And that the safety and efficacy of plant drugs depend on the use of proper plant part and its biological potency, which in turn depends upon the presence and nature of required active compounds/secondary metabolites, the use of plant drugs demands correct identification and characterization (Vinoth et al., 2011; Zafar et al., 2005). Evaluation of plant drugs is the correct identification of plants, determination of their quality and purity, and detection of adulterants. Evaluation of crude drug is necessary because of three main reasons: biochemical variations in the drug, deterioration due to treatment and storage, substitution and adulteration as a result of carelessness, ignorance or fraud (Ali, 2008; Kokate et al., 2012). WHO acknowledged that pharmacognostical standards should be proposed as a protocol for the diagnosis of herbal drugs, and authenticated raw material is the basic starting point in developing a botanical product (Nasreen et al., 2010; Smillie and Khan, 2010). Pharmacognosy has played a pivotal role in the discovery and development of new drugs and therapies, and has been continuing to do so even today. The word Pharmacognosy is derived from two Greek words, ‘Pharmakon’ meaning ‘drug’ and ‘gnosis’ meaning ‘knowledge’. Therefore, Pharmacognosy literally means knowledge of drugs (Ajay et al., 2012; Sarker, 2012). It is a scientific study of the structural, physical, chemical and sensory characters of crude drugs of plant, animal and mineral origins. The crude drugs are that they exist as they occur naturally and are not compounded or mixed with other substances (Wallis, 1999).

Methods:

The guidelines set by WHO for authentication and quality evaluation of medicinal plants from the pharmacognostical study point of view are summarized below:

Collection:

The most convenient time of collection is the period during which the drug is highest in its contents of active principles. Roots and rhizomes are usually collected when their aerial growths or the vegetative processes have ceased, i.e., their tissues are fully stored with reserved foods (it being assumed that medicinal constituents will be most abundant at this time). The time for collection of bark is usually spring or early summer when the sap is rising in the stem and the cambium is active, as it is easy to detach them from the stem. If there is a rainy season, it is during that period that the bark is most easily collected. The usual time for the collection of leaves is when the flowers are just beginning to expand, or the flowering is just arriving at its height. At this time it is reasonable to assume that the whole plant has arrived at its condition of maximum vigour and that the leaves are in the healthiest state containing optimum products of the plant’s metabolism and, therefore, should be at this period of their development suited to exert the most desirable therapeutic action. Flowers should be collected prior to or just at the time of pollination under fine, dry weather, because petals which are damp when gathered become badly discoloured during drying. Fruits are collected either ripe or half-ripe, but full grown. Seeds should be collected when fully matured and if possible before the fruits have opened. A complete specimen possesses all parts of the plant including the root system. The unorganized drugs such as resin, gum, latex, etc. are collected as soon as they ooze out of the plant. Acacia gum is collected 2-3 weeks after making incision on the tree bark and when it is sufficiently hard; opium and Papaya lattices are collected after coagulation of latex, Turpentine oleoresin and balsam of peru are collected when the plant is about 8-10 years old, and so on (Kokate et al., 2012; Pandey, 1993; Wallis, 1999).

Identification and Classification:

The plant material collected at an appropriate stage of its growth is well authenticated by detailed taxonomical study and the correct botanical identity is established. The widely accepted Natural System of Classification of Bentham and Hooker (1862-1883) is usually followed for identification and classification of plants. The botanical identity – scientific name (genus, species, subspecies/variety, author, and family) – of each medicinal plant should be verified and recorded. If available, the local and English common names should also be recorded (Anonymous, 2003).

Organoleptic (Sensory) Analysis:

Organoleptic characters (i.e., impression on the organs) such as the evaluation of colour under normal light (vision), odour or smell (olfaction), taste (gustation), texture-rough or smooth (tactility/tactition), etc. are carried out by means of the organs of sense (Anonymous, 2011; Wallis, 1999).

Macroscopic Analysis:

Morphological or macroscopical study of plant parts used as drugs, e.g., stem, fruit, stem bark, root, etc. is done by observing them with the naked eyes or with the aid of a magnifying lens. In this description, size, shape, markings, the sound or ‘snap’ of their fracture, etc. are referred to (Anonymous, 2011; Wallis, 1999).

