Abstract
Cordycepin is successfully isolated and purified from Cordyceps millitaris in two-step purification by high-speed countercurrent chromatography. Two solvent systems, ethyl acetate–1-butanol–water (3:2:5, v/v/v) and trichloromethane–methanol–1-butanol–water (2:1:0.25:1, v/v/v/v), were used for the two-step purification. The purity of the prepared cordycepin was 98.1% according to the high-performance liquid chromatography analysis.
Keywords: High-speed countercurrent chromatography, Cordycepin, Cordyceps millitaris, HPLC
INTRODUCTION
Cordyceps millitaris is one of the traditional Chinese medicinanl fungi which have been widely used in traditional Chinese medicine. The studies have shown that cordycepin is one of the most important effective constituents [1–3]. Because of the importance of its biological properties, a large quantity of pure materials is urgently needed for further studies [4–5]. However, the preparative separation and purification of cordycepin from other constituents of the fungus C. millitaris by traditional methods are tedious, requiring multiple chromatographic steps resulting in low recovery. High-speed countercurrent chromatography (HSCCC) is a unique liquid–liquid partition technique that uses no solid support matrix. HSCCC eliminates the irreversible adsorptive loss of samples onto the solid support matrix used in the conventional chromatographic column. HSCCC has been successfully used for the preparative separation of natural products such as traditional Chinese medicinal herbs [6–9]. No report has been seen on the use of HSCCC solely for isolation and purification of cordycepin directly from a crude extract of the fungