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Determination and assay validation of luteolin and apigenin in human urine after oral administration of tablet of Chrysanthemum morifolium extract by HPLC

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Abstract

A simple, selective, precise, and accurate RP-HPLC assay for simultaneous analysis of luteolin and apigenin in human urine was developed and validated. Prior to HPLC analysis, urine samples were incubated with β-glucuronidase/sulfatase. Separation and quantification were achieved on an Agilent C18 column under isocratic conditions using a mobile phase (methanol:0.2% phosphoric acid aqueous solution 55:45, v/v) maintained at 1.0 ml/min at 30 °C. The standard curves were linear over the range of 0.0975–7.800 and 0.1744–13.95 μg/ml for luteolin and apigenin, respectively (r > 0.999). The assay recoveries for luteolin and apigenin were above 85.7%. The intra-day and inter-day precision (R.S.D.) for luteolin were below 2.2 and 4.0%, respectively, and for apigenin were less than 2.8 and 5.4%, respectively. Stability studies showed three concentration of luteolin and apigenin in urine quality control samples were stable undergoing three freeze–thaw cycles, storage at room temperature for 4 h, and at −20 °C for 3 days. The limit of quantitation was 39.20 ng/ml (n = 5) for luteolin and 31.45 ng/ml (n = 5) for apigenin in human urine. The method developed was employed successfully to determine luteolin and apigenin in urine samples obtained from eight healthy volunteers following oral administration of tablet of Chrysanthemum morifolium extract (CME).

Introduction

The flower of Chrysanthemum morifolium Ramat. (CM) has been used as healthy food and folk medicine for sudorific, antidote, and clearing away pathogenic heat in China. The previous study of our Lab indicated CM attenuated contractile function and coronary flow reduction of isolated rat heart caused by ischemia/reperfusion [1]. Our study also showed CM induced both endothelium dependent and independent relaxation of rat thoracic aorta caused by phenylephrine or high level of K+ [2]. Clinical study demonstrated CM had beneficial effect on coronary heart disease and could prevent of the cardiovascular diseases [3]. Studies showed flavonoids such as luteolin-7-O-β-d-glucoside, apigenin-7-O-β-d-glucoside were the main components of CM [4]. It is widely considered that flavonoid glucosides can be hydrolyzed in the gastrointestinal tract by the enzymes of intestinal bacteria [5] or the corresponding liver enzymes [6] followed by absorption and conjugation of their aglycones, which subsequently are excreted in urine mainly as sulfuric acid/glucuronic acid conjugates and to a lesser extent as free aglycones [7], [8]. Our study has showed luteolin-7-O-β-d-glucoside and apigenin-7-O-β-d-glucoside in C. morifolium extract (CME) could be hydrolyzed when CME incubated with intestinal bacteria, which could explain why luteolin-7-O-β-d-glucoside and apigenin-7-O-β-d-glucoside were not be detected in plasma of dog even if 15 g/kg of CM was administrated orally. As we speculated, luteolin and apigenin were detected in dog [9], rabbit, rat, and human plasma.

Since luteolin and apigenin possess the same effect as CM, such as vasodilatation [10], [11] and antioxidation [12], we think they are the actual bioactive constituents of CM. The determination of luteolin and apigenin in plasma and the pharmacokinetics in dog after oral dose of CME have been reported [9]. However, the urine excretion of luteolin and apigenin after oral administration of CME or luteolin and apigenin or their glucosides contained drug has not been reported. Therefore, the aims of the present study were to develop and validate a simple and sensitive HPLC method for simultaneous analysis of luteolin and apigenin in human urine, and apply the developed method for determination of luteolin and apigenin in human urine of volunteers following oral administrated of CME tablet.

Section snippets

Materials

Luteolin and apigenin (purity > 99%) were obtained from J & K-ACIoS (serial number: 62696) and Sigma–Aldrich Company (Lot 111K1520), respectively. C. morifolium extract (CME) tablet (Lot 040515), containing 7.13 mg of luteolin and 5.42 mg of apigenin in each tablet determined by HPLC after being hydrolyzed with hydrochloric acid, was provided by the Institute of Medicine, Zhejiang University, China. β-Glucuronidase (Type IX-A, from E. coli) and sulfatase (Type H-1, from pomatia) were purchased from

Chromatographic selectivity

This simple procedure afforded efficient separation and quantification of luteolin and apigenin in human urine, without interference of peaks of endogenous constituents from urine and reagents. Fig. 1 showed the typical chromatograms of blank urine, blank urine spiked with standard substances (0.1872 μg/ml luteolin and 0.3348 μg/ml apigenin), and urine sample after oral administration of CME tablet for 2 h. Luteolin and apigenin were eluted in approximately 10.5 and 16.0 min, respectively (Fig. 1).

Linearity and range

Chromatographic conditions

To achieve good separation of luteolin and apigenin from interfering compounds in the human urine, methanol, and 0.2% phosphoric acid at different ratio (55:45, 52:48, 50:50, v/v) had been used as mobile phase. The results revealed that no interference peaks from endogenous constituents presented in urine and reagents were observed at the retention time of luteolin and apigenin. Due to the reasonable retention times with the symmetrical peaks and good separation, a mixture of

Conclusion

In the present study, a simple, selective, precise, and accurate HPLC assay was established and employed to simultaneously analyze luteolin and apigenin in human urine of eight healthy volunteers following a single oral dose of CME tablets. It also can be applied to determine luteolin and apigenin in urine of experimental animals after administration of Chinese traditional medicine or nature drug containing luteolin, apigenin or their glucosides.

Acknowledgements

This study was supported by the Committee of Science and Technology of Zhejiang Province, China (No. G20020578) and Zhejiang Nature Science Foundation, China (No. Y204379).

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