………Dry and wash Residue ……………. Mix in chloroform …………. Evaporate solvent……….. Solasodine solid residue is obtained. METHOD. 2 Powdered drug + ethanol-------- Soxhlation 6 hrs.------------- Solvent distilled off…………… Concentrated to syrupy mass ---------Add 5 ml HCl Boil …….. Reflux for 2 hr……………. Cool it Filter………… Residue + Boil water………. Adjust pH-9 by NH 3 10 …………. Boil under reflux for 2 hrs ……… Cool Filter…..Dry Ppt ……….. Solasodine solid residue is obtained. UTILIZATION OF SOLASODINE Used as a precursor for steroidal synthesis. It is first converted to 16- dehydropregnalone acetate which acts as a precursor for steroidal synthesis like Corticosteroids Pregnane Used in synthesis of Sex hormones and Oral contraceptives. Shows Antispermatogenic Activity Used as Hypocholestremic Agent Used as Antiatherosclerotic Agent . Quinine Biological Sources It is obtained from the bark of Cinchona calisaya Wedd Cinchona officinalis Linn. belonging to family Rubiaceae. Thalleioquin Test: Add to 2-3 ml of a weakly acidic solution of a quinine salt a few drops of bromine- water followed by 0.5 ml of strong ammonia solution a distinct and characteristic emerald green colour is produced. The coloured product is termed as thalleioquin the chemical composition of which is yet to be established. This test is so sensitive that quinine may be detected to a concentration as low as 0.005. Basic Structures of Cinchona Alkaloids The various quinoline alkaloids which possess potent medicinal activities are namely: quinine quinidine cinchonine and cinchonidine.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Identification test1.Vitali – Morin reaction: - Alkaloid/ atropine 1µg + Drop of H SO 2 4 Evaporate to dryness Indicates Which produce Add 0.3ml of 3 solution of presence of atropine bright purple color KOH in methyl alcohol2. On addition of AgNO3 solution to solution Yellowish of hyoscine

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY hydrobromide white ppt Insoluble – HNO3 Soluble – Dil. NH3 T.B.E.K.B TLC• 1 solution of atropine dissolved in 2N acetic acid isspotted over silica gel G plate and eluted in the solventsystem of strong NH3 solution – methanol : 5 : 100.• TLC plate is spread with an acidified iodoplatinate solution.•Rf – 0.18.•Solvent system – Acetone – 0.5 sosium chloride solution.•Spraying reagents – Dragondroff‟s reagent. Chemistry Properties•Melting point – 115oC to 116oC.•Molecular formula – C17H23NO3. Identification test for Citric Acid: Add a few milligrams of your substance to a solution containing 15mL of pyridine and 5mL of acetic anhydride. If citric acid is present a bright red color is produced. Manufacturing Process Technology: The leaves of the "Mentha Arvenisis" are subjected to steam distillation the distillation products are condensed and separated into peppermint oil and water. The crude mint oil then obtained is refined by vacuum filteration and then chilled to about 5-10 degree C to obtain Menthol Crystals. The crystals thus formed are centrifuged and obtain about 45 yield of menthol. The spent oil is treated with sodium hydroxide and Boric Acid while crystalline borate esters which are formed are separated and decomposed by steam. The Menthol thus released is recovered by crystallisation under reduced temperatures and centrifuging. The mother liquor is distilled to obtain dementholised peppermint oil. The overall yield of menthol is about 50 and an equal amount of dementhonised oil is obtained as co-product. Another commercial process is the Haarmann-Reimer process. This process starts from m-cresol which is alkylated with propene to thymol. This compound is hydrogenatedin the next step. Racemic menthol is isolated by fractional distillation. The enantiomers are separated by chiral resolution in reaction with methyl benzoate selective crystallisation followed by hydrolysis. Racemic menthol can also be formed by hydrogenation of pulegone. Chemical Tests 1. When 10 mg crystals menthol are first dissolved in 4 drops of concentrated sulphuric acid and then a few drops of vanillin sulphuric acid reagent are added it shows an orange yellow colouration that ultimately changes to violet on the addition of a few drops of water.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Definition of Electrophoresis Electrophoresis is a separations technique that is based on the the mobility of ions in an electric field. Positively charged ions migrate towards a negative electrode and negatively-charged ions migrate toward a positive electrode.For safety reasons one electrode is usually at ground and the other is biased positively or negatively. Ions have different migrationrates depending on their total charge size and shape and can therefore be separated. Instrumentation An electrode apparatus consists of a high-voltage supply electrodes buffer and a support for the buffer such as filter paper cellulose acetate strips polyacrylamide gel or a capillary tube. Open capillary tubes are used for many types of samples and the other supports are usually used for biological samples such as protein mixtures or DNA fragments. After a separation is completed the support is stained to visualize the separated components. Resolution can be greatly improved using isoelectric focusing. In this technique the support gel maintains a pH gradient. As a protein migrates down the gel it reaches a pH that is equal to its isoelectric point. At this pH the protein is netural and no longer migrates i.e it is focused into a sharp band on the gel. Schematic of zone electrophoresis apparatus Specific electrophoretic techniques  disc electrophoresis  capillary electrophoresis  gel electrophoresis SDS-PAGE

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY PREPARATION OF ALLERGENIC EXTRACT: · Grinding · Defatting · Extraction · Clarification · Dialysis · Concentration · Sterilization · Lypholization · Testing · Standardization · Storage GRINDING: The material to be extracted must be ground or subdivided in order to effect efficient extraction of the allergens. Household blenders or small plant mills can be used for dried materials while juicers or food grinders can be used for those containing much moisture. Materials such as hairs feathers and textiles should be divided finely with shears. DEFATTING:

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Many allergenic substances including all pollens should be defatted before final extraction. Ether and petroleum ether are used most commonly for this purpose but alcohols occasionally may be included in the menstruum. Defatting provides a clearer final extract and also removes irritants found in large amounts in some substances e.g. Coffee tea cottonseed pepper mustard ginger. The extract obtained in the defatting process may be used in the preparation of some patch-testing substances. EXTRACTION: The extraction procedures in current use are based upon the assumption that allergens are water-soluble proteins or glycoproteins although the identity of only a few is known. Extraction is carried out normally for 24 to 72 hours in a cold room using sterile pyrogen-free buffered saline cocas solution or similar aqueous menstruum of pH 8. Buffered Saline Sodium chloride 5 gm Monobasic potassium phosphate 0.36 gm Dibasic sodium phosphate anhydrous 7 gm Phenol crystals 4 gm Water for injection USP to make 1000 mL Coca‟s Solution Sodium chloride 5 gm Phenol crystals 5 gm Sodium bicarbonate 2.5 gm Water for injection USP to make 1000 mL CLARIFICATION: After extraction the mixture is clarified by coarse filtration. DIALYSIS: Some extracts are dialyzed against saline or running tap water to remove irritants or coloring agents. Most pollens require no dialysis but some substances e.g. house dust mustard potato spinach beets give nearly universally positive reactions unless dialyzed. CONCENTRATION: Concentration of the extract where required may be achieved by a number of methods but care should be taken not to alter the allergens. STERILIZATION: The processed extract is sterilized by filtration usually through a cellulose membrane filter. Prefilters usually are required but asbestos should not be used since it may adsorb some immunogens and may be carcinogenic. LYPHOLIZATION: Freeze-dried pollen extracts are prepared essentially as described above except that water rather than electrolyte solution is used as the extracting medium. The lyophilized products are reconstituted with buffered saline at time of use. TESTING:

