Pharmacognostic Identification Studies On Four Sources Of Manis Spp.

The traditional Chinese medicine "pangolin scales" are the scales of Manis pentadactyla Linnaeus, the animals of Manidae. They have the quality of Xian and little Han. They can Tongjingxiaru, xiaozhongpainong and Soufengtongluo. They are common clinical medicines. There are 7 species of pangolin in the world. Manis pentadactyla), Manis crassicaudata and Manis javanica live in Ansia; Manis gigantea, Manis tricuspis, Manis temminckii and Manis tetradactyla live in Africa. The pangolins living in China are listed as Two National Key Protected Wild Animals. All the species of pangolin are listed as Appendix Ⅱ of CITES.In the past, the domestic identification studies of pangolin scales mainly concentrated on experience. As the development of technology, some new methods such as microscopic, TLC, CE, IR, X-ray, DNA, etc, have gradually been used in qualitative and quantitative analysis of pangolin scales. However, the identification studies mainly focus on counterfeits such as pig nails and sheep nails. The identification studies depend on the experience and sense of operaters. The cost of DNA technology is very high because it has a high requirement in quality of operaters and the environment. These methods are not on the basis of the ingredients of the animals of Manidae. They can not response their intrinsic material basis. They do not focus on the difference among different species. So, it is very necessary to establish a simple, convenient economical and reliable method, and to make up the deficiency of experience and DNA identification.We collect 16 samples of scales, which are from four species of Manidae. We test four methods, which are character, microscopic TLC and HPLC. We establish a quick identification of pangolin scales, and provide a scientific basis for the identification of pangolin scales of different species.Character identification: Collect 20 scales from different species and different parts randomLy and measure the appearance data, including the weight, length, width and thickness, then carry out SPSS analysis.Observe the outside, inside and fracture surface of the scales, and carry out micro morphological observation.The results of appearance data measuring: As to the weight, length and width, there are significant differences between Manis gigantean and the other 3 species (P<0.01),and the differences among the other 3 species are not significant. As to the width, there are significant differences between Manis tricuspis and the other 3 species (P<0.01). As to the thickness, there are significant differencesamong the four species (P<0.01). Manis gigantean is the thickest one, Manis tricuspisis the thinnest one, and Manis pentadactyla is thicker than Manis javanica. It can be a basis of identification.The results of micro morphological observation:As to the back scales, Manis pentadactyla are flat round or concave, Manis javanica are convex, and Manis tricuspis have three tips. It can be a basis of identification among the three species. There are transverse shrinking textures on the scales of Manis tricuspis, and it is a significant difference from other 3 speciesThe results of color comparison: As to the color of scales, Manis pentadactyla Is dark brown, Manis javanica is grey-black, Manis tricuspis is red-brown or brown-yellow and Manis gigantean is grey-yellow. It can be the elementary basis for identification.The results of TLC methodology study (the best TLC Chromatographic conditions):The developing solvent is Petroleum ether- Ethyl ethe- Glacial acetic acid (10:10:0.5) and Hexane-Ethyl acetatee- Glacial acetic acid (7:2:0.5). Launch two times chromatography, view in 365nm, and then colored with 10% ethanol, heat in 105℃, view in 365nm. The scanning wavelength before coloring is 280nm, and after coloring is 400nm.The results of TLC research:All the 4 species have the main spot of cholesterol and the other spots are mostly matched. The Chromatograms of Manis pentadactyla and Manis javanica are similar, and they are significantly different from the other 2 species. Manis gigantean has two characteristic spots (Rf=0.43,0.25), and the spot of Rf=0.43 shows strong absorption under the scanning of 280nm. It is a significant difference from other species. Manis tricuspis misses two main spots ((Rf=0.56,0.62), and it is also a significant difference from other speciesThe results of HPLC research: As the reducing of solvent polarity (water,50% ethanol, anhydrous ethanol), the chromatographic peaks reduce, too. The extract of anhydrous ethanol after degreased by petroleum ether has the fewest chromatographic peaks.The difference among fingerprints of water extract is very small, and the common the chromatographic peaks are significant. It indicates that we can find the characteristic peaks of pangolin scales with this method. The fingerprints of Manis pentadactyla (the extract of 50% ethanol) is significantly different from other 3 species. There are significant differences among the fingerprints of extract of anhydrous ethanol from different species.