Fresh works samples were collected during the field trips in different countries of Pakistan. Detailed morphological ( macro & A ; microscopic ) scrutiny was carried out by utilizing binocular stereo rapid climb light microscope ( Model Kyowa SZF ( 0.75x-3.4x ) utilizing oculus piece, WF 10 x 10/20 ) . The descriptions of works species were besides compared by utilizing different Floras ( Nasir & A ; Ali, 1982, Hooker, 1875 ; Tutin & A ; Heywood, 1972 ; Hooker & A ; K.C.S.I. , 1885 ; 1894 and Saldanha & A ; Nicolson, 1976 ) .
Fresh stuff of different medicative works species ( Datura metel solanium nigrum, withania coagulans, Cassia angustifolia, Cassia occidentalis, Dalbergia obovata Calendula officinalis Parthenium integrifolium, Silybum eburneum, ) were collected from different vicinities of Pakistan and were used for foliage epidermal survey. Leaf samples were prepared harmonizing to the modified method of Cotton ( 1974 ) , who followed Clark`s ( 1960 ) technique but with a small alteration. ( Shaheen et al. , 2010 ) . The foliages were placed in a tubing filled with 88 % Lactic acid kept hot in boiling H2O bath ( Model, Memmert-91126-FRG, Germany ) for approximately 15 to 30 proceedingss. Lactic acid softens the tissues of foliage due to which it is possible to grate the leaf surface with crisp scalpel. Slides of both dorsal and ventral surface of foliage were prepared and mounted in clean 88 % lactic acid. Features i.e. form, size of cuticular cells, wall thickness, smooth or rippling wall, trichome ( form and construction ) , agreement of pore in cuticle, or the presence of tissues with characteristic cells were studied which are used in the microscopic hallmark of herbal drugs.
Fresh polliniferous stuff was utilized for palyno-morph survey harmonizing to the modified method of Wodehouse technique ( Ronald, 2000 ) . Pollen was acetolized and stained by utilizing glycerin jelly. Glycerin jelly was prepared harmonizing to modified method ( Ahmad et al. , 2003 ) . Common adhesive, crystalline finger nail gloss was used to seal off the borders of mention slides. Reference slides were clearly labeled with full designation, vicinity, and day of the month.
Qualitative characters studied under light microscope for pollen morphology were type of pollen, form in polar & A ; equatorial position, presence or absence of colpi & A ; spinal columns, form of pore ( ora ) and sculpturing. The quantitative characters were polar & A ; equatorial diameter, P/E ratio, figure of spinal columns ( In instance of echinate pollen ) , figure of spinal columns between colpi ( In instance of echinate pollen ) figure of colpi, length & A ; breadth of colpi, figure of pores, spine length and exine thickness was observed and measured.
188.8.131.52 Pollen Fertility Analysis
The pollen birthrate appraisal was carried out by using the techniques used by Meo and Khan ( 2004 ) . A mature undehiscened anther was squashed in a bead of acetocarmine. Debris was removed gently and a screen faux pas was placed over the discoloration. The slides were observed at low magnification. The figure of stained and unstained pollen grains was tabulated. Fully stained pollen were considered fertile while the lightly stained pollens, unstained pollens were considered unfertile.
Light Microscopic Photographs ( LM )
The LM of dorsal and ventral cuticle of foliage samples and pollen was carried out. The microphotographs were taken on the 40x, 60x and 100x lens.
Scaning Electron Microscopic Photographs ( SEM )
The foliages and pollen samples were sent to Centralized Science Laboratory, Karachi for acquiring the exposure. 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 stores and collected from the field. All parts of herbal drugs including, wood bark, roots, rootstocks, foliages, stems, fruit, flowers and seeds of debatable medicative workss were identified by analyzing macro-morphological characters. Organoleptic analysis involved the usage of sight, odor, gustatory sensation, touch and microscopy of petroleum drugs to measure works stuffs frequently comparing the belongingss of a known sample with those of a mention criterion.
