QUALITY CONTROL &STANDARDIZATION OF HERBAL

DRUGS

Presented byK. Anupama

Shilpi Bhatnagar

Quality Control of Herbal Drugs

• Quality control for efficacy and safety of herbal products isof paramount importance.

• Quality can be defined as the status of a drug that isdetermined by identity, purity, content, and other chemical,physical, or biological properties, or by the manufacturingprocesses.

• Quality control is a term that refers to processes involved inmaintaining the quality and validity of a manufacturedproduct.

• Quality control for efficacy and safety of herbal products isof paramount importance.

• Quality can be defined as the status of a drug that isdetermined by identity, purity, content, and other chemical,physical, or biological properties, or by the manufacturingprocesses.

• Quality control is a term that refers to processes involved inmaintaining the quality and validity of a manufacturedproduct.

What are Herbal Drugs?

• The term “herbal drugs” denotes plants or plant parts thathave been converted into phytopharmaceuticals by means ofsimple processes involving harvesting, drying, and storage.

• A practical addition to the definition is also to include othercrude products derived from plants, which no longer showany organic structure, such as essential oils, fatty oils,resins, and gums.

• Derived or isolated compounds in the processed state suchas extracts or even isolated purified compounds (e.g.strychnine from Strychnos nux-vomica) or mixtures ofcompounds (e.g. abrin from Abrus precatorius) are, as arule, not included in the definition.

• The term “herbal drugs” denotes plants or plant parts thathave been converted into phytopharmaceuticals by means ofsimple processes involving harvesting, drying, and storage.

• A practical addition to the definition is also to include othercrude products derived from plants, which no longer showany organic structure, such as essential oils, fatty oils,resins, and gums.

• Derived or isolated compounds in the processed state suchas extracts or even isolated purified compounds (e.g.strychnine from Strychnos nux-vomica) or mixtures ofcompounds (e.g. abrin from Abrus precatorius) are, as arule, not included in the definition.

Quality Control

• In general, quality control is based on threeimportant Pharmacopoeial definitions:

1. Identity: Is the herb the one it should be?

2. Purity: Are there contaminants, e.g., in the form of otherherbs which should not be there?

3. Content or assay: Is the content of active constituentswithin the defined limits?

• In general, quality control is based on threeimportant Pharmacopoeial definitions:

1. Identity: Is the herb the one it should be?

2. Purity: Are there contaminants, e.g., in the form of otherherbs which should not be there?

3. Content or assay: Is the content of active constituentswithin the defined limits?

• It is obvious that the content is the most difficult one to assess,since in most herbal drugs the active constituents are unknown.Sometimes markers can be used which are, by definition,chemically defined constituents that are of interest for controlpurposes, independent of whether they have any therapeuticactivity or not.

• To prove identity and purity, criteria such as type ofpreparation, physical constants, adulteration, contaminants,moisture, ash content and solvent residues have to be checked.

• The correct identity of the crude herbal material, or thebotanical quality, is of prime importance in establishing thequality control of herbal drugs.

• It is obvious that the content is the most difficult one to assess,since in most herbal drugs the active constituents are unknown.Sometimes markers can be used which are, by definition,chemically defined constituents that are of interest for controlpurposes, independent of whether they have any therapeuticactivity or not.

• To prove identity and purity, criteria such as type ofpreparation, physical constants, adulteration, contaminants,moisture, ash content and solvent residues have to be checked.

• The correct identity of the crude herbal material, or thebotanical quality, is of prime importance in establishing thequality control of herbal drugs.

Identity

• It can be achieved by macro- and microscopical examinations.Voucher specimens are reliable reference sources. Outbreaks ofdiseases among plants may result in changes to the physicalappearance of the plant and lead to incorrect identification. Attimes an incorrect botanical quality with respect to the labeling canbe a problem.

• For example, in the 1990s, a South American product labeled as“Paraguay Tea” was associated with an outbreak of anticholinergicpoisoning in New York. Subsequent chemical analysis revealedthe presence of a class of constituents that was different from themetabolites normally found in the plant from which Paraguay tea ismade.

• It can be achieved by macro- and microscopical examinations.Voucher specimens are reliable reference sources. Outbreaks ofdiseases among plants may result in changes to the physicalappearance of the plant and lead to incorrect identification. Attimes an incorrect botanical quality with respect to the labeling canbe a problem.

• For example, in the 1990s, a South American product labeled as“Paraguay Tea” was associated with an outbreak of anticholinergicpoisoning in New York. Subsequent chemical analysis revealedthe presence of a class of constituents that was different from themetabolites normally found in the plant from which Paraguay tea ismade.

Purity

• It is closely linked with the safe use of drugs and deals withfactors such ash values, contaminants (e.g. foreign matter inthe form of other herbs), and heavy metals.

• However, due to the application of improved analyticalmethods, modern purity evaluation also includes microbialcontamination, aflatoxins, radioactivity, and pesticideresidues.

• Analytical methods such as photometric analysis, thin layerchromatography (TLC), high performance liquidchromatography (HPLC), and gas chromatography (GC) canbe employed in order to establish the constant composition ofherbal preparations.

• It is closely linked with the safe use of drugs and deals withfactors such ash values, contaminants (e.g. foreign matter inthe form of other herbs), and heavy metals.

• However, due to the application of improved analyticalmethods, modern purity evaluation also includes microbialcontamination, aflatoxins, radioactivity, and pesticideresidues.

• Analytical methods such as photometric analysis, thin layerchromatography (TLC), high performance liquidchromatography (HPLC), and gas chromatography (GC) canbe employed in order to establish the constant composition ofherbal preparations.

Content or Assay

• It is the most difficult area of quality control to perform, since inmost herbal drugs the active constituents are not known.Sometimes markers can be used.

• In all other cases, where no active constituent or marker canbe defined for the herbal drug, the percentage extractablematter with a solvent may be used as a form of assay, anapproach often seen in pharmacopeias.

• The choice of the extracting solvent depends on the nature ofthe compounds involved, and might be deduced from thetraditional uses.

• It is the most difficult area of quality control to perform, since inmost herbal drugs the active constituents are not known.Sometimes markers can be used.

• In all other cases, where no active constituent or marker canbe defined for the herbal drug, the percentage extractablematter with a solvent may be used as a form of assay, anapproach often seen in pharmacopeias.

• The choice of the extracting solvent depends on the nature ofthe compounds involved, and might be deduced from thetraditional uses.

• For example, when a herbal drug is used to make a tea, the hotwater extractable matter, expressed as milligrams per gram ofair-dried material, may serve this purpose.

• A special form of assay is the determination of essential oils bysteam distillation. When the active constituents (e.g.sennosides in Senna) or markers (e.g. alkylamides inEchinacea) are known, a vast array of modern chemicalanalytical methods such as ultraviolet/visible spectroscopy(UV/VIS), TLC, HPLC, GC, mass spectrometry (MS), or acombination of GC and MS (GC/MS), can be employed.

• For example, when a herbal drug is used to make a tea, the hotwater extractable matter, expressed as milligrams per gram ofair-dried material, may serve this purpose.

• A special form of assay is the determination of essential oils bysteam distillation. When the active constituents (e.g.sennosides in Senna) or markers (e.g. alkylamides inEchinacea) are known, a vast array of modern chemicalanalytical methods such as ultraviolet/visible spectroscopy(UV/VIS), TLC, HPLC, GC, mass spectrometry (MS), or acombination of GC and MS (GC/MS), can be employed.

Several problems not applicable to syntheticdrugs influence the quality of herbal drugs:

1. Herbal drugs are usually mixtures of many constituents.

2. The active principle(s) is (are), in most cases unknown.

3. Selective analytical methods or reference compounds maynot be available commercially.

4. Plant materials are chemically and naturally variable.

5. The source and quality of the raw material are variable.

6. The methods of harvesting, drying, storage, transportation,and processing (for example, mode of extraction and polarityof the extracting solvent, instability of constituents, etc.) havean effect.

1. Herbal drugs are usually mixtures of many constituents.

2. The active principle(s) is (are), in most cases unknown.

3. Selective analytical methods or reference compounds maynot be available commercially.

4. Plant materials are chemically and naturally variable.

5. The source and quality of the raw material are variable.

6. The methods of harvesting, drying, storage, transportation,and processing (for example, mode of extraction and polarityof the extracting solvent, instability of constituents, etc.) havean effect.

• Strict guidelines have to be followed for the successfulproduction of a quality herbal drug. Among them areproper botanical identification, phytochemical screening,and standardization.

• Strict guidelines have to be followed for the successfulproduction of a quality herbal drug. Among them areproper botanical identification, phytochemical screening,and standardization.

WHO Guidelines

• Owing to long standing and time proven uses of herbal drugsalong with higher safety margins, WHO has taken necessarysteps to ensure Quality Control of herbal drugs with moderntechniques and application of suitable standards for thispurpose.

• The pharmacopoeias of different countries includemonographs indicating quality parameters and standards forvarious herbal drugs and for some of their products.

• The objective put forth are provisions for recommendedgeneral test methods and also the general limits for thecontaminants for herbal drugs.

• Owing to long standing and time proven uses of herbal drugsalong with higher safety margins, WHO has taken necessarysteps to ensure Quality Control of herbal drugs with moderntechniques and application of suitable standards for thispurpose.

• The pharmacopoeias of different countries includemonographs indicating quality parameters and standards forvarious herbal drugs and for some of their products.

• The objective put forth are provisions for recommendedgeneral test methods and also the general limits for thecontaminants for herbal drugs.

Evaluation Parameters:

Different techniques involved in standardization of crudedrugs :1. Macroscopic methods2. Microscopic methods3. Physical methods4. Chemical methods5. Biological methods

Different techniques involved in standardization of crudedrugs :1. Macroscopic methods2. Microscopic methods3. Physical methods4. Chemical methods5. Biological methods

STANDARDIZATION OFHERBAL DRUGS

• Standardization of drug means confirmation of its identityand determination of its quality and purity and detectionof nature of adulterant by various parameters likemorphological, microscopical, physical, chemical andbiological observations.

• Standardized extracts are high-quality extractscontaining consistent levels of specified compounds, andthey are subjected to rigorous quality controls during allphases of the growing, harvesting, and manufacturingprocesses.

• Standardization of drug means confirmation of its identityand determination of its quality and purity and detectionof nature of adulterant by various parameters likemorphological, microscopical, physical, chemical andbiological observations.

• Standardized extracts are high-quality extractscontaining consistent levels of specified compounds, andthey are subjected to rigorous quality controls during allphases of the growing, harvesting, and manufacturingprocesses.

TYPES OF STANDARDIZATION

There are two types of standardization :

In the first category, “true” standardization, a definite phytochemical orgroup of constituents is known to have activity. Ginkgo with its 26%ginkgo flavones and 6% terpenes is a classic example. These productsare highly concentrated and no longer represent the whole herb, andare now considered as phytopharmaceuticals. In many cases they arevastly more effective than the whole herb.

The other type of standardization is based on manufacturersguaranteeing the presence of a certain percentage of markercompounds; these are not indicators of therapeutic activity or quality ofthe herb.

There are two types of standardization :

In the first category, “true” standardization, a definite phytochemical orgroup of constituents is known to have activity. Ginkgo with its 26%ginkgo flavones and 6% terpenes is a classic example. These productsare highly concentrated and no longer represent the whole herb, andare now considered as phytopharmaceuticals. In many cases they arevastly more effective than the whole herb.

The other type of standardization is based on manufacturersguaranteeing the presence of a certain percentage of markercompounds; these are not indicators of therapeutic activity or quality ofthe herb.

TECHNIQUES OFSTANDARDIZATION

Different techniques involved in standardization of crudedrugs :

1. Macroscopic methods2. Microscopic methods3. Physical methods4. Chemical methods5. Biological methods

Different techniques involved in standardization of crudedrugs :

1. Macroscopic methods2. Microscopic methods3. Physical methods4. Chemical methods5. Biological methods

MACROSCOPIC METHODS• ORGANOLEPTIC EVALUATION: Organoleptic evaluation of drugs

refers to the evaluation of a drug by colour, odour, size, shape, tasteand special features including touch, texture etc. Since the majorityof information on the identity, purity and quality of the material canbe drawn from these observations, they are of primary importancebefore any further testing can be carried out.

• For this purpose authentic specimen of the material under study andsamples of pharmacopoeial quality should be available to serve as areference.

• This evaluation procedure provides the simplest and quickestmeans to establish the identity and purity and thereby ensure qualityof a particular sample.

• ORGANOLEPTIC EVALUATION: Organoleptic evaluation of drugsrefers to the evaluation of a drug by colour, odour, size, shape, tasteand special features including touch, texture etc. Since the majorityof information on the identity, purity and quality of the material canbe drawn from these observations, they are of primary importancebefore any further testing can be carried out.

• For this purpose authentic specimen of the material under study andsamples of pharmacopoeial quality should be available to serve as areference.

• This evaluation procedure provides the simplest and quickestmeans to establish the identity and purity and thereby ensure qualityof a particular sample.

PHYSICAL STANDARDIZATION

PHYSICAL STANDARDIZATION OF HERBAL DRUGS:• Viscosity• Melting point• Solubility• Moisture content and volatile matter• Specific gravity• Density• Optical rotation• Refractive index• Bitterness value• Hemolytic activity• Swelling index• Foaming index• Ash value

PHYSICAL STANDARDIZATION OF HERBAL DRUGS:• Viscosity• Melting point• Solubility• Moisture content and volatile matter• Specific gravity• Density• Optical rotation• Refractive index• Bitterness value• Hemolytic activity• Swelling index• Foaming index• Ash value

VISCOSITY:Viscosity of a liquid is constant at a given temperature and isan index of its composition. Hence, it can be used as a meansof standardizing liquid drugs.

MELTING POINT:In case of pure photochemical, melting points are very sharpand constant.

The crude drugs from plant or animal origin, containing themixed chemicals, are described with certain range of meltingpoint.

Their purity can be ascertained by determining their meltingpoints in that range for e.g. Colophony- 75-80˚c Cocoa butter-30-33˚c

VISCOSITY:Viscosity of a liquid is constant at a given temperature and isan index of its composition. Hence, it can be used as a meansof standardizing liquid drugs.

MELTING POINT:In case of pure photochemical, melting points are very sharpand constant.

The crude drugs from plant or animal origin, containing themixed chemicals, are described with certain range of meltingpoint.

Their purity can be ascertained by determining their meltingpoints in that range for e.g. Colophony- 75-80˚c Cocoa butter-30-33˚c

SOLUBILTY:The presence of adulterant could be indicated by solubilitystudies. E.g., pure Asafoetida is soluble in carbon disulphide

MOISTURE CONTENT:The moisture content of the drug should be minimized inorder to prevent decomposition of crude drug either due tochemical change or microbial contamination.

The moisture content is determined by heating a drug at105˚c in an oven to a constant weight. For the drugscontaining volatile constituents, toluene distillation method isused. E.g. Aloe should have moisture content not more than10% w/w.

SOLUBILTY:The presence of adulterant could be indicated by solubilitystudies. E.g., pure Asafoetida is soluble in carbon disulphide

MOISTURE CONTENT:The moisture content of the drug should be minimized inorder to prevent decomposition of crude drug either due tochemical change or microbial contamination.

The moisture content is determined by heating a drug at105˚c in an oven to a constant weight. For the drugscontaining volatile constituents, toluene distillation method isused. E.g. Aloe should have moisture content not more than10% w/w.

OPTICAL ROTATION: Optically active compounds have theproperty of rotating the plane of polarized light. This property isknown as optical rotation. Normally, the optical rotation isdetermined at 25˚c using sodium lamp as the source of light. E.g.castor oil has optical rotation from +3.5˚to +6˚

REFRACTIVE INDEX: When a ray of light passes from onemedium to another of different density, then the ratio of velocityof light in vacuum to its velocity in substance is termed asrefractive index of second medium. It is constant for a pure drugand varied with wavelength of incident light, temperature andpressure E.g., Castor oil has refractive index 1.4758-1.527

OPTICAL ROTATION: Optically active compounds have theproperty of rotating the plane of polarized light. This property isknown as optical rotation. Normally, the optical rotation isdetermined at 25˚c using sodium lamp as the source of light. E.g.castor oil has optical rotation from +3.5˚to +6˚

REFRACTIVE INDEX: When a ray of light passes from onemedium to another of different density, then the ratio of velocityof light in vacuum to its velocity in substance is termed asrefractive index of second medium. It is constant for a pure drugand varied with wavelength of incident light, temperature andpressure E.g., Castor oil has refractive index 1.4758-1.527

ASH VALUES AND EXTRACTIVES: The residueremaining after incineration is the ash content of drug.

Total ash method is used to measure the total amount ofmaterial remaining after incineration.

Acid insoluble ash is the residue obtained after boilingthe total ash with dil. HCl and igniting the remaininginsoluble matter.

Water soluble ash is the difference in weight betweentotal ash and residue after treatment of total ash withwater.

ASH VALUES AND EXTRACTIVES: The residueremaining after incineration is the ash content of drug.

Total ash method is used to measure the total amount ofmaterial remaining after incineration.

Acid insoluble ash is the residue obtained after boilingthe total ash with dil. HCl and igniting the remaininginsoluble matter.

Water soluble ash is the difference in weight betweentotal ash and residue after treatment of total ash withwater.

DETERMINATION OF EXTRACTABLE MATTER:HOT EXTRACTION: Place 4 gms powdered material in a conical flask. Add water and weigh to obtain total weight. Shake and allowed to stand for 1hr. attach the reflux

condenser and boil for 1hr. Re-adjust to the original weight with solvent. Shake and filter. Transfer the filter to a flat bottomed disk

and evaporate to dryness on a water bath. Dry at 105˚ c for 6hrs, cool and weigh immediately. Calculate the content of extractable matter in mg per g of air

dried material.

DETERMINATION OF EXTRACTABLE MATTER:HOT EXTRACTION: Place 4 gms powdered material in a conical flask. Add water and weigh to obtain total weight. Shake and allowed to stand for 1hr. attach the reflux

condenser and boil for 1hr. Re-adjust to the original weight with solvent. Shake and filter. Transfer the filter to a flat bottomed disk

and evaporate to dryness on a water bath. Dry at 105˚ c for 6hrs, cool and weigh immediately. Calculate the content of extractable matter in mg per g of air

dried material.

COLD MARCERATION: Place the powdered material in a conical flask. Macerate with 100ml of solvent specified for 6hrs, shake

then allowed to stand for 18hrs. Filter and transfer the filtrate to flat bottomed disk and

evaporate to dryness on a water bath. Dry at 105ºc for 6hrs, cool and weigh immediately. Calculated the content of extractable matter in mg per g

of air dried material.

COLD MARCERATION: Place the powdered material in a conical flask. Macerate with 100ml of solvent specified for 6hrs, shake

then allowed to stand for 18hrs. Filter and transfer the filtrate to flat bottomed disk and

evaporate to dryness on a water bath. Dry at 105ºc for 6hrs, cool and weigh immediately. Calculated the content of extractable matter in mg per g

of air dried material.

BITTERNESS VALUE:• Medicinal plants having strong bitter taste are therapeutically used as

appetizing agents.• The bitterness is determined by comparing the threshold bitter

concentration of an extract material with that of quinine hydrochloride.• The bitterness value is expressed as units equivalent to the bitterness of

a solution containing 1gm of quinine hydrochloride in 2000ml.• 0.1gm of quinine hydrochloride is dissolved in 100ml drinking water and

the stock solution is prepared.• Then it is diluted and tested and compared with drug.• Bitterness value in unit per gm = 2000*C

A*B• Where, A = concentration of stock solution B = volume of test solution in

tube with threshold bitter concentration C = quantity of quininehydrochloride in the tube with threshold bitter concentration

BITTERNESS VALUE:• Medicinal plants having strong bitter taste are therapeutically used as

appetizing agents.• The bitterness is determined by comparing the threshold bitter

concentration of an extract material with that of quinine hydrochloride.• The bitterness value is expressed as units equivalent to the bitterness of

a solution containing 1gm of quinine hydrochloride in 2000ml.• 0.1gm of quinine hydrochloride is dissolved in 100ml drinking water and

the stock solution is prepared.• Then it is diluted and tested and compared with drug.• Bitterness value in unit per gm = 2000*C

A*B• Where, A = concentration of stock solution B = volume of test solution in

tube with threshold bitter concentration C = quantity of quininehydrochloride in the tube with threshold bitter concentration

HAEMOLYTIC ACTIVITY: Haemolytic activity of plant material is determined by comparison with

that of reference material, Saponin R, having haemolytic activity of1000units/g.

