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In the different areas of Pakistan, the fresh plant samples were collected during the field trips Detailed morphological (the macro & the microscopic) examination was done by using the binocular light microscope. The descriptions of the plant specimens were also given by comparing data with the different Floras (Nasir & Ali, 1982, Hooker, 1875; Tutin & Heywood, 1972; Hooker & K.C.S.I., 1885; 1894 and Saldanha & Nicolson, 1976).
Fresh material of the different medicinal plant species (Datura metel, solanium nigrum, withania coagulans, Cassia angustifolia, Cassia occidentalis, Dalbergia obovata Calendula officinalis, Parthenium integrifolium, Silybum eburneum,) were collected from different localities of the Pakistan and also were used for the leaf epidermal study. Leaf samples were prepared by the method modified by the Cotton (1974), which followed the technique of Clark`s (1960) but with the bit modification of Shaheen et al., 2010. The leave specimens were put in a tube which is filled with the 88 percent of Lactic acid that is kept hot in a boiling water bath, for about 25 to 40 minutes, Lactic acid is that which softens tissues of the leaves and made it possible that scrapes the surface of the leaf with the sharp scalpel. Both slides (the abaxial and the adaxial leaf surfaces) were then prepared and then mounted in the clean 80% of lactic acid, Features such as the shape, the size of the epidermal cells, the wall thickness, the smooth or the undulating wall, the trichome (shape and structure), the arrangement of the stomata in the epidermis, or the presence of the tissues with the characteristic cells were studied which are now used in the microscopic authentication of herbal drugs.
The fresh polliniferous material was utilized for palyno-morph study according to the method modified by the Wodehouse technique, (Ronald, 2000). Pollen was first acetolized and then stained by using the glycerin jelly. The glycerin jelly, was prepared by the method modified by Ahmad et al., 2003. Common adhesive and the transparent (finger) nail polish was then used to seal out the edges of the reference slides. The reference slides then were labeled clearly with the full identification, date and, the locality.
Qualitative characters studied under light microscope for pollen morphology were type of pollen, shape in polar & equatorial view, absence/ presence of colpi and the spines, the shapes of pore and the sculpturing. The quantitative characters were the polar & the equatorial diameter, P/E ratio, number of spines (In case when pollen is echinate), number of spines between the colpi (In case when pollen is echinate) number of the colpi, the length & the width of the colpi, the number of pores, the spine length and the exine thickness was observed and measured.
22.214.171.124 Pollen Fertility Analysis
The pollen fertility estimation was carried out by employing the techniques used by Meo and Khan (2004). A mature undehiscened anther was squashed in a drop of acetocarmine. Debris was removed gently and a cover slip was placed over the stain. The slides were observed at low magnification. The number of stained and unstained pollen grains was tabulated. Fully stained pollen were considered fertile while the lightly stained pollens, unstained pollens were considered sterile.
Light Microscopic Photographs (LM)
The LM of the abaxial and the adaxial epidermis of the leaf samples and the pollen was done. The microphotographs then were taken on the 40, 60 and the 100x lens.
Scanning Electron Microscopic Photographs (SEM)
The leaves and pollen samples were sent to Centralized Science Laboratory, Karachi for getting the photographs. The method was followed by the Terrel and Wergin (1979) and Hilu and Wright (1984).
3.1.6 Organoleptic Analysis
Material for organoleptic analysis was procured from herbal shops and collected from the field. All parts of herbal drugs including, wood bark, roots, rhizomes, leaves, stems, fruit, flowers and seeds of problematic medicinal plants were identified by examining macro-morphological characters. Organoleptic analysis involved the use of sight, smell, taste, touch and microscopy of crude drugs to evaluate plant materials often comparing the properties of a known sample with those of a reference standard.
3.1.7 UV, IR and visible lights Photographs
Photography of the herbal drugs under the short wavelength of the UV Lamp and IR lamp and the under visible lights were taken by the Digital camera. This photography which has high-resolution gives the authentic approach, toward the identification of the doubtful and the problematic plant species used in herbal drugs.