3.1.7 UV, IR and seeable visible radiations Photographs
Photography of herbal drugs under short wavelength of UV ( UVGL-58 Lamp, 254/365 nanometer ) , IR and seeable visible radiations were taken by Digital camera ( Sony, DSC-W50 ) . This high-resolution picture taking provides reliable attack toward the designation of dubious and debatable works species used in herbal drugs.
3.2 CHEMICAL METHODS FOR AUTHENTICATION
Fluorescence and Solubility Analysis
The simple method to find the fluorescence of powdery drug was adopted. 5 gm powdered drug was assorted in 20 milliliters sulfuric acid, hydrochloric acid, acetic acid, and H2O. Each trial tubing was shaken and boiled. Method followed was that of Evers and Smith ( 1955 ) . The solubility and keeping of original coloring material of powdery stuffs was noted in assorted dissolvers in cold and hot conditions. Filter paper was besides used to happen out alteration in coloring material.
3.2.2 Chemical Analysis
This probe was confined to acid hydrolysis of flavonoids, sensing of alkaloids, glycosides, tannic acids, amylum grains, anthraquinones, saponins and volatile and fixed oils, allelopathic and extremist violet ( UV ) & A ; Infra-red ( IR ) analysis.
184.108.40.206 Acid Hydrolysis
For the extraction of flavonoid aglycones a little sum of dried works stuff was treated with 2 normal ( 2N ) hydrochloric acid ( HCl ) and heated for one hr in a H2O bath at about 100oC. By this intervention usually all flavonoids-O-glycosides were converted to flavonoids aglycones, anthocyanins to anthocyanidins whereas the C-glycosides remain unaffected ( Fig 2 ) . After chilling, the flavonoid aglycones were extracted with diethyl quintessence ( Et2O ) from the aqueous stage. A 2nd series of extraction by n-butanol quantitatively removes the anthocyanidins.
220.127.116.11 Detection of alkaloid
15g of each powdered drug were macerated with ethyl alcohol ( 50ml ) in conelike flasks covered by a cotton palpebra of 24 hours. The stuff was shaken and so filtered by filter papers.3-4 beads of this infusion were taken in a trial tubing and 5ml of distilled H2O acidify with 1-2 beads of 2M HCL was assorted and 1ml of Dragendorff reagent was added in to this mixture and was shaken. Presence of orange or orange ruddy ppt indicated the presence of alkaloid ( British Pharmacopoeia 1999 ) .
18.104.22.168 Detection of glycoside
Those acrimonious species, which gave negative trials to alkaloidal reagent, were considered hence perchance to incorporate glycosides.
22.214.171.124 Detection of tannic acids
10g of each powdered drug were macerated with distilled H2O ( 40-50ml ) in 100ml conelike flasks covered by cotton palpebra for 8-10 hours. Then filtered by filter documents. 1ml of this infusion was taken in a trial tubing and added 1ml of “ ferrous chloride solution ” into it and was shaken. The presence of bluish, bluish black or chocolate-brown colour indicated the presence of tannic acids.
126.96.36.199 Detection of amylum grains
1-2g of each powered was taken on glass slides. One bead of 0.5M Iodine solution was added to it, the presence of bluish or black colour indicated the presence of amylum grains.
188.8.131.52 Detection of Anthraquinones
2-3g of each powdery drug was macerated in diethyl quintessence ( 5-6ml ) in trial tubing for 10-12 proceedingss. Then it was filtered by filter paper.2-3 beads of 20 % Na hydrated oxide ( acerb sodium carbonate ) , ( 20g of NaOH + 80ml of distilled H2O ) were added, and shaken. The presence of pink, violet or ruddy colour in the aqueous bed indicated the presence of anthraquinone ( British Pharmacopoeia 1999 ) .
184.108.40.206 Detection of saponin
2-3g of each powdery drug was foremost taken in a trial tubing. 5-6ml of distilled H2O was added into it, and was shaked. Presence of any pronounced frothing indicated the presence of saponin.
220.127.116.11 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 proceedingss. The presence of any oily discoloration on filter paper showed the presence of oil. If the discoloration 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 discoloration disappeared so it indicated the presence of volatile oil. ( British Pharmacopoeia 1999 ) .