Method of preparation of standard: Fill a glass stopper flask to 1/10 of itsvolume with sodium citrate. Add sufficient volume of blood freshlycollected from healthy ox and shake, this can be stored for about 8 daysat 2-4̊ c. Place 1ml of citrated blood in a volumetric flask with phosphatebuffer pH 7.4.

Haemolytic activity = 1000* a/bWhere, 1000 = defined haemolytic activity of Saponin standard a =quantity of saponin standard that produce total haemolysis(g) b =quantity of plant material that produce total haemolysis (g)

HAEMOLYTIC ACTIVITY: Haemolytic activity of plant material is determined by comparison with

that of reference material, Saponin R, having haemolytic activity of1000units/g.

Method of preparation of standard: Fill a glass stopper flask to 1/10 of itsvolume with sodium citrate. Add sufficient volume of blood freshlycollected from healthy ox and shake, this can be stored for about 8 daysat 2-4̊ c. Place 1ml of citrated blood in a volumetric flask with phosphatebuffer pH 7.4.

Haemolytic activity = 1000* a/bWhere, 1000 = defined haemolytic activity of Saponin standard a =quantity of saponin standard that produce total haemolysis(g) b =quantity of plant material that produce total haemolysis (g)

SWELLING INDEX: The swelling index is the volume in ml taken up by theswelling of 1gm of plant material under specified conditions. Itsdetermination is based on addition of water or a swelling agent asdescribed in test procedure.

FOAMING INDEX: The foaming ability of an aqueous decoction of plantmaterial and their extracts is measured in terms of foaming index.

WATER AND VOLATILE MATTER: Azeotropic method is used to directlymeasure the water present in a material. Loss on drying In order tomeasure volatile matter, plant is diluted with water and distillate iscollected in a graduated tube. The aqueous portion separates andreturns to distillation flask. A solvent of low mass density with a suitableboiling point may be added to measuring tube to easily separate thevolatile oil.

SWELLING INDEX: The swelling index is the volume in ml taken up by theswelling of 1gm of plant material under specified conditions. Itsdetermination is based on addition of water or a swelling agent asdescribed in test procedure.

FOAMING INDEX: The foaming ability of an aqueous decoction of plantmaterial and their extracts is measured in terms of foaming index.

WATER AND VOLATILE MATTER: Azeotropic method is used to directlymeasure the water present in a material. Loss on drying In order tomeasure volatile matter, plant is diluted with water and distillate iscollected in a graduated tube. The aqueous portion separates andreturns to distillation flask. A solvent of low mass density with a suitableboiling point may be added to measuring tube to easily separate thevolatile oil.

CHEMICAL METHODS OF STANDARDIZATION OF HERBALDRUGS:

• It comprises of different chemical tests and assays.• The isolation, purification and identification of active constituents are

chemical methods of evaluation.• Quantitative chemical tests such as acid value, saponification value

etc., are also covered under this technique.• Qualitative chemical tests are used in detection of adulteration.• The chemical evaluation also covers phytochemical screening

carried out for establishing chemical profile of a drug.

CHEMICAL METHODS OF STANDARDIZATION OF HERBALDRUGS:

• It comprises of different chemical tests and assays.• The isolation, purification and identification of active constituents are

chemical methods of evaluation.• Quantitative chemical tests such as acid value, saponification value

etc., are also covered under this technique.• Qualitative chemical tests are used in detection of adulteration.• The chemical evaluation also covers phytochemical screening

carried out for establishing chemical profile of a drug.

CHEMICAL EXAMINATION: Detection of alkaloids Detection of carbohydrates and glycosides Detection of phytosterols Detection of fixed oils and fats Detection of saponins Detection of phenolic compounds and tannins Detection of protein and free amino acids Detection of gums and mucilage Detection of volatile oils

CHEMICAL EXAMINATION: Detection of alkaloids Detection of carbohydrates and glycosides Detection of phytosterols Detection of fixed oils and fats Detection of saponins Detection of phenolic compounds and tannins Detection of protein and free amino acids Detection of gums and mucilage Detection of volatile oils

BIOLOGICAL/TOXICOLOGICAL STANDARDIZATION: Drugswhich cannot be assayed by chemical, or physical means areevaluated by biological methods.

