| Objective: To measure the content of total flavonoids of Xinjiang Glycyrrhiza inflate Bat(GIB), by comparing those of Glycyrrhiza glabra L(GGL) as reference; To observe the distribution and measure the content of Licochalcone A(Lico A) from whole plant of GIB; To establish the process of preparing Lico A reference product; To study on the anti-cervical cancer activity of flavonoid extracts of the two species of Xinjiang licorice; To preliminary study the GIB cervical cancer suppository formula, and optimizing the ingredients composition. Methods: 1. Using ultrasonic assisted ethanol extraction method was used for preparing the crude extract of two kinds of licorice; the polyamide and macroporous resin column chromatography was used for purifieng the total flavonoids; The UV method was used for determination of the content of crude and purified total flavonoids in two kinds of licorice extracts. 2. The qualitative scaning was used within thin layed chromatographic method for identifieng the species specific flavonoid substance Lico A in the whole plant of GIB, and HPLC method was used to determine the content of Lico A in GIB roots and stems. 3. The silica gel column chromatography was used for progressive purification of Lico A from the extract of GIB root, and study on the preparation technology of Lico A reference substance. 4. The MTT assay was used for study on the anticancer activities of four kinds of licorice extracts(crude and purified) and monomer Lico A towards human Si Ha and He La cervical cancer cells proliferation, cisplantin was used as positive control; the target objects were intervening for the cells in 24 h, 48 h and 72 h. The apoptotic effects were measured by flow cytometry; 5. GIB cervical cancer suppository formula was initially prepared within GIB ethanolic extract as main component,and the qualitive identification was performed.Results: 1. The contents of total flavonoids in the crude extracts of GIB and GGL were 5.6% and 3.6%, and those in the purified flavonoid extracts were 56.4% and 49.7%, respectively. The special TLC spot in GIB ethanolic extract was identified as Licochalcone A(Lico A), which was only found in GIB root and stem, but not found in leaf and seed. 2. By using the HPLC method, the content of Lico A in the root of GIB was 0.468%, and in the stem was 0.254%. 3. The yield of Lico A reference product prepared by different chromatographic purification method was 3.37mg/g(by polyamide), 3.05mg/g(by macroporous resin) and 3.56mg/g(by silica gel) respectively. 4. The four kinds of licorice extracts showed different degrees of anti-cervical cancer activity. Their inhibition rates were respectively about 10.7%-92%(Si Ha cells) and 13.1%-93.7%(He La cells), when the concentration range was at 25~175μg/m L. Among them, the inhibitory effect of GIB purified flavonoid extract was much strong than those of GGL. The target object have the time-dependent maner. The experimental results show that their apoptotic effect towards Si Ha and He La cells were 26.9% and 38.7% at the concentration of 125μg/m L and 100μg/m L. 5. Furthermore, the GIB cervical cancer suppository formula was initially studied, and the initial quality standards was established. Conclusion: 1. The content of total flavonoids in GIB was higher than those in GGL; the co-utilization of polyamide and macroporous resin was an effective method for flavonoid purification than that of single polyamide method. This method allows us to reach easily to the standard of traditional Chinese medicine extract containing 50% of total flavonoids. 2. Lico A is mainly distributed in the root and stem of GIB, this is the first time to proposed the GIB stems can be used as a new resource for acquisition of Lico A. Repeated silica gel column chromatographic method could be the effective way for high performance preparation of Lico A in the laboratory condition. 3. The flavonoid extracts of two kinds of licorice showed, at a certain range of concentration, different degree of cervical cancer cell inhibition and promote apoptosis activities. Among them, the anticancer activity of GIB flavonoid extract was higher than the GGL. The results of this study could lay an important base for the progressive research and development of Xinjiang GIB licorice resources. |
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