Factor affecting Herb Quality Comparing with conventional preparations, herbal product represent a number of unique problems when quality aspects are considered, these are because of the nature of the herbal ingredients present there in, which are complex mixtures of secondary metabolites that can vary depending on environment and genetic factor. The constituents responsible for the claimed therapeutic effect are unknown or partly explained. Further complication use of combinations of herbal ingredients as are use traditionally.

Quality standards of Herbal Products 1.Structural standard: comprises the structure form of the crude drug which limits amount of certain parts of organism concern 2.Analytical standards: constituents of material 3.Standards relating to physical constant, e.g. density, refractive index ….. etc.

Factors relating to quality of Herbal drug To ensure reproducible quality of the herbal remedy, proper control of starting material is almost essential. To control the quality of starting material, the following aspects need to be considered: 1.Authentication and reproducibility of herbal ingredients by macroscopic & microscopic characters and comparison with authentic herb. 2.Inter/Intra species variation in plants. 3.Environmental factors. 4.Plant part used. 5.Time of harvesting. 6.Post harvesting factors 7.Contaminants of herbal ingredients. 8.Pesticides, fumigants and other toxic materials

Sampling procedures 1.Assessment of the quality of herbal drugs is directly dependent on the selection of sample for examination, the sample must be truly representative of material undergoing analysis. 2.Because herbal materials are an aggregate of individual plants and/or different parts of the same plant and thus have an element of heterogeneity, sampling should be carried out with special care by personnel with the necessary expertise. 3.Number of sample according to number of package in shipment.

4. Sampling material in bulk:  Inspection each container for conformity with pharmacopoeia monographs or other requirements regarding packaging and labeling  Check integrity of outer package and note any defect  Damaged container sample d separately

 After opening container inspect the follwing: 1.Organoleptic character (color, texture & odor) 2.Presentation of material 3.Presence of admixtures, forign matter, moulds or sign of decay 4.Presence of insects 5.Presence of packaging material

Morphological Examinations 1.Organoleptic evaluation 2.Macromorphological Evaluation 3.Cytomorpholgical evaluation

1. Organoleptic Evaluation Evaluation of drug by color, odor, size, shape, taste, and special features including surface characteristic (touch, texture,etc). Since the majority of information on the identity, purity and quality of material can be drawn from these observations, they are of primary importance before any further testing can be carried out

2. Macromorphological Evaluation Morphology is the study of the form of an object, whilst morphography is the description of that form Interpretations of the morphological characteristics based on different parameters, for all the plant organs give us a first hand tool to know the features of whole or powdered drugs and adulteration of commercial significance. It is most useful for only a part of plant to be used either because the active constituent is found in a particular part or because of economic consideration. e.g. anthraquinone in bark of cascara sagrada so there is little point in collecting material other than bark

3. Cytomorphological Evaluation Examination of the cell form and arrangement of different cells in a drug Cytomorphological characters play a major role in drug identification The plant drugs contain some basic cell types e.g. Parenchyma, collenchymas, sclarenchema …..etc along with some cell inclusion characteristics i.e. the presence of ergastic substances like starch, calcium oxalate, silica ….etc. Analysis of the plant drugs based on the distribution of these various cell types within different organs is important to ensure the identity and quality of herbal drugs.

Microscopical Evaluation Microscopical technique provide detailed information about the crude drugs by virtue of its main analytical uses. Used to visualize fine structure of minute objects and thereby confirm the structural details of the plant drugs under evaluation. These techniques can be used in the determination of the optical as well as micro- chemical properties of the crude drug specimens under study.

Microscopical inspection of crude drugs from plant origin is essential for identification of the grounded on powdered material. It can provide supporting evidence which in combination with other analytical parameters can used to obtain full evidence for standardization and evaluation of herbal drug

Instruments for microscopical study 1.Camera-Lucida 2.Photomicrography 3.Modified light microscope 4.Polarizing microscopy 5.Phase contrast microscopy 6.Electron microscopy 7.Ultraviolet microscopy

Microscopical Methods 1.Microchemical testing of herbal drug: use different chemical reagents, known as cleaning reagents or bleaching agent, e.g. phlorglucinol, Iodine water 2.Micro-chemical precipitation: when amount available material is strictly limited, with a view to obtain precipitate of characteristically formed microscope crystals, used for alkaloid. 3.Microsublimation: applied to crude drugs that contain any volatile crystalline principle, crude drug under study is kept in small thin glass tube and sealed, the tube is incorporate in a method of heating sample is sublimed

Development of Standardization parameters Out of the numerous practical applications of pharmacognosy, the great importance for the pharmaceutical industry is in the evaluation of the crude drug. This involve the determination of identity, purity and quality. Purity depends upon the absence of foreign matter whether organic or inorganic. Quality refers essentially to the concentration of the active constituents in the drugs that make it valuable to medicine. By virtue of these constituents or components, the product is used and it’s economic and commercial value is essential.

