| Summary: | 碩士 === 國立臺灣師範大學 === 化學研究所 === 90 === Capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) are currently the most commonly used methods for analyzing the contents of constituents of Chinese herb drugs. In this study we have developed the CE conditions for analyzing Curcumae Rhizoma and used HPLC to identify the source of Rhei Rhizoma and detect its constituents in the living body.
This study is divided into four parts. The first part is on the development of CE analysis method. Curcumae Rhizoma is a commonly used Chinese herb drug derived from the dried rhizome of the Zingiberaceous plant Curcuma long L. The drug possesses antiphlogistic, antibacterial, antioxidation, antitumor and hypoglycemic effects. This study used CE to analyze the eight important pharmacologically active constituents, a-turmerone, b-tumerone, ar-turmerone, curcumin, demethoxycurcumin, bisdemethoxycurcumin, p-tolymethyl carbinol and curcumenol. Experimental results show that by means of the micellar electrokinetic capillary chromatography (MEKC) with the addition of the borate acid solution containing the surfactant SC (sodium cholate) as a buffer, we are able to analyze six of the constituents within 25 min. Whereas, a-turmerone and b-tumerone are inseparable because of their similarity in structure.
The second part discusses the differences of contents of sennoside B (SB), sennoside A (SA), rhein (RH), aloe-emodin (AE), emodin (EM) and chrysophanol (CH) in the formula Coptis and Rhubarb Combination extracted by different methods; and changes of Rhei Rhizoma constituents in artificial gastric fluid and artificial intestinal fluid for different compositions of the formula made up of the ingredients Rhei Rhizoma, Scutellariae Radix and Coptidis Rhizoma in different ratios. The constituents of Rhei Rhizoma undergo almost no changes in artificial gastric fluid. However, in artificial intestinal fluid, the Rhei Rhizoma constituents can inhibit the hydrolysis of the glycosides from Scutellariae Radix. Hence, in the presence of Scutellariae Radix, or when a larger amount of Rhei Rhizoma is incorporated in the formula, the various constituents of Rhei Rhizoma are consumed in a higher rate. This condition is most conspicuous in the formula composed of Rhei Rhizoma/Scutellariae Radix/Coptidis Rhizoma in the ratios of 2 : 1 : 1. Compared on the basis of equal amount of drug material, the extraction-yield ratios of the various constituents are SB 25%, SA 6%, RH 29%, AE 16%, EM 43% and CH 12%.
The third part compares the absorption and metabolism conditions with the formula extracted by different methods, and develops some suitable pretreatment method for the formula to be fed to mice whose blood and urine are then sampled and analyzed for differences in the contents of the various constituents of Rhei Rhizoma. This study uses Oasisâ Cartridge for the pretreatment, by which interfering substances can be effectively eliminated and the sample condensed. The recovery rates of the constituents are all above 90%, except AL, which is 79%. After pretreatment, the blood and urine samples have various LC peaks that do not present serious overlapping. The peaks corresponding to CH, RH, EM and AE can be clearly identified, and the column does not get clogged so easily. This study compares the formula extracted with water, 50% ethanol and mixed-decoction, and has found that the formula extracted by the mixed-decoction mode yields lower contents of the various constituents in the urine of the mice. Moreover, the tendency (pharmacokinetic) graphs of the various constituents show that the constituents from the formula prepared by mixed concoction appear earliest in the blood stream of the mice and remain there for the longest time, showing a prominent tendency of decline only after 12 hours.
The final part is on the collection of 31 samples derived from Rheum officinale Baillon, R. palmatum L. and R. tanguticum Maxim. which were analyzed with HPLC for nineteen constituents from these samples in order to establish a rule for identifying the sources of Rhei Rhizoma. The results show that the peak area ratios between the internal standard (IS) and 6 aloe-emodin-8-O-b-D-(6”-O-galloyl)- glucopyranoside, 12 aloe-emodin-w-O-b-D-gluco-pyranoside, 13 physcion-8-O-b-D- glucopyranoside, 15 aloe-emodin, 16 rhein, 18 chrysophanol and 19 physcion can serve as an indicator in identifying the source of Rhei Rhizoma, whereby we have found that the horse-hoof Rhei Rhizoma (Rheum officinale) has 15/IS < 0.24, 16/IS < 0.67, 18/IS < 0.38 and 19/IS < 0.1.While the Tangutic Rhei Rhizoma (Rheum tanguticum) and the palm-leafed Rhei Rhizoma (Rheum palmatum), which along with the horse-hoof Rhei Rhizoma all belong to the same grade known as “silk-grained Rhei Rhizoma”, have 15/IS > 0.25, 16/IS > 0.67, 18/IS > 0.49 and 19/IS > 0.08. The way to discriminate the Tungutic from the palm-leafed Rhei Rhizoma is that the former has 6/IS . 0.37, 12/is > 0.08 and 13/is > 0.1; and the latter, 6/IS < 0.36, 12/IS < 0.03 and 13/IS < 0.07 (The internal standard is prepared mixing 1 ml of 0.504 mg/ml methyl-2,4-dihydroxybenzoate solution with 0.2 g of sample in 45 ml of 70% methanol and making to 50ml. A 10-ml aliquot of the solution is then injected into LC for analysis). In addition, the Tungutic Rhei Rhizoma has 16/6 < 3.3, 18/6 < 2.0, 19/6 < 0.5; and the palm-leafed Rhei Rhizoma has 16/6 > 3.5, 18/6 > 3.4 and 19/6 > 0.6. Based on the above data, we are able to identify the sources of Rhei Rhizoma samples.
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