QUALITY CONTROL & STANDARDIZATION OF HERBAL DRUGS

Presented by K. Anupama Shilpi Bhatnagar

Quality Control of Herbal Drugs

Quality control for efficacy and safety of herbal products is of paramount importance. Quality can be defined as the status of a drug that is determined by identity, purity, content, and other chemical, physical, or biological properties, or by the manufacturing processes. Quality control is a term that refers to processes involved in maintaining the quality and validity of a manufactured product.

What are Herbal Drugs?

The term herbal drugs denotes plants or plant parts that have been converted into phytopharmaceuticals by means of simple processes involving harvesting, drying, and storage. A practical addition to the definition is also to include other crude products derived from plants, which no longer show any organic structure, such as essential oils, fatty oils, resins, and gums. Derived or isolated compounds in the processed state such as extracts or even isolated purified compounds (e.g. strychnine from Strychnos nux-vomica) or mixtures of compounds (e.g. abrin from Abrus precatorius) are, as a rule, not included in the definition.

Quality Control
In general, quality control is based on three important Pharmacopoeial definitions:

1. Identity: Is the herb the one it should be? 2. Purity: Are there contaminants, e.g., in the form of other herbs which should not be there? 3. Content or assay: Is the content of active constituents within 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 control purposes, independent of whether they have any therapeutic activity or not. To prove identity and purity, criteria such as type of preparation, physical constants, adulteration, contaminants, moisture, ash content and solvent residues have to be checked. The correct identity of the crude herbal material, or the botanical quality, is of prime importance in establishing the quality control of herbal drugs.

Identity
It can be achieved by macro- and microscopical examinations. Voucher specimens are reliable reference sources. Outbreaks of diseases among plants may result in changes to the physical appearance of the plant and lead to incorrect identification. At times an incorrect botanical quality with respect to the labeling can be a problem. For example, in the 1990s, a South American product labeled as Paraguay Tea was associated with an outbreak of anticholinergic poisoning in New York. Subsequent chemical analysis revealed the presence of a class of constituents that was different from the metabolites normally found in the plant from which Paraguay tea is made.

Purity
It is closely linked with the safe use of drugs and deals with factors such ash values, contaminants (e.g. foreign matter in the form of other herbs), and heavy metals. However, due to the application of improved analytical methods, modern purity evaluation also includes microbial contamination, aflatoxins, radioactivity, and pesticide residues. Analytical methods such as photometric analysis, thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and gas chromatography (GC) can be employed in order to establish the constant composition of herbal preparations.

Content or Assay
It is the most difficult area of quality control to perform, since in most herbal drugs the active constituents are not known. Sometimes markers can be used. In all other cases, where no active constituent or marker can be defined for the herbal drug, the percentage extractable matter with a solvent may be used as a form of assay, an approach often seen in pharmacopeias. The choice of the extracting solvent depends on the nature of the compounds involved, and might be deduced from the traditional uses.

 For example, when a herbal drug is used to make a tea, the hot water extractable matter, expressed as milligrams per gram of air-dried material, may serve this purpose. A special form of assay is the determination of essential oils by steam distillation. When the active constituents (e.g. sennosides in Senna) or markers (e.g. alkylamides in Echinacea) are known, a vast array of modern chemical analytical methods such as ultraviolet/visible spectroscopy (UV/VIS), TLC, HPLC, GC, mass spectrometry (MS), or a combination of GC and MS (GC/MS), can be employed.

Several problems not applicable to synthetic drugs 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 may not 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 polarity of the extracting solvent, instability of constituents, etc.) have an effect.

 Strict guidelines have to be followed for the successful production of a quality herbal drug. Among them are proper botanical identification, phytochemical screening, and standardization.

WHO Guidelines
Owing to long standing and time proven uses of herbal drugs along with higher safety margins, WHO has taken necessary steps to ensure Quality Control of herbal drugs with modern techniques and application of suitable standards for this purpose. The pharmacopoeias of different countries include monographs indicating quality parameters and standards for various herbal drugs and for some of their products. The objective put forth are provisions for recommended general test methods and also the general limits for the contaminants for herbal drugs.

Evaluation Parameters:
Different techniques involved in standardization of crude drugs : 1. Macroscopic methods 2. Microscopic methods 3. Physical methods 4. Chemical methods 5. Biological methods

STANDARDIZATION OF HERBAL DRUGS

Standardization of drug means confirmation of its identity and determination of its quality and purity and detection of nature of adulterant by various parameters like morphological, microscopical, physical, chemical and biological observations. Standardized extracts are high-quality extracts containing consistent levels of specified compounds, and they are subjected to rigorous quality controls during all phases of the growing, harvesting, and manufacturing processes.

TYPES OF STANDARDIZATION
There are two types of standardization :
In the first category, true standardization, a definite phytochemical or group of constituents is known to have activity. Ginkgo with its 26% ginkgo flavones and 6% terpenes is a classic example. These products are highly concentrated and no longer represent the whole herb, and are now considered as phytopharmaceuticals. In many cases they are vastly more effective than the whole herb. The other type of standardization is based on manufacturers guaranteeing the presence of a certain percentage of marker compounds; these are not indicators of therapeutic activity or quality of the herb.

