Evaluation of changes of lungs in asthmatic rats treated with aqueous

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Senthil K K K et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-62 2018 442-452 442 IJAMSCR |Volume 6 | Issue 2 | Apr - Jun - 2018 www.ijamscr.com Research article Medical research Evaluation of changes of lungs in asthmatic rats treated with aqueous and non aqueous extract of clerodendrum serratum and theophylline Dr.K.K. Senthil Kumar M.Sivakumar S.Kannan R.Somu 1 HOD Associate Professor Department of Pharmaceutical Analysis 2 Assistant Professor Department of Pharmacology 3 Associate Professor Department of Pharmaceutical Biotechnology 4 Professor Department of Pharmaceutical Chemistry J.K.K.Munirajah Institute of Health Sciences College of Pharmacy TN-Palayam-638506 Tamilnadu India. Corresponding Author: Dr.K.K. Senthil Kumar Email id: kksenthil.agmail.com ABSTRACT The aim of the present work is help the researchers for the development of an alternative methods rather than inhalers and oral anti asthmatic drugs for the treatment of asthma and COPD which will minimize the complication. Many plants obtained from the natural source play a significant role in the health care system. Literature survey on herbal drugs has shown significant anti asthmatic activity which has not shown any remarkable side effect. The pharmacological mechanism which the phyto constituents producing the anti asthmatic activity are not clearly understood till date. The several herbal formulation have derived from the Ayurveda traditional system of Indian medicine and its additional system of medicine yet to be scientifically validated that they have exhibited pharmacological action against Asthmatic. Only less number of scientific data of traditional medicines is available for the treatment of Asthmatic. INTRODUCTION Pharmacognosy has been basically evolved as an applied science pertaining to the study of all types of the drugs of natural origin. However its subject matter is directed towards the modern allopathic medicine. During the course of development many civilizations has raised and perished but the systems of medicines developed by them in various parts of the world are still practised and are also popular as the alternative system of medicines. These are alternative systems in the sense that modern allopathic system has been globally acclaimed as the principal system of the medicine and so all the other systems prevent and practised in various parts of the world are supposed to be alternative systems. The philosophy and the basic principles of these so called alternative systems might differ significantly from each other but the fact cannot be denied that these systems have served the humanity for the treatment and management of disease and also for maintenance of good health 1-5. About 80 of the world population still rely and use the medicines of traditional systems. Asthma is a hyper reactive airway disease running ISSN:2347-6567 International Journal of Allied Medical Sciences and Clinical Research IJAMSCR

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Senthil K K K et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-62 2018 442-452 443 a chronic course it has worldwide prevalence and is a common cause hospitalization in children. It is estimated that currently 300 million peoples suffer from asthma with a possibility of an additional 100 million likely to suffer from the disease over the next 15-20 yea There are two types of asthma 1. Allergic or extrinsic 2. Idiosyncratic or intrinsic. Allergic asthma is a result of an antigen/antibody reaction on mast cells in the respiratory tract. This type is often associated with a family history of atopy allergic diseases such as eczema. Idiosyncratic asthma or intrinsic asthma is a result of neurological imbalances in the autonomic system in which the system in which the sympathetic and parasympathetic systems are not properly coordinated. 6-10 PATHOPHYSIOLOGY Chronic inflammatory reaction is the characteristic features in the pathogenesis of bronchial asthma which results in intermittent airflow obstruction and bronchial hyper responsiveness. The airway obstruction in asthma is due to factors that includes bronchospasm oedema of the airway increased mucus secretion cellular infiltration of the airway walls and injury to the airway epithelium. Repetition of the inflammatory events in asthma can cause irreversible functional changes in the airway passages a process called remodelling. The remodelled airway passages are persistently narrow as the disease progresses and become less and less responsive to drug treatment. The mechanism of inflammation in asthma may be acute sub acute or chronic. 11-15 Inflammatory reaction An attack of asthma begins when the allergens is inhaled. The allergen binds to IgE antibodies that have binding side for the allergens mast cells in the lungs. Binding of allergen to IgE triggers exocytosis of the mast cells with the release of histamine leukotrienes. Leukotrienes cause the smooth muscle cells of the bronchi to contract narrowing the lumen of the bronchi. This is the early phase. Next they attract on accumulation of the inflammatory cells especially eosinophils-leading to production of mucus. This is the late phase which occurs 4-6hrs after the early phase 16-20. MATERIALS AND METHODS Collection of plant A novel approach to plant selection is a computerized selection method or literature information selection technique LIST that correlates biological activity botanical facts and chemotoxanomical information using NAPRALERT database. Based on the literature review. the plant was collected from eastern part of the Ooty. Clerodendrum serratum is a small tree belonging to family verbenaceae. The plant was taxonomically identified by the botanist Dr.A.Balasubramanian ABS Botanical Conservation Research and Training Centre KAARIPATTI SALEM -636003 T.N. INDIA. Extraction of plant The leaf of Clerodendrum serratum were shade dried at room temperature for 10 to 15 days. After shade drying of leaves which was converted in to coarse powder form by using the mechanical mixer. Powdered leaf material was defatted using petroleum ether. Defatted plant material was extracted in soxhlet apparatus. Further extract with 90 of ethanol and concentrated by using desiccators for the removal of remaining moisture. The final amount of solid residue was 35 w/w.