Microscopic Analysis:

The different parts of the drug plants are subjected to microscopic examination. Sections are normally cut by hand with the help of a sharp razor. But for more detailed and critical study, the use of microtome is desirable in which case the materials are to be killed, fixed in various fixatives and embedded in paraffin before cutting the sections. In case of leaves, surface preparation and transverse section, preferably through midrib, are made. For cylindrical plant parts like stem, root, rhizome, etc., sections are cut at right angles to the axis (transverse or cross section) or parallel to the axis (longitudinal section). The study also includes measurement of cells, tissues, etc. which are of diagnostic value for identification of the sample. The microscopic descriptions are supplemented with camera lucida drawings/ photomicrographs wherever necessary (Anonymous, 2011; Brain and Turner, 1975; Johansen, 1940; Trease and Evans, 2002).

Powdered-drug Analysis:

Analysis of powdered drug is, by and large, a microscopic investigation to provide better diagnostic characters for rapid and accurate identification. The drug sample is dried, grounded to powder in an electric grinder, treated with a clearing agent, mostly chloral hydrate solution and kept overnight. It is then mounted in chloral hydrate solution and glycerin solution (9:1). For better clearing action, the slide can be slightly heated before placing the cover slip on it. Thereafter, the investigation is done under the microscope with reference to the presence or absence of particular diagnostic characters such as starch grains, calcium oxalate crystals, fibres, etc.

Preliminary observations for the following characteristics of powdered drugs are also performed: (1) colour and taste, (2) the powder, mixed with a small quantity of water is warmed and the odour observed, (3) fineness (coarse/moderately coarse/moderately fine/fine/very fine) and degree of uniformity of the particles, (4) sensation of smoothness and grittiness when the powder is gently rubbed on the hand, (5) surface appearance by reflected light, shining particles such as hairs, fibres and calcium oxalate crystals, and (6) the powder is pressed between the folds of a piece of paper and look for any oily stain (Anonymous, 2011; Pharmacopoeia of India, 2001; Wallis, 1999).

Fluorescence Analysis (inside UV chamber):

The powdered drugs are treated with various reagents like ethyl alcohol, methanol, petroleum ether, etc. and their distinctive colours observed inside UV chamber at 254 nm and 366 nm (Chase and Pratt, 1948; Harborne, 1973; Khandelwal, 2005).

Physico-chemical Analysis:

The following physico-chemical analyses are carried out according to the procedures as given in the Indian Pharmacopoeia (2001), WHO Guidelines on Quality Control Methods for Herbal Materials (Anonymous, 2011) and Raman (2006):

(1) Determination of Foreign Matter:

The plant sample (500 gm in case of barks, roots, rhizomes and 200 gm in case of leaves, flowers, seeds, fruits) is spread out in a thin layer and macroscopic analysis done with the naked eyes or with the aid of a magnifying lens to remove foreign matter such as soil, stone, sand, mould, insect, etc. other than source plant. The removed foreign matter is weighed and the percentage present calculated.

(2) Determination of Moisture Content (Loss on Drying): About 4 gm of the powdered drug is dried to a constant weight by heating in an oven at 100-105⁰C for 5 hours, cooled in a dessicator and weighed. The loss of moisture content is expressed in percentage by the equation:

A-B

% of moisture = —— x 100

Where,

A = Weight of wet sample

B = Weight of dry sample

(3) Determination of Total Ash:

2gm of the powdered drug is spread in a crucible in an even layer, ignited/incinerated by gradually increasing the heat to 500-600ºC until it is white indicating the absence of carbon, cooled in a desiccator and weighed (If a carbon-free ash cannot be obtained in this manner, cool the crucible and moisten the residue with about 2ml of water or a saturated solution of ammonium nitrate reagent. Dry on a water bath, then on a hot plate and ignite to constant weight. Allow the residue to cool in a suitable desiccator for 30 minutes and weigh without delay). The percentage of total ash with reference to the air-dried material is calculated.

(4) Determination of Acid-Insoluble Ash:

To the crucible containing the total ash, 25ml of dilute HCL (1:5=acid:water) is added, covered with a watch glass and boiled gently for 5 minutes. Rinse the watch glass with 5 ml of hot water and add this liquid to the crucible. The insoluble matter/residue is then collected by pouring into a conical flask through an ashless filter paper (Whatman No. 41). The filter paper containing the insoluble matter is transferred to the original crucible, dried on a hot plate and ignited to constant weight. The residue is then allowed to cool in a desiccator for 30 minutes and thereafter weighed without delay. The percentage of acid-insoluble ash with reference to the air dried material is calculated.

