Material Basis For The Anti-inflammatory And Analgesic Effects Of Laportea Bulbifera And Its Quality Control

Objective: To clarify the effective fractions and material basis of Laportea bulbifera for its anti-inflammatory and analgesic activity and to establish methods for its quality control. Methods: The best extraction solvent was determined by examining the extraction efficiency of the chemical constituents of L.bulbifera and the anti-inflammatory and analgesic activity of the extracts. D101 macroporous resin column chromatography(CC) was used for the preparation of fractions of different polarities. The active fractions were determined by testing their corresponding biological effects on the classical inflammatory and analgesic models, the chemical components of the active fractions were isolated and purified by various CCs including MCI gel CC, silica gel CC, reversed phase C18 CC and Sephadex LH-20 CC. Their structures were identified by combined analysis of their physicochemical properties and spectral data. The components with higher contents were then subjected to purity tests. Multi-marker determination and tannins determination methods were established by HPLC-UV and UV, respectively, for the quality control of L.bulbifera. In addition, items related to the quality control of L.bulbifera were determined. These items are moisture, total ash, acid insoluble ash content, alcohol soluble extract, organic chlorine pesticide residues, heavy metals and harmful elements and so on. Results: 70% aqueous ethanol was employed to extract the compounds from L.bulbifera, subsequent separation of the obtained solvent-free extract on a D101 macroporous resin CC eluted with H2 O, 50% aqueous ethanol and 95% aqueous ethanol afforded fractions W, 50 E, and 95 E, respectively. Fractions 50 E and 95 E were then determined to be the anti-inflammatory and analgesic fractions of L.bulbifera. 15 compounds were isolated and analyzed from the active fractions. Six compounds were isolated from the active fraction 50 E and were identified as(-)-gallocatechin(1),(±)-epigallocatechin(2),(+)-catechin(3),(-)-epicatechin(4), phloroglucinol(5) and daucosterol(6), one compound(i.e, β-sitosterol, 7) was isolated and 8 volatile chemical constituents were identified by GC-MS from fraction 95 E. These volatile constituents are: hexadecanoic acid, methyl ester(8), methyl hexadec-9-enoate(9), hexadecanoic acid, ethyl ester(10), 8,11-Octadecadienoic acid, methyl ester(11), 9-Octadecenoic acid(Z)-, methyl ester(12), methyl stearate(13), linoleic acid ethyl ester(14), ethyl Oleate(15).(-)-Gallocatechin(1),(±)-epigallocatechin(2),(+)-catechin(3) and(-)-epicatechin(4), the four chemical ingredients with higher contents in L.bulbifera were chosen as reference substances and chemical markers for establishing an HPLC-UV method for the multi-marker determination of L.bulbifera. Their contents varied among the 28 samples in the ranges of 0.3103~1.246 mg·g-1, 0.3176~1.886 mg·g-1, 0.1276~0.5408 mg·g-1, 0.0869~0.7491 mg·g-1, respectively. A UV method was established for the simutaneous determination of the total tannis and condensed tannis, and the results showed that the contents of total tannins in L.bulbifera was in the range of 92.34~178.1 mg·g-1 and those for condensed tannins were 72.79~141.4 mg·g-1, which suggested condensed tannins to be the main structural type of tannins. The contents of moisture, total ash, acid-insoluble ash and the alcohol soluble extract in L.bulbifera were 7.71%~15.8%, 5.36%~10.6%, 0.618~4.10% and 13.5~27.5%, respectively. Levels of all heavy metals and harmful elements of the 28 samples except for two whose lead levels exceeds to the standard. The nine organochlorine pesticide residues including BHC and DDT were not detected in the samples. Conclusion: Fractions 50 E and 95 E were the anti-inflammatory and analgesic fractions of L.bulbifera. Among the 15 compounds isolated or identified from the active fractions, compounds 1~5 were isolated from the genus Laportea for the first time. The multi-marker determination and tannins determination methods established in this paper have good accuracy, sensitivity and repeatability, and could be used for the quality control of L.bulbifera. The study provides evidence and experimental methods for further research and development of L.bulbifera and its related products.