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Senthil K K K et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-62 2018 442-452 444 Plant profile Clerodendrum serratum Linn native to tropical and warm temperate regions of the world with most of the species occurring in tropical Africa and southern Asia but some in the tropical Americas and northern Australia and a few extending north into the temperate zone in eastern Asia. Clerodendrum serratum Linn Verbenaceae.is a slightly woody shrub with blunty stems and branches. These trees are about 2-8 ft high. It is annual or perennial usually aromatic. Chemical constituents Leaf extracts contains: Stigma sterolspinasterol Luteolin Luteolin-7-0 glucuronide Apigenin Baicalin Suctellarin 7-0 glucuronide Traditional use Clerodendrum serratum as effective treatment against asthma bodyache cholera eye disorder ulcers snake-bite wound tuberculosis and epilepsy. It has antibacterial antihistaminic hepatoprotective antipyretic antinociceptive and anti-inflammatory. Root is pungent bitter acrid dry heating anti-inflammatory digestive carminative depurative expectorant antispasmodic stimulant appetizer and anthelmintic. Leaves are used in fever and hiccough. Its boiled leaves are used in cephalgia and opthalmia where as its boiled seeds in butter milk is used as aperients in dropsy and in catarrhal affection of lungs Shah 2003. Clerodendrum serratumits methnolic extract exhibit significant anticancer activity as compared to aqueous extract. Zalke et al. 2010.Antibacterial activity of Clerodendrum serratum .L Vidya S.M et al. 2010. PRELIMINARY PHYTOCHEMICAL STUDIES The pharmacological and therapeutic action of crude drug is determined by the nature of its constituents. Thus the plant species may be considered as a biosynthetic laboratory not only for the chemical compounds e.g. carbohydrates proteins and fats that are utilized as a food by humans and animals but also for a magnitude of compounds including alkaloids flavonoids glycosides etc. which exert definite physiological effects. Tests for carbohydrates and glycosides A small quantity of the extracts was dissolved separately in 4ml of distilled water and filtered. The filtrate was subjected to various tests to detect the presence of Carbohydrates. Molisch’s test 1. Filtrate was treated with 2-3 drops of 1 alcoholic _- napthol solutions and 2ml of Con.