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY The extracts are thermolabile and must be sterilized by filtration and sterility tests for both aerobic and anaerobic microorganisms must be performed on the finished products. Toxicity testing usually is performed in guinea pigs and recommended particularly for autogenous extracts where unknown toxic constituents may be present. Recent concerns for possible mycotoxin contaminants in mold extracts or from mold contamination of other substances have resulted in more intensive efforts to detect and eliminate these toxins. STANDARDIZATION: Most allergenic extracts carry the statement “No US standard of potency”. Although the first standardized allergenic extract was licensed in 1982 and there has been much progress realized in this area there are still no completely satisfactory means of assaying allergenic extracts and expressing their potency. The two most common measures of allergenic potency are by weight/volume w/v and the protein nitrogen unit PNU. Weight/volume is the weight of allergenic substances extracted per volume of extracting fluid. For example a 1:50 extract is prepared by extracting 1 g of substance with 50 mL of solvent and decimal dilutions of this extract provide 1:500 1:5000 etc concentrations. The protein nitrogen units also are listed often along with the w/v concentration on commercial products and 1 mg of protein nitrogen equals 100000 PNU. The allergenic protein is virtually always a small and variable part of the total protein and neither the PNU nor weight/volume standards correlate consistently with each other or clinical potency. Units of Potency for Allergenic Extracts Unit Description Used Weight/volume w/v Allergen g per volume mL of extracting fluid Worldwide Protein Nitrogen Unit PNU 1 mg protein N 100000 PNU Worldwide Allergy Unit AU Skin testing to endpoint US Biological Unit BU Skin testing relative to histamine Europe Three general methods are used to estimate potency better in the preparation of standardized allergenic extracts. Specific allergens in an extract are compared to those in the reference standard by immunoelectrophoresis. Two systems of bioassay based upon skin testing in patients sensitive to the particular extract are used presently o establish the potency of a reference standard. The Nordic system is used in Europe and potency is expressed in biological unit BU. The American system adopted by FDA expresses potency in terms of allergy unit AU. Radioallergosorbent-inhibition RAST-inhibiton tests are used widely to evaluate allergenic extracts. RAST-inhibition not skin testing is the main methods of comparing different batches of standardized allergenic extracts with reference standards. Standard extracts represent a major improvement in allergenic extracts and in general probably are more potent than the conventional extracts. However a standardized extract can be either more or less potent than the corresponding w/v extract and the two should never be used interchangeably. The same general principles of administration apply to both standardized and conventional extracts. Because allergenic extracts are not standardized completely the appropriate dosage for immunotherapy must be determined clinically. The initial dilution of extract starting dose and progression of dosage must be determined clinically. The initial dilution of extract starting dose and progression of dosage must be determined carefully on the basis of the patient‟s history and sensitivity tests. Because dilute extracts tend to lose activity more rapidly the first dose from a more concentrated vial generally should be the same or less than the previous dose. Also it is common to reduce the dose whenever a new lot of extract is started and then build the dose back to the maintenance level over a period of several weeks.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY STORAGE: Allergenic extracts tend to show reduced potency within a matter of weeks or months after their preparation but there have been few detailed studies on the stability of these products. Both high temperatures and freezing usually have deleterious effects and the latter may cause agglomeration of adjuvant extracts. Some extracts also contain proteolytic enzymes and these may contribute to decomposition of the allergens. Both glycerinated and lyophilized products are more stable than aqueous extracts. Very dilute extracts tend to lose potency by adsorption to the surfaces of containers and syringes and thus usually are prepared close to the time of use. Several studies have shown that the inclusion of Tween 80 Teen 20 or human serum albumin reduces or adsorption but a more-complete investigation of this problem is required. The adjuvant extracts should not be diluted with either phosphate buffered saline or Coca‟s solution since these may cause partial release of allergen normal saline containing 0.4 phenol is a satisfactory diluents. The adjuvant extracts may be mixed with one another but should not be mixed with other types of extracts. All allergenic extracts should be refrigerated at 2 to 8 ̊ and freezing should be avoided. The expiration date for aqueous extracts is usually 18 months while for glycerinated scratch test and bulk extracts is usually 3 years. Lyophilized products have an expiration date of 4 years or 18 months after reconstitution so long as the time falls within the original 4 years. Care must be exercised in changing to new lots or different dilutions of extracts because of possible variations in potency. It generally is recommended that quantities of extract sufficient to last the patient for 1 year be prepared to avoid frequent changes in extracts. Classification of allergens in plants 1.1 Inhalent allergens Inhalent allergens from grass or tree pollens house dust mite and animal dander are the major substances that are capable of provoking type I hyperresponsiveness. Among those allergens one of the most common ones is pollen of plants3. An individual who has hypersensitivity to pollen often suffers from seasonal allergic rhinitis or extrinsic asthma. Weeds grasses and trees are common sources of pollen and high concentrations of these pollen allergens in the air surrounding us correspond well to pollen-related hypersensitivity disease. The major and most widespread allergenic components of pollen is the group I allergens. Thus the allergy caused by these allergens is often termed "seasonal". These allergenic proteins in pollens with molecular weight about 30 kD are quickly and profusely released by grass pollen upon hydration4. In recent years research in this area has focused on the characterization of relevant grass pollen allergens because as many as approximately 40 of allergic individuals start their symptoms immediately after contacting with grass pollens5. 1.2 Ingestent allergens Ingestent allergens often refer to substances inducing allergy after the sensitized individuals eating a certain food. Typical symptoms of this type allergy include mouth or throat itching and lip swelling. In recent years an increase in tree nut and peanut allergy has been reported in Europe and in US. For example peanut and/or tree nut allergy affect approximately 1.1 of US population corresponding to 3 million individuals at risk of adverse reaction to these foods6. Of these individuals 50 considered in the survey performed by Sicherer et al.7 were reactive to peanut 30 to walnut and 10 to almond while only 4 were reactive to both peanut and tree nut. In previous reports the percentage of allergic individuals symptomatically reactive to two or more nuts has been found to be nearly 10 which corresponds to at least half a million individuals in the world reactive to two related nuts. On the other hand a recent study reported that approximately 35 patients with pollen allergy were also sensitive to fresh fruits and vegetables8. 1.3 Contactent allergens Latex is the most important contactent allergens in plants. Since the late 1980s this immediate-type allergy provoked by natural rubber products has been reported around the world9. It is now known as latex allergy. It also can be induced by wide-ranging latex products. In addition allergy to exotic fruit is

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY frequently reported in studies on latex-allergic subjects. Subjects suffering from the latex-fruit syndrome become primarily sensitized to latex and then develop food allergy as a result of cross-reacting IgE against protein such as in banana and avocado10. Nowadays plant defense-related proteins induced by stress were reported as a main kind of latex allergen. 2 The biological functions of allergens in plants In last decade with the implementation of molecular biological techniques in the field of allergen characterization the sequence nature and three-dimensional structure of several important allergens have been revealed. Application of molecular cloning techniques also enable us to understand the natural functions of the IgE-binding proteins in plants. There are at least three major biological functions for the allergens in plants. 2.1 Calcium-binding protein In plant molecular biology calcium in pollen is recognized as an essential constituent of in vitro pollen- germination media and a potential chemoattractant guiding pollen growth. In 1999 Rozwadowski et al.11 characterized calcium-binding protein from Brassica and Arabidopsis pollen. By sequence comparison the protein was revealed as a part of a family of pollen allergens identified recently in several evolutionarily distant dicot and monocot plants. The protein also has strong immunoreactivity to IgE from a human subject allergic to Brassica pollen12. In addition the members in the two EF-hand allergen families share an average sequence identity of 77 which is of comparable magnitude within and outside the calcium- binding domain. In fact several kinds of plant allergens with EF-hand calcium-binding domains have been identified in birch13 Bermuda grass14 and rapeseed12. Calcium binding plant proteins have now been discovered as relevant cross-reactive allergens and the EF-hand domain is the major epitope for antibody reorganization in those allergens15. 2.2 Pathogenesis-related protein PR protein PR proteins which represent an important group of human allergens can be up-regulated in plants in response to stressors such as freezing drought temperature fungi viruses or bacteria infection. So far several allergenic PR proteins have been biochemically characterized. They belong to different PR protein groups there are 10 groups of PR proteins in nature. For example Jun a 3 the allergen in mountain cedar was found to be homologous to the PR-5 protein group. Plant allergens Bet v 1 in birch Mal d1 in apple and Dau c 1 in carrot are members of PR protein 10 group16. Similarly the major allergen in rubber Hev b617 and its metabolic products Bar r 2 in turnip18 and Pers a 1 in avocado19 have the properties of chitinase and belong to the PR protein 4 group. Investigation of potential common functions and structures of PR-proteins will uncover some "law" of allergens in plants and will explain the reason for cross-reaction phenomena in plant allergens. 2.3 Expansins A cell wall-loosening agent is extracellular protein that promotes plant cell wall enlargement by disrupting noncovalent bonding between cellulose microfibrils and matrix polymers20. When the first expansin complementary DNA was sequenced BLAST searches in GenBank revealed a distant sequence similarity to a group of grass allergens called group-1 allergen. It was characterized further that group-1 allergens in plants were indeed structurally and functionally related to expansin and that their vegetative homologs comprise a second family of expansins such as Lol pIin ryegrass Ory s I in rice and Zea m I in maize4. But different from the original group of expansin this group of expansin in pollens could only induce extension in the cell walls of grass and was not effective on the walls from dicotyledons21. Recently the cell wall-loosening agents in pollens have been named as β-expansin family in order to distinguish it from the original group of expansins which are now called α-expansins. In type I hyperresponsiveness there are varieties of cross-reaction between allergens in different plants22. The common conservative domain and/or isotope among different allergens in plants are the radical cause of these phenomena. Thus research on identification and characterization of allergens and their structures and biological functions will be benefit for the diagnosis and treatment of pollen related allergic diseases. 3 Progress in gene cloning and recombinant protein production of plant allergens

