Aims. The objectives of this study were (i) to isolate and characterize by conventional column chromatography, structurally diverse triterpenes from G. lucidum to serve as chemical markers; (ii) to develop a high performance liquid chromatography (HPLC) method for the quality control and/or standardization of Lingzhi-containing products; (iii) to utilize and optimize operating conditions for the newer extraction technology: supercritical fluid extraction (SFE), in order to maximize yields of bioactive triterpenes, and to reduce time and costs. / Background. The dried fruiting body of Ganoderma lucidum, commonly known as Lingzhi, has been used extensively as a traditional Chinese medicine (TCM) for many centuries not only in China, but also in other countries such as Japan and Korea. In recent years, Lingzhi has also become a popular health supplement in many Western countries. The chemical composition of Lingzhi is complex, but it has been well documented that the lipophilic triterpenoid class of compounds possess a range of biological effects that include antitumor, immunomodulatory, cardiovascular, respiratory and antihepatotoxic activity. A major drawback in TCM research has been the lack of authentic chemical standards, and efficient methods for the extraction and analysis of bioactive fractions and/or single components. Conventional extraction methods for G. lucidum are time-consuming and laborious, and often result in low yields of useful chemical constituents. / Conclusion. This study enabled the development of a method for the simultaneous analysis of structurally diverse triterpenes with remarkably different chromatographic profiles. The isolated triterpenes, as chemical markers, and the HPLC method can readily be used for quality control and/or standardization purposes in evaluating Lingzhi-containing products. Optimization of operating conditions for SFE facilitated the rapid and selective extraction of acidic triterpenes from raw G. lucidum in significantly higher yields. / Methods. Raw material of G. lucidum was extracted with 80% ethanol; subjected to repeated column chromatography to purify triterpenes; and characterized by NMR (1H and 13C) and mass spectroscopy. Isolated lipophilic triterpenes were qualitatively and quantitatively analyzed by reversed-phase HPLC using an ODS column (150 x 4.6 mm) and PDA detection at 256 nm. The assay was validated over appropriate concentration ranges and benzophenone was used as an internal standard. Supercritical fluid extraction of G. lucidum was carried out using a commercial supercritical fluid extractor system Thar, SFE-1000M. Briefly, the raw powder of G. lucidum was soaked in ethanol containing 10% aqueous ammonia for 30 minutes prior to extraction. Extractions were performed at temperatures of 40, 50 and 60°C; and pressures of 200, 250, 300 and 350 bar; 5% ethanol was used as the co-solvent; and the flow rate of CO 2 was set at 20 g/min. / Results. Eight compounds were isolated and identified from G. lucidum: four triterpenes; namely, lucidenic acid N, ganoderic acid B, ganodermanontriol, and ganodermadiol; two steroids; ergosterol-7, 22-dien-3beta-ol and ergosterol peroxide; and two fatty acids, oleic acid and tetracosanoic acid. The four triterpenes were utilized as chemical markers, and the developed HPLC method was able to simultaneously analyze the structurally diverse components with good resolution. The total analysis run time was 110 min, and retention times (tR) were 14.88, 18.96, 63.88 and 90.73 min respectively, and the eluting system was a mixture of three solvents, methanol (A), acetonitrile (B) and 2% acetic acid solution (C): 0-22 min, 5% A, 25% B and 70% C; 22-85 min, gradient elution, the ratio changed gradually to 5% A, 80% B and 15% C; 85-110 min, gradient elution, the ratio changed gradually to 5% A, 85% B and 10% C. The validated HPLC method and the isolated chemical markers were effectively applied to determine the triterpenoid contents in a variety of commercial Lingzhi products. Supercritical fluid extraction conditions of: pressure 300 bar and temperature 50°C, gave the highest yields of triterpene-containing extracts. HPLC analysis of the SFE extracts showed predominantly acidic triterpenes such as lucidenic acid N and ganoderic acid B. / Hong Xin. / "December 2006." / Advisers: Ho Yee Ping; Albert H. L. Chow. / Source: Dissertation Abstracts International, Volume: 68-09, Section: B, page: 5968. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 149-175). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_343826 |
| Date | January 2006 |
| Contributors | Hong, Xin, Chinese University of Hong Kong Graduate School. Division of Pharmacy. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, theses |
| Format | electronic resource, microform, microfiche, 1 online resource (xxii, 209 p. : ill.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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