INDICATION FOR BIOLOGICAL EVALUATION:This is true for the substances having an Interfering obstaclesWhen quantity is too small, no specific chemical test isavailable. When the action of drug is due to a mixture ofsubstance, purification of drug is not possible.

BIOLOGICAL/TOXICOLOGICAL STANDARDIZATION: Drugswhich cannot be assayed by chemical, or physical means areevaluated by biological methods.

INDICATION FOR BIOLOGICAL EVALUATION:This is true for the substances having an Interfering obstaclesWhen quantity is too small, no specific chemical test isavailable. When the action of drug is due to a mixture ofsubstance, purification of drug is not possible.

BIOASSAY : When the estimation of crude drug or its preparation isdone by means of its effect on living organism like bacteria, fungi, oranimal tissue or entire animal it is known as BIOASSAY.

TYPES OF BIOASSAYQUANTAL: It is all or none phenomenonGRADED: Based on observation that there is a proportionate increase in

the observed response with increase in concentration or dose.• Graded bioassay can be performed by using any of the following

techniques Matching bioassay Interpolation Method Bracketing Method Multiple point bioassay

BIOASSAY : When the estimation of crude drug or its preparation isdone by means of its effect on living organism like bacteria, fungi, oranimal tissue or entire animal it is known as BIOASSAY.

TYPES OF BIOASSAYQUANTAL: It is all or none phenomenonGRADED: Based on observation that there is a proportionate increase in

the observed response with increase in concentration or dose.• Graded bioassay can be performed by using any of the following

techniques Matching bioassay Interpolation Method Bracketing Method Multiple point bioassay

TOXICOLOGICAL STANDARDIZATION: Determination of pesticides. Determination of arsenic and heavy metals Determination radioactive contamination Determination of aflatoxins.

TOXICOLOGICAL STANDARDIZATION: Determination of pesticides. Determination of arsenic and heavy metals Determination radioactive contamination Determination of aflatoxins.

DETERMINATION OF HEAVY METALS

• Contamination by toxic metals can either be accidental orintentional. Contamination by heavy metals such as mercury,lead, copper, cadmium, and arsenic in herbal remedies canbe attributed to many causes, including environmentalpollution, and can pose clinically relevant dangers for thehealth of the user and should therefore be limited.

• A simple, straightforward determination of heavy metals canbe found in many pharmacopeias and is based on colorreactions with special reagents such as thioacetamide ordiethyldithiocarbamate, and the amount present is estimatedby comparison with a standard.

• Contamination by toxic metals can either be accidental orintentional. Contamination by heavy metals such as mercury,lead, copper, cadmium, and arsenic in herbal remedies canbe attributed to many causes, including environmentalpollution, and can pose clinically relevant dangers for thehealth of the user and should therefore be limited.

• A simple, straightforward determination of heavy metals canbe found in many pharmacopeias and is based on colorreactions with special reagents such as thioacetamide ordiethyldithiocarbamate, and the amount present is estimatedby comparison with a standard.

Radioactive contamination:The exposure cannot be avoided because of many naturallyoccurring sources including radionucleotides occurring in groundand atmosphere. Health risk depend on– Specific radionucleotides– Level of contamination– Quantity of food

Aflatoxins:Aflatoxins are the poisonous substance in the spores of the fungusAspergillus flavus. The toxin is known to produce cancer in humanbeings living in warm and humid region of the world. Stored nuts andcereals are contaminated by the fungus. They should therefore bedetermined after using a suitable clean up procedure.

Radioactive contamination:The exposure cannot be avoided because of many naturallyoccurring sources including radionucleotides occurring in groundand atmosphere. Health risk depend on– Specific radionucleotides– Level of contamination– Quantity of food

Aflatoxins:Aflatoxins are the poisonous substance in the spores of the fungusAspergillus flavus. The toxin is known to produce cancer in humanbeings living in warm and humid region of the world. Stored nuts andcereals are contaminated by the fungus. They should therefore bedetermined after using a suitable clean up procedure.

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