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Allergen-specific immunotherapy SIT represents one of the few curative approaches toward type I hyperresponsiveness23. But there are three major problems associated with SIT: first presently SIT is performed with natural allergen extracts containing mixtures of allergens nonallergenic and/or toxic proteins and other macromolecules which are hard to standardize. Second systemic administration of allergen can cause severe IgE-mediated side effects during the treatment on patients and third therapeutically effective dose often cannot be achieved because of non-standardized extracts or side effects. With the clarification of the nature sequence and three-dimensional structure of several important allergens molecular level recognization of allergens and IgE antibodies will become available. To date cDNA sequence of 60 pollen allergens from 27 plant species have been deposited in the allergen databank Since pure and standardized recombinant allergens can be formulated to replace natural extracts using genetic engineered allergens for SIT become a possible and promising method for immunotherapy. In last decade a variety of recombinant allergens from plants mites molds mammals and insects have been expressed using various systems such as E.coli24 Pichia25 and plants26. Moreover the recombinant allergens can be engineered to reduce the risk of the IgE-mediated side effects. The molecules with reduced allergenicity hypoallergen would not lead to anaphylactic reaction upon injection and would allow higher-dose administration of allergen which has showed to be more effective in symptom reduction than low dose. In this way high dose of allergen can be administered to allergic patients which increases the efficacy of the treatment. Based on this consideration site-directed mutant and comformation has been applied in the recombination of hypoallergens27 28. The clinical use of these products may lead to not only improve diagnostic specificity and sensitivity but also safer and more effective immunotherapy. 4 Summary As the most widespread species on the earth plant is a part of the human normal life. It is hard to avoid plant allergens from trees grasses and weeds. Although specific immunotherapy represents a curative approach toward allergy the mechanism operating in SIT still remains not completely understood. In recent statistics there has been a significant increase in the prevalence of allergic disease over the past 2 to 3 decades. Currently more than 130 million people suffer from the asthma and the numbers are increasing29. There is a research considering that air pollutants from industry and automobiles are cofactors contributing to recent increase in allergic disease and asthma30. On the other hand man cannot ignorance transgenic plants are widespread in the modern world it could be the source for new kind of allergens. Hallucinogen Hallucinogens are a general group of pharmacological agents that can be divided into three broad categories: psychedelics dissociatives and deliriants. These classes of psychoactive drugs have in common that they can cause subjective changes in perception thought emotion and consciousness. Unlike other psychoactive drugs such as stimulants and opioids these drugs do not merely amplify familiar states of mind but rather induce experiences that are qualitatively different from those of ordinary consciousness. These experiences are often compared to non-ordinary forms of consciousness such as trancemeditation dreams or insanity. L. E. Hollisters criteria for establishing that a drug is hallucinogenic is:  in proportion to other effects changes in thought perception and mood should predominate  intellectual or memory impairment should be minimal  stupor narcosis or excessive stimulation should not be an integral effect  autonomic nervous system side effects should be minimal and  addictive craving should be absent. Not all drugs produce the same effect and even the same drug can produce different effects in the same individual on different occasions.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Dissociatives Dissociatives produce analgesia amnesia and catalepsy at anesthetic doses. 10 They also produce a sense of detachment from the surrounding environment hence "the state has been designated as dissociative anesthesia since the patient truly seems disassociated from his environment." 11 Dissociative symptoms include the disruption or compartmentalization of "...the usually integrated functions of consciousness memory identity or perception." 12p. 523 Dissociation of sensory input can cause derealization the perception of the outside world as being dream-like or unreal. Other dissociative experiences include depersonalization which includes feeling detached from ones body feeling unreal feeling able to observe ones actions but not actively take control being unable to recognize ones self in the mirror while maintaining rational awareness that the image in the mirror is the same person. 131415 Simeon 2004 offered "...common descriptions of depersonalisation experiences: watching oneself from a distance similar to watching a movie candid out-of-body experiences a sense of just going through the motions one part of the self acting/participating while the other part is observing...." 16 The primary dissociatives achieve their effect through blocking the signals received by the NMDA receptor set NMDA receptor antagonism and include ketamine phencyclidinePCP dextromethorphan DXM and nitrous oxide. 171819 However dissociation is also remarkably administered by salvinorin As the active constituent in Salvia divinorumshown to the left potent κ-opioid receptor agonism 20 and is notably the most potent psychoactive chemical harnessed directly from the plant kingdom. citation needed Some dissociatives can have CNS depressant effects thereby carrying similar risks as opioids which can slow breathing or heart rate to levels resulting in death when using very high doses. DXM in higher doses can increase heart rate and blood pressure and still depress respiration. Inversely PCP can have more unpredictable effects and has often been classified as a stimulant and a depressant in some texts along with being as a dissociative. While many have reported that they "feel no pain" while under the effects of PCP DXM and Ketamine this does not fall under the usual classification of anesthetics in recreational doses anesthetic doses of DXM may be dangerous. Rather true to their name they process pain as a kind of "far away" sensation pain although present becomes a disembodied experience and there is much less emotion associated with it. As for probably the most common dissociative nitrous oxide the principal risk seems to be due to oxygen deprivation. Injury from falling is also a danger as nitrous oxide may cause sudden loss of consciousness an effect of oxygen deprivation. Because of the high level of physical activity and relative imperviousness to pain induced by PCP some deaths have been reported due to the release of myoglobin from ruptured muscle cells. High amounts of myoglobin can induce renal shutdown. 21 Along with most if not all of the chemicals in this article none of the dissociatives have any physically addictive properties though psychological addiction has been observed. Many users of dissociatives have been concerned about the possibility of NMDA antagonist neurotoxicity NAN. This concern is partly due to William E. White the author of the DXM FAQ who claimed that dissociatives definitely cause brain damage. 22 The argument was criticized on the basis of lack of evidence 23 and White retracted his claim. 24 Whites claims and the ensuing criticism surrounded original research by John Olney. In 1989 John Olney discovered that neuronal vacuolation and other cytotoxic changes "lesions" occurred in brains of rats administered NMDA antagonists including PCP and ketamine. 25 Repeated doses of NMDA antagonists led to cellular tolerance and hence continuous exposure to NMDA antagonists did not lead to cumulative neurotoxic effects. Antihistamines such as diphenhydramine barbiturates and even diazepam have been found to prevent NAN. 26 LSD and DOB have also been found to prevent NAN. 27

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Deliriants Deliriants as their name implies induce a state of delirium in the user characterized by extreme confusion and an inability to control ones actions. They are called deliriants because their subjective effects are similar to the experiences of people with delirious fevers. Included in this group are such plants as Atropa belladonna deadly nightshade Brugmansia species Angels Trumpet Datura stramonium Jimson weed Hyoscyamus nigerhenbane Mandragora officinarum mandrake and Myristica fragrans nutmeg as well as a number of pharmaceutical drugs when taken in very high doses such asdiphenhydramine Benadryl and its close relative dimenhydrinate Dramamine. Uncured tobacco is also a deliriant due to its intoxicatingly high levels of nicotine. 28 In addition to the dangers of being far more disconnected from reality than with other drugs and retaining a truly fragmented dissociation from regular consciousness without being immobilized the anticholinergics are toxic carry the risk of death by overdose and also include a number of uncomfortable side effects. These side effects usually includedehydration and mydriasis dilation of the pupils. Most modern-day psychonauts who use deliriants report similar or identical hallucinations and challenges. For example diphenhydramine as well as dimenhydrinate when taken in a high enough dosage often are reported to evoke vivid dark and entity-like hallucinations peripheral disturbances feelings of being alone but simultaneously of being watched and hallucinations of real things ceasing to exist. Deliriants also may cause confusion or even rage and thus have been used by ancient peoples as a stimulant before going into battle. Traditional use Psychedelics have a long history of traditional use in medicine and religion where they are prized for their perceived ability to promote physical and mental healing. In this context they are often known asentheogens. Native American practitioners using mescaline-containing cacti most notably peyote San Pedro and Peruvian torch have reported success against alcoholism and Mazatec practitioners routinely use psilocybin mushrooms for divination and healing. Ayahuasca which contains the powerful psychedelic DMT is used in Peru and other parts of South America for spiritual and physical healing as well as in religious festivals. Taxonomy Hallucinogens can be classified by their subjective effects mechanisms of action and chemical structure. These classifications often correlate to some extent. In this article they are classified as psychedelicsdissociatives and deliriants preferably entirely to the exclusion of the inaccurate word hallucinogen but the reader is well advised to consider that this particular classification is not universally accepted. The taxonomy used here attempts to blend these three approaches in order to provide as clear and accessible an overview as possible. Almost all hallucinogens contain nitrogen and are therefore classified as alkaloids. THC and salvinorin A are exceptions. Many hallucinogens have chemical structures similar to those of human neurotransmitters such as serotonin and temporarily modify the action of neurotransmitters and/or receptor sites.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Causes: Causes of teratogenesis can broadly be classified as:  Toxic substances such as for humans drugs in pregnancy and environmental toxins in pregnancy.  Vertically transmitted infection  Lack of nutrients. For example lack of folic acid in the nutrition in pregnancy for humans can result in spina bifida.  Physical restraint. An example is Potter syndrome due to oligohydramnios in humans.  Genetic disorders PLANT TOXIC PART SYMPTOMS HOUSE PLANTS Hyacinth Narcissus Daffodil Bulbs Nausea vomiting diarrhea. May be fatal. Oleander Leaves branches Extremely poisonous. Affects the heart produces severe digestive upset and has caused death. Dieffenbachia Dumb Cane Elephant Ear All parts Intense burning and irritation of the mouth and tongue. Death can occur if base of the tongue swells enough to block the air passage of the throat. Rosary Pea Castor Bean Seeds Fatal. A single Rosary Pea seed has caused death. One or two Castor Bean seeds are near the lethal dose for adults. FLOWER GARDEN PLANTS Larkspur Young plant seeds Digestive upset nervous excitement depression. May be fatal. Monkshood Fleshy roots Digestive upset and nervous excitement. Autumn Crocus Star of Bethlehem Bulbs Vomiting and nervous excitement. Lily-of-the-Valley Leaves flowers Irregular heart beat and pulse usually accompanied by digestive upset and mental confusion. Iris Underground stems Severe-but not usually serious-digestive upset. Foxglove Leaves Large amounts cause dangerously irregular heartbeat and pulse usually digestive upset and mental confusion. May be fatal. Bleeding Heart Foliage roots May be poisonous in large amounts. Has proved fatal to cattle. VEGETABLE GARDEN PLANTS Rhubarb Leaf blade Fatal. Large amounts of raw or cooked leaves can cause convulsions coma followed rapidly by death. ORNAMENTAL PLANTS Daphne Berries Fatal. A few berries can kill a child.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Wisteria Seeds pods Mild to severe digestive upset. Many children are poisoned by this plant. Golden Chain Bean-like capsules in which the seeds are suspended Severe poisoning. Excitement staggering convulsions and coma. May be fatal. Laurels Rhododendrons Azaleas All parts Fatal. Produces nausea and vomiting depression difficult breathing prostration and coma. Jasmine Berries Fatal. Digestive disturbance and nervous symptoms. Lantana Camara Red Sage Green berries Fatal. Affects lungs kidneys heart and nervous system. Grows in the southern U.S. And in moderate climates. Yew Berries foliage Fatal. Foliage more toxic than berries. Death is usually sudden without warning symptoms. TREES AND SHRUBS Wild and cultivated cherries Twigs foliage Fatal. Contains a compound that releases cyanide when eaten. Gasping excitement and prostration are common symptoms. Oaks Foliage acorns Affects kidneys gradually. Symptoms appear only after several days or weeks. Takes a large amount for poisoning. Elderberry All parts especially roots Children have been poisoned by using pieces of the pithy stems for blowguns. Nausea and digestive upset. Black Locust Bark sprouts foliage Children have suffered nausea weakness and depression after chewing the bark and seeds. PLANTS IN WOODED AREAS Jack-in-the-Pulpit All parts especially roots Like Dumb Cane contains small needle-like crystals of calcium oxalate that cause intense irritation and burning of the mouth and tongue. Moonseed Berries Blue purple color resembling wild grapes. May be fatal. Mayapple Apple foliage roots Contains at least 16 active toxic principles primarily in the roots. Children often eat the apple with no ill effects but several apples may cause diarrhea. Mistletoe Berries Fatal. Both children and adults have died from eating the berries. PLANTS IN SWAMP OR MOIST AREAS Water Hemlock All parts Fatal. Violent and painful convulsions. A number of people have died from hemlock.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY PLANTS IN FIELDS Buttercups All parts Irritant juices may severely injure the digestive system. Nightshade All parts especially the unripened berry Fatal. Intense digestive disturbance and nervous symptoms. Poison Hemlock All parts Fatal. Resembles a large wild carrot. Jimson Weed Thorn Apple All parts Abnormal thirst distorted sight delirium incoherence and coma. Common cause of poisoning. Has proved fatal. First Aid Workers who have come in contact with poisonous plants should:  Immediately rinse skin with rubbing alcohol specialized poison plant washes degreasing soap such as dishwashing soap or detergent and lots of water. o Rinse frequently so that wash solutions do not dry on the skin and further spread the urushiol.  Scrub under nails with a brush.  Apply wet compresses calamine lotion or hydrocortisone cream to the skin to reduce itching and blistering. o Follow the directions on any creams and lotions. Do not apply to broken skin such as open blisters. o Oatmeal baths may relieve itching.  An antihistamine such as diphenhydramine Benadryl can be taken to help relieve itching. o Follow directions on the package. o Drowsiness may occur. o If children come in contact with work clothing contaminated with urushiol a pediatrician should be contacted to determine appropriate dosage.  In severe cases or if the rash is on the face or genitals seek professional medical attention. Treatment Severe allergic reactions anaphylaxis need to be treated with a medicine called epinephrine which can be life saving when given right away. If you use epinephrine call 911 and go straight to the hospital. The best way to reduce symptoms is to avoid what causes your allergies. This is especially important for food and drug allergies. There are several types of medications to prevent and treat allergies. Which medicine your doctor recommends depends on the type and severity of your symptoms your age and overall health. Illnesses that are caused by allergies such as asthma hay fever and eczema may need other treatments. Medications that can be used to treat allergies include: ANTIHISTAMINES

