Simultaneous quantification of six major phenolic acids in the roots of Salvia miltiorrhiza and four related traditional Chinese medicinal preparations by HPLC–DAD method

doi:10.1016/j.jpba.2005.10.021

Journal of Pharmaceutical and Biomedical Analysis

Volume 41, Issue 1, 11 April 2006, Pages 48-56

https://doi.org/10.1016/j.jpba.2005.10.021 Get rights and content

Abstract

A high-performance liquid chromatographic method was applied to the determination of danshensu, protocatechuic aldehyde, rosmarinic acid, lithospermic acid, salvianolic acid B and salvianolic acid A in the roots of Salvia miltiorrhiza and four related traditional Chinese medicinal preparations. The six phenolic acids were simultaneously analyzed with a Zorbax Extend C₁₈ column by gradient elution using 0.026% (v/v) phosphoric acid and acetonitrile as the mobile phase. The flow rate was 1 ml min⁻¹, and detection wavelength was set at 288 nm. The recovery of the method was in the range of 95.1–104.8%, and all the compounds showed good linearity (r > 0.9997) in a relatively wide concentration range. This assay was successfully applied to the determination of six major phenolic acids in 32 samples. The results indicated that the developed HPLC assay could be readily utilized as a quality control method for S. miltiorrhiza and its related traditional Chinese medicinal preparations.

Introduction

The dried roots of Salvia miltiorrhiza (Danshen), a traditional Chinese medicine, are widely used to treat coronary heart disease, cerebrovascular disease, bone loss, hepatitis, hepatocirrhosis and chronic renal failure [1], [2], [3], [4], [5], [6]. There are a number of traditional Chinese medicinal preparations (TCMPs)-containing Danshen such as Fufang Danshen tablet (FDT), Compound Danshen dripping pills (CDDP), Danshen injection (DSI), Xiangdan injection (XDI), etc., among which Danshen is the major component. The TCMPs-containing Danshen were mainly used to treat coronary heart disease, heart-stroke, cerebrovascular diseases and cardiovascular diseases [7], [8], [9], [10], [11], [12], [13]. To ensure their clinical efficacy, quality control of S. miltiorrhiza and its related TCMPs is of great importance.

According to the pharmacological investigations, the active constituents of Danshen are divided into two groups: phenolic acids which are water soluble and tanshinones which are lipophilic [14], [15], [16]. In the past 20 years, the research attention has been focused on the phenolic acids of Danshen due to their notable pharmacological activities [17], [18], [19], [20]. Up to now, quantitative determination of water soluble constituents in Danshen has been only focused on one or two compounds, which could not reflect the overall quality of Danshen. Based on the pharmacological research, protocatechuic aldehyde, rosmarinic acid and lithospermic acid have such biological activities as anti-atherosclerosis, anti-phlegmonosis, anti-oxidation and protecting myocardial damage [21], [22], [23], [24], [25], [26]. While danshensu, salvianolic acid B and salvianolic acid A not only have the actions described above, but also are the characteristic constituents of S. miltiorrhiza. Therefore, quantitative analysis of these constituents is significant for the quality control of S. miltiorrhiza and its related TCMPs.

Earlier we reported the metabolic studies of the total phenolic acids from the roots of S. miltiorrhiza and the major constituent—salvianolic acid B [27], [28], [29]. In the present paper, we intend to report the simultaneous quantification of the major phenolic acids (danshensu, protocatechuic aldehyde, rosmarinic acid, lithospermic acid, salvianolic acid B and salvianolic acid A) (see Fig. 1) in S. miltiorrhiza and related TCMPs including FDT, CDDP, DSI and XDI (see Table 1) by reversed phase liquid chromatography. The results suggested that contents of the six major phenolic acids could be used to evaluate the quality of S. miltiorrhiza and its related TCMPs.

Section snippets

Materials

All the samples were purchased from various drugstores around China (see Table 5). The roots of S. miltiorrhiza used to separate the standard compounds were obtained from Zhongjiang Danshen Cultivation Base in Sichuan province.

Chemicals and standards

HPLC grade acetonitrile and methanol (E. Merck, Darmstadt, Germany) were used for the HPLC analysis. Deionized water was purified by Milli-Q system (Millipore, Bedford, MA, USA). Phosphoric acid was of analytical grade from Beijing Beihua Fine Chemicals Co. Ltd. (Beijing,

Chromatographic conditions

A good separation condition should satisfy the need that the analyzed peaks have baseline separation with adjacent peaks within a short analysis time as far as possible. To obtain the chromatograms with the good separation, fixed phase, mobile phase, column temperature, detection wavelength and flow rate were, respectively, investigated.

For phenolic acids, the fixed phase of Zorbax Extend C₁₈ column was better than BDS-Hypersil C₁₈, YMC-Pack ODS-A C₁₈ and Luna C₁₈ columns. Various mixtures of

Conclusion

This was the first report on the simultaneous determination of six major phenolic acids in the samples of roots of S. miltiorrhiza and four Danshen-containing TCMPs. A simple, rapid and accurate assay method was successfully established. The results suggested that this HPLC method could be considered as good quality criteria to control the quality of S. miltiorrhiza and its related TCMPs.

Acknowledgements

We thank the Ministry of Science and Technology of China (2002BA906A, 2002DEA 20021 and 2001BAC01A56) and National Administration of Traditional Chinese Medicine (2004ZX01) for financial support of this work.

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Simultaneous quantification of six major phenolic acids in the roots of Salvia miltiorrhiza and four related traditional Chinese medicinal preparations by HPLC–DAD method

Abstract

Introduction

Section snippets

Materials

Chemicals and standards

Chromatographic conditions

Conclusion

Acknowledgements

J. Hepatol.

Cancer Lett.

Life Sci.

Biochim. Biophys. Acta

J. Pharma. Biomed. Anal.

J. Chromatogr. A