Studies On The Chemical Constituents Of Fibrous Roots Of Ophiopogon Japonicus

The tuber of Ophiopogon japonicus (Thunb.) Ker-Gawl.(Liliaceae), is recorded in the Pharmacopoeia of the People's Republic of China(2005), vol. I. The distribution and cultivation of Ophiopogon in China are very widespread, the famous place of production are Hangzhou of Zhejiang and Santai of Sichuan. Traditionally, it is used its tuber for medicine, the fibrous are always discarded. Studies have shown that the chemical constituents of fibrous and tubers are basically same, therefore, the development of fibrous roots of Ophiopogon has great practical significance, in order to understanding more of the chemical composition of fibrous roots of Ophiopogon, and providing chemical basis for the utilization of resources of Ophiopogon, the chemical constituents of fibrous roots of Ophiopogon were studied in this paper.Firstly, summary on chemical constituents, steroids and homoisoflavones are the main constituents of Ophiopogon, it has been studied from 1978, up to now, there are 47 steriodes have been founded in Ophiopogon, their basic structure are spirostane and furostan, more over, only the herbs of O. japonicus contain homoisoflavones, and there are 38 homoisoflavonoids of Ophiopogon been reported, all of them are existed in the form of aglycone. Studies have shown that the steroids and pharmacological effects are similar between fibrous roots and tubers of Ophiopogon. Even the content of some main constituents in fibrous roots is more than those in tubers. As one of the main effective constituents, steroids of Ophiopogon has the obvious anti-hypoxic, anti-mutagenic and anti-tumor activity, homoisoflavonoids has the activity of anti-inflammatory, the inhibition of the phosphorylation, anti-estrogen, anti-mutagenesis, anti-tumor, anti-fungus and mycobacteria, antitussive, immunomodulation, improve blood condition, inhibition of the growth of melanocytes and anti-angiogenesis. In addition, polysaccharides are able to promoting humoral immunity and cellular immunity, and inducing a variety of cytokines. Plant polyphenol has the activity of anti-oxidant and anti-tumor. It can also inhibit platelet adhesiveness and induce vasodilation, so as to prevent coronary artery disease and angiosclerosis. Thus, more and more researchers pay attention to the total phenols of Ophiopogon .Secondly, a series of study of the chemical constituents of O. japonicus have been done in our lab, and five compounds have been identified, they are: methylophiopogonanone A, methylophiopogonanone B,β-sitosterol, ophiopogonin D and ophiopogonin D′. In this article, four known compounds have been isolated from the fibrous roots of O. japonicus, with the application of extraction of solvent, purification of macroporous resin, silica gel column chromatography, reversed-phase column chromatography and Sephadex chromatography. The process of extraction and isolation was as follows: The dried fibrous roots of O. japonicus (30 kg) obtained in Sichuan Province in China were extracted exhaustively with hot ethanol (85 %). The extract was combined and concentrated to dryness. The residue was dissolved in water and extracted with n-BuOH saturated with water to afford n-BuOH extract. The n-BuOH extract (400 g) was then chromatographed on a silica gel column, and then repeatedly chromatographed on reversed-phase column chromatography to afford four compound (1-3); The dried fibrous roots of O. japonicus (15 kg) obtained in Sichuan Province in China were extracted exhaustively with hot ethanol (85 %). The extract was combined and concentrated to dryness. The residue was dissolved in water and absorbed by macroporous resin, eluted with water to remove carbohydrates. After that eluted with lye and then neutralizate it with acid. Finally, the neutralization liquid was absorbed by macroporous resin again, then eluted with ethanol to afford total phenols. The total phenols (66 g) was then chromatographed on a silica gel column, and then repeatedly chromatographed on reversed-phase column chromatography and Sephadex LH-20 column chromatography to afford compound (4). The chemical structures of these compounds were elucidated by the basis of physicochemical properties and NMR spectroscopy. They are: ophiogenin 3-O-α-rhamnopyranosyl(1→2)-β-D-glucopyranoside (1); ophiogenin 3-O-α-L-[rhamnopyran -osyl(1→2)][β-D-xylopyranosyl(1→3)]-β-D-glucopyranoside(2); ruscogenin [2-O-acetyl-α-L -rhamnopyranosyl(1→2)][β-D-xylopyranosyl(1→3)]-β-D-fucopyranoside (3); N-(2-(4-hydrx -ylphenyl)ethyl)-4-hydroxycinnamide (4).Finally, we prepare the total phenols of fibrous roots of Ophiopogon japonicus by macroporous resin, The reference substance of N-(2-(4-hydroxyphenyl)ethyl)-4-hydroxycinn -amide is prepared here, establish a simple HPLC method for the determination of total phenols in fibrous roots of O. japonicus. The condition of HPLC are as follows: Kromasil C18 column (4.6 mm×250 mm, 5μm), mobile phase were acetonitrile and water (24 : 76) at a flow rate of 1.0 mL/min, the column tempreture was 25°C. Detection wave was 254 nm. Conclusion: The linear range is 0.6~3.6μg, r= 0.9996, the quantity of N-(2-(4-hydroxyphenyl) ethyl)-4-hydroxycinnamide in total phenols of fibrous roots of O. japonicus is 0.36 %, the content of the total phenols in fibrous roots of O. japonicus which is calculated by N-(2-(4-hydroxyphenyl)ethyl)-4-hydroxycinnamide is 0.016‰. The method is simple, accurate, rapid, sensitive. And it provided the research foundation for the further study of the quality standard of the total phenols in fibrous roots of O. japonicus.In conclusion, the study on the consititutes of fibrous roots of O. japonicus is contributed to the futher exploitation the resource of herbs, and it can promote the rational use of traditional Chinese herbs, thus the research has a certain theoretical significance and practical value.