(5) Determination of Water-Soluble Ash:

To the crucible containing the total ash is added 25ml of distilled water and boiled for 5 minutes. The insoluble matter is collected in an ashless filter paper, washed with hot water and ignited in a crucible for 15 minutes at a temperature not exceeding 450º. The weight of the insoluble matter is substracted from the weight of the total ash (the difference in weight represents the water-soluble ash). The percentage of water-soluble ash with reference to the air-dried material is calculated.

(6) Determination of Alcohol-Soluble Extractive:

5g of the air-dried material is weighed accurately and poured into a glass-stoppered conical flask. 100 ml of distilled alcohol (90%) added, shaken occasionally for 6 hours and then allowed to stand for 18 hours. Filtered (using filter paper) rapidly and transferred 25 ml of the filtrate to a beaker/tared flat-bottomed dish and evaporated to dryness on a hot plate. Cooled in a desiccator for 30 minutes and weighed without delay. The percentage of alcohol-soluble extractive with reference to the air-dried material is calculated.

(7) Determination of Water-Soluble Extractive: Performed as directed for the determination of alcohol-soluble extractive, using chloroform water instead of alcohol.

(8) Determination of Ether-Soluble Extractive:

Performed as directed for the determination of alcohol-soluble extractive, except that ether is used for maceration.

Quantitative Microscopy:

As a part of quantitative microscopy, stomatal index, palisade ratio, stomatal number and vein-islet number are determined following the methods advocated by Wallis (1999) and Indian Pharmacopoeia (2001) by taking fresh leaves of the plants.

(1) Stomatal Index:

The stomatal index, which is the percentage of the number of stomata formed by the total number of epidermal cells (including the stomata, each stoma being counted as one cell) is calculated as per the formula:

I = —— x 100

E+S

Where,

I = Stomatal Index

S = No.of stomata per sq. mm. area of leaf

E = No.of epidermal cells in the same area of leaf

In this method, leaf fragments of about 5×5 mm in size (cut from the central part of the lamina/interneural lamina, i.e., halfway between the midrib and the margin) are placed in a test tube containing about 5 ml of chloral hydrate solution and heated in a boiling water-bath for 15-30 minutes or until the fragments become transparent. A fragment is then transferred to a microscopic slide, prepared the mount in chloral hydrate solution and put a small drop of glycerol solution on one side of the cover glass to prevent the preparation from drying. It is then examined with a 40x objective and a 6x eyepiece, to which a microscopical drawing apparatus is attached. A cross (x) for each epidermal cell and a circle (o) for each stoma are marked on the drawing paper, and the numbers of epidermal cells and stomata in the field of vision counted.

(2) Palisade Ratio:

It is the average number of palisade cells beneath one upper epidermal cell, and is obtained by dividing the total number of palisade cells by the number of epidermal cells. Leaf fragments are prepared and examined under the microscope as mentioned above for stomatal index. Four adjacent epidermal cells and then the palisade cells lying beneath the epidermal cells are traced on paper. The number of palisade cells under the four epidermal cells (where a cell is intersected, include it in the count if more than half of it is within the area of the epidermal cells) is counted. The total number of palisade cells divided by four gives the palisade ratio.

(3) Stomatal Number:

It is the average number of stomata per square millimetre of epidermis. Leaf fragments are prepared and examined under the microscope as mentioned above for stomatal index. A cross (x) for each stoma is marked on the drawing paper and the average number of stomata per square millimetre is calculated.

(4) Vein-Islet Number:

Vein-islet is the minute area of photosynthetic tissue encircled by the ultimate branches of the conducting strands; and the number of vein-islets per square millimeter is termed the ‘Vein-Islet Number’. Leaf fragments are prepared as mentioned above for stomatal index by boiling for 30-60 minutes or until the fragments become transparent. A stage micrometer is placed on the microscope stage and examined with 4x objective and a 6x eye piece to draw a line representing 1 mm on a sheet of paper by means of a microscopical drawing apparatus, and the square of this 1 mm is taken. The stage micrometer is replaced by a slide containing the mounted sample, and the veins are traced through the camera lucida. The number of vein-islets within the square (including those overlapping on two adjacent sides and excluding those intersected by the other two sides) is counted.

