Simultaneous extraction of epimedin A, B, C and icariin from Herba Epimedii by ultrasonic technique
Introduction
Herba Epimedii, the aerial parts of species of Epimedium L. (Berberidaceae), is one of the most famous Chinese herbal medicines listed in the Pharmacopoeia of the People’s Republic of China [1]. Herba Epimedii has been utilized to treat cardiovascular diseases and other chronic illness (infertility, amnesia and neurasthenia, impotence, senile functional diseases, etc.) in China for over 2000 years [2]. It is also grown as a herb for various medicinal purposes in Japan, Korea and the Mediterranean region [3], [4]. The major active constituents of Herba Epimedii are flavonoids and more than 60 kinds of flavonoids have been identified [5], [6], [7]. Among them, epimedin A, B, C and icariin are considered major bioactive components that make up more than 52% of total flavonoids in Herba Epimedii [8]. Epimedin B, C and icariin were found being able to promote the proliferation of the osteoblast-like cells and having potential activity against osteoporosis [2]. Recent researches also demonstrated that epimedin A, C and icariin exhibit anti-tumor and immunoregulatory effects [6], [9], [10], [11], [12]. Epimedin A and icariin are observed to improve the function of the cardiovascular system [6], [12]. Besides, icariin is considered to be a potential source of antioxidants, antidepressants and antiobesity agents [3], [13], [14]. Therefore, epimedin A, B, C and icariin possess important pharmacological activities and may become promising phytopharmaceuticals or nutraceuticals. It is of great interest to extract these medicinally active constituents from Herba Epimedii simultaneously.
Extraction and determination of epimedin A, B, C and icariin in Herba Epimedii are also essential for the quality evaluation of the traditional Chinese medicine. A large number of studies found that the quality of Herba Epimedii often varied considerably according to its species, harvest season, storage, geographic origin or other growing conditions [15], [16], [17]. Epimedin A, B, C and icariin are usually used as the marker compounds for evaluating Herba Epimedii by high-performance liquid chromatography (HPLC) [8], [11], [17] and capillary zone electrophoresis [18]. Though determining the contents of epimedin A, B, C or icariin have been widely described [8], [18], no study has been reported on the extraction of the four flavonoids simultaneously. Conventional Soxhlet extraction and boiling extraction are very time-consuming and require relatively high extraction temperature, which have been used for several decades. Thus, there is an increasing demand for a novel extraction technique with shortened extraction time, reduced extraction temperature, and increased extraction efficiency.
Ultrasonic technique is being used widely in analytical chemistry, facilitating different steps in the analytical process, particularly in sample preparation [19], [20], [21]. Ultrasonic-assisted extraction (UAE) is an expeditious, inexpensive and efficient alternative to traditional extraction techniques and, in some cases, even to supercritical fluid and microwave-assisted extraction, which has been demonstrated by application to both organic and inorganic analytes in a wide variety of samples [22]. UAE of icariin from Herba Epimedii has been performed, but the recovery of icariin was not satisfactory [23]. Although total flavonoids of Herba Epimedii have also been extracted by means of ultrasonication, the recoveries of the four marker flavonoids (i.e. epimedin A, B, C and icariin) have not been determined [24]. Actually, total flavonoids are composed of not only bioactive constituents (e.g., icariin), but also various pigments without significant medicinal activities [15]. So far, there is no report for the simultaneous extraction of epimedin A, B, C and icariin using ultrasonic technique. The goals of this study are to optimize the UAE condition for extracting epimedin A, B, C and icariin from Herba Epimedii, compare UAE with boiling extraction and Soxhlet extraction, characterize the photostability of these four flavonoids in the crude extract, and measure the contents of these four flavonoids in 20 Epimedium species by HPLC method coupled with UAE method.
Section snippets
Reagents and materials
Standards of epimedin A, B, C and icariin were purchased from ChromaDex (USA). Ethanol and glacial acetic acid of analytical grade (Tianjin Bodi Chemicals Co. Ltd., China) were filtrated through 0.45 μm microporous membranes before use. Acetonitrile of HPLC grade (Fisher Chemicals, USA), ultrapure water obtained from a Milli-Q Plus system (Millipore, USA), and macroporous adsorption resin (Shandong Dongda Chemical Industry Company, China) were also used in this study.
A total of 20 species of
Effects of extraction variables
Both methanol and ethanol have been successfully used as the solvent for extracting icariin and total flavonoids in Herba Epimedii [23]. However, methanol was regarded as not in compliance with good manufacturing practice (GMP) due to its high toxicity [27]. Ethanol was thus adopted as the solvent for UAE in this study. Fig. 1 shows that the extraction yields of epimedin A, B, C and icariin with aqueous ethanol solution as the solvent are higher than those with water (0% ethanol solution) as
Conclusions
The UAE method reported here can offer an effective alternative for simultaneous extraction of epimedin A, B, C and icariin from Herba Epimedii. It can be applied to sample preparation for the quality control of Herba Epimedii. Additionally, it might serve as a promising industrial extraction protocol of the four flavonoids. Compared with conventional boiling extraction and Soxhlet extraction, UAE is an easy, efficient and inexpensive method with low toxicity and high reproducibility. The
Acknowledgements
This research was partially funded by the National Natural Science Foundation of China (Grant No. 30570171), 100 Talents Program of Chinese Academy of Sciences (05045112) and the Directional Project of Chinese Academy of Sciences. The authors would like to thank Prof. Wang Yuchun from Institute of Process Engineering, Chinese Academy of Sciences for critical review of the manuscript.
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