| Summary: | 博士 === 國立臺灣師範大學 === 化學研究所 === 89 === The quality of Chinese herbal drugs is determined by either chemical assay or bioassay, and the former is usually performed by capillary electrophoresis (CE) or high-performance liquid chromatography (HPLC). In this study, a number of analytical methods, including CE and HPLC, were developed to evaluate the quality of Cinnamomi Ramulus, Puerariae Radix and Ephedrae Herba, and to postulate the origin of Puerariae Radix and the bioavailability of Cinnamomi Ramulus.
Cinnamomi Ramulus contains coumarin, cinnamyl alcohol, cinnamaldehyde, cinnamic acid, 2-methoxycinnamaldehyde and cinnamyl acetate as its bioactive constituents. Here, we developed an HPLC and a CE method for determining the six cinnamomi constituents simultaneously. In micellar electrokinetic chromatography (MEKC), a buffer solution containing 18 mM sodium borate, 30 mM sodium dodecyl sulfate (SDS) and 12.5% isopropanol (v/v) was found to be the most suitable approach to determine the contents of these marker substances within 50 minutes. In HPLC, a buffer solution containing 30 mM sodium acetate, methanol and acetonitrile was applied to analyze these marker substances within 35 minutes.
An HPLC method and a CE method for the separation of seven components (puerarin, daidzin, 6,7-dimethoxycoumarin, daidzein, genistein, formononetin and biochanin A) in Puerariae Radix were developed. Detection at 254 nm with a linear gradient elution system of 30 mM dihydrogenphosphate buffer in HPLC or with 40 mM SDS and 10 mM sodium dihydrogenphosphate in CE was found to be able to separate these components well. Contents of the individual components in an unpretreated Puerariae Radix extract could be easily determined within 65 min by HPLC or 50 min by CE.
Two simple methods for simultaneous determination of six ephedrine alkaloids ((-)-l-ephedrine), (+)-d-pseudoephedrine, (-)-l-methylephedrine, (+)-d-methylpseudoephedrine, (-)-l-norephedrine and (+)-d-norpseudoephedrine) in Ephedrae Herba by HPLC were developed. The first one was carried out by using a Cosmosil 5C18-MS column with a gradient solvent system consisted of 50 mM phosphate buffer and acetonitrile, and the contents of alkaloids in non-pretreated Ephedra extracts could easily be determined in 50 min. Alternatively, the alkaloids could be determined by using a Cosmosil 5C18-MS column with an isocratic solvent system of 25 mM SDS - acetonitrile solution within 35 minutes.
A total of 25 samples of Puerariae Radix, 14 samples from the markets and 11 samples from Sun Ten Pharmaceuticals, were collected and assayed. Results showed that the samples bought from Taiwan herbal markets were all belonged to P. thomsonii. The two kinds of Puerariae Radix can be distinguished by the ratios of puerarin / daidzein or the contents of puerarin. The ratio puerarin / daidzein was higher than 11.92 in P. lobata, but less than 6.89 in P. thomsonii. In addition, the contents of puerarin were higher than 2.5 mg/g in P. lobata, but less than 2.0 mg/g in P. thomsonii. From data of chemical analysis of the herbal constituents, the origin and quality of a herb, could be postulated.
The bioavailability of cinnamomi constituents can be determined by assaying the contents of compounds existed in animal blood with HPLC. Experimental results showed that the major component in the blood of the mice was cinnamic acid regardless which were fed with cinnamic acid, cinnamaldehy or cinnamyl alcohol. Values for the content of cinnamic acid and the time of maximal concentration were calculated. The relative bioavailability of Cinnamomi Ramulus was higher than that of pure standards and also mixed marker substances. In vitro experiment, analytical data showed that the oxidating rate of cinnamaldehyde with mice blood in test tube is much slower than that in animals.
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