Thin Layer Chromatography (TLC):

A straight line is drawn across a TLC plate/chromatoplate (referred to as the stationary phase) with pencil, approximately 1 cm from the bottom edge, and a small spot of solution containing the sample/analyte is applied to the centre of this line using a very thin glass pipette. The TLC plate is then placed carefully into a glass beaker or any other suitable separation chamber (containing solvent/elutant known as the mobile phase to a depth of less than 1 cm) so that the spot of the sample does not touch the surface of the elutant in the chamber. As the solvent moves up the plate by capillary action, it carries the sample up the plate (elutes the sample). After the separation is complete, the R_f values of individual compounds appearing as spots separated vertically are calculated by the formula, R_f = distance travelled by sample/distance travelled by solvent (Anonymous, 2011; Egon Stahl, 2005; Kokate, 1994).

DISCUSSION:

Plant materials and herbal remedies derived from them represent a substantial proportion of the global drug market, and in this respect internationally-recognized guidelines for their quality assessment are necessary. It is well known that the quantitative concentration of biologically active constituents varies with the stage of plant growth and development. This also applies to non-targeted toxic or poisonous indigenous plant ingredients. Medicinal plant materials should, therefore, be collected during the appropriate season or time period to ensure the best possible quality of both source materials and finished products. The best time for collection (quality peak season or time of day) should be determined according to the quality and quantity of biologically active constituents rather than the total vegetative yield of the targeted medicinal plant parts. According to WHO guidelines, medicinal plant materials are categorized according to sensory, macroscopic and microscopic characteristics. An examination to determine these characteristics is the first step towards establishing the identity and degree of purity of such materials, and should be carried out before any further tests are undertaken. Wherever possible, authentic specimens of the materials in question and samples of pharmacopoeia quality should be available to serve as a reference. The gross morphological characters of plant parts such as root, stem, leaf, etc. provide the best basis for identification and classification of plants, and also frequently reveal the presence of contaminants or deterioration in a sample. The microscopical or anatomical evaluation which is carried out with the help of microscope is also of fundamental importance as it gives a preliminary idea about the nature and disposition of cells, tissues and cell inclusions such as starch, crystals, fixed oils, etc., and thus helps understand where the compounds of interest are located. It is mostly used for qualitative evaluation of organized crude drugs in entire and powdered forms. Linear measurement of cells and tissues further provides better diagnostic characters for accurate identification. For instance, the diameter of starch grains will assist in distinguishing Cassia bark from Cinnamon, the length of stamota in leaves of Barosma betulina will exclude leaves of other Barosma species, the height of sclerenchymatous cells in the testa of Cinnamon will indicate the presence of certain inferior varieties, and so on. Quantitative microscopy is one the most important tools for the study of crude drugs, mainly for the standardization of leaf drugs and their powders. The numbers of vein-islet per unit area of leaf, stomatal index and palisade ratio of plant leaves remain constant. They can be used as a distinguishing characteristic to differentiate between different species of the same plant or different plants. In roots, the type and arrangement of the principal conducting cells of vascular tissues may be diagnostic of the species. The presence, type, and arrangement of fibers, sclereids and other tissues, and the presence and location of ergastic material may also be diagnostic features. Roots can morphologically be distinguished from rhizomes (underground stems) primarily by the absence of nodes and internodes which are present in rhizomes. In the case of stems, several external macroscopic features that may be diagnostic of the species include the attributes of the nodes, internodes, leaf scars, vascular bundle scars, lenticles, buds; position and arrangement of the leaves along the stem, and the presence of tendrils, spines, thorns or prickles. As in the roots, the type and arrangement of the principal conducting cells of vascular tissues; the presence, type, and arrangement of fibers, sclereids and other tissues; and the presence and location of ergastic material may also be diagnostic features. Several macroscopic features of leaves that may be diagnostic of the species include the attributes of the leaf blade, petiole, stipules and phyllotaxy. Microscopic diagnostic features of epidermal cells include the cuticle thickness and markings, the shape and arrangement of stomata and guard cells, the arrangement and size of subsidiary cells, stomatal number, stomatal index, etc. Additional features useful in the identification of leaf material also include type and arrangement of trichomes, mesophyll, vascular tissues and conducting cells of the vascular tissues; palisade mesophyll ratio; presence and appearance of accessory tissues such as parenchymatous or sclerenchymatous bundle sheaths, endodermis and transfusion tissue; presence, type, and arrangement of fibers, sclereids and other tissues; and presence, location and physical appearance of ergastic material. Flowers are the best diagnostic morphological features of any flowering