Evaluation of Crude Drugs

Chapter 11

Evaluation of Crude Drugs

INTRODUCTION

• Evaluation of a drug ensures the identity of a drug and determines the quality and purity of drugs. The main reasons behind the need for evaluation of crude drugs are biochemical variation in the drug, effect of treatment and storage of drugs, and the adulterations and substitutions.

• Improvements in analytical methods have definitely led to improvements in harvesting schedules, cultivation techniques, storage, activity, stability of active compounds, and product purity. All of these gains have resulted in tremendous improvements in the quality of herbal preparations now available.

• Methods currently employed in evaluating herbs are organoleptic, microscopic, physical, chemical, and biological parameters.

ORGANOLEPTIC EVALUATION

• Organoleptic evaluation means the study of drugs using organs of senses. It refers to the methods of analysis like colour, odour, taste, size, shape, and special features, such as: touch, texture, etc. Obviously, the initial sight of the plant or extract is so specific that it tends to identify itself. If this is not enough, perhaps the plant or extract has a characteristic odour or taste. Organoleptic analysis represents the simplest, yet the most human form of analysis.

• Talka gum, which is used as a substitute for acacia gum could be identified by its colour and form. Talka gum is usually broken and also some tears are brown in colour and other colourless, whereas acacia is white to yellow in colour. Mangosteen fruits are a substitute for bael fruits and can be identified by darker rind and the wedge-shaped radiate stigmas. Cuprea Bark (Remijia pedupiculata) differs in its morphological character with cinchona. Blood Root used as an adulterant for hydrastis is dark reddish-brown in colour, whereas hydrastis is yellow in colour. Rheum rhaponticum are much smaller than those of the Chinese rhubarb and are easily distinguished.

• Ginger and capsicum have pungent taste, whereas gentian and chirata have bitter taste. Morphological differentiation of leaves and pods of Indian senna and Alexandrian senna, sweet taste of liquorice, odours of umbelliferous fruits, disc-shaped structure of nux vomica, conical shape of aconite, quills of cinnamon, etc. are few examples of this organoleptic evaluation.

MICROSCOPICAL EVALUATION

• Microscopic evaluation is indispensable in the initial identification of herbs, as well as in identifying small fragments of crude or powdered herbs, and in the detection of adulternts (e.g. insects, animal faeces, mold, fungi, etc.) as well as identifying the plant by characteristic tissue features. Every plant possesses a characteristic tissue structure, which can be demonstrated through study of tissue arrangement, cell walls, and configuration when properly mounted in stains, reagents, and media. Lignin stains red or pink with a drop of phloroglucinol and concentrated hydrochloric acid. Mucilage is stained pink with rhuthenium red, and N/50 iodine solution stains starch and hemicellulose blue.

• Stomatal index: It is the percentage which the numbers of stomata form to the total number of epidermal cells, each stoma being counted as one cell. Stomatal index can be calculated by using the following formula:

• Stomatal Index (S.I.) = S × 100 E + S

• where, S = number of stomata per unit area and E = number of epidermal cells in the same unit area.

• Timmerman (1927) and Rowson (1943) were amongst the first few to investigate leaf drugs for stomatal number and stomatal index.

• Vein-islet number: It is defined as the number of vein islets per sq. mm of the leaf surface midway between the midrib and the margin. It is a constant for a given species of the plant and is used as a characteristic for the identification of the allied species. Levin in 1929 determined vein-islet numbers of several dicot leaves.

• Veinlet termination number: It is defined as the number of veinlet termination per sq. mm of the leaf surface midway between midrib and margin. A vein termination is the ultimate free termination of veinlet. Hall and Melville in 1951 determined veinlet termination number of distinguishing between Indian and Alexandrian Senna.

• Palisade ratio: It is defined as the average number of palisade cells beneath each epidermal cell. Unlike veinislet number for the determination of which an unbroken portion of the leaf is required, palisade ratio can be determined with the powdered drug. The technique of palisade ratio determination was introduced by Zorning and Weiss (1925) in their studies on Compositae. 

• One example is vein-islet number of Alexandrian senna is 25–29.5, whereas Indian senna is 19.5–22.5. Stomatal index of Alexandrian senna is 10–15, whereas that of Indian Senna is 14–20.

Quantitative Microscopy (Lycopodium Spore Method)

• This is an important technique employed in identification of crude drug when chemical and physical methods are inapplicable. Using this, one can determine the proportions of the substances present by means of the microscope, using the Lycopodium spore method.

