Compedial method for evaluaion of "crude drug"

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it is seminar for quality assurance sem 2, it is only for evaluation of perticular crude drug...

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COMPENDIAL METHODS FOR EVALUATION OF CRUDE DRUGS :

COMPENDIAL METHODS FOR EVALUATION OF CRUDE DRUGS GUIDED BY: - PREPARED BY:- Mr. Nisarg Patel Sanket . D. Patel Assistant Professor M. Pharm SEM-II Q.A Departmen

Contents:

Introduction Determination of ash Determination of extractable matter Determination of water and volatile matter Determination of volatile oils Determination of bitterness value Determination of haemolytic activity Determination of tannins Determination of swelling index Determination of foaming index Determination of pesticide residues Determination of arsenic and heavy metals Determination of microorganisms References Contents

Definition:

A Crude Drug is naturally occurring, unrefined substance derived from organic or inorganic sources such as plant, animal, bacteria, organs or whole organisms intended for use in the diagnosis, cure, treatment, or prevention of disease in man or other animals. Definition

The Q. C. Parameter for evaluation of crude drug:

A) Physical Parameters: Viscosity Volatile matter Density Refractive index Hemolytic activity Foaming index Melting point Solubility The Q. C. Parameter for evaluation of crude drug Moisture content Specific gravity Optical rotation Bitterness value Swelling index Ash value

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B) Chemical Parameter Detection of alkaloids. Detection of carbohydrates and glycosides. Detection of phytosterols . Detection of fixed oils and fats. Detection of saponins Detection of protein and free amino acids. Detection of gums and mucilage. Detection of volatile oils. Detection of phenolic compounds and tannins .

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C) Botanical Parameter Microscopical parameters Macroscopical parameters D) Biological / toxicological parameter Determination of pesticides. Determination of arsenic and heavy metals. Determination radioactive contamination. Determination of a flatoxins .

Determination of Ash:

Ashing involves an oxidation of the component of the product. A high ash value is indicative of contamination, Substitution, adulteration or carelessness in preparing the crude drug for marketing. Three type of Ash : Total Ash Acid-insoluble Ash Water Soluble Ash Determination of Ash

Total Ash:

Take a 2-3 gm of the air dried crude drug in the tared platinum or silica dish and incinerate at temp. not exceeding 450°C. If carbon free Ash can not obtained, Then Collect the residue on ash less filter paper. Until Free from carbon, cool and Weigh. Incinerate the residue and filter paper until the Ash is white. Calculate the % of Ash with Reference to air dried drug. Total Ash

Acid Insoluble Ash:

To the crucible containing the total ash, add 25 ml of hydrochloric acid. cover with a watch glass and boil gently for 5 minutes. Rinse the watch-glass with 5 ml of hot water and add this liquid to the crucible. Collect the insoluble matter on an ashless filter paper and wash with hot water until the filtrate is neutral. Acid Insoluble Ash

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Transfer the filter-paper containing the insoluble matter to the original crucible, dry on a hot-plate and ignite to constant weight. Allow the residue to cool in a suitable desiccator for 30 minutes, then weigh without delay. Calculate the content of acid insoluble ash in mg per g of air-dried material.

Water Soluble Ash::

Boil the Ash obtained in Total Ash with 25 ml of water 5 min. Collect the insoluble matter on an Ashless filter paper. Wash with hot water, ignite for 15 min. at a temperature not exceeding 450°C. Subtract the Wt. of the insoluble matter from the Wt. of the Ash taken. Calculate the % of water soluble Ash with Reference to air dried drug. Water Soluble Ash:

Determination of Extractable matter:

This method determines the amount of active constituents extracted with solvents from a given amount of medicinal plant material. It is employed for materials for which as yet no suitable chemical or biological assay exists. Determination of Extractable matter

Method.1 - Hot extraction:

Place about 4.0g of coarsely powdered air-dried material, accurately weighed, in a glassstoppered conical flask. Add 100ml of water and weigh to obtain the total weight including the flask. Shake well and allow to stand for 1 hour. Attach a reflux condenser to the flask and boil gently for 1 hour; cool and weigh. Readjust to the original total weight with the solvent specified in the test procedure for the plant material concerned. Method. 1 - Hot extraction

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Shake well and filter rapidly through a dry filter. Transfer 25 ml of the filtrate to a tarred flat-bottomed dish and evaporate to dryness on a water-bath. Dry at 105°C for 6 hours, cool in a desiccator for 30 minutes, then weigh without delay. Calculate the content of extractable matter in mg per g of air-dried material.

Method.2 - Cold maceration:

4.0g of coarsely powdered air-dried material, accurately weighed, in a glassstoppered conical flask. Macerate with 100ml of the solvent specified for the plant material concerned for 6 hours, shaking frequently, and then allow to stand for 18 hours. Filter rapidly taking care not to lose any solvent, transfer 25 ml of the filtrate to a tared flat-bottomed dish and evaporate to dryness on a water-bath. Dry at 105°C for 6 hours, cool in a desiccator for 30 minutes and weigh without delay. Calculate the content of extractable matter in mg per g of airdried material. Method. 2 - Cold maceration

Determination of Water and Volatile matter:

An excess of water in medicinal plant materials will encourage microbial growth, the presence of fungi or insects, and deterioration following hydrolysis. This is especially important for materials that absorb moisture easily or deteriorate quickly in the presence of water. Determination of Water and Volatile matter

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Two Methods Azeotropic method (toluene distillation). Loss on drying (gravimetric determination).

a). Azeotropic method (toluene distillation) :

a). Azeotropic method (toluene distillation) Distillation Assambly

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Weigh accurately a quantity of the material expected to give about 2-3 ml of water and transfer to the flask. (For weighing material with a paste-like character, use a boat of metal foil.) Add a few pieces of porous porcelain and heat the flask gently for 15 minutes. When boiling begins, distil at a rate of 2 drops per second until most of the water has distilled over, and then increase the rate of distillation to about 4 drops per second. As soon as the water has been completely distilled, rinse the inside of the condenser tube with toluene R. Continue the distillation for 5 more minutes, remove the heat, allow the receiving tube to cool to room temperature and dislodge any droplets of water adhering to the walls of the receiving tube by tapping the tube.

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Allow the water and toluene layers to separate and read off the volume of water (second distillation). Calculate the content of water as a percentage using the formula: 100 (n1-n)/w Where, w = the weight in g of the material being examined n = the number of ml of water obtained in the first distillation n 1 = the total number of ml of water obtained in both

b). Loss on drying (gravimetric determination):

Place about 2-5g of the prepared air-dried material, or the quantity specified in the test procedure for the plant material concerned, accurately weighed, in a previously dried and tared flat weighing bottle. Dry the sample by one of the following techniques: - in an oven at 100-105°C; - in a desiccator over phosphorus pentoxide R under atmospheric pressure or reduced pressure and at room temperature. Dry until two consecutive weightings do not differ by more than 5mg, unless otherwise specified in the test procedure. Calculate the loss of weight in mg per g of air-dried material. b). Loss on drying (gravimetric determination)

Determination of Volatile oils:

Volatile oils are characterized by their odour, oil-like appearance and ability to volatilize at room temperature. Chemically, they are usually composed of mixtures of, for example, monoterpenes , sesquiterpenes and their oxygenated derivatives. Determination of Volatile oils

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Apparatus used for determination of volatile oil

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The plant material is distilled with water and the distillate is collected in a graduated tube. The aqueous portion separates automatically and is returned to the distillation flask. If the volatile oils possess a mass density higher than or near to that of water, or are difficult to separate from the aqueous phase owing to the formation of emulsions, a solvent with a low mass density and a suitable boiling-point may be added to the measuring tube. The dissolved volatile oils will then float on top of the aqueous phase.

Determination of Bitterness Value:

The bitter properties of the plant material are determined by comparing the threshold bitter concentration of an extract of the materials with that of a dilute solution of quinine hydrochloride R. The bitterness value is expressed in unit equivalent to the bitterness of a solution containing 1 g of quinine hydrochloride R in 2000 ml. Determination of Bitterness Value

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Preparation of Stock & diluted quinine hydrochloride solutions:- Dissolve 0.1 g of quinine hydrochloride R in sufficient safe drinking water to produce 100 ml. further dilute 5 ml of this solution to 500 ml with safe drinking water. This stock solution of quinine hydrochloride (SQ) contains 0.01 mg/ ml. Use nine test tubes for the serial dilution for the intial test as indicated in Table.

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Serial dilution for the initial test for the determination of bitterness value

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Stock & diluted solutions of the Plant Material :- Prepare the solution as specified in the test procedure for the given plant material ( Sr ). Use 10 test tubes for the serial dilution for the test. Serial dilution for the second test for the determination of bitterness value

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After rinsing the mouth with safe drinking water taste 10 ml of the most dilute solution swirling it in the mouth mainly near the base of tongue for 30 seconds If the bitter sensation is no longer felt in the mouth after 30 sec. spit out the solution & wait for 1 min on ascertain whether this is due to delayed sensitivity. The next higher concentration should not be tasted until at least 10 min have passed. The threshold bitter concentration at which a material continues to provoke sensation after 30 sec.