Based on concentration and nature of the constituents, a crude drug may conform to all official standards of purity and be of good quality. Naturally occurring inorganic or contaminants, beside other contaminants affect purity of crude drug which need proper assessment and detection of different parameters indicate their acceptability by criteria determined.

Standardization involves adjusting the herbal drug preparation to a defined content of a constituent or a group of substances with known therapeutic activity by adding excipients or by mixing herbal drugs or herbal drug preparations. Botanical extracts made directly from crude plant material show substantial variation in composition, quality, and therapeutic effects.

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.

No regulatory definition exists for standardization of dietary supplements. As a result, the term “standardization” may mean many different things. Some manufacturers use the term standardization incorrectly to refer to uniform manufacturing practices; following a recipe is not sufficient for a product to be called standardized. Therefore, the presence of the word “standardized” on a supplement label does not necessarily indicate product quality. When the active principles are unknown, marker substance(s) should be established for analytical purposes and standardization.

Marker substances are chemically defined constituents of a herbal drug that are important for the quality of the finished product. Ideally, the chemical markers chosen would also be the compounds that are responsible for the botanical’s effects in the body.

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. Ex. 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.

Standardization of Herbal drug Determination of solvent extraction values Determination of Ash values Determination of Total solids Determination of Crude fiber Determination of Moisture content Determination of Essential oil in crude drug Microbial infestations and their determination Determination of Bitterness value Determination of Hemolytic activity Determination of Swelling index Determination of Foaming index Determination of Tannins Determination of Arsenic & Heavy metals Determination of Pesticides Radioactive contamination

1. Determination of solvent extraction values Determine the amount of active constituents in a given amount of medicinal plant material when extracted with solvent. It is employed for that material for which no chemical or biological assay method exist. As mentioned in different official books (BP). The determination of water soluble and alcohol soluble extractives, is used as a mean of evaluating crude drugs which are not readily estimated by other means.

Method used 1.Determination of water soluble extraction. 2.Determination of alcohol soluble extraction 3.Solvent Hexane soluble extraction 4.Volatile ether soluble extraction 5.Nonvolatile ether soluble extraction

2. Determination of Ash values The ash of any material is composed of their non-volatile inorganic components. Controlled incineration of crude drugs result in an ash residue consisting of an inorganic material (metallic salt and silica). The value varies within fairly wide limits and therefore important parameter for the purpose of evaluation of crude drugs.

In certain drug, the percentage variation of the weight of ash from sample to sample is very small and any marked difference indicates a change in quality. Unwanted parts of drugs, sometimes posses a character that will raise the ash value. Direct contamination, such as by sand or earth, is immediately detected by the ash value. Determined by 3 method: Total Ash, Acid insoluble Ash, Water soluble ash

3. Determination of total solids Total solid is the residue obtained when the prescribed amount of the preparation is dried to constant weight under the condition specified in method.

4. Determination of crude fiber Estimation of crude fiber denotes the measurement of the content of cellulose, lignin and cork cell in the plant tissue. Material is de-fatted and boiled with dilute acid to eliminate the soluble material, dried and weighed, excess of the crude fiber indirect adulteration with woody tissue like kernels.

5. Determination of moisture content Moisture is an inevitable component of crude drugs, which must be eliminated as far as practicable. The drying process should reduce the moisture content of the drug below this critical, or threshold level. It is difficult to state a precise upper limit of moisture that can be permitted in crude drug. USP and NF make no commitment in this regard in most cases. Not only is the ultimate dryness of the drug is important, equally important is the rate at which the moisture is removed and the conditions under which it is removed. Method of determination: Loss on drying, Azeotropic distillation, Karl Fischer

Azeotropic volumetric method Measurement of water or other volatile constituents present in crude drug. Sample is distilled together with immiscible solvent such as toluene, xylene. Water present in the sample is absorbed by the solvent. Water and solvent is distilled together and separated in the receiving tube on cooling. It is based on the fact that water and benzene, toluene will form azeotropic mixture.

Apparatus used to determine water content by the azeotropic method (dimensions in mm)

7. Determination essential oil in crude drug Volatile oils are characterized by their odor, oil-like appearance and ability to volatilize at room temperature. Because they are considered to be the "essence" of the plant material, and are often biologically active, they are also known as "essential oils". The term "volatile oil" is preferred because it is more specific and describes the physical properties. Only 2000 species are characterized by the volatile oil. e.g. citrus family, parsley family and mints. Volatile oil comprise a diverse group of compounds, embodying principally terpene, sesquiterpene and diterpene.