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Antihistamines are available over-the-counter and by prescription. They are available in many forms including:  Capsules and pills  Eye drops  Injection  Liquid  Nasal spray CORTICOSTEROIDS Anti-inflammatory medications corticosteroids are available in many forms including:  Creams and ointment for the skin  Eye drops  Nasal spray  Lung inhaler Patients with severe allergic symptoms may be prescribed corticosteroid pills or injections for short periods of time. DECONGESTANTS Decongestants can help relieve a stuffy nose. Do not use decongestant nasal spray for more than several days because they can cause a "rebound" effect and make the congestion worse. Decongestants in pill form do not cause this problem. People with high blood pressure heart problems or prostate enlargement should use decongestants with caution. OTHER MEDICINES Leukotriene inhibitors are medicines that block the substances that trigger allergies. Zafirlukast Accolate and montelukast Singulair are approved for people with asthma and indoor and outdoor allergies. ALLERGY SHOTS Allergy shots immunotherapy are sometimes recommended if you cannot avoid the allergen and your symptoms are hard to control. Allergy shots keep your body from over-reacting to the allergen. You will get regular injections of the allergen. Each dose is slightly larger than the last dose until a maximum dose is reached. These shots do not work for everybody and you will have to visit the doctor often. Allergy Tests Allergy testing involves having a skin or blood test to find out what substance orallergen may trigger an allergic response in a person. Skin tests are usually done because they are rapid reliable and generally less expensive than blood tests but either type of test may be used. Skin tests A small amount of a suspected allergen is placed on or below the skin to see if a reaction develops. There are three types of skin tests:  Skin prick test. This test is done by placing a drop of a solution containing a possible allergen on the skin and a series of scratches or needle pricks allows the solution to enter the skin. If the skin develops a red raised itchy area called a wheal it usually means that the person is allergic to that allergen. This is called a positive reaction.  Intradermal test. During this test a small amount of the allergen solution is injected into the skin. An intradermal allergy test may be done when a substance does not cause a reaction in the skin prick test but is still suspected as an allergen for that person. The intradermal test is more sensitive than the skin prick test but is more often positive in people who do not have symptoms to that allergen false-positive test results.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY  Skin patch test. For a skin patch test the allergen solution is placed on a pad that is taped to the skin for 24 to 72 hours. This test is used to detect a skin allergy called contact dermatitis. Blood test Allergy blood tests look for substances in the blood called antibodies. Blood tests are not as sensitive as skin tests but are often used for people who are not able to have skin tests. The most common type of blood test used is the enzyme-linked immunosorbent assay ELISA EIA. It measures the blood level of a type of antibody called immunoglobulin E or IgE that the body may make in response to certain allergens. IgE levels are often higher in people who have allergies or asthma. Other lab testing methods such as radioallergosorbent testing RAST or an immunoassay capture test ImmunoCAP UniCAP or Pharmacia CAP may be used to provide more information. Challenge testing: Challenge testing is when small amounts of a suspected allergen are introduced to the body orally through inhalation or other routes. Except for testing food and medication allergies challenges are rarely performed. When this type of testing is chosen it must be closely supervised by an allergist. Elimination/Challenge tests: This testing method is used most often with foods or medicines. A patient with a suspected allergen is instructed to modify his/her diet to totally avoid that allergen for determined time. If the patient experiences significant improvement he/she may then be “challenged” by reintroducing the allergen to see if symptoms can be reproduced. Patch testing: Patch testing is used to help ascertain the cause of skin contact allergy or contact dermatitis. Adhesive patches usually treated with a number of common allergic chemicals or skin sensitizers are applied to the back. The skin is then examined for possible local reactions at least twice usually at 48 hours after application of the patch and again two or three days later. Unreliable tests: There are other types of allergy testing methods that the that are unreliable including applied kinesiology allergy testing through muscle relaxation cytotoxicity testing urine autoinjection skin titration Rinkel method and provocative and neutralization subcutaneous testing or sublingual provocation. How the Test Is Performed There are three common methods of allergy skin testing. The skin prick test involves:  Placing a small amount of substances that may be causing your symptoms on the skin most often on the forearm upper arm or back.  Then the skin is pricked so the allergen goes under the skins surface.  The health care provider closely watches the skin for swelling and redness or other signs of a reaction. Results are usually seen within 15-20 minutes.  Several allergens can be tested at the same time. The intradermal skin test involves:  Injecting a small amount of allergen into the skin.  Then the health care provider watches for a reaction at the site.  This test is more likely to be used to find out if you are allergic to something specific such as bee venom or penicillin. Patch testing is a method to diagnose the cause of skin reactions that occur after the substance touches the skin.  Possible allergens are taped to the skin for 48 hours.  The health care provider will look at the area in 72 - 96 hours.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Differential diagnosis Before a diagnosis of allergic disease can be confirmed other possible causes of the presenting symptoms should be considered. Vasomotor rhinitis for example is one of many maladies that shares symptoms with allergic rhinitis underscoring the need for professional differential diagnosis. Once a diagnosis of asthma rhinitis anaphylaxis or other allergic disease has been made there are several methods for discovering the causative agent of that allergy. ALTERNATIVE SYSTEM OF MEDICINES Siddha Principles Like all other medicine systems Siddha system of medicine also has some underlying principles and concepts. These fundamental principles bear resemblance to that of ancient Ayurveda. According to Siddha system the human body food and the drugs are the replica of the universe irrespective of their origin. Moreover they believe that the universe holds two main entities namely matter and energy. Siddhars call them Siva male and Shakti female. The two are inseparable and co-exist as matter cannot subsist without the energy in it and vice versa. They are also the primordial elements Bhutas known as Munn solid Neer fluid Thee radiance Vayu gas and Veli ether. These are present in every substance in varied proportions. Also Earth Water Fire Air and Ether are the manifestations of these elements. Even the human body is made up of these five elements in different permutations. It also considers that it is an assortment of three humours seven basic tissues and the waste products produced by the body such as faeces urine and sweat. The food ingested by the humans is regarded as the elementary building material of the body which in turn is converted into humours body tissues and waste products. Besides the food and drugs also contains mixture of five elements. However when the equilibrium of humors considered as health is disturbed it leads to disease or sickness. Drugs constituting varying proportion of the elements are responsible for therapeutic actions and results. Apart from this Siddha system also lays down the concept of salvation in life. The exponents of this system emphasize on achievement of this state via medicines and meditation. Principles of Ayurveda Ayurveda is a holistic healing science which comprises of two words Ayu and Veda. Ayu means life and Vedameans knowledge or science. So the literal meaning of the word Ayurveda is the science of life. Ayurveda is a science dealing not only with treatment of some diseases but is a complete way of life. Ayurveda aims at making a happy healthy and peaceful society. The two most important aims of Ayurveda are: + To maintain the health of healthy people + To cure the diseases of sick people A Person is seen in Ayurveda as a unique individual made up of five primary elements.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY These elements are ether space air firewater and earth. Just as in nature we too have these five elements in us. When any of these elements are imbalanced in the environment they will in turn have an influence on us. The foods we eat and the weather are just two examples of the influence of these elements . While we are a composite of these five primary elements certain elements are seen to have an ability to combine to create various physiological functions. The elements combine with Ether and Air in dominence to form what is known in Ayurveda as Vata Dosha. Vatagoverns the principle of movement and therefore can be seen as the force which directs nerve impulses circulation respiration and elemination etc. The elements with Fire and Water in dominence combine to form the Pitta Dosha . The Pitta Dosha is responsible for the process of transformation or metabolism. The transformation of foods into nutrients that our bodies can assimilate is an example of a Pitta function. Pitta is also responsible for metabolism in the organ and tissue systems as well as cellular metabolism. Finally it is predominantly the water and earth elements which combine to form the Kapha Dosha. Kapha is responsible for growth adding structure unit by unit. It also offers protection for example in form of the cerebral-spinal fluidwhich protects the brain and spinal column. The mucousal lining of the stomach is another example of the function of Kapha Dosha protecting the tissues. We are all made up of unique proportions of VataPitta and Kapha. These ratios of the Doshas vary in each individual and because of this Ayurveda sees each person as a special mixture that accounts for our diversity. Ayurveda gives us a model to look at each individual as a unique makeup of the three doshas and to thereby design treatment protocols that specifically address a persons health challenges. When any of the doshas become accumulated Ayurveda will suggest specific lifestyle and nutritional guidelines to assist the individual in reducing the dosha that has become excessive. Also herbal medicines will be suggested to cure the imbalance and the disease. Understanding this main principle of Ayurveda it offers us an explanation as to why one person responds differently to a treatment or diet than another and why persons with the same disease might yet require different treatments and medications. Other important basic principles of Ayurveda which are briefly mentioned here are: 1. Dhatus- These are the basic tissues which maintain and nourish the body. They are seven in number namely- rasachyle rakthablood mamsamusclesmedafatty tissue asthibone majjamarrow and suklareprodutive tissue. Proper amount of each dhatu and their balanced function is very important for good health. 2. Mala- These are the waste materials produced as a result of various metabolic activities in the body. They are mainly urine feaces sweat etc. Proper elimination of the malas is equally important for good health. Accumulation of malas causes many diseases in the body. 3. Srotas- These are different types of channels which are responsible for transportation of