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In order to save time in the second test, it is advisable first whether the solution in tube no.5 gives a bitter sensation. If so, find the threshold bitter concentration of the material by testing the dilutions in tubes 1-4 . If the solution in the tube no. 5 does not give a bitter sensation, find the threshold bitter concentration by tasting the dilutions in tubes 6-10.

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Calculate the bitterness value in units per gm using following formula, 2000 × C A × B Where, A = the concentration of the stock solution ( Sr ) mg/ml, B = the volume of Sr ( in ml ) in the tube with threshold bitter concentration C = the quantity of quinine hydrochloride R in the tube with threshold bitter concentration

Determination of Haemolytic activity:

Saponin having detergent property, frothing & hemolytic activity that is ability to cause haemolysis, when added to a suspension of blood, Saponins produce changes in erythrocyte membranes, causing hemoglobin to diffuse into the surrounding medium. The hemolytic activity of plant materials, or a preparation containing saponins is determined by comparison with that of a reference material, saponin R, which has a haemolytic activity of 1000 units per gm. Determination of Haemolytic activity

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Method: Preliminary test: Prepare serial dilution of plant material extract with phosphate buffer 7.4 and blood suspension (2%) using four Test tube as follow:

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Mix, avoiding formation of foam, allow to stand for 6 hr at room temperature. Examine tube & record dilution for total haemolysis & proceed as follows : If total haemolysis is observed only in tube no. 4, use original extract. If observed in 3, 4, prepare 2 fold dilution of original extract. If observed in 2, 3, 4, prepare 5 fold dilution of original extract. If all tubes contain, clear, red solution, prepare 10 fold dilution of original extract.

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Main test: Prepare serial dilution of plant material extract diluted / undiluted with phosphate buffer 7.4 and blood suspension (2%) using 13 Test tube as follow:

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Mix, observe result after 24 hr. Calculate amount material in gm that produce complete haemolysis. Prepare similar dilutions of saponin R & calculate quantity of saponin R in gm that produce complete haemolysis. Calculate haemolytic activity of material using following formula: 1000 × a / b where, 1000 = defined haemolytic activity of saponin R on ox blood, a = Quantity of saponin R that produce total haemolysis, b = Quantity of material that produce total haemolysis.

Determination of Tannins:

What is Tannins? Tannins are substances capable of turning animal hides into leather by binding proteins to form water-insoluble substances that are resistant to proteolytic enzymes. Chemically, tannins are complex substances; they usually occur as mixtures of polyphenols that are difficult to separate and crystallize They are easily oxidized, polymerized in solution, form colloidal solution with water & non crystalline compound. Types of tannins: Hydrolysable : Eg . Gallic acid, pyrogallol , ellagic acid etc. Condensed : Eg . Catechutannic acid, catechin etc. Determination of Tannins

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Weighed accurately a quantity of powder into a conical flask. Add 150 ml of water and heat over a boiling water-bath for 30 minutes. Cool, transfer the mixture to a 250-ml volumetric flask and dilute to volume with water. Allow the solid material to settle and filter the liquid through a filter-paper, discarding the first 50 ml of the filtrate. To determine the total amount of material that is extractable into water, evaporate 50.0 ml of the plant material extract to dryness, dry the residue in an oven at 105°C for 4 hours and weigh (W 1 ).

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To determine the amount of plant material not bound to hide powder that is extractable into water, take 80.0ml of the plant material extract, add 6.0g of hide powder R and shake well for 60 minutes. Filter and evaporate 50.0ml of the clear filtrate to dryness. Dry the residue in an oven at 105°C and weigh (W 2 ). To determine the solubility of hide powder, take 6.0 g of hide powder R, add 80.0ml of water and shake well for 60 minutes. Filter and evaporate 50.0ml of the clear filtrate to dryness. Dry the residue in an oven at 105°C and weigh (W 3 ).

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Calculate the quantity of tannins as a percentage using the following formula: [T 1- ( T 2- T 0)] x500 w Where, w = the weight of the plant material in grams.

Determination of Swelling Index:

The swelling index is the volume in ml taken up by the swelling of 1 g of plant material under specified conditions. Determination of Swelling Index

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METHOD Carry out simultaneously no fewer than 3 determinations for any given material. Introduce the specified quantity of the plant material, previously reduced to the required fineness & accurately weighed into a 25 ml glass stoppered measuring cylinder. Unless otherwise specified in the test procedure, add 25 ml of water & shake the mixture thoroughly every 10 minutes for 1 hour. Allow to stand for 3 hours at room temperature, or as specified. Measure the volume in ml occupied by the plant material, including any sticky mucilage. Calculate the mean value of the determinations, related to 1 gm of plant material.