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Senthil K K K et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-62 2018 442-452 445 Sulphuric acid was added along the sides of the test tube. Appearance of brown ring at the junction of two liquids shows the presence of carbohydrates. 2. Another portion of the extract was hydrolysed with hydrochloric acid for few hours on a water bath and the hydrolysate was subjected to Legal’s and Borntrager’s test to detect the presence of different glycosides. Legal’s test To the hydrolysate I ml of pyridine and few drops of sodium nitropruside solutions were added and then it was made alkaline with sodium hydroxide solution. Appearance of pink to red colour shows the presence of glycosides Bortanger’s test Hydrolysate was treated with choloroform and then the chloroform layer was separated. To this equal quantity of dilute ammonia solution added. Ammonia layer acquires pink color showing the presence of glycosides. TEST FOR ALKALOIDS  A small portion of the solvent free alcohol and aqueous extracts were stirred separately with few drops of dilute hydrochloric acid and filtered. The filtrate was tested with various reagents for the  Presence of alkaloids.  Mayer’s reagent - Cream ppt  Dragendroff’s reagent - Orange brown ppt.  Harger’s reagent - Yellow ppt  Wagner’s reagent - Reddish brown ppt TEST FOR PHYTOSTEROL The extract was refluxed with solution of alcoholic potassium hydroxide till complete saponification has taken place. The mixture was diluted and extracted with ether. The ether layer was evaporated and the residue was tested for the presence of phytosterol. Libermann burchard test The residue was dissolved in few drops of dil. Acetic acid 3 ml of acetic anhydride was added followed by few drops of Con. Sulphuric acid. Appearance of bluish green color shows the presence of phytosterol. TESTS FOR FIXED OILS Spot test Small quantities of various extracts were separately pressed between two filter papers. Appearance of oil stain on the paper indicates the presence of fixed oil. Few drops of 0.5N alcoholic potassium hydroxide were added to small quantity of various extracts along with a drop of phenolphthalein. The mixture was heated on a water bath for 1-2 hours. Formation of soap or partial neutralization of alkali indicates the presence of fixed oils and fats. Test for gums and mucilages Small quantities of the extracts were added separately to 25 ml of absolute alcohol with constant stirring and filtered. The precipitate was dried in air and examined for its swelling properties for the presence of carbohydrates. Test for saponins The extract was diluted with 20ml of distilled water and it was agitated in a graduated cylinder for 15 minutes. The formation of 1cm layer of foam shows the presence of saponins. Test for proteins and free amino acids Small quantities of the extracts were dissolved in few ml of water and treated with following reagents.  Appearance of red color shows the presence of protein and free amino acid  Appearance of purple color shows the presence of proteins and free aminoacid  Equal volumes of 5 sodium hydroxide solution 1 copper sulphate solution was added.  Appearance of pink or purple color shows the presence of proteins and free amino acids. Test for phenolic compounds and tannins. Small quantities of the extracts were taken separately in water and test for the presence of phenolic compounds and tannins was carried out with the following reagents. 1. Dil. Ferric Chloride solution 5 – Violet Color. 2. 1 solution of gelatin containing 10 sodium chloride – White ppt. 3. 10 lead acetate solution – White ppt.

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Senthil K K K et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-62 2018 442-452 446 TEST FOR FLVAVONOIDS With aqueous sodium hydroxide solution Blue to violet color anthocyanins Yellow color flavones yellow to orange flavonones With concentrating sulphuric acid Yellow orange color anthocyanins yellow to orange color flavones orange to crimson flavonones Shinoda’s test Small quantities of the extract were dissolved in alcohol to them piece of magnesium followed by Conc. Hydrochloric acid drop wise added and heated. Appearance of magenta color shows the presence of flavonoids. TLC METHOD Aluminium sheets and glass backed TLC plates were used for the isolation of compounds. The plates were divided in to size of 10 cm x 1.5 cm. A light pencil was drawn 1cm from the bottom and top edge of the chromatographic plate. 6mg of column fraction sample was dissolved in 60 ml of absolute methanol and placed as preparatory on two TLC plates 10x1.5 cm using a 10 micro litre of capillary which delivers approximately 10 microgram spot on to the place until each plate contains 150 microgram and subsequently placed in the eluting solvent. Chloroform: methanol: water 5:4:1 in a TLC tank which was fill to a depth of 0.5 cm the solvent migrated upwards on to the TLC plate until the pencil line drawn across the top edge solvent front was reached. The plates were removed from the chamber and air dried. A portion of the plates 1 cm was cut off using a glass cutter and sprayed with a detecting reagent diphenyl ethantamine glycosides in order to visualize the constituents on the eluted plates after heating for three minutes at 110o c in on oven. The plates were also visualized under UV light at 360 nm and 254 nm and the fluorescence 360 nm or quenching 254 nm. Compounds were marked and the spot layer were out lined with the pencil. Sample 1: 5/7 0.7142 Sample 2: 4.7/7 0.6714. Report The RF value of sample 1 and sample 2 were coinciding with standard values. RF value for sample 1 0.7142 RF value for sample 2 0.6714. ACUTE ORAL TOXICITY STUDIES Procedure Preparation of animals The animals are randomly selected marked to permit individual identification and kept in their cages for at least 5 days prior to dosing to allow for acclimatisation to the laboratory conditions.