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY food dhatusmalas and doshas. Proper functioning of srotas is necessary for transporting different materials to the site of their requirement. Blockage of srotas causes many diseases. 4. Agni- These are different types of enzymes responsible for digestion and transforming one material to another. All these factors should function in a proper balance for good health. They are inter-related and are directly or indirectly responsible for maintaining equilibrium of the tridoshas. Balance and Harmony of the Three Doshas When the three Doshas are well harmonised and function in a balanced manner it results in good nourishment and well-being of the individual . But when there is imbalance or disharmony within or between them it will result in elemental imbalance leading to various kinds of ailments. The Ayurvedic concept of physical health revolves round these three Doshas and its primary purpose is to help maintain them in a balanced state and thus to prevent disease.This humoral theory is not unique to the ancient Indian Medicine : The Yin and Yang theory in chinese medicine and the Hippocratic theory of four humours in Greek medicine are also very similar. The Qualities of the Three Doshas The three Doshas possess qualities and their increase or decrease in the system depends upon the similar or antagonistic qualities of everything ingested. Vata is : dry cold light mobile clear rough subtle Pitta is : slightly oily hot intense light fluidfree flowing foul smelling. Kapha is: oily cold heavy stable viscid smooth soft Both Vata and Pitta are light and only Kapha is heavy. Both Vata and Kapha are cold and only Pitta is hot. Both Pitta and Kapha are moist and oily and only Vata is dry. Anything dry almost always increases Vata anything hot increases Pittaand anything heavy Kapha. Puffed rice is dry cold light and rough - overindulgence in puffed rice therefore is likely to increase Vata in the overindulger. Mustard oil is oily hot intense fluid strong-smelling and liquid and increases Pitta in the consumer. Yoghurt which being creamy cold heavy viscid smooth and soft is the very image of Kapha adds to the bodys Kapha when eaten. All Five elemets as expressed through Vata Pitta and Kapha are essential to life working together to create health or produce disease. No one dosha can produce or sustain life - all three must work together each in its own way.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Principles of Unani The Unani system recognises that disease is an unnatural process and symptoms of a disease are bodys reaction to noxious factors from its surroundings. The chief function of the physician is to aid the natural force of the body which is termed as Tabi‟at Physis. Unani medicine is based on four Humor theories which are Dam Blood Balgham Phlegm Safra Yellow bile and Sauda black bile. The body has the power of self-control to maintain an optimum balance of these humors which is called as Quwwat-e- Mudabbirah-e-Badan Medicatrix Naturae. The essential constituents and the working principles of the body according to Unani system of medicine can be classified into seven main groups: 1. Arkaan Elements 2. Mizaj Temperament 3. Akhlaat Humors 4. A‟za Organs 5. Arwaah. Pneuma 6. Quwa Faculties of Power 7. Afa‟al Actions Unani system of medicine believes that Arkaan elements which are broadly divided in four categories i.e. Earth Arz Water Maa‟ Air Hawa and Fire Naar are bricks of human structure and have their own temperament. After mixing and interaction of these principle elements in a particular ratio a new structure comes into existence having its own temperament Mizaj. Mizaj Temperament of each and every individual varies widely as per composition as well as other surrounding factors and circumstances. Normal temperament is defined as a condition in which a person survives comfortably with all symptoms of healthy life. Akhlaat Humors are liquid components of body which run through different channels inside the body and provide nutrition to the whole tissues of the body and maintain normal health. These humors have been named according to their colour as Dam Blood Balgham Phlegm Safra Yellow bile and Sauda black bile which are red white yellow and black in colour respectively. Equilibrium of these humors is mainly responsible for health. Any alteration/deviation in quality or quantity from optimum position may lead to disease. A‟zaa Organs are solid components of the body which are composed of different types of tissues. Organs collectively form a system. Through these organized systems body performs its routine activity. Certain organs are specified as vital organs Azaa-e- Raeesah of the body which include Heart Brain Liver Testes/ Ovaries and these are responsible for vital functioning of the body and play major role in continuity and propagation of life. Arwah Pneuma are the gaseous components of the body mainly consisting of Naseem Oxygen which runs in the body through blood in dissolved form. It is the basic source of life which provides energy for all body activities.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Quwa Faculties of Power are nothing but ability to perform. All organs have been assigned a particular type of action on the basis of their nature and compositions. The main four faculties of power are Quwwat- e-Haiwaniyah Quwwat-e-Nafsaaniyah Quwwat-e-Tab‟iyah and Quwwat-e-Tanaasuliyah and the four vitals organs i.e. Heart Brain Liver Testes/ Ovaries are responsible for these powers respectively. Afa‟al Functions are bodily activities essential for fulfilling the objectives of the body. The organs and also testimony perform these to the presence of power in them. Principles of Homeopathy Similia Similibus Curanter This is the law of similars. It states that that which can cause can cure. The onion which produces tears in the eye and irritation similar to a cold can be used as a homeopathic medicine to cure colds which have irritating tears. The early Indians recognised this principle and states that Vishasya Vishamevam Aushadam and Samaha Samena Shantihi but it was Dr.Samuel Hahnemann who through his studies and experiments on the various medicines available in nature practically proved the law. Simplex Similimum Minimum This principle consists of three words. The first is Simplex i.e : simple medicines not compound should be prescribed. This is the doctrine of single remedy. Mixture of medicines or polypharmacy is not allowed. Only one medicine must be given at a time. Similimum - As discussed previously the totality of symptoms of the patient must be taken. This will yield a picture which corresponds to one medicine the similimum which must be given. That medicine which has been tested on various provers and has produced similar symptoms as that of the patient is the similar remedy. Minimum - A low dosage of medicine is recommended. In homeopathy less is more so medicines of low potency and given at long intervals have a better impact. Hahnemann in fact used to give just one dose of the medicine and wait to see the reaction over a period of time. Principle of Individualisation Treat the patient not the disease. This is the most important doctrine of homeopathy. Not two human beings are alike and so the medicines used for their treatment need not be alike. Homeopathic medicines are prescribed based on the totality of symptoms of that individual. So the name of the disease is not important to the doctor who tries to get a complete picture of the patient - his symptomsthe modalities of symptoms his likes and disliked his environment etc to arrive at the individualised remedy - which is the similimum. Principle of Potentisation Homeopathic medicines are diluted in alcohol or milk-sugarlactose to make them more palatable and also to reduce the harmful effects. It has been found that the more the medicine is diluted the more effective and powerful it becomes. So the process of the dilution is called as potentisation and the medicines are referred to as potencies.The crude homeopathic medicineeg : Cinchona/Lachesis is triturated in alcohol to yield the mother tincture. The mother tincture is denoted by the symbol ø. Potency : 1x potency of the medicine signifies 1 part of mother tincture diluted with 9 parts of alcohol / milk sugar. 2x potency is 1x of medicine diluted with 9 parts of sugar milk / alcohol. 1C potency is mother tincture diluted with 99 parts. 1M potency is mother tincture diluted with 999 parts. Low potency : 1x 3x 6x 3c 12x 6c

