Studies On The Chemical Constituents And Content Determination Of Fibrous Roots Of Ophiopogon Japonicus

The tuber of Ophiopogon japonicus (Thunb.) Ker-Gawl. (Liliaceae), a traditional Chinese medicine, is recorded in the Pharmacopoeia of the People's Republic of China (2005), vol.I. It has been used in combination with Panax ginseng and Schisandra chinensis for pharmacological effects on caidiovascular diseases and inflammatory. Phytochemical researches have shown that this plant mainly includes steroidal saponins, homoisoflavonoids, polysaccharides and so on. In order to develop medical resources of this crude drug, the chemical constituents and content determination of fibrous roots of O. japonicus were studied in this present paper.Firstly, advances on chemical constituents, pharmacological effects and content determination of Ophiopogon Ker-Gawl. (Liliaceae) were reviewed. 43 steroidal saponins, 38 homoisoflavonoids and many other constituents have been found since 1968. Moreover, 26 steroidal saponins and 35 homoisoflavo- noids were isolated from O. japonicus. Pharmacological investigations performed so far on O. japonicus have shown that this herb has several biological activities, such as antithrombosis, protection of myocardial ischemia, anti-inflammatory, antitumor, prevention of Sjogren's syndrome, hypoglyeemie effect and antitussive action.Many research efforts were made to determine the contents of steroidal saponins and homoisoflavonoids in O. japonicus, including ophiopogonin D, ophiopogonin B, methylophiopogonanone A and methylophiopogonanone B, respectively. Several methods, including HPLC-UV and HPLC-ELSD, have been reported. Much attention has been paid to the fibrous roots of O. japonicus since 1990. The studies on chemical constituents, pharmacological effects and content determination revealed that the fibrous roots and tubers of O. japonicus are similar. However, the content of ophiopogonin D was 6 times higher in fibrous roots than that in tubers of O. japonicus.Secondly, GC/MS was used to compare the chemical constituents of fibrous roots and tubers of O. japonicus obtained in Sichuan Province in China. 61 peaks were found in fibrous roots of O. japonicus and 42 peaks were found in tubers of O. japonicus, respectively. In addition, 36 compounds were identified and confirmed by analysis of MS spectra and indexing of NIST 98 spectral database. Comparing with literatures, 29 new compounds, such asβ-elemene,α-caryophyllene,β-caryophyllene, longifolene-(V4), butylated hydroxytoluene, n-hexadecanoic acid, squalene and many other constituents have been found in this herb.Five known compounds have been isolated from the fibrous roots of O. japonicus. The process of extraction and isolation was as follows: The dried fibrous roots of O. japonicus (15kg) obtained in Sichuan Province in China were extracted exhaustively with hot ethanol (85%). The extract was concent- rated to dryness. The residue was dissolved in water and extracted with Et2O and n-BuOH saturated with water to afford Et2O extract and n-BuOH extract, respectively. The Et2O extract (50g) was then chromatographed on a silica gel column and eluted with gradient mixtures of petroleum ether and ethyl acetate (14:1, 10:1, 5:1, 0:1) to afford four fractions. The 10:1 fraction was further subjected to repeated silica gel column chromatography using petroleum ether and ethyl acetate (8:1) as eluents to affordβ-sitosterol (24mg). The 5:1 fraction passed over a Sephadex LH-20 column eluted with absolute ethanol to give methylophiopogonanone A (83mg) and methylophiopogonanone B (72mg), respectively. The n-BuOH extract (50g) was submitted to column chromatography over silica gel with chloroform, acetone and methanol to afford three fractions. The acetone fraction eluted was subjected to on a silica column chromatography gel using chloroform-methanol-ethyl acetate-water (2:2:4:1, lower layer) as eluents to afford four fractions (I-IV) which were combined according to TLC monitoring. The fraction IV was then chromatographed on a silica gel and eluted with chloroform-methanol-water (6:5:1, lower layer) to give ophiopogon D (38mg) and ophiopogon D′(21mg), respectively. The chemical structures of these compounds were identified by NMR spectroscopy.Finally, a simple HPLC method was established for simultaneous determination of methylophiopogonanone A (MOPA) and methylophiopogon- anone B (MOPB) in fibrous roots of O. japonicus obtained in Sichuan Province in China. Meanwhile, the content of MOPA and MOPB in tubers and leaves of O. japonicus and 16 samples purchased from different stores in Changchun City were determined and compared with that of fibrous of fibrous roots of O. japonicus. The separation of the two homoisoflavonoids was achieved on a stainless steel Kromasil C18 column (4.6mm×250mm, 5μm) at 25℃. The solvents used for HPLC separation of the two homoisoflavonoids in samples were carried out with acetonitrile and 0.2% acetic acid (50:50) at a flow rate of 1.0mL/min. Detection wave was set at 296nm. The results indicated that the content of MOPA and MOPB and the total content of the two homoisoflavonoids in fibrous roots and tubers of O. japonicus were higher than that in market samples. The content of MOPA and MOPB in fibrous roots were 1.8 times and 2.5 times higher than that in tubers, respectively. The total content of MOPA and MOPB in fibrous roots was 2 times higher than that in tubers. The variation trend of the content of the two homoisoflavonoids among 7 market samples was not obvious. However, the two homoisoflavonoids in leaves and 9 market samples were not detected.In conclusion, this present paper provided valuable experimental data for further development of fibrous roots of O. japonicus.