• Thus, if spent ginger is the adulterant, one knows that ginger contains 286,000 starch grains per mg, and the amount used as an adulterant can be calculated by using this figure. The percentage purity of an authentic powdered ginger is calculated using the following equation:

CHEMICAL EVALUATION

• The chemical evaluation includes qualitative chemical tests, quantitative chemical tests, chemical assays, and instrumental analysis. The isolation, purification, and identification of active constituents are chemical methods of evaluation. Qualitative chemical tests include identification tests for various phytoconstituents like alkaloids, glycosides, tannins, etc. The procedures for the identification tests of various phytoconstituents are given under their respective chapters in the text, where it could be referred. Examples of identification of constituents are: copper acetate used in the detection of colophony present as an adulterant for resins, balsams, and waxes; Holphen’s test for cottonseed oil and Baudouin’s test for sesame oil in olive oil; the test with acetic and nitric acids for Gurjun balsam in copaiba; Van Urk’s reagent for ergot; Vitali’s morins reaction for tropane alkaloids; iodine for starch; murexide test for purine bases, etc. are examples of this evaluation.

• Instrumental analyses are used to analyse the chemical groups of phytoconstituents using chromatographic and spectroscopic methods. Chromatographic methods include paper chromatography, thin-layer chromatography, gas chromatography, high-performance liquid chromatography, and high-performance thin-layer chromatography. Spectroscopic methods include ultraviolet and visible spectroscopy, infrared spectroscopy, mass spectroscopy, and nuclear magnetic spectroscopy.

PHYSICAL EVALUATION

• In crude plant evaluation, physical methods are often used to determine the solubility, specific gravity, optical rotation, viscosity, refractive index, melting point, water content, degree of fibre elasticity, and other physical characteristics of the herb material.

Solubility

• Drugs specific behaviours towards solvents are taken into consideration. This is useful for the examination of many oils, oleoresins, etc. Few examples are the solubility of colophony in light petroleum, the solubility of balsam of Peru in solution of chloral hydrate, the solubility of castor oil in half its volume of light petroleum and the turbidity produced with two volumes of the solvent; the solubility of balsam of Peru in an equal volume of alcohol, 90%, and the production of a turbidity with a larger volume; castor oil is soluble only in three volumes of 90% alcohol, while the adulterated form it shows good solubility in alcohol. Alkaloidal bases are soluble in organic solvents and alkaloidal salts are soluble in polar solvents.

Optical Rotation

• Anisotropic crystalline solids and samples containing an excess of one enantiomer of a chiral molecule can rotate the orientation of plane-polarized light. Such substances are said to be optically active, and this property is known as optical rotation. The enantiomer that rotates light to the right, or clockwise when viewing in the direction of light propagation, is called the dextrorotatory (d) or (+) enantiomer, and the enantiomer that rotates light to the left, or counterclockwise, is called the levorotatory (l) or () enantiomer. Few examples of drugs with this property are eucalyptus oil (0° to +10°), honey (+3° to 15°), Chenopodium oil (30° to 80°), etc.

Refractive Index

• Refractive index is defined as the property of a material that changes the speed of light, computed as the ratio of the speed of light in a vacuum to the speed of light through the material. When light travels at an angle between two different materials, their refractive indices determine the angle of transmission refraction of the light beam. In general, the refractive index varies based on the frequency of the light as well; thus, different colours of light travel at different speeds. High intensities can also change the refractive index. This could be used as a parameter in evaluating the herbal drugs; for example castor oil 1.4758 to 1.527, clove oil 1.527 to 1.535, etc.

Specific Gravity

• It is also known as relative density. The ratio of the mass of a solid or liquid to the mass of an equal volume of distilled water at 4°C (39°F) or of a gas to an equal volume of air or hydrogen under prescribed conditions of temperature and pressure. Some examples of specific gravity of drugs are cottonseed oil 0.88–0.93, coconut oil 0.925, castor oil 0.95, etc.

Viscosity

• Viscosity is the resistance of a fluid to flow. This resistance acts against the motion of any solid object through the fluid and also against motion of the fluid itself past stationary obstacles. Viscosity of a liquid is constant at a given temperature and is an index of its composition. Viscosity also acts internally on the fluid between slower- and faster-moving adjacent layers. Since it is constant at a given temperature, it is used as an evaluation parameter; for example, pyroxylin kinematic viscosity, 1100–2450 centistokes.

Melting Point

• The melting point of a solid is the temperature at which it changes state from solid to liquid. Plant constituents have very sharp and constant melting points. As far as crude drugs are concerned, melting point range has been fixed due to mixed chemicals. The following drugs could be evaluated using this parameter; for example, beeswax 62–65°C, wool fat 34–44°C, agar melts at 85°C, etc.