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Medium potency : 12x 30x 30c High potency : 200c 1M 20 M CM LM etc. Law of Direction The law of direction of cure proposed by Dr.Constantine Hering states that - "As a patient recovers from a disease the symptoms move from within outwards from above downwardsfrom centre to circumference and disappear in the reverse order of their appearance" A patient suffering from a skin disease may use various medicines which suppress this disorder and send it into the body and it may manifest as athma. So when this patient takes homeopathic medicine the asthma is replaced with the skin infection and then finally the skin infection leaves to yield a cure. Three-legged stool This principle attributed to the elder LippeDr.Adolph Lippe states that while prescribing a medicine three leading symptoms of that medicine should match the symptoms of the patient. Just as a stool with three legs is more stable than a stool with one leg medicine given on the basis of atleast three key symptoms is more reliable than that treated with one symptom. Thus a careful study is required to apply this law. Use of Materia Medica The Materia Medica is a dictionary of homeopathic medicines and their symptoms. It is a book which is the final authority on homeopathy. The materia medica contains the list of symptoms experienced by provers of the medicine. The symptoms are arranged in a systematic order - Mind symptoms related to mind/mental Head Eyes etc.. It is not required for a doctor to memorise or remember all the contents of the Materia Medica. What is required is to understand the nature / keynotes of each remedy. A number of materia medicas have been authored. Prominent among them are KentsLectures Herings Guiding Symptoms Allens Keynotes etc. Repertorisation The repertory is an index to the Materia Medica. It is a book containing all possible symptoms arranged in alphabetical order for each of the organs of the body. The physican has to regularly refer this book to find out the medicines which have produced in a prover the symptoms of the patient. Only through correct usage of the repertory can the job of prescription be made easier.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Types of formulation used in alternative system of medicine. Definition : Ayurvedic medicines are all the medicines intended for internal or external use for or in the diagnosis treatment mitigation or prevention of disease or disorder in human beings or animal and manufactured exclusively in accordance with the formulae described in the authorative books of Ayurvedic Systems of medicine specified in the first schedule of the Drug and Cosmetic act 1940. Ayurvedic Dosage Forms Liquid Asava Arishta Arka Kwaha Taila Dravaka Netrabindu Semisolides Avaleha Lepa Matras Kalka Swarasa Kajjali Praash Solid Vatika Gutika Churna Bhasma Ksharas Nasyas Sattva

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY HERBALS AND THEIR FORMULATIONS Tinctures are concentrated herbal extracts that are made using alcohol and chopped herbs. The tincture is especially effective in drawing out the essential compounds of plants especially those that are fibrous or woody and from roots and resins. 1 Since this method ensures that the herbs and their nutrients can be preserved for a long time it is often mentioned in herbal books and remedies as a preferred way of using herbs. In addition many herbalists love tinctures for other beneficial reasons such as their being easy to carry their utility for long-term treatments and their ability to be absorbed rapidly as well as allowing for immediate dosage changes. 2 As well should the tincture prove bitter its easily added to juice to disguise the flavor. Another benefit of tinctures is that they keep nutrients from the plants in a stable soluble form and they retain the volatile and semi-volatile ingredients that are otherwise lost in heat-treatment and processing of dry herbal extracts. Fresh Herb • Finely chop or grind clean herb to release juice and expose surface area. • Fill jar 2/3 to 3/4 with herb. OR Fill jar 1/4 to ½ with roots. • Pour alcohol over the herbs. Cover completely • Jar should appear full of herb but herb should move freely when shaken. Dried Herb • Use finely cut herbal material. • Fill jar 1/2 to 3/4 with herb OR Fill jar 1/4 to 1/3 with roots. • Pour alcohol over the herbs. Cover completely • Roots will expand by ½ their size when reconstituted Purchase quality alcohol. The preferred type of alcohol for producing a tincture isvodka. 3 This is owing to its being colorless odorless and fairly flavorless. If you cannot obtain vodka brandy rum or whiskey can be substituted. Whatever alcohol is chosen it must be 80 proof namely 40 alcohol to prevent mildewing of the plant material in the bottle.It is also possible to make a tincture from quality apple cider vinegar or glycerin. 4 The alternatives may work better where the patient refuses alcohol. Use a suitable container. The container for the tincture should be glass or ceramic. Avoid using metallic or plastic containers because these can react with the tincture or leach dangerous chemicals over time. Items such as a Mason jar a glass bottle with an attached stopper etc. are ideal for steeping a tincture. In addition you will need to get some small dark glass tincture bottles for storing the tincture in once it has been made these bottles should have a tight screw-on or tight clip-on lid to prevent air intrusion during storage but to allow for ease of use. Ensure that all containers are both washed clean and sterilized prior to use. Prepare the tincture. You can prepare a tincture by measurement or by sight it really depends on your level of comfort with simply adding herbs and judging by eye or whether you feel more comfortable adding them by measured weight. Also you should know whether you want to add fresh powdered or dried herbs to the tincture. Some suggestions for adding the herbs in the order of fresh powdered or dried are as follows:  Add enough fresh chopped herbs to fill the glass container. Cover with alcohol. 5  Add 4 ounces 113g of powdered herb with 1 pint 473ml of alcohol orvinegar/glycerin. 6  Add 7 ounces 198g of dried herb material to 35 fluid ounces 1 liter of alcohol or vinegar/glycerin. Using a butter knife stir around the edge of the glass container to ensure that air bubbles are broken. Seal the container. Place it into a cool dark area a cupboard shelf works best. The container should be stored there for 8 days to a month. 7

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY  Shake the container regularly. Humbart Santillo recommends shaking it twice a day for 14 days 8 while James Wong recommends shaking it occasionally. 9  Be sure to label the steeping tincture so that you know what it is and the date on which it was made. Keep it out of the reach of children and pets. Strain the tincture. Once the steeping time is finished either the tincture instructions youre following will inform you of this or youll know already from experience but if not about two weeks is a good steeping time strain the tincture as follows:  Place a muslin cloth across a sieve. Place a large bowl underneath to catch the strained liquid.  Gently pour the steeped liquid through the muslin-lined sieve. The muslin will capture the plant material and the liquid will pass through into the bowl underneath.  Press the herb material with a wooden or bamboo spoon to squeeze out some more liquid and lastly twist the muslin to extract any leftover liquid from the herbs. Burdock Root Extract Natural healers use this herb as an effective blood purifier believing that it rids the body of toxins. Excellent for arthritis and applied externally for skin problems. Burdock is still used today as a diuretic and to support the healing of chronic acne and psoriasis. Butchers Broom Extract For centuries European herbalists have used this herb to relieve water retention and to treat the discomfort and pain caused by poor circulation in the legs. This plant contains steroid-like compounds that may constrict veins and reduce inflammation caused by arthritis and rheumatism. Capsicum Tincture Capsicum tincture produces a local stimulant and analgesic effect. Use in cases of pain along the spinal nerves and other nerve endings nerve root syndrome inflammation of the voluntary muscles lower back pain and pain in the hips. Do not use in case of hypersensitivity. How to Make Herbal Syrups Herbal syrups are not hard to make and are a good alternative way to prepare some mixtures especially some of the herbs that are really bitter. Some of the herbs are bitter which serves a natural purpose – it keeps us from overusing and also stimulates digestive juices. The bitterness can also make us not want to take the medicine especially children. Syrups help in this area and also it can extend the storage life of the herb. Syrups are good to use for colds and flu and to soothe a sore throat. Make sure you never use honey for children younger than one to two years old. First decide which herb you want to use For a basic syrup you can use a infusion or decoction that you have made. Put 1 part infusion or decoction to 1 part honey or sugar in a saucepan Gently heat this until the sugar or honey is completely dissolved. Cool slightly and pour into clean glass or ceramic jars or bottles Keep in the refrigerator for three to six months You can use 1 to 2 teaspoons of the syrup up to three times a day. A really popular herbal syrup is elderberry syrup you can make it with fresh or dried berries. There are red and blue elderberries the red ones have seeds that are toxic so use the blue ones. Or just buy some dried elderberries. It is good for cold and flu among other things.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Put a cup of fresh elderberries or a half cup of dried berries in a saucepan Pour in 3 cups of water Bring it to a boil then simmer for 30 minutes Smash the berries Strain the berry mixture with a strainer and cheesecloth Stir in the honey Pour into a bottle and label Store in the refrigerator for a few month Take 1 teaspoon every 2-3 hours while sick. Some use it regularly with food as you would syrup. Elder is VERY high in bioflavonoids and is a great antioxidant. They say for children under 2 years you can add the syrup to hot water and it will kill any microbes in the honey that might make them sick or just use sugar. What is a cream Creams generally consist of two basic components an oil phase and an aqueous phase. A cream is formed when the oil phase is successfully emulsified into the aqueous phase producing an oil in water emulsion of stable and solid consistency at room temperatures. Functions of a cream. A cream can be successfully used to deliver and hold nutrients and medications on the skins surface. Both the oil and aqueous components can be used as a carrier. The skin has a limited capacity to absorb many oils and some chemical compounds and is responsive to surface medications such as herbal extracts and to vibrational energies such as Flower essences. Procedure 1. Make the CREAM BASE by measuring the oil components into the larger jug and the aqueous components into the smaller jug. The oil phase must contain the emulsifier. Heat both in the water bath saucepans so they do not come in direct contact with the heating element and are thus protected from being over-heated. 2. Stir the components regularly with the spatula to distribute the heat and use the stick thermometer to measure the temperature. The usual temperature before mixing for the making of a stable emulsion is 80C for the aqueous component and 70C for the oils. 3. When the aqueous and oil components are at the required temperature and any waxes have melted mix the two together by removing both jugs out of the baths and away from the heating elements and pouring the water component into the oil. Use vigorous stirring or preferably a hand-held Bamix type stick blender to make an emulsion. 4. Do this for 1-2 minutes to allow the emulsion to form. Avoid blending air into the liquid pulse the blender and keep the blender head well under the liquid. 5. Quickly cool the mix to around 55C by sitting the jug in the cold-water bath as you stir the emulsion. 6. Add the remaining ingredients including any tinctures and specialized oils and omega 3 and fragrance oils at this time with constant stirring. Remove any set cream from the sides and bottom of the jug. Use a little gentle water bath heat if required. Blend again and avoid blending air into the emulsion.. 7. Allow the liquid emulsion to sit for a minute or two and tap the base of the jug to remove air bubbles. 8. When at 44C or showing signs of thickening i.e. starting to set usually around 42C pour into ready uncapped jars. Attention is needed as the cream can set quickly and a little hot water bath heat may be required to finish the pouring.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Capping and labeling 1. Allow the cream to cool until "cold to touch" before capping as condensation can occur on the inside of the lid and drop onto the surface of the cream and lead to mould growth. 2. Before capping check both the cream in the jar and the cap for any surface contaminants. 3. When the cream has set apply a label including the name for the cream date of expiry storage advice batch number and it is best to include details of the ingredients and how the cream is to be used. Assessment of quality Pharmaceutical assessment This should cover all important aspects of the quality assessment of herbal medicines. It should be sufficient to make reference to a pharmacopoeial monograph if one exists. If no such monograph is available a monograph must be supplied and should be set out as in an official pharmacopoeia. All procedures should be in accordance with good manufacturing practices. Crude plant material The botanical definition including genus species and authority should be given to ensure correct identification of a plant. A definition and description of the part of the plant from which the medicine is made e.g. leaf flower root should be provided together with an indication of whether fresh dried or traditionally processed material is used. The active and characteristic constituents should be specified and if possible content limits should be defined. Foreign matter impurities and microbial content should be defined or limited. Voucher specimens representing each lot of plant material processed should be authenticated by a qualified botanist and should be stored for at least a 10-year period. A lot number should be assigned and this should appear on the product label. Plant preparations Plant preparations include comminuted or powdered plant materials extracts tinctures fatty or essential oils expressed juices and preparations whose production involves fractionation purification or concentration. The manufacturing procedure should be described in detail. If other substances are added during manufacture in order to adjust the plant preparation to a certain level of active or characteristic constituents or for any other purpose the added substances should be mentioned in the manufacturing procedures. A method for identification and where possible assay of the plant preparation should be added. If identification of an active principle is not possible it should be sufficient to identify a characteristic substance or mixture of substances e.g. “chromatographic fingerprint” to ensure consistent quality of the preparation. Finished product The manufacturing procedure and formula including the amount of excipients should be described in detail. A finished product specification should be defined. A method of identification and where possible quantification of the plant material in the finished product should be defined. If the identification of an active principle is not possible it should be sufficient to identify a characteristic substance or mixture of substances e.g. “chromatographic fingerprint” to ensure consistent quality of the product. The finished product should comply with general requirements for particular dosage forms.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY For imported finished products confirmation of the regulatory status in the country of origin should be required. The WHO Certification Scheme on the Quality of Pharmaceutical Products Moving in International Commerce should be applied. Stability The physical and chemical stability of the product in the container in which it is to be marketed should be tested under defined storage conditions and the shelf-life should be established. Assessment of safety This should cover all relevant aspects of the safety assessment of a medicinal product. A guiding principle should be that if the product has been traditionally used without demonstrated harm no specific restrictive regulatory action should be undertaken unless new evidence demands a revised risk - benefit assessment. A review of the relevant literature should be provided with original articles or references to the original articles. If official monograph/review results exist reference can be made to them. However although long- term use without any evidence of risk may indicate that a medicine is harmless it is not always certain how far one can rely solely on long-term usage to provide assurance of innocuity in the light of concern expressed in recent years over the long-term hazards of some herbal medicines. Reported side-effects should be documented according to normal pharmaco-vigilance practices. Toxicological studies Toxicological studies if available should be part of the assessment. Literature should be indicated as above. Documentation of safety based on experience As a basic rule documentation of a long period of use should be taken into consideration when assessing safety. This means that when there are no detailed toxicological studies documented experience of long- term use without evidence of safety problems should form the basis of the risk assessment. However even in cases of drugs used over a long period chronic toxicological risks may have occurred but may not have been recognized. The period of use the health disorders treated the number of users and the countries with experience should be specified. If a toxicological risk is known toxicity data must be submitted. The assessment of risk whether independent of dose or related to dose should be documented. In the latter case the dosage specification must be an important part of the risk assessment. An explanation of the risks should be given if possible. Potential for misuse abuse or dependence must be documented. If long-term traditional use cannot be documented or there are doubts on safety toxicity data should be submitted. Assessment of efficacy This should cover all important aspects of efficacy assessment. A review of the relevant literature should be carried out and copies provided of the original articles or proper references made to them. Research studies if they exist should be taken into account. Activity The pharmacological and clinical effects of the active ingredients and if known their constituents with therapeutic activity should be specified or described.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Evidence required to support indications The indications for the use of the medicine should be specified. In the case of traditional medicines the requirements for proof of efficacy should depend on the kind of indication. For treatment of minor disorders and for non-specific indications some relaxation in requirements for proof of efficacy may be justified taking into account the extent of traditional use. The same considerations may apply to prophylactic use. Individual experiences recorded in reports from physicians traditional health practitioners or treated patients should be taken into account. Where traditional use has not been established appropriate clinical evidence should be required. Combination products As many herbal remedies consist of a combination of several active ingredients and as experience of the use of traditional remedies is often based on combination products assessment should differentiate between old and new combination products. Identical requirements for the assessment of old and new combinations would result in inappropriate assessment of certain traditional medicines. In the case of traditionally used combination products the documentation of traditional use such as classical texts of Ayurveda traditional Chinese medicine Unani Siddha and experience may serve as evidence. An explanation of a new combination of well known substances including effective dose ranges and compatibility should be required in addition to the documentation of traditional knowledge of each single ingredient. Each active ingredient must contribute to the efficacy of the medicine. Clinical studies may be required to justify the efficacy of a new ingredient and its positive effect on the total combination. Intended use Product information for the consumer Product labels and package inserts should be understandable to the consumer or patient. The package information should include all necessary information on the proper use of the product. The following elements of information will usually suffice: • name of the product • quantitative list of active ingredients • dosage form • indications - dosage if appropriate specified for children and the elderly - mode of administration - duration of use - major adverse effects if any - overdosage information

