Quality evaluation of Flos Lonicerae through a simultaneous determination of seven saponins by HPLC with ELSD

https://doi.org/10.1016/j.chroma.2005.02.031Get rights and content

Abstract

A new HPLC coupled with evaporative light scattering detection (ELSD) method has been developed for the simultaneous quantitative determination of seven major saponins, namely macranthoidin B (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-d-glucopyranosyl(6→1)-O-β-d-glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-l-arabinopyranosyl(2→1)-O-α-l-rhamnopyranoside (7) in Flos Lonicerae, a commonly used traditional Chinese medicine (TCM) herb. Simultaneous separation of these seven saponins was achieved on a C18 analytical column. The mobile phase consisted of (A) acetonitrile–acetic acid (95:0.5) and (B) 0.5% aqueous acetic acid using a gradient elution of 29%A at 0–10 min, 29–46%A at 10–25 min and 46%A at 25–30 min. The drift tube temperature of ELSD was set at 106 °C, and with the nitrogen flow-rate of 2.6 l/min. All calibration curves showed good linear regression (r2 > 0.9922) within test ranges. This method showed good reproducibility for the quantification of these seven saponins in Flos Lonicerae with intra- and inter-day variations of less than 3.0% and 6.0%, respectively. The validated method was successfully applied to quantify seven saponins in five sources of Flos Lonicerae, which provides a new basis of overall assessment on quality of Flos Lonicerae.

Introduction

Flos Lonicerae (Jinyinhua in Chinese), the dried buds of several species of the genus Lonicera (Caprifoliaceae), is a commonly used traditional Chinese medicine (TCM) herb. It has been used for centuries in TCM practice for the treatment of sores, carbuncles, furuncles, swelling and affections caused by exopathogenic wind-heat or epidemic febrile diseases at the early stage [1]. Though four species of Lonicera are documented as the sources of Flos Lonicerae in China Pharmacopeia (2000 edition), i.e. L. japonica, L. hypoglauca, L. daystyla and L. confusa, other species such as L. similes and L. macranthoides have also been used on the same purpose in some local areas in China [2]. So it is an important issue to comprehensively evaluate the different sources of Flos Lonicerae, so as to ensure the clinical efficacy of this Chinese herbal drug.

Chemical and pharmacological investigations on Flos Lonicerae resulted in discovering several kinds of bioactive components, i.e. chlorogenic acid and its analogues, flavonoids, iridoid glucosides and triterpenoid saponins [3]. Previously, chlorogenic acid has been used as the chemical marker for the quality evaluation of Flos Lonicerae, owing to its antipyretic and antibiotic property as well as its high content in the herb. But this compound is not a characteristic component of Flos Lonicerae, as it has also been used as the chemical marker for other Chinese herbal drugs such as Flos Chrysanthemi and so on [4], [5]. Moreover, chlorogenic acid alone could not be responsible for the overall pharmacological activities of Flos Lonicerae [6]. On the other hand, many studies revealed that triterpenoidal saponins of Flos Lonicerae possess protection effects on hepatic injury caused by Acetaminophen, Cd, and CCl4, and conspicuous depressant effects on swelling of ear croton oil [7], [8], [9], [10], [11]. Therefore, saponins should also be considered as one of the markers for quality control of Flos Lonicerae. Consequently, determinations of all types of components such as chlorogenic acid, flavonoids, iridoid glucosides and triterpenoidal saponins in Flos Lonicerae could be a better strategy for the comprehensive quality evaluation of Flos Lonicerae.

Recently an HPLC-ELSD method has been established in our laboratory for qualitative and quantitative determination of iridoid glucosides in Flos Lonicerae [12]. But no method was reported for the determination of triterpenoidal saponins in Flos Lonicerae. As a series studies on the comprehensive evaluation of Flos Lonicerae, we report here, for the first time, the development of an HPLC-ELSD method for simultaneous determination of seven triterpenoidal saponins in the Chinese herbal drug Flos Lonicerae, i.e. macranthoidin B (1), macranthoidin A (2), dipsacoside B (3), hederagenin-28-O-β-d-glucopyranosyl(6→1)-O-β-d-glucopyranosyl ester (4), macranthoside B (5), macranthoside A (6), and hederagenin-3-O-α-l-arabinopyranosyl(2→1)-O-α-l-rhamnopyranoside (7) (Fig. 1).

Section snippets

Samples, chemicals and reagents

Five samples of Lonicera species, L. japonica from Mi county, HeNan province (LJ1999-07), L. hypoglauca from Jiujang county, JiangXi province (LH2001-06), L. similes from Fei county, ShanDong province (LS2001-07), L. confusa from Xupu county, HuNan province (LC2001-07), and L. macranthoides from Longhu county, HuNan province (LM2000-06), respectively, were collected in China. All samples were authenticated by Dr. Ping Li, professor of department of Pharmacognosy, China Pharmaceutical

Results and discussions

The temperature of drift tube and the gas flow-rate are two most important adjustable parameters for ELSD, they play a prominent role to an analyte response. In our previous work [12], the temperature of drift tube was optimized at 90 °C for the determination of iridoids. As the polarity of saponins are higher than that of iridoids, more water was used in the mobile phase for the separation of saponins, therefore the temperature for saponins determination was optimized systematically from 95 °C

Conclusions

This is the first report on validation of an analytical method for qualification and quantification of saponins in Flos Lonicerae. This newly established HPLC-ELSD method can be used to simultaneously quantify seven saponins, i.e. macranthoidin B, macranthoidin A, dipsacoside B, hederagenin-28-O-β-d-glucopyranosyl(6→1)-O-β-d-glucopyranosyl ester, macranthoside B, macranthoside A, and hederagenin-3-O-α-l-arabinopyranosyl(2→1)-O-α-l-rhamnopyranoside in Flos Lonicerae. As the saponin profile alone

Acknowledgements

This project is financially supported by Fund for Distinguished Chinese Young Scholars of the National Science Foundation of China (30325046) and the National High Tech Program (2003AA2Z2010).

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