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Senthil K K K et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-62 2018 442-452 447 Preparation of doses In general test substances should be administered in a constant volume over the range of doses to be tested by varying the concentration of the dosing preparation. Where a liquid end product or mixture to be tested however the use of the undiluted test substances i.e. at a constant concentration may be more relevant to the subsequent risk assessment of the substances and is a requirement of some regulatory authorities. In either case the maximum dose volume for administration must not be exceeded. The maximum volume of the liquid that can be administrated at one time depends on the size of the test animal. In rodents the volume should not normally exceed 1ml/100 gm of body weight. However in the case of aqueous solution 2ml/100 gm body weight can be considered. With respect to the formulation the dosing preparation by use of an aqueous solution/ suspension/ emulsion is recommended wherever possible followed in order of preference by solution/suspension/emulsion in oil ex. Cornoil possible solution in other vehicle. For vehicles other then water the toxicological characteristics of the vehicle should be known. Doses must be prepared shortly prior to administration unless the stability of the preparation over the period during which it will be used is known and shown to be acceptable. Administration of the dose The test substance is administered in a single dose by gavages by using a stomach tube or suitable incubation cannula. In the unusual circumstance that a single dose is not possible the dose may be given in small fraction over period not exceeding 24 hours. Animal should be fasted prior to dosing e.g. with rat food but not water should be withheld over night with the mouse food but not water should be withheld for 3-4 hours following the period of fasting the animal should be weighed and the test substances is administered. After the substance has been administered food may be withheld for a further 3-4 hours in rats or 1-2 hours in mice. Where a dose is administered infraction over a period it may be necessary to provide the animals with food and water depending on the length of period. Number of the Animal and Dose Levels Three animals are used for each step. The dose level to be used as the starting dose is selected from one hour of four fixed levels 5 50 300 2000 mg/kg body weight. The starting dose level should be that which is most likely to produce mortality in some of the dosed animals. When available information suggests that mortality is unlikely at the highest starting dose level 2000 mg/kg body weight then a limit test should be conducted. When there is no information on a substance to be tested for animal Exceptionally and only when one justified by specific regularity needs the use of additional upper dose level 5000 mg/kg body weight may be considered. For reason of animal welfare concern testing of animals in GHS category 5 ranges 2000 – 5000 mg/kg is discouraged and should only be considered and when there is a strong likelihood that results of such a test have direct relevance for protecting human or animal health or the environment. welfare reasons it recommended using the starting dose of 300 mg/kg body weight. The time interval between treatment groups is determined by the onset duration and severity of toxic signs. Treatment of animals at the next dose should be delayed until one is confident of survival of the previously dosed animals. SCREENING METHODS FOR ANTI ASTHMATICS Animals Adult male albino rats weighing 150 – 200 g were used for the present study. Animals were acclimated for 15 days in our disease free animal house prior to the start of the experiment. The animals were kept in clean and dry plastic cages with 12 h light: 12 h dark cycle at 25+20 c temperature and 45 – 60 relative humidity. Animals were given free access to standard feed and water and libitum. For experimental purpose the animals were kept on overnight fasting but allowed free access to water. The research conducted under the guidelines of CPCSEA and approved by Institutional Animal Ethics Committee in ref. No. M.Pharma/2013/09. Invivo method Overnight fasted Rats were divided into six groups 1. Inducer control IC Acetylcholine + Citric acid 0.2 spray 2. STD received Theophylline 200 mg/kg 3. AECS 100mg/kg 200mg/kg 4. NAECS 200mg/kg 400mg/kgp.o. Bronchospasm was induced in rats by exposing them to Acetylcholine Citric acid 0.2