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY - contraindications warnings precautions and major drug interactions - use during pregnancy and lactation • expiry date • lot number • holder of the marketing authorization. Identification of the active ingredients by the Latin botanical name in addition to the common name in the language of preference of the national regulatory authority is recommended. Sometimes not all information that is ideally required may be available so drug regulatory authorities should determine their minimal requirements. Promotion Advertisements and other promotional material directed to health personnel and the general public should be fully consistent with the approved package information. Utilization of these guidelines These guidelines for the assessment of herbal medicines are intended to facilitate the work of regulatory authorities scientific bodies and industry in the development assessment and registration of such products. The assessment should reflect the scientific knowledge gathered in that field. Such assessment could be the basis for future classification of herbal medicines in different parts of the world. Other types of traditional medicines in addition to herbal products may be assessed in a similar way. The effective regulation and control of herbal medicines moving in international commerce also requires close liaison between national institutions that are able to keep under regular review all aspects of production and use of herbal medicines as well as to conduct or sponsor evaluative studies of their efficacy toxicity safety acceptability cost and relative value compared with other drugs used in modern medicine. Extraction techniques of Medicinal plants Extraction as the term is used pharmaceutically involves the separation of medicinally active portions of plant or animal tissues from the inactive or inert components by using selective solvents in standard extraction procedures. The products so obtained from plants are relatively impure liquids semisolids or powders intended only for oral or external use. These include classes of preparations known as decoctions infusions fluid extracts tinctures pilular semisolid extracts and powdered extracts. Such preparations popularly have been called galenicals named after Galen the second century Greek physician. The purposes of standardized extraction procedures for crude drugs are to attain the therapeutically desired portion and to eliminate the inert material by treatment with a selective solvent known as menstruum.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY The extract thus obtained may be ready for use as a medicinal agent in the form of tinctures and fluid extracts it may be further processed to be incorporated in any dosage form such as tablets or capsules or it may be fractionated to isolate individual chemical entities such as ajmalicine hyoscine and vincristine which are modern drugs. Thus standardization of extraction procedures contributes significantly to the final quality of the herbal drug. Circularly extraction Methods of Extraction of Medicinal Plants Maceration In this process the whole or coarsely powdered crude drug is placed in a stoppered container with the solvent and allowed to stand at room temperature for a period of at least 3 days with frequent agitation until the soluble matter has dissolved. The mixture then is strained the marc the damp solid material is pressed and the combined liquids are clarified by filtration or decantation after standing. Infusion Fresh infusions are prepared by macerating the crude drug for a short period of time with cold or boiling water. These are dilute solutions of the readily soluble constituents of crude drugs. Digestion This is a form of maceration in which gentle heat is used during the process of extraction. It is used when moderately elevated temperature is not objectionable. The solvent efficiency of the menstruum is thereby increased. Decoction In this process the crude drug is boiled in a specified volume of water for a defined time it is then cooled

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY and strained or filtered. This procedure is suitable for extracting water-soluble heat-stable constituents. This process is typically used in preparation of Ayurvedic extracts called “quath” or “kawath”. The starting ratio of crude drug to water is fixed e.g. 1:4 or 1:16 the volume is then brought down to one-fourth its original volume by boiling during the extraction procedure. Then the concentrated extract is filtered and used as such or processed further. Percolation This is the procedure used most frequently to extract active ingredients in the preparation of tinctures and fluid extracts. A percolator a narrow cone-shaped vessel open at both ends is generally used. The solid ingredients are moistened with an appropriate amount of the specified menstruum and allowed to stand for approximately 4 h in a well closed container after which the mass is packed and the top of the percolator is closed. Additional menstruum is added to form a shallow layer above the mass and the mixture is allowed to macerate in the closed percolator for 24 h. The outlet of the percolator then is opened and the liquid contained therein is allowed to drip slowly. Additional menstruum is added as required until the percolate measures about three-quarters of the required volume of the finished product. The marc is then pressed and the expressed liquid is added to the percolate. Sufficient menstruum is added to produce the required volume and the mixed liquid is clarified by filtration or by standing followed by decanting. Hot Continuous Extraction Soxhlet In this method the finely ground crude drug is placed in a porous bag or “thimble” made of strong filter paper which is placed in chamber E of the Soxhlet apparatus Figure 2. The extracting solvent in flask A is heated and its vapors condense in condenser D. The condensed extractant drips into the thimble containing the crude drug and extracts it by contact. When the level of liquid in chamber E rises to the top of siphon tube C the liquid contents of chamber E siphon into fl ask A. This process is continuous and is

