Studies On The Chemical Contents And The Assaying Of Polysaccharide In Thladiantha Dubia Bunge Fruit

The fruit of Thladiantha dubia Bunge (FTDB), one plant of the family of Cucurbitaceae, is commonly used for the regulating vital energy, promoting blood flow, calming the adverse-rising energy, and so on. It is well known in a northeast and Northern China. People lived in Cheng-De use FTDB to cure the pain in waist and leg, or strain in the lumbar , and the curative effect is pefect. Pharmacology experiment showed that FTDB exhibites strong effect on relieve pain, antiinflammatory and anti-anaphylaxis. However limited study has been carried on the chemical components of the FTDB. Nagao T isolated 6 compounds from the root of Thladiantha dubia Bunge (TDB), which are duibiosides A, B, C, D, E, F. Lilanfang analysed the volatile oil of the FTDB by using GC-MS. Inorder to look for active constituent on pain relieving and antiinflammatory, also to exploit new drug, we studied the FTDB including extraction , isolation and structure identification. Besides, we had measured the contents of polysaccharide in FTDB.Objective: To study components in Thladiantha dubia Bunge, using silica gel column chromatogram, polymide colum chromatogram, and Sephadex LH-20 column chromatogram. To extract fractionate, isolate and purify the ingredient and to get pure compound. To establish the structures of compounds isolated from FTDB with the aids of spectrosopic methods including UV, IR, 1H-NMR, 13C-NMR, MS. To study the anti-inflammation action of water extracts. To determine the the contents of polysaccharide by colourimetry with sulfuric acid-phenol as colorimetric reagent.Methods: 1 Pre-test of composition of Thladiantha dubia Bunge fruit(TDBF): Air-dried fruits of TDBF were extracted with acidic ethanol, water, methanol and petroleum ether, respectiely. Thses four extracts were investigated. Checking the ingredients in the fruits of FTDB following the ingredients pre-test.2 Extraction, isolation, and structure identification of TDBF: Dried TDBF (2Kg) were extracted with 95% ethanol three times and the solvent was evaporated under reduced pressure, yielded 1220.7 g extract. The extract was diluted with water and extracted with petroleum ether, ether, ethyl acetate and n-butanol, respectively, and after evaporated the solvent, we got each fraction and emulsifying fraction. Petro-fraction resolved in methanol was fractionated by silica gel column chromatography to obtain white particle crystalⅣ, white particle crystalⅠ, white needle crystalⅡ. Methanol elution was further separated by silica gel column chromatography again to obtain white particle crystalⅢ. Petro-fraction insoluble substance in methanol was isolated by silica gel column chromatography to obtain white needle crystalⅤ, white solidⅥ(which was a mixture as midicated by GC-MS analysis), white lamellar crystalⅦ. Et2O-fraction, which was insoluble in acetone, was purified by silica gel column chromatography to obtain white particle crystal, which was similar to compoundⅢ. Emulsifying fraction was isolated by silica gel column chromatography and futher purified with Sephadex LH-20 to obtain white particle crystalⅧ, n-butanol fraction was isolated by silica gel column chromatography and futher purified with Sephadex LH-20 to obtain white needle crystalⅨ.3 Inspect the determination method of content:The purified polysaccharides is determined by colourimetry with sulfuric acid-phenol as colourimetric reagent: (1) Extraction and purification of polysaccharides: TDBF was refluxed by hot water to extract polysaccharides purified by degrease and deproteinization. (2) Preparation of phenol: After distillation, the phenol was diluted with water, reserved in refrigerator. (3) Preparation of sample: The sample was extracted with water by supersound, concentrated and metered volumn by water. (4) Preparation of standard curve: Prepared appropriate concentration reference and choosed suitable coloration reagent, wave and determine absorbility. (5) Experiment of reproducibility: the sample was injected 5 times sequencely and determined the absorbability. (6) Experiment of recovery: weighed out sample, added the polysaccharide respectively, determined the absorbability and calculated recovery. (7) Experiment of precision: Weighed out 5 shares of TDBF to make 5 samples, determined the absorbability and calculated recovery. (8) Experiment of stability: In one hour, determined the absorbability of the sample solution, polysaccharides solution, and glucose solution.Results: 1 Pre-test results of composition shows that the fruits of Thladiantha dubia Bunge contain alkaloid, steroids, coumarin, organic acids, tannin, glycosides, sugers, protein, amino acids and lipids.2 On chemical constituents study of TDBF: eight compounds and one mixture were isolated by using column gel chromatography, six of the compounds were identified by chemical and spectroscopic methods, the mixture was identified by GC-MS. Six structures were elucidated as: palmitic acid (Ⅰ),α-spinasterol (Ⅱ),α-spinastero-3-O-glucoside (Ⅲ), triacontanol (Ⅳ),α-spinasteryl-3-O-β-D-glucoside-6′-palmitate (Ⅶ), manicol (Ⅸ). The mixture (Ⅵ) were made up by tetracosane, pentacosane, hexacosane, heptacosane, octacosane, nonacosane and triacontane.3 The results on the content of the polysaccharide: Sulfuric acid-phenol was choose as coloration reagent, the wave length was 388 nm. The regression equation was A = 12.862C+0.0069, r = 0.9992, (n=5). The content of polysaccharide in TDBF was 3.426 %, reproducibility: RSD = 3.44 %; recovery: RSD=1.81 %, stability of sample solution: RSD = 1.86 %.Conclusion: The results of our experiment showed that the solvents and methods of extraction used in these experiments are practicable. With silica gel, polymide and Sephadex LH-20 column chromatogram, the components in TDBF were successively separated and purified. Six compounds were isolated and identified. Of them five compounds were identified as new constituents for the first time in the plant of Thladiantha Bunge, which will provide information for TDBF and to invertigate pharmacological activity. Eatablished method is easy, reproductive and accurate to identify TDBF with polysaccharides and glucose as reference by UV-spectrophotometry. These methods were simple, specific and repoductive.