Author: To, Man-ling
Title: Application of chromatographic and chemometrics techniques to enhance chemical analysis of sheng mai san, cinnamomi cortex, radix ligustici chuanxiongand other Chinese herbal medicines
Degree: Ph.D.
Year: 2004
Subject: Hong Kong Polytechnic University -- Dissertations
Chromatographic analysis
Medicinal plants -- China -- Analysis
Materia medica -- China -- Analysis
Chemistry, Analytic
Department: Department of Applied Biology and Chemical Technology
Pages: xxii, 225 leaves : ill. (some col.) ; 30 cm
Language: English
Abstract: Chromatographic techniques are the most common analytical methods applied in many fields of chemistry. It is also applied to the Traditional Chinese medicines (TCM) which is widely used as therapeutic alternative within Chinese communities. In the past decade, the rapid development of computer technology and the chemometrics based methodologies enhance the data processing techniques of chromatography. Nowadays, the data analysis of chromatographic data becomes more accurate, reliable and user-friendly. In this research study, chromatographic studies together with chemometrics were applied for both qualitative and quantitative studies of TCMs including Sheng Mai San, Cinnamomi Cortex, RDLP and Radix Ligustici Chuanxiong. Sheng Mai San (SMS) is one of the tonic prescriptions with a compound formulation in traditional Chinese medicine (TCM) comprising of three herbs of Radix Ginseng (RG), Fructus Schisandrae (FS) and Radix Ophiopogonis (RO). It is used to replenish qi, restore the normal pulse, nourish yin, promote the production of body fluid, astringe yin-fluid and arrest sweat. For most analyses of the traditional Chinese patent formulas, the herbal compositions are determined separately and it is time-consuming in so doing. To tackle this problem, the three associated herbs of SMS were studied under the same experimental conditions using both Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC) techniques with modification of the solvent systems as developed in this work. In this approach, the identification of the three herbs within SMS was successful by carried out in such a simple way. The extracting solvent utilized for the samples was methanol. The solvent system for TLC analysis was ethyl acetate-hexane- methanol- chloroform (1:2:1:1.5 v/v) and the mobile phase for HPLC study was 32.5% of acetonitrile with 67.5% of buffer solution (0.01M (NH4)H2PO4 in water) under gradient elution. In this way, the three herbs in SMS can be identified and distinguished easily. TCM and its related products are rapidly accepted by many nations as alternative medicines. However, it is difficult to make a conclusion on the quality of the herbs by focusing only on the amount of a few active ingredients as TCM is a complex mixture with multiple components and compositions. Chromatographic fingerprint is the most effective approach to assess authenticity and quality of TCM. A single herb, Cinnamomi Cortex, is one of the important TCM herbs for medical uses and also a common flavoring for food, baked goods and beverages. Twenty-seven Cinnamomi Cortex samples from P.R. China, Vietnam and Burma were collected and their volatile oil contents of Cinnamomi Cortex were analyzed by using Gas Chromatography-Mass Spectroscopy (GC-MS). Eighteen common components were found and 16 of them were identified by the MS library (Wiley138) and they are benzaldehyde, benzenepropanal, cis-cinnamaldehyde, cinnamaldehyde, alpha-copaene, (-)-beta-elemene, (+)-sativen, zingiberene, trans-caryophyllene, gamma-muurolene, (-)-alpha-muurolene, gamma-cadinene, delta-cadinene, 3,8-triene-cadala- 1 [10], cubenol and cadalin. The GC-MS procedure developed in this investigation is good for authentication of a suspected Cinnamomi Cortex sample by checking whether all those eighteen peaks are observed. Besides, the chromatographic fingerprint of Cinnamomi Cortex was established by using the computer aided similarity evaluation (CASE), which is a chemometric based method, applied to the whole GC-MS chromatograms of all the twenty-seven samples. In the Principal Component Plot (PCA) of these chromatograms, the quality of Cinnamomi Cortex samples can be easily categorized into three types according to their relative percentages of the active ingredient cinnamaldehyde. Type one has the relative content over 70%, while types two and three within 50-70% and less than 50% respectively. Through the procedure and results obtained, the authenticity and quality of the Cinnamomi Cortex samples can be verified in a simple, reliable and effective way. In addition, a rapid, simple and nondestructive method for analyzing of Cinnamomi Cortex according to the volatile oil content of cis-cinnamaldehyde, cinnamaldehyde and alpha-copaene was developed using near-infrared diffuse reflectance (NIRR) spectroscopy. GC-MS method was employed as the reference method in the examination. The NIRR calibration equations obtained by the Partial Least-Squares (PLS) regression with the pre-treatment of standard normal variate (1100-2500 nm) and the second derivative (segment = 10 nm, gap = 3 nm) gave very good correlation with those of GC-MS method. The coefficient of determination for the calibration equations (R2) of cis-cinnamaldehyde, cinnamaldehyde and alpha-copaene are 0.91, 0.90 and 0.96 respectively. The overall performance showed that it is good for future analysis of volatile oil of Cinnamomi Cortex samples by using the NIRR spectroscopy. Qualitative and quantitative analyses of the active ingredients are the most common method for TCM investigation. However, the standards of the active ingredients are usually not commercially available. Preparative high performance liquid chromatography (PHPLC) is a potential tool to solve the problem. Preparative chromatography technique is able to isolate milligram to gram quantities of pure substances for structural studies, bioassays, pharmacological tests, reference substances and standards for quantitative determinations. However, some practical problems were found during the scale-up process. The pilot study of the collection of RDLP, a TCM formulation which has been used for more than 20 years to treat liver cancer, explored the practical concerns in various aspects during scale-up relating to sample introduction, pump, column, detector, solvent recycler, fraction collector and rotary evaporator. Although PHPLC is an expensive method, the results obtained can be excellent. It is worth while to do so because TCM standards are rare and very expensive. Moreover, Radix Ligustici chuanxiong is one of the herbs that regulate the blood. It is a very effective herb for invigorating the blood and promoting its circulation. The standard of the active ingredients are not available in the commercial market. PHPLC technique was applied to isolate the main constituents of Radix Ligustici chuanxiong. The multisimplex treatment with the use of the MultiSimplex(R) software was employed to optimize the experimental condition in HPLC analysis. It gave the optimum condition through eleven trials for Radix Ligustici chuanxiong. In the n-hexane extracts of L. chuanxiong, five active components were isolated successfully in the PHPLC system and they are senkyunolide A, butylphthalide, neocnidilide, z-ligustilide and z-butylidienephthalide. All of them are good for qualitative purpose because of their significant in the chromatograms. The purity of butylphthalide, z-ligustilide and z-butylidenephthalide are over 94% in both HPLC and GC-MS studies. They are good to serve as the standards for quantitative analysis.
Rights: All rights reserved
Access: open access

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