3.2 METHODS FOR CHEMICAL AUTHENTICATION
Fluorescence and Solubility Analysis
The simple method to determine the fluorescence of the powdered drug first was adopted, 5 gram of the powdered drug was first mixed in the 20 ml of sulphuric acid, the hydrochloric acid, the acetic acid, and the water. Each of the test tube then was shaken, and boiled. The method which was followed was of Evers and Smith (1955). The solubility and retention of original colour of powdered materials was then noted in the various solvents present in the cold and the hot conditions. Then filter paper was used so to find out the change in the colour.
3.2.2 Chemical Analysis
This investigation was confined to acid hydrolysis of flavonoids, detection of alkaloids, glycosides, tannins, starch grains, anthraquinones, saponins and volatile and fixed oils, allelopathic and ultra violet (UV) & Infra-red (IR) analysis.
126.96.36.199 Acid Hydrolysis
For the extraction of the flavonoid aglycones small amount of the dried plant material first was treated with the two normal (2N), hydrochloric acid, (HCl), and then heated for about one hour in the water bath for about, 100oC, By doing this, normally all the flavonoids,-O-glycosides, were then converted to the flavonoids aglycones, anthocyanins to the anthocyanidins whereas the C-glycosides remained unaffected (Fig 2). After the cooling, the flavonoid aglycones then were extracted with the diethyl ether (Et2O) from the aqueous phase. Then a second series of the extraction by the n-butanol, quantitatively, removes the anthocyanidins.
188.8.131.52 Detection of alkaloid
Fifteen gram of each of powdered drug were first macerated with the ethanol, (50ml), in the conical flasks which were covered by the cotton lid of twentyfour hours. The material was then shaken and then filtered by the filter papers. Three-four drops of this extract were then taken in test tube and then five ml of the distilled water acidified with one-two drops of two M HCL was then mixed and one ml of the Dragendorff reagent, was added into this mixture and then was shaken. Presence of the orange or orange red ppt, indicated the presence of the alkaloid. (British Pharmacopoeia 1999).
184.108.40.206 Detection of glycoside
Those species which are bitter, gave the negative tests to the alkaloidal reagents, were then considered, therefore possibly to contain the glycosides.
220.127.116.11 Detection of tannins
Ten gram of each of powdered drug was macerated with the distilled water (40/50ml) in the 100ml of the conical flasks, covered by the cotton lid for about 8-10, hours. Then filtered by the filter papers. One ml of the extract was then taken in test tube, and added one ml of "ferric chloride solution" into it and then was shaken. The presence of the blue, blue/ black or the brownish color indicated the presence of tannins.
18.104.22.168 Detection of starch grains
One to two g of each of powered was taken on the glass slides. One drop of the 0.5M, Iodine solution was first added to it, then presence of blue or black color indicated the presence of starch grains.
22.214.171.124 Detection of Anthraquinones
2-3g of each of powdered drug was first macerated in diethyl ether, (5-6ml), in test tube, for 10-12 minutes. Then it was filtered by filter paper.2-3, drops of 20% sodium hydroxide, (caustic soda), (20g, of NaOH + 80ml, of distilled water) were then added, and shaken. The presence of the pink, violet or red color in the aqueous layer indicated the presence of anthraquinone (British Pharmacopoeia 1999).
126.96.36.199 Detection of saponin
2-3g of each powdered drug was first taken in a test tube. 5-6ml of distilled water was added into it, and was shaked. Presence of any marked frothing indicated the presence of saponin.
188.8.131.52 Detection of voltile and fixed oils
8-10g of each powered drugs were taken on a filter paper, covered by another filter paper. It was placed under a mechanical presser and was pressed for 5 minutes. The presence of any oily stain on filter paper showed the presence of oil. If the stain on filter paper was still present after heating it in an oven for 2-3 hours at 90C. It indicated the presence of fixed oil. If the stain disappeared then it indicated the presence of volatile oil. (British Pharmacopoeia 1999).