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY carried out until a drop of solvent from the siphon tube does not leave residue when evaporated. The advantage of this method compared to previously described methods is that large amounts of drug can be extracted with a much smaller quantity of solvent. This effects tremendous economy in terms of time energy and consequently financial inputs. At small scale it is employed as a batch process only but it becomes much more economical and viable when converted into a continuous extraction procedure on medium or large scale. Aqueous Alcoholic Extraction by Fermentation Some medicinal preparations of Ayurveda like asava and arista adopt the technique of fermentation for extracting the active principles. The extraction procedure involves soaking the crude drug in the form of either a powder or a decoction kasaya for a specified period of time during which it undergoes fermentation and generates alcohol in situ this facilitates the extraction of the active constituents contained in the plant material. The alcohol thus generated also serves as a preservative. If the fermentation is to be carried out in an earthen vessel it should not be new: water should first be boiled in the vessel. In large-scale manufacture wooden vats porcelain jars or metal vessels are used in place of earthen vessels. Some examples of such preparations are karpurasava kanakasava dasmularista. In Ayurveda this method is not yet standardized but with the extraordinarily high degree of advancement in fermentation technology it should not be difficult to standardize this technique of extraction for the production of herbal drug extracts. Counter-current Extraction In counter-current extraction CCE wet raw material is pulverized using toothed disc disintegrators

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY to produce a fine slurry. In this process the material to be extracted is moved in one direction generally in the form of a fine slurry within a cylindrical extractor where it comes in contact with extraction solvent. The further the starting material moves the more concentrated the extract becomes. Complete extraction is thus possible when the quantities of solvent and material and their flow rates are optimized. The process is highly efficient requiring little time and posing no risk from high temperature. Finally sufficiently concentrated extract comes out at one end of the extractor while the marc practically free of visible solvent falls out from the other end. This extraction process has significant advantages: iii A unit quantity of the plant material can be extracted with much smaller volume of solvent as compared to other methods like maceration decoction percolation. iv CCE is commonly done at room temperature which spares the thermolabile constituents from exposure to heat which is employed in most other techniques. v As the pulverization of the drug is done under wet conditions the heat generated during comminution is neutralized by water. This again spares the thermolabile constituents from exposure to heat. vi The extraction procedure has been rated to be more efficient and effective than continuous hot extraction. Ultrasound Extraction Sonication The procedure involves the use of ultrasound with frequencies ranging from 20 kHz to 2000 kHz this increases the permeability of cell walls and produces cavitation. Although the process is useful in some cases like extraction of rauwolfia root its large-scale application is limited due to the higher costs. One disadvantage of the procedure is the occasional but known deleterious effect of ultrasound energy more than 20 kHz on the active constituents of medicinal plants through formation of free radicals and consequently undesirable changes in the drug molecules.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Supercritical Fluid Extraction Supercritical fluid extraction SFE is an alternative sample preparation method with general goals of reduced use of organic solvents and increased sample throughput. The factors to consider include temperature pressure sample volume analyte collection modifier cosolvent addition flow and pressure control and restrictors. Generally cylindrical extraction vessels are used for SFE and their performance is good beyond any doubt. The collection of the extracted analyte following SFE is another important step: significant analyte loss can occur during this step leading the analyst to believe that the actual efficiency was poor. There are many advantages to the use of CO2 as the extracting fluid. In addition to its favorable physical properties carbon dioxide is inexpensive safe and abundant. But while carbon dioxide is the preferred fluid for SFE it possesses several polarity limitations. Solvent polarity is important when extracting polar solutes and when strong analyte-matrix interactions are present. Organic solvents are frequently added to the carbon dioxide extracting fluid to alleviate the polarity limitations. Of late instead of carbon dioxide argon is being used because it is inexpensive and more inert. The component recovery rates generally increase with increasing pressure or temperature: the highest recovery rates in case of argon are obtained at 500 atm and 150° C. The extraction procedure possesses distinct advantages: i The extraction of constituents at low temperature which strictly avoids damage from heat and some organic solvents. ii No solvent residues. iii Environmentally friendly extraction procedure. The largest area of growth in the development of SFE has been the rapid expansion of its applications. SFE finds extensive application in the extraction of pesticides environmental samples foods and fragrances essential oils polymers and natural products. The major deterrent in the commercial application of the extraction process is its prohibitive capital investment.

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY Phytonics Process A new solvent based on hydrofluorocarbon-134a and a new technology to optimize its remarkable properties in the extraction of plant materials offer significant environmental advantages and health and safety benefits over traditional processes for the production of high quality natural fragrant oils flavors and biological extracts. Advanced Phytonics Limited Manchester UK has developed this patented technology termed “phytonics process”. The products mostly extracted by this process are fragrant components of essential oils and biological or phytopharmacological extracts which can be used directly without further physical or chemical treatment. The properties of the new generation of fluorocarbon solvents have been applied to the extraction of plant materials. The core of the solvent is 1122-tetrafluoroethane better known as hydrofluorocarbon- 134a HFC-134a. This product was developed as a replacement for chlorofluorocarbons. The boiling point of this solvent is -25° C. It is not flammable or toxic. Unlike chlorofluorocarbons it does not deplete the ozone layer. It has a vapor pressure of 5.6 bar at ambient temperature. By most standards this is a poor solvent. For example it does not mix with mineral oils or triglycerides and it does not dissolve plant wastes. The process is advantageous in that the solvents can be customized: by using modified solvents with HFC-134a the process can be made highly selective in extracting a specific class of phytoconstituents. Similarly other modified solvents can be used to extract a broader spectrum of components. The biological products made by this process have extremely low residual solvent. The residuals are invariably less than 20 parts per billion and are frequently below levels of detection. These solvents are neither acidic nor alkaline and therefore have only minimal potential reaction effects on the botanical materials. The processing plant is totally sealed so that the solvents are continually recycled and fully recovered at the end of each production cycle. The only utility needed to operate these systems is electricity and even then they do no consume much energy. There is no scope for the escape of the solvents. Even if some solvents do escape they contain no chlorine and therefore pose no threat to the ozone layer. The waste biomass from these plants is dry and “ecofriendly” to handle Advantages of the Process • Unlike other processes that employ high temperatures the phytonics process is cool and gentle and its products are never damaged by exposure to temperatures in excess of ambient. • No vacuum stripping is needed which in other processes leads to the loss of precious volatiles. • The process is carried out entirely at neutral pH and in the absence of oxygen the products never suffer acid hydrolysis damage or oxidation. • The technique is highly selective offering a choice of operating conditions and hence a choice of end

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY products. • It is less threatening to the environment. • It requires a minimum amount of electrical energy. • It releases no harmful emissions into the atmosphere and the resultant waste products spent biomass are innocuous and pose no effluent disposal problems. • The solvents used in the technique are not flammable toxic or ozone depleting. • The solvents are completely recycled within the system. Applications The phytonics process can be used for extraction in biotechnology e.g for the production of antibiotics in the herbal drug industry in the food essential oil and flavor industries and in the production of other pharmacologically active products. In particular it is used in the production of top quality pharmaceutical- grade extracts pharmacologically active intermediates antibiotic extracts and phytopharmaceuticals. However the fact that it is used in all these areas in no way prevents its use in other areas. The technique is being used in the extraction of high-quality essential oils oleoresins natural food colors flavors and aromatic oils from all manner of plant materials. The technique is also used in refining crude products obtained from other extraction processes. It provides extraction without waxes or other contaminants. It helps remove many biocides from contaminated biomass. Parameters for Selecting an Appropriate Extraction Method i Authentication of plant material should be done before performing extraction. Any foreign matter should be completely eliminated. ii Use the right plant part and for quality control purposes record the age of plant and the time season and place of collection. iii Conditions used for drying the plant material largely depend on the nature of its chemical constituents. Hot or cold blowing air flow for drying is generally preferred. If a crude drug with high moisture content is to be used for extraction suitable weight corrections should be incorporated. iv Grinding methods should be specified and techniques that generate heat should be avoided as much as possible. v Powdered plant material should be passed through suitable sieves to get the required particles of uniform size. vi Nature of constituents: a If the therapeutic value lies in non-polar constituents a non-polar solvent may be used. For example lupeol is the active constituent of Crataeva nurvala and for its extraction hexane is

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T.B.EKNATH BABU T.B.E.K.B STUDENT AT A.K.C.P ADVANCED PHARMACOGNOSY generally used. Likewise for plants like Bacopa monnieri and Centella asiatica the active constituents are glycosides and hence a polar solvent like aqueous methanol may be used. b If the constituents are thermolabile extraction methods like cold maceration percolation and CCE are preferred. For thermostable constituents Soxhlet extraction if nonaqueous solvents are used and decoction if water is the menstruum are useful. c Suitable precautions should be taken when dealing with constituents that degrade while being kept in organic solvents e.g. flavonoids and phenyl propanoids. d In case of hot extraction higher than required temperature should be avoided. Some glycosides are likely to break upon continuous exposure to higher temperature. e Standardization of time of extraction is important as: • Insufficient time means incomplete extraction. • If the extraction time is longer unwanted constituents may also be extracted. For example if tea is boiled for too long tannins are extracted which impart astringency to the final preparation. f The number of extractions required for complete extraction is as important as the duration of each extraction. viii The quality of water or menstruum used should be specified and controlled. ix Concentration and drying procedures should ensure the safety and stability of the active constituents. Drying under reduced pressure e.g. using a Rotavapor is widely used. Lyophilization although expensive is increasingly employed. x The design and material of fabrication of the extractor are also to be taken into consideration. xi Analytical parameters of the final extract such as TLC and HPLC fingerprints should be documented to monitor the quality of different batches of the extracts. Source: Sukhdev Swami Handa Suman Preet Singh Khanuja Gennaro Longo Dev Dutt Rakesh. 2008. Extraction technologies for medicinal and aromatic plants International centre for science and high technology.

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