The terpene skeleton structure is subjected to many modification, alcohol,aldhyde,ketone,phenol,ether,lactone and ester structures are all found among different volatile oils. Most volatile oil are isoprenoid compounds formed through fusion of isoprene units. in order to determine the volume of oil, the plant material is distilled with water and distillate is collected in graduated tube, the aqueous portion separates automatically and returned to distillation flask, volatile oil will float on top of aqueous phase. Quality of oil determined by other analytical procedures.

Apparatus used to determine volatile oils (dimensions in mm)

8. Microbial infestation and its determination Medicinal plant materials normally carry a great number of bacteria and moulds, often originating in soil and environment. While a large range of bacteria and fungi form the naturally occurring microflora of herbs, aerobic spore-forming bacteria frequently predominate. Current practices of harvesting, handling and production may cause additional contamination and microbial growth. The Determination of Escherichia coli and moulds may indicate the quality of production and harvesting practices.

Methods for decontamination are restricted. For example, the use of ethylene oxide has been forbidden within countries of the European Union. Treatment with ionizing irradiation is also forbidden or requires a special registration procedure in some countries. In addition, the presence of aflatoxins in plant material can be hazardous to health if absorbed even in very small amounts. They should therefore be determined after using a suitable clean-up procedure.

Test for specific microorganisms The conditions of the test for microbial contamination are designed to minimize accidental contamination of the material being examined; the precautions taken must not adversely affect any microorganisms that could be revealed. Main contaminate are E.coli, Salmonella, Pseudomonas aeruginosa, staphylococcus tested by different subcultures method The total viable aerobic count of the material being examined is determined, as specified in the test procedure, for the plant material concerned using one of the following methods: membrane-filtration, plate count or serial dilution. The test procedure have to be validated on the microbial strain as specified by WHO.

8. Microbial infestation and its determination Medicinal plant materials normally carry a great number of bacteria and moulds, often originating in soil and environment. While a large range of bacteria and fungi form the naturally occurring microflora of herbs, aerobic spore-forming bacteria frequently predominate. Current practices of harvesting, handling and production may cause additional contamination and microbial growth. The Determination of Escherichia coli and moulds may indicate the quality of production and harvesting practices.

Methods for decontamination are restricted. For example, the use of ethylene oxide has been forbidden within countries of the European Union. Treatment with ionizing irradiation is also forbidden or requires a special registration procedure in some countries. In addition, the presence of aflatoxins in plant material can be hazardous to health if absorbed even in very small amounts. They should therefore be determined after using a suitable clean-up procedure.

Test for specific microorganisms The conditions of the test for microbial contamination are designed to minimize accidental contamination of the material being examined; the precautions taken must not adversely affect any microorganisms that could be revealed. Main contaminate are E.coli, Salmonella, Pseudomonas aeruginosa, staphylococcus tested by different subcultures method The total viable aerobic count of the material being examined is determined, as specified in the test procedure, for the plant material concerned using one of the following methods: membrane-filtration, plate count or serial dilution. The test procedure have to be validated on the microbial strain as specified by WHO.

Limit specified by WHO for the microbial contamination in Herbal drugs The WHO has specified total microbial contamination limits for the medicinal plant materials. Different limits are set according to the use of the material and the material itself. For contamination of "crude" plant material intended for further processing (including additional decontamination by a physical or chemical process) the limits, adapted from the provisional guidelines established by an international consultative group, are given for untreated plant material harvested under acceptable hygienic conditions: - Escherichia coli, maximum 104 per gram; - mould propagules, maximum 105 per gram.

For plant materials that have been pretreated (e.g. with boiling water as used for herbal teas and infusions) or that are used as topical dosage forms: - aerobic bacteria, maximum 107 per gram; - Yeasts and moulds, maximum 104 per gram; - Escherichia coli, maximum 102 per gram; - other enterobacteria, maximum 104 per gram; - salmonellae, none. For other plant materials for internal use: - aerobic bacteria, maximum 105 per gram; - yeasts and moulds, maximum 103 per gram; - Escherichia coli, maximum 10 per gram; - other enterobacteria, maximum 103, per gram; - salmonellae, none.

Presence and detection of Aflatoxin Aflatoxin is a toxin from Aspergillus flavus and Aspergillus parasiticus having the chemical formula C17H12O6, which may cause hepatic carcinoma in human beings. The plant species may be contaminated with this toxin. The test for aflatoxin as prescribed by WHO for the herbal drugs is designed to detect the possible presence of B1,B2,G1&G2 which are dangerous contaminants in any plant material of plant origin.

Detection of aflatoxin by TLC method