27 CHAPTER 2 2.1 Introduction Pharmacognosy as a subject of pharmaceutical curriculum focused on those natural products employed in the allopathic system of medicine. Coincident with the increasing attractiveness of alternative therapies and the tremendous range of herbal products now available to the public, regulatory requirements covering therapeutic plant are introduced by many countries in order to control the quality of these products. Monograph are now available giving description, test for identity and purity and assay of the active constituents. Pharmacognosy being concerned with the description and identification and it History, Commerce, Collection, Preparation and Storage. It is still of fundamental important particularly for pharmacopoeial identification and quality control purpose. Pharmacognosy is the study of medicine derived from natural source. It is the study of the physical, chemical, biochemical and biological properties of drug substances of natural origin as well as the search of new drug from natural origin. Pharmacognosy is interdisciplinary include ethanobotany, ethanopharmcology, phytochemistry and phytotherapy. The pharmacognostical studies of plant drugs involves the sources of drug, the morphological character, histological characters, chemical constituents and their qualitative test various physicochemical test and pharmacological action of the drug or the active constituents. It also includes the commercial varieties, substitutes, adulterants and any other quality control of the drugs.
28 To ensure reproducible quality of herbal plant, authentication is essential. The pharmacognostical study not only gives the authentication but also quality, purity and standard of the plant drug. According to WHO [38] the macroscopical and microscopical description of a medicinal plant is the first step towards establishing the identity and the degree of purity of such material. The pharmacognostical parameters are major reliable and inexpensive criteria for conformation of the crude drugs. The present work therefore attempts to report various necessary pharmacognostical standards of bark and leaf of the plant Thespesia populnea.
29 2.2 Material and methods Collection of plant material The plant Thespesia populnea soland ex.correa belonging to family Malvaceae are widely distributed throughout tropical regions and coastal forest in India. The bark and leaf of Thespesia populnea soland ex.correa were collected from Selaiyur university campus in Chennai, Tamil Nadu in the month of January 2007. The species for the proposed study was identified and authenticated by Director, Plant Anatomy Research Centre, Chennai. A voucher specimen (PARC/236/07) has been deposited in the herbarium of the same department. The shade dried bark and leaves were cut it into small pieces and made into coarsely powdered using mechanical grinder and preserved in air tight container. The fresh bark and leaf were used for macroscopical and microscopical study. The fresh leaf is utilized for quantitative microscopy. The powders of the plant material were used for physicochemical determination.
30 2.3 Macroscopy The fresh barks of Thespesia populnea were used for macroscopical study. The size, shape, color, taste, odour, and the fracture surface of bark were observed. The freshly leaves of the plant were collected and investigated in different organoleptic features by repeated observations. Morphological studies, such as shape, size, apex, surface, base, margin, venation, taste and odour of leaves, were carried out. 2.4 Microscopy In microscopical study the following procedure had been adopted. Preparation of histological specimen: The bark and leaf were cut into required size and fixed in FAA (Formalin 5 ml + Acetic acid 5 ml + 70% Ethanol 90 ml). After 24 hr of fixing, the specimens were dehydrated with graded series of tertiary butyl alcohol. Infiltration of the specimens was carried by gradual addition of paraffin wax (melting point 58-60 0 C) until tertiary butyl alcohol solution attained super saturation. The specimens were cased into paraffin blocks. Sectioning [39,40,41] The paraffin embedded specimen was sectioned with the help of rotary microtome. The thickness of the section was 10-12 µm. After dewaxing the sections were stained with toluidine blue. Since toluidine blue is a polychromatic stain, the staining results were remarkably good and some phytochemical reactions were obtained. The dye rendered pink color to the cellulose walls, blue to the lignified cells, dark green to suberin, violet to the mucilage, blue to the protein
31 bodies etc., wherever necessary sections also stained with safranin and fast green and iodine (For starch). Photomicrographs [42,43,44,45] Microscopic descriptions of tissues are supplemented with micrographs wherever necessary. Photographs of different magnifications were taken with Nikon Labphot-2 microscope units. For normal observations bright fields was used. For the study of crystals, starch grains and lignified cells polarized light were employed. Since these structures have birefringent property, under polarized light they appear bright against dark back ground. The prepared sections were observed through the microscope and distribution of various types of tissues was noted.
32 2.5 Quantitative microscopy The important identifying characteristic of leaf constants like Stomatal Number, Stomatal Index, Vein-islet number, Vein termination number were found out and tabulated (Table No:8). Stomatal number [38,39] leaf. It is the average number of stomata per square mm of the epidermis of the Procedure: Clear the piece of the leaf (middle part) by boiling with chloral hydrate solution or alternatively with chlorinated soda. Peel out upper and lower epidermis separately by means of forceps. Keep it on slide and mount in glycerin water. Arrange a camera lucida and drawing board for making the drawings to scale. Draw a square of 1mm by means of stage micrometer. Place the slide with cleared leaf (epidermis) on the stage. Trace the epidermis cell and stomata. Count the number of stomata present in the area of 1 sq. mm. Include the cell if at least half of its area lies within the square. Record the result for each of the ten fields and calculate the average number of stomata per sq. mm.