Determination of Foaming Index:

Many medicinal plant materials contain saponins that can cause a persistent foam when an aqueous decoction is shaken. The foaming ability of an aqueous decoction of plant materials and their extracts is measured in terms of a foaming index. Determination of Foaming Index

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Weigh accurately 1 gm coarse powder, transferred to 5ooml conical flask containing 100ml boiling water. Boil for 30 min. Cool, filter to 100 ml volumetric flask & make up volume to 100ml. Take 10 T. T., Mark with 1 to 10, add 1ml, 2ml, 3ml etc. solution to T. T. Make up volume with water to 10 ml in each T. T. Shake them in a lengthwise motion for 15 seconds, two shakes per second. Allow to stand for 15 minutes and measure the height of the foam. METHOD

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Result given as : If height of foam is less than 1 cm, foaming index is less than 1000. If height of foam is more than 1 cm, foaming index is more than 1000. If a height of foam is 1 cm in any tube, then according to volume of filterate added in tube is used to determine index. Foaming index is calculated by formula : 1000 / a Where, a = volume of filterate added to test tube

Determination of Pesticide Residues:

Pesticides are chemicals derived from synthetic and natural sources which are effective in small concentrations against pest. Determination of Pesticide Residues

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A capillary gas chromatography with an ECD is used for the measurement. Helium is used as carrier gas & a mixture of argon methane as an auxiliary gas for the detection. First separation system : Use a silica column, 30 m long with an internal diameter of 0.25 mm, packed with a chemically bound phase of 5% phenyl, 95% methyl- polysiloxane .

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Temp. programme Heat at 60 ºC for 0.5 minutes; Increase the temperature at a rate of 30ºC per minute to 160ºC & maintain this temperature for 2 minutes; Increase the temperature at a 2 ºC per minute to 250ºC & maintain this temperature for 5 minutes. Use a ‘ split/split-free’ injector to inject the sample & maintain the injection port at a temperature of 240 ºC . Inject a volume of 1 µl at a rate of 30 seconds . The detector temperature should be 300 ºC.

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Second separation system Packed with a chemically bound phase of 7%cynopropyl, 86% methyl-polysiloxane,7% phenyl. Temp programme:- Heat at 60 ºC for 0.2 minutes; Increase the temperature at a rate of 30ºC per minute to 180ºC & maintain this temperature for 1 minutes; Increase the temperature at a 2 ºC per minute to 250ºC & maintain this temperature for 5 minutes. Use a column injector to inject a volume of 1 µl at a rate of 30 seconds. The detector temperature should be 300 ºC.

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Use the external standard method for the qualitative & quantitative evaluation of the organochlorine pesticides In the test solutions with reference solutions of The following pesticides: Α-,β-,γ- & δ- hexachlorocyclohexane (HCH) Hexachlorobenzene; Quintozine. Measure the peak height of the pesticides obtained in the chlomatograms.

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Calculate the concentration of the residues in mg/kg using following formula: h t × 10 W r w h r Where, h t = peak height obtained for the test solution in mm. W = quantity of sample in ng in the purified extract (g). w r = quantity of pesticide in the reference solution injected, h r = peak height obtained for the reference solution in mm.

Determination of Arsenic and Heavy metals:

Metal: luster, malleability, high electric and thermal conductivity; chemically form bases which can react with acids Heavy Metal: Metal of High Specific Gravity: - Cadmium 112 - Lead 207 - Mercury 200 - Zinc 65 (Not so heavy) - Aluminum 27 (Light) -Arsenic 75 - Selenium 79 Heavy Metals – Sources: In general: Worldwide: associated with mining industry. Others: - Environmental pollution, - Accidental inclusion in processing, - Contamination from containers. Determination of Arsenic and Heavy metals

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Excess heavy metal accumulation in soil is toxic to humans and other animals. Exposure to heavy metals is normally chronic. (exposure over a longer period of time), due to food chain transfer. Acute (immediate) poisoning from heavy metals is rare through ingestion or dermal contact, but is possible. Chronic problems associated with long-term heavy metal exposures are: Lead – mental lapse. Cadmium – affects kidney, liver, and GI tract. Arsenic – skin poisoning, affects kidneys and CNS.