TECHNIQUES OF STANDARDIZATION
Different techniques involved in standardization of crude drugs : 1. Macroscopic methods 2. Microscopic methods 3. Physical methods 4. Chemical methods 5. Biological methods

MACROSCOPIC METHODS
ORGANOLEPTIC EVALUATION: Organoleptic evaluation of drugs refers to the evaluation of a drug by colour, odour, size, shape, taste and special features including touch, texture etc. Since the majority of information on the identity, purity and quality of the material can be drawn from these observations, they are of primary importance before any further testing can be carried out. For this purpose authentic specimen of the material under study and samples of pharmacopoeial quality should be available to serve as a reference. This evaluation procedure provides the simplest and quickest means to establish the identity and purity and thereby ensure quality of 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

VISCOSITY: Viscosity of a liquid is constant at a given temperature and is an index of its composition. Hence, it can be used as a means of standardizing liquid drugs. MELTING POINT: In case of pure photochemical, melting points are very sharp and constant. The crude drugs from plant or animal origin, containing the mixed chemicals, are described with certain range of melting point. Their purity can be ascertained by determining their melting points in that range for e.g. Colophony- 75-80c Cocoa butter30-33c

SOLUBILTY: The presence of adulterant could be indicated by solubility studies. E.g., pure Asafoetida is soluble in carbon disulphide MOISTURE CONTENT: The moisture content of the drug should be minimized in order to prevent decomposition of crude drug either due to chemical change or microbial contamination. The moisture content is determined by heating a drug at 105c in an oven to a constant weight. For the drugs containing volatile constituents, toluene distillation method is used. E.g. Aloe should have moisture content not more than 10% w/w.

OPTICAL ROTATION: Optically active compounds have the property of rotating the plane of polarized light. This property is known as optical rotation. Normally, the optical rotation is determined at 25c using sodium lamp as the source of light. E.g. castor oil has optical rotation from +3.5to +6 REFRACTIVE INDEX: When a ray of light passes from one medium to another of different density, then the ratio of velocity of light in vacuum to its velocity in substance is termed as refractive index of second medium. It is constant for a pure drug and varied with wavelength of incident light, temperature and pressure E.g., Castor oil has refractive index 1.4758-1.527

ASH VALUES AND EXTRACTIVES: The residue remaining after incineration is the ash content of drug. Total ash method is used to measure the total amount of material remaining after incineration. Acid insoluble ash is the residue obtained after boiling the total ash with dil. HCl and igniting the remaining insoluble matter. Water soluble ash is the difference in weight between total ash and residue after treatment of total ash with water.

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 105c 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 quinine hydrochloride 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 of 1000units/g. Method of preparation of standard: Fill a glass stopper flask to 1/10 of its volume with sodium citrate. Add sufficient volume of blood freshly collected from healthy ox and shake, this can be stored for about 8 days at 2-4 c. Place 1ml of citrated blood in a volumetric flask with phosphate buffer pH 7.4. Haemolytic activity = 1000* a/b Where, 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 the swelling of 1gm of plant material under specified conditions. Its determination is based on addition of water or a swelling agent as described in test procedure. FOAMING INDEX: The foaming ability of an aqueous decoction of plant material and their extracts is measured in terms of foaming index. WATER AND VOLATILE MATTER: Azeotropic method is used to directly measure the water present in a material. Loss on drying In order to measure volatile matter, plant is diluted with water and distillate is collected in a graduated tube. The aqueous portion separates and returns to distillation flask. A solvent of low mass density with a suitable boiling point may be added to measuring tube to easily separate the volatile oil.

CHEMICAL METHODS OF STANDARDIZATION OF HERBAL DRUGS: 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

BIOLOGICAL/TOXICOLOGICAL STANDARDIZATION: Drugs which cannot be assayed by chemical, or physical means are evaluated by biological methods. INDICATION FOR BIOLOGICAL EVALUATION: This is true for the substances having an Interfering obstacles When quantity is too small, no specific chemical test is available. When the action of drug is due to a mixture of substance, purification of drug is not possible.

BIOASSAY : When the estimation of crude drug or its preparation is done by means of its effect on living organism like bacteria, fungi, or animal tissue or entire animal it is known as BIOASSAY. TYPES OF BIOASSAY QUANTAL: It is all or none phenomenon GRADED: 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.

DETERMINATION OF HEAVY METALS

Contamination by toxic metals can either be accidental or intentional. Contamination by heavy metals such as mercury, lead, copper, cadmium, and arsenic in herbal remedies can be attributed to many causes, including environmental pollution, and can pose clinically relevant dangers for the health of the user and should therefore be limited. A simple, straightforward determination of heavy metals can be found in many pharmacopeias and is based on color reactions with special reagents such as thioacetamide or diethyldithiocarbamate, and the amount present is estimated by comparison with a standard.

Radioactive contamination: The exposure cannot be avoided because of many naturally occurring sources including radionucleotides occurring in ground and atmosphere. Health risk depend on Specific radionucleotides Level of contamination Quantity of food Aflatoxins: Aflatoxins are the poisonous substance in the spores of the fungus Aspergillus flavus. The toxin is known to produce cancer in human beings living in warm and humid region of the world. Stored nuts and cereals are contaminated by the fungus. They should therefore be determined after using a suitable clean up procedure.

THANK YOU