plant and their diagnostic features include type of inflorescence; presence, number and appearance of the primary floral parts (sepals, petals, stamens and carpels); type of symmetry displayed by the floral parts; relative position of the ovaries in regards to other parts of the flower; number of ovules per ovary; type of placentation; physical appearance of pollen grains; presence of nectaries; presence of covering or glandular trichomes; and physical features of accessory structures such as the receptacle and bracts. For fruits, the identifying characteristics are the number of pistils found in the fruit, the number of carpels within each pistil, the number of seeds within each carpel, the placentation of the fruit, and whether the fruit is dehiscent, indehiscent or fleshy. Additional diagnostic features include the number of sutures in a dehiscent fruit, the seeds are fused to or free from the pericarp wall, physical features of the three layers of pericarp of fleshy fruits (epicarp, mesocarp and endocarp), and presence and physical appearance of accessory tissues such as the receptacle and bracts. The characteristics of the seeds within the fruit are also diagnostic features of the species. The macroscopic features of seeds used in identification include the shape and size of the seed, appearance of the seed-coat, position of hilum and micropyle, and presence of accessory structures of seed coat such as arils, caruncle or oil bodies. Physical features of the embryo such as its size, shape, position, the number and appearance of the cotyledons, etc. are also diagnostic of the species. The physico-chemical analysis is helpful in judging identity and purity even from the crushed or powdered plant material, and fluorescence analysis under various reagents help in fulfilling the inadequacy of physical and chemical methods for identification. Every herb has a characteristic mineral content and corresponding typical ash content of the drug. So, when vegetable drugs are incinerated, they leave an inorganic ash which in the case of many drugs varies within fairly wide limits, and these values are of significance for the purpose of plant drug evaluation. This includes both ‘physiological ash’, which is derived from the plant tissue itself, and ‘non- physiological ash’, which is the residue of the extraneous matter (e.g. sand and soil) adhering to the plant surface. Hence, the quantitative test of total ash helps in determining both physiological and non-physiological ash. The total ash value is also useful to exclude drugs which have been coated with chalk, lime or calcium sulphate to improve their appearance, as is done with nutmegs and ginger. Acid-insoluble ash is the residue obtained after boiling the total ash with dilute HCL, and igniting the remaining insoluble matter. This measures the amount of silica present, especially as sand and siliceous earth. Water-insoluble ash is the difference in weight between the total ash and the residue after treatment of the total ash with water. The determination of extractive matter with a solvent reveals the amount of active constituents present in a given amount of medicinal plant material. Such extractive values are indications of the extent of polar, medium polar and non-polar components present in the drug. The method of water-soluble extractive is applied to drugs which contain water-soluble active constituents of crude drugs such as tannins, sugars, plant acids, mucilage, glycosides, etc.; alcohol-soluble extractive is ideal for the extraction of various alcohol-soluble constituents such as tannins, resins and alkaloids; ether-soluble extractive is applied for the extraction of volatile oils, fixed oils and resins. Checking moisture content helps reduce error in the estimation of actual weight of drug material. Low moisture suggests better stability against degradation of product. Limits for water content should be set for every given plant material. This is especially important for materials that absorb moisture easily or deteriorate quickly in the presence of water. Determination of foreign matter enables to get the drug in pure form. Herbal drugs should be made from the stated part of the plant and be devoid of other parts of the same plant or other plants. They should be entirely free from mould or insect, including excreta and visible contaminant such as sand and stones, poisonous and harmful foreign matter, chemical residues, etc. Macroscopic examination can easily be employed to determine the presence of foreign matter, although microscopy is indispensable in certain special cases (for example, starch deliberately added to dilute the plant material). TLC is the common fingerprint method for herbal analysis because of its simplicity, rapidity and economy. It is widely adopted for the rapid and positive analysis of plant drugs and, therefore, Pharmacopoeias are using TLC as a means for assessing quality and purity of herbal drugs. It provides semi-quantitative information about the chief constituents of the plant drug and, thus, enables easy assessment of the drug quality. Rf value is characteristic and the less polar compounds move higher up the plates resulting in higher R_f values. So identification of individual compounds appearing as spots can be done by comparing with R_f standard references. And when foreign matter of plant drugs consists, for example, of a chemical residue, TLC is often needed to detect the contaminants. Furthermore, TLC provides drug fingerprint. Thus, the importance of Pharmacognosy in research on plant-based medicines cannot be overemphasized.

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Received on 26.02.2013

Modified on 02.03.2013

Accepted on 04.03.2013

Research Journal of Pharmacognosy and Phytochemistry. 5(2): March-April 2013, 77-83