Moisture Content

• The moisture content of a drug will be responsible for decomposition of crude drugs either producing chemical change or microbial growth. So the moisture content of a drug should be determined and controlled. The moisture content is determined by heating a drug at 105°C in an oven to a constant weight. Following are the examples of two crude drugs with their moisture content limit: the moisture content of Digitalis and Ergot should not be more than 5% w/w and 8% w/w, respectively.

Ultraviolet Light

• Certain drugs fluoresce when the cut surface or the powder is exposed to ultraviolet radiation, and it is useful in the identification of those drugs. Some pieces of rhapontic, Indian, and Chinese rhubarb are very difficult to distinguish, and it is very difficult in powdered form, but examination in ultraviolet light gives such marked differences in fluorescence that the varieties can be easily distinguished from each other.

Ash Values

• The determination of ash is useful for detecting low-grade products, exhausted drugs, and excess of sandy or earthy matter. Different types of ash values are used in detection of crude drugs like, total ash, acid-insoluble ash, watersoluble ash, and sulphated ash.

• Total ash is useful in detecting the crude drugs that are mixed with various mineral substances like sand, soil, calcium oxalate, chalk powder, or other drugs with different inorganic contents to improve their appearance, as is done with nutmegs and ginger. The maximum temperature used for total ash should be not more than 450°C because alkali chlorides that may be volatile in higher temperatures would be lost.

• The water-soluble ash is used to detect the presence of material exhausted by water. Sulphated ash is done by addition of sulphuric acid in order to get sulphate salts, and the percentage ash is calculated with reference to the air-dried drug. The temperature used for this is above 600°C. The total ash and acid-insoluble ash values of Guduchi are not more than 16 and 3%, respectively. The total ash value and water-soluble ash values of ginger are 6 and 1.7%, respectively.

Extractive Values

• The extracts obtained by exhausting crude drugs with different solvents are approximate measures of their chemical constituents. Various solvents are used according to the type of the constituents to be analysed. Water-soluble extractive is used for crude drugs containing water-soluble constituents like glycosides, tannins, mucilage, etc.; alcohol-soluble extractive is used for crude drugs containing tannins, glycosides, resins, etc.; and ether-soluble extractives are used for drugs containing volatile constituents and fats.

Extractive Values of Some Crude Drugs

Foreign Organic Matters

• The parts of the organ or organs other than those parts of drugs mentioned in the definition and description of the drug are known as foreign organic matters. They may be insect, moulds, earthy material, animal excreta, etc. Each and every vegetable drug has their own limits. Few examples of such limits are: garlic should not contain more than 2%, saffron should not contain more than 2%, satavari should not contain more than 1%, etc.

BIOLOGICAL EVALUATION

• The plant or extract can then be evaluated by various biological methods to determine pharmacological activity, potency, and toxicity. The biological evaluation would serve better than the physical and chemical evaluation for drugs that could not be satisfactorily assayed by these last two methods. Moreover, this is an important method, the crude drugs are considered important only because of their biological effects and this evaluation would conclude the effect. These methods are considered to be less precise, more time-consuming and more expensive. Bioassays should be as simple as possible, and attempts should be made to have access to a large number of different tests so that many biological properties can be screened. The bioassay methods are of three types they are, toxic, symptomatic and tissue or organ methods. Different animals are used in toxic and symptomatic method and isolated organ or tissue is used in the third method.

• Oxytocic activity of vasopressin injection is tested on guinea pigs, and oxytocic injection is assayed on young domestic chickens by injecting into an exposed crural or brachial vein and noticing the changes in blood pressure. Pigeons are used to assay Digitalis glycosides by transfusing the drug through the alar vein to the blood stream and observing the lethal effects. Depressor activities and mydriatic effects of certain drugs are tested in cats and cat’s eye, respectively. Anthelmintic drugs are evaluated on worms.

• The drugs that have an effect in eyes are assayed on rabbit’s eyes. Dogs are used to assay the drugs that exhibit cardiac and gastrointestinal activities. Effects of Ergot are carried out on cock’s comb or rabbit’s intestine or its uterus. Next to the animals, the studies are carried out in human beings also. In some instances, the effects that are observed from animal studies would be different when tested in humans. The tested biological activities include hepatoprotective activity, hypoglycaemic activity, antiinflammatory activity, antiulcer activity, immunomodulatory activity, etc.

• Microbiological assays are carried out to determine the effects of drug in various microorganisms, and this is employed in the identification of antimicrobial drugs. The methods used in this type of assays are agar well-diffusion method, disc-diffusion method, and turbidimetric method. In other microbiological methods, the living bacteria yeast moulds are used for assaying vitamins.