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Senthil K K K et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-62 2018 442-452 448 spray produced by an ultra sound nebulizer in an aerosol chamber 241424 cm made of Perspex glass. The time required for appearance of pre convulsive dyspnoea caused by the Acetylcholine Citric acid 0.2 spray was recorded for each animal. Prior to drug treatment each animal was placed on Histamine chamber and exposed to Acetylcholine + Citric acid 0.2 aerosol. The preconvulsive time PCT i.e the time of aerosol exposure to the onset of dyspnoea leading to the appearance of convulsion was noted. As soon as the preconvulsion dyspnoea PCD was noted the animals were removed from the chamber and placed in fresh air to recover as basal value. Rats were then allowed to recover from dyspnoea for 24 hrs. After 24 hrs the animals of STD received Theophylline 200 mg/kg AECS 100mg/kg 200mg/kg NAECS 200mg/kg 400mg/kg. These animals were again subjected to Acetylcholine + Citric acid 0.2 aerosol later at an interval of 1hr 4hrs and 24 hrs to determine preconvulsion time PCT. The protection offered by the treatment was calculated by using the following formula Percentage protection 1 – T1/T2 100 Where T1 the mean of PCT before administration of test drugs and T2 the mean of PCT after administration of test drugs at 1 hr 4 hrs and 24 hrs. Statistical analysis All the values were expressed as mean + - SEM. The results were analyzed for statistical significance by using one-way ANOVA followed by Dunnett’s test. P0.05 was considered significant. INVITRO METHOD Parameters for investigation The other major type of blood cells are the white blood cells WBC’s which are also referred to as leukocytes. There are many more RBC’s than there are WBC’s. For every leukocyte present in a sample there will normally be 600 to 700 RBC’s. The major role of the white blood cells is to defend the body against invading organisms such as bacteria viruses and fungi. There are different types of leukocytes and a white blood count WBC is a total of all the various kinds. The normal range for a WBC count in the dog would be between 6000 and 17000 per microliter and in the cat 4900-20000/μ l. The number of WBCs is typically elevated when the body is fighting a severe infection or stressed by metabolic toxins a patient that was in kidney failure with waste products building up in its body would normally have an elevated WBC. In addition when extremely excited if we overly excite or frighten the animal when drawing the blood sample white blood cells will be released into the blood and the levels will rise. The WBC count will be lower than normal if an animal has been weakened from a prolonged debilitating disease and in some viral infections. WBC’s are divided into two groups depending on how they react to the stains that are used to better observe them under a microscope. There are granulocytes they are WBCs with granules that absorb the stain and the agranulocytes those that do not absorb the stain. The granulocytes include the neutrophils eosinophils and the basophils while the agranulocytes are the lymphocytes and monocytes Histopathological examination After 14 days experimental period and the last blood sampling the whole Lungs were removed after sacrificing the animal and were fixed in 10 formalin for histopathological examination. Sections were cut and stained by hematoxylin and eosin HE for histological examination. Statistical analysis All the values were expressed as mean + - SEM. The results were analyzed for statistical significance by using one-way ANOVA followed by Dunnett’s test. P0.05 was considered significant. RESULTS AND DISCUSSIONS Asthma is common respiratory disease. The morbidity and the mortality of the disease is increasing and making a global concern. The syndrome of bronchial asthma is characterized by wide spread narrowing of the bronchial tree due to contraction of the smooth muscle in response to multiple stimuli resulting in the release of chemical mediators such as Ach and Citric acid. In the presence study c.serratum..... Significantly inhibited the Ach and Citric acid induced anti asthmatic properties of the plant. Ach and Citric acid induced Bronchoconstriction is the traditional

