Study On The Methodology Of Quality Control Of Isoflavones From Rhizoma Belamcandae

Rhizoma Belamcandae (Shegan in Chinese), derived from the rhizomes of Belamacanda chinensis (L.) DC, and firstly recorded in Sheng Nong's Herbal Classic, has been used as a kind of folk medicine for the treatment of coughing and pharyngitis in China. It has many important pharmacologic actions, such as anti-inflammatory, anti oxidation, clear free radicle, anti-cancer, and so on.The constituents of Belamacanda chinensis include isoflavonoids, quinines, phenols, terpenoids, steroids and some micro-ingredients. Isoflavonoids are the major active compounds, such as Tectoridin, Iridin, Tectorigenin, Irigenin, Irisflorentin, etc.In the present paper, microwave-assisted extraction has been first used for the extraction of isoflavonoids. the orthogonal design L9(34)was used to select the optimum extraction process. To qualitative analyze nine main isoflavonoids by high performance liquid chromatography– diode array detector / electro spray ionization-mass spectrometry/mass spectrometry (HPLC-DAD/ESI-MS/MS), we developed a two-step HSCCC method to isolate and purify five main isoflavonoids from Belamacanda chinensis. A two-dimensional column-switching system for rapid isolation isoflavonoids in Belamacanda chinensis was established. Nine isoflavonoids in samples purchased from sixteen different locations were quantitative analyzed according to their finger prints.1. Study on extraction and assaying of isoflavonoids in Belamacanda chinensis.Soxhlet extraction, reflux extraction, ultrasonic extraction and microwave-assisted extraction were compared in extracting isoflavonoids from Belamacanda chinensis. Results showed that ultrasonic extraction and microwave-assisted extraction had a higher yield of isoflavonoids than soxhlet extraction and reflux extraction. Microwave-assisted extraction has been used to extract isoflavonoids from Belamacanda chinensis. The orthogonal design L9(34)was used to select the optimum extraction process. In this study, we stuied on the influence of different extraction solvents, the ratio of the weight of sample to the volume of solvent, time and temperature of extraction. As a result, the optimum extraction condition was: 60% ethanol as the extraction solvent, the ratio of the weight of sample to the volume of solvent to be 1:12, extraction time to be 10 min, and extraction temperature to be 75℃.2. Rapid identification of isoflavonoids in Belamacanda chinensis by HPLC-DAD/ESI-MS/MSA novel method including separation and identification of isoflavonoids from Belamacanda chinensis by high performance liquid chromatography– diode array detector / electrospray ionization-mass spectrometry/mass spectrometry (HPLC-DAD/ESI-MS/MS) was established. Initially, an analytical method based on nine of the major constituents of Belamacanda chinensis was characterized based on their retention behavior obtained on-line by their UV spectra and the HPLC-DAD/ESI-MS/MS. Nine isoflavonoids were preliminarily identificated as tectoridin, iristectorin B, iridin, irilin D, tectorigenin, iristectorigenin B, irigenin, irisflorentin and dichtomitin.3. Separation and purification of five isoflavonoids using HSCCC method.Five isoflavonoids including two isoflavonoid glycosides and three isoflavonoid aglycones were isolated and purified by a two-step high-speed counter-current chromatography (HSCCC). After four continuous injections, HSCCC solvent system composed of n-hexane–n-butanol–methanol–water (1:4:1.5:6, v/v/v/v) as enrichment step. The stationary phase composed mainly of aglycon components were reclaimed and further purified by another HSCCC with solvents systems: chloroform–methanol–water (4:2.7:2, v/v/v). The lower phase was used as the mobile phase in the head to tail elution mode. By injecting 800 mg of the enriched extract of B. chinensis, the 1st step HSCCC procedure yielded 17.6 mg of tectoridin and 23.6 mg of iridin with the purity of 98.2% and 98.5%, respectively. The 2nd HSCCC isolated 15.6 mg of Irisflorentin, 22.4 mg of irigenin and 5.2 mg of tectorigenin with the purity all over 98%, The chemical structure identification of all pure fractions was confirmed by MS analysis, 1H NMR and 13C NMR.4. Establish of two-dimensional column-switching system for rapid isolation of active compounds in Belamacanda chinensis.The introduction of six-port switching value instead of sample loop assured 100% recovery from the first dimension to the second one, and the injection volumes of the second dimension could reach 20 mL. The two-dimensional column-switching system without loop trapping was successfully applied for the isolation and purification of the five major isoflavonoids from Belamacanda chinensis. 87.2 mg of tectoridin, 108.9 mg of iridin, 24.2 mg of tectorigenin, 81.8 mg of irigenin and 53.7 mg of irisflorentin were yielded from 1.5 g crude extract power, and purities were all over 98.6% by HPLC.5. Quantitative determination of nine isoflavonoids in Belamacanda chinensis of different location by HPLC.A HPLC method for determining the contents of nine isoflavones in isoflavonoid extract of Belamacanda chinensis was set up. Nine isoflavonoids in samples purchased from sixteen different locations were analyzed. It was found that there were remarkable differences, in term of concentrations of the nine isoflavonoids among different places. The contents of Belamacanda chinensis from Shanxi, Hunan, Guangxi, Yunnan, etc. were found to be higher than other locations, and drugs from these locations could be better than other locations.