Limit Test For Arsenic:

Principle The amount of arsenic in the medicinal plant material is estimated by matching the depth colour with that of a standard stain. Reactions: H 2 AsO 4 KI H 3 AsO 3 Arsenic acid arsenious acid H 3 AsO 3 + 3H 2 AsH 3 + H 2 O Arsenious acid arsine gas AsH 3 + mercuric bromide yellow stain Limit Test For Arsenic

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Apparetus for limit test of Arsenic

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Preparation of the sample (by acid digestion) Place35-70 g of coarsely ground material, accurately weighed, in a Kjeldahl flask, capacity 800-1000 ml. +(10-25 ml) water + (25-50 ml) nitric acid + carefully add (20 ml) sulphuric acid. Heat Gradually add nitric acid (~1000 g/l), drop by drop, until all the organic matter is destroyed. a clear solution with copious vapours of sulfur trioxide is obtained. Cool add (75 ml) water + (25 ml) ammonium oxalate. Heat again until sulfur trioxide vapours develop. Cool, transfer to a 250-ml volumetric flask and dilute to volume with water.

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Preparation of standard stain: Add (10 ml) hydrochloric acid + 1 ml dilute arsenic As to 50 ml water. The resulting solution, when treated as described in the general test yields a stain on mercuric bromide paper. standard stain (10 μg of As).

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Method: Take an aliquot (25-50 ml) of the test solution +1 g of potassium iodide + l0 g of granulated zinc in the wide mouthed bottle place the prepared glass tube assembly quickly in position. reaction:-40minutes,40’c temp. Compare any yellow stain that is produced on the mercuric bromide paper with a standard stain produced in a similar manner with a known quantity of dilute arsenic. The contents of lead and cadmium may be determined by atomic absorption spectrophotometry Maximum amount in dried plant materials: -lead: 10mg/kg. -cadmium: 0.3mg/kg.

Microscopical Examination:

detailed examination of a drug Use - To identify the organised drugs by their known histological character. This mostly used for qualitative evaluation of organised crude drugs in entire and powdered forms and can be used to distinguish cellular structure. For the efective results, various reagents can be used to distinguish cellular structure. eg . A drop of phloroglucinol and conc. Hcl gives red stain with lignin. Mucilage is stained with ruthenium red to give pink color. Microscopical Examination

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Parameter For Evaluation:- a). Stomatal index is the percentage which give the number of stomata to total number of epidermal cells. Procedure : Place leaf fragments of about 5 × 5 mm in size in a test tube containing about 5 ml of chloral hydrate solution and heat in a boiling water-bath for about 15 minutes or until the fragments become transparent. Transfer a fragment to a microscopic slide and prepare the mount, the lower epidermis uppermost, in chloral hydrate solution and put a small drop of glycerol-ethanol solution on one side of the cover-glass to prevent the preparation from drying. Examine with a 40x objective and a 6x eye piece, to which a microscopical drawing apparatus is attached. Mark on the drawing paper a cross (x) for each epidermal cell and a circle (o) for each stomata. It is calculated by following equation : S. I. = S / E + S × 100 where, S = no. of stomata per unit area, E = no. of epidernal cells in unit area.

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b). Stomatal no.: is the average no. of stomata present per sq mm of epidermal cell. c). Palisade ratio : is the average no. of palisade cells beneath each epidermal cell. d). Vein islet & veinlet termination number : is the no. of vein islets & veinlet termination per sq mm of the leaf surface midway between midrib and margin.

Official Drugs And Their Evaluation Methods:

Methods For Evaluation Of Crude Drugs Name Of Crude Drugs HPLC Amalaki,Amara,Arjuna,Ashvagandha,Bhibhitaki , Bhringraj , Brahmi , Coleus, Garcinia , Gokhru , Gudmar , Guduchi , Guggul resin, Gugulipid , Haridra , Haritaki , Kalmegh , Kunduru , Kutki , Lausna , Mendukaparni , Manjistha , Maricha , Opium, Pippali , Punarnava , Senna Pods, Shatavari , Shati , Shunti,Vasaka,yasti GC Clove Oil,Tulsi TLC Artemisa , Shatavari , TITRATION Belladona leaf, Papain , Tolu Blasam Official Drugs And Their Evaluation Methods

REFERENCES:

Quality Control of Herbal Drugs, Pulok K Mukherjee , Business Horizons Pharmaceutical publishers, 2002,186-245. Indian Pharmacopoeia-2007, Ministry of health and Family Welfare, Controller of Publication, Delhi, Vol -III, page no.2015-2073. Quality Control Methods for Medicinal Plant Materials, World Health Organization, Geneva,1998 REFERENCES