33 Stomatal index [38,39] Stomatal index is the percentage which the number of stomata forms to the total number of epidermal cells, each stomata being counted as one cell. Stomatal index can be calculated by using following equation. S I = 100 E + S I = Stomatal index, S = No. of stomata per unit area, E = No. of epidermal cells in the same unit area. Procedure: Clear the piece of the leaf (middle part) by boiling with chloral hydrate solution or alternatively with chlorinated soda. Peel out upper and lower epidermis separately by means of forceps. Keep it on slide and mount in glycerin water. Arrange a camera lucida and drawing board for making the drawings to scale. Draw a square of 1mm by means of stage micrometer. Place the slide with cleared leaf (epidermis) on the stage. Trace the epidermis cell and stomata. Count the number of stomata, also the number of epidermal cells in each field. Calculate the stomatal index using the above formula. Determine the values for upper and lower surface (epidermis) separately.
34 Vein-islet number [38,39] A vein-islet is the small area of green tissue surrounded by the veinlets. The vein-islet number is the average number of vein-islets per square millimeter of a leaf surface. It is determined by counting the number of vein-islets in area of 4 sq. mm. of the central part of the leaf between the midrib and the margin. Procedure: Clear a piece of the leaf by boiling in choral hydrate solution for about thirty minutes. Arrange camera lucida and drawing board for making drawings to scale. Place stage micrometer on the microscope and using 16 mm objectives, draw a line equivalent to 1mm as seen through the microscope. Construct a square on this line. Move the paper so that the square is seen in the eye piece, in the centre of the field. Place the slide with the cleared leaf (epidermis on the stage). Trace off the veins which are included within the square, completing the outlines of those islets which overlap two adjacent sides of the square. Count the number of vein islets in the square millimeter. Where the islets are intersected by the sides of the square, include those on two adjacent sides and exclude those islets on the other sides. (To obtain a critical result for a leaf, 4 sq. mm. should be used, preferably in one large area of 4 sq. mm.). Find the average number of vein islets from the four adjoining squares, to get the values for one sq. mm.
35 Veinlet termination number [38,39] Veinlet termination number is defined as the number of veinlet termination per sq. mm of the leaf surface, midway between midrib of the leaf and its margin. A vein termination is the ultimate free termination of veinlet. Procedure: Clear a piece of the leaf by boiling in choral hydrate solution for about thirty minutes. Arrange camera lucida and drawing board for making drawings to scale. Place stage micrometer on the microscope and using 16 mm objectives, draw a line equivalent to 1mm as seen through the microscope. Construct a square on this line. Move the paper so that the square is seen in the eye piece, in the centre of the field. Place the slide with the cleared leaf (epidermis on the stage). Trace off the veins which are included within the square, completing the outlines of those islets which overlap two adjacent sides of the square. Count the number of veinlet terminations present within the square. Find the average number of veinlet termination number from the four adjoining squares, to get the values for one sq. mm.
36 2.6 Physicochemical constants [46] Ash values The residue remaining left after incineration of the crude drug is designated as ash. The residue obtained usually represents the inorganic salts naturally occurring in the drug and adhering to it. It varies with in definite limits according to the soils. It may also include inorganic matter deliberately added for the purpose of adulteration. Hence, an ash value determination furnishes the basis for judging the identity and cleanliness of any drug and gives information relative to its adulteration/contamination with inorganic matter, thus ash values are helpful in determining the quality and purity of drug. The total ash of a crude drug reflects the care taken in its preparation. The acid insoluble ash is a part of the total ash that is insoluble in dilute hydrochloric acid. A higher limit of acid-insoluble ash is imposed, especially in cases where silica may be present or when the calcium oxalate content of the drug is higher Procedure given in Indian Pharmacopoeia was used to determine the different ash values such as total ash, acid insoluble ash, and water-soluble ash value. a) Determination of Total Ash Value Accurately weighed about 3 grams of air dried powdered drug was taken in a tarred silica crucible and incinerated by gradually increasing the temperature to make it dull red hot until free from carbon. Cooled and weighed, repeated for constant value. Then the percentage of total ash was calculated with reference to the air-dried drug.
37 b) Determination of Acid Insoluble Ash Value The ash obtained as directed under total ash was boiled with 25 ml of 2N hydrochloric acid for 5 minutes. The insoluble matter was collected on an ash less filter paper, washed with hot water, dried the filter paper, ignited and weighed. Then calculate the percentage of acid insoluble ash with reference to the air-dried drug. c) Determination of Water soluble Ash Value The total ash obtained was boiled with 25 ml of water for 5 minutes. The insoluble matter was collected on an ash less filter paper, washed with hot water and ignited for 15 minutes at a temperature not exceeding 450 0 C. The weight of insoluble matter was subtracted from the weight of total ash. The difference in weight represents the water-soluble ash. The percentage of water-soluble ash was calculated with reference to the air-dried drug. d) Determination of Loss on Drying Weigh about 1.5 gm of the powdered drug into a weighed flat and thin porcelain dish. Dry in the oven at 100 0 C. Cool in a desiccators and watch. The loss in weight is usually recorded as moisture. The values were tabulated (Table 9 and 10).