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Fresh plant samples were collected during the field trips in different areas of Pakistan. Detailed morphological (macro & microscopic) examination was carried out by using binocular stereo zoom light microscope (Model Kyowa SZF (0.75x-3.4x) using eye piece, WF 10 x 10/20). The descriptions of plant species were also compared by using different Floras (Nasir & Ali, 1982, Hooker, 1875; Tutin & Heywood, 1972; Hooker & K.C.S.I., 1885; 1894 and Saldanha & Nicolson, 1976).
Fresh material of 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 Pakistan and were used for leaf epidermal study. Leaf samples were prepared according to the modified method of Cotton (1974), who followed Clark`s (1960) technique but with a little modification. (Shaheen et al., 2010). The leaves were placed in a tube filled with 88% Lactic acid kept hot in boiling water bath (Model, Memmert-91126-FRG, Germany) for about 15 to 30 minutes. Lactic acid softens the tissues of leaf due to which it is possible to scrape the leaf surface with sharp scalpel. Slides of both abaxial and adaxial surface of leaf were prepared and mounted in clean 88% lactic acid. Features i.e. shape, size of epidermal cells, wall thickness, smooth or undulating wall, trichome (shape and structure), arrangement of stomata in epidermis, or the presence of tissues with characteristic cells were studied which are used in the microscopic authentication of herbal drugs.
Fresh polliniferous material was utilized for palyno-morph study according to the modified method of Wodehouse technique (Ronald, 2000). Pollen was acetolized and stained by using glycerin jelly. Glycerin jelly was prepared according to modified method (Ahmad et al., 2003). Common adhesive, transparent finger nail polish was used to seal off the edges of reference slides. Reference slides were clearly labeled with full identification, locality, and date.
Qualitative characters studied under light microscope for pollen morphology were type of pollen, shape in polar & equatorial view, presence or absence of colpi & spines, shape of pore (ora) and sculpturing. The quantitative characters were polar & equatorial diameter, P/E ratio, number of spines (In case of echinate pollen), number of spines between colpi (In case of echinate pollen) number of colpi, length & width of colpi, number of pores, spine length and exine thickness was observed and measured.
220.127.116.11 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 abaxial and adaxial epidermis of leaf samples and pollen was carried out. The microphotographs were taken on the 40x, 60x and 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 herbal drugs under short wavelength of UV (UVGL-58 Lamp, 254/365 nm), IR and visible lights were taken by Digital camera (Sony, DSC-W50). This high-resolution photography provides authentic approach toward the identification of doubtful and problematic plant species used in herbal drugs.
3.2 CHEMICAL METHODS FOR AUTHENTICATION
Fluorescence and Solubility Analysis
The simple method to determine the fluorescence of powdered drug was adopted. 5 gram powdered drug was mixed in 20 ml sulphuric acid, hydrochloric acid, acetic acid, and water. Each test tube was shaken and boiled. Method followed was that of Evers and Smith (1955). The solubility and retention of original colour of powdered materials was noted in various solvents in cold and hot conditions. Filter paper was also used to find out change in 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.
18.104.22.168 Acid Hydrolysis
For the extraction of flavonoid aglycones a small amount of dried plant material was treated with 2 normal (2N) hydrochloric acid (HCl) and heated for one hour in a water bath at about 100oC. By this treatment normally all flavonoids-O-glycosides were converted to flavonoids aglycones, anthocyanins to anthocyanidins whereas the C-glycosides remain unaffected (Fig 2). After cooling, the flavonoid aglycones were extracted with diethyl ether (Et2O) from the aqueous phase. A second series of extraction by n-butanol quantitatively removes the anthocyanidins.
22.214.171.124 Detection of alkaloid
15g of each powdered drug were macerated with ethanol (50ml) in conical flasks covered by a cotton lid of 24 hours. The material was shaken and then filtered by filter papers.3-4 drops of this extract were taken in a test tube and 5ml of distilled water acidify with 1-2 drops of 2M HCL was mixed and 1ml of Dragendorff reagent was added in to this mixture and was shaken. Presence of orange or orange red ppt indicated the presence of alkaloid (British Pharmacopoeia 1999).
126.96.36.199 Detection of glycoside
Those bitter species, which gave negative tests to alkaloidal reagent, were considered therefore possibly to contain glycosides.
188.8.131.52 Detection of tannins
10g of each powdered drug were macerated with distilled water (40-50ml) in 100ml conical flasks covered by cotton lid for 8-10 hours. Then filtered by filter papers. 1ml of this extract was taken in a test tube and added 1ml of "ferric chloride solution" into it and was shaken. The presence of blue, blue black or brownish color indicated the presence of tannins.
184.108.40.206 Detection of starch grains
1-2g of each powered was taken on glass slides. One drop of 0.5M Iodine solution was added to it, the presence of blue or black color indicated the presence of starch grains.
220.127.116.11 Detection of Anthraquinones
2-3g of each powdered drug was 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 added, and shaken. The presence of pink, violet or red color in the aqueous layer indicated the presence of anthraquinone (British Pharmacopoeia 1999).
18.104.22.168 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.
22.214.171.124 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).