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Senthil K K K et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-62 2018 442-452 449 immunological model of the antigen induced air way obstruction. Ach and Citric acid when inhaled causes hypoxia and leads to convulsion in rats and causes very strong smooth muscle contraction profound hypotension capillary dilation in cardio vascular system a prominent effect caused by histamine leads to severe Bronchoconstriction in rats that causes asphyxia and death. Bronchodilator can delay the occurrence of these symptoms. The results of the study confirmed the bronchodilator properties of the plant justifying its traditional claim in the treatment of asthma. Drugs effective in the asthma are mostly steroidal and also flavonoids in nature. Phytochemical profile of the plant reveals the presence flavonoids Apigenin steroidal nucleus _-spinasterol in the form of triterpenoids. The anti asthmatic activity showed by leaves of the plant extract Apigenin and _-spinasterol may be because of the chemical moieties. However this claims demands for further research and studies are in fact underway to isolate and characterized the active principles responsible for the anti asthmatic activity. Table:1 PHYTOCHEMICAL CONSTITUENTS OF DIFFERENT EXTRACTS OF LEAVES OF CLERODENDRUM SERRATUM PhytoChemical Constituents Aqueous extract Non aqueous extract Carbohydrates - - Glycosides - - Alkaloids - - Flavonoids + + Phenols + + Fixed oils - - Steroids + + Saponins - - Gums mucilage - - Proteins free amino acids - - + - Positive - – Negative Mortality result of sighting study starting dose in main study is decided and carried out with six animals per dose level 1000mg/kg.Based on the mortality result on 14h day of observation the doses for in vivo study were selected. Table:2 PRE CONVULSION DYSNOPEA TREATMENT DOSE BEFORE 1 Hr 4 Hrs 24 Hrs Negative control Ach + Citric acid 18.2 + 2.2739 18.2 +0.1250 17.4 +0.091 17.2+0.110 Positive control Ach + Citric acid + Theophylline 18.2 +0.1250 55.8 +1.315 64.3 +1.548 34 +1.472 AECS 100 mg/kg 17.5 + 0.1371 41 +1.291 43 +1.291 22.8+0.478 AECS 200 mg/kg 17.5 +0.2955 57.8 +1.750 59.8 +1.750 33 +1.225 NAECS 200 18.0 +0.4328 38.3 +1.250 40 +1.250 21.8+0.478

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Senthil K K K et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-62 2018 442-452 450 mg/kg NAECS 400 mg/kg 17.5 +0.5282 40 +1.291 41.5 +1.258 27.1+0.314 Values are represented as mean ± S.E.M n6 One-way ANOVA followed by Student-Newman-Keuls post test P 0.001 HISTOPATHOLOGICAL REPORT Control Group:1 Control Group:2 Negative control group:1 Negative group:2 Positive control group:1 Positive control group:2 Aq.Extract 100 mg/kg Treated group Aq.Extract 200 mg/kg Treated group

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Senthil K K K et al / Int. J. of Allied Med. Sci. and Clin. Research Vol-62 2018 442-452 451 Non Aq.Extract 200 mg/kg Treated group Non Aq.Extract 400 mg/kg Treated group CONCLUSION On the basis of the result in these experiments this may be stated that the aqueous extract of Clerodendrum serratum 200mg/kg has a beneficial effect in asthmatic patients. It will reduce the alveolar thickness and Eosinophils counts in blood further studies are required to purify the active principle and to study the molecular mechanism of the exact pathway. Non aqueous extract of Clerodendrum serratum 400mg/kg having fewer amounts of steroids and flavonoids because it will filter the all compounds and reduce the quantity of chemical moieties. But aqueous extract of Clerodendrum serratum 200mg/kg having higher amount of chemicals when compare to Non aqueous extract of Clerodendrum serratum 400mg/kg so it will produce anti asthmatic at equal to standard drug. In lungs alveolar thickness reduced by the AECS 200mg/kg as equal to standard drug and also it’s same as normal lungs. So aqueous extract of Clerodendrum serratum having good beneficial effect for the asthmatic patients. This information’s will be useful for the development of alternative method rather than anti asthmatic agents Inhalers Tablets Injections Nasal sprays Respules for the treatment of Asthmatic patients. This will minimize the wheezing and or asthmatic symptoms. REFFERENCE 1. The U.S. Food and Drug Administration Center for food safety and Applied Nutrition. Economic Characterization of the Dietary Supplement Industry Final Report. 1999 2. Barz W. and Ellis E. Potential of plant cell cultures for pharmaceutical production. In Natural products as medicinal agents. eds. Beal J.L. and Reinhard E. Suppl. to Planta Medica Hippokrates Verlag Stuttgart. pp 1981 471-508. 3. Cassady J.M. Chang C.-J. and McLaughlin J.E. Recent advances in the isolation and structural elucidation of antineoplastic agents in higher plants. In Natural products as medicinal agents. eds. Beal J.L. and Reinhard E. Suppl. Planta Medica. Hippokrates Verlag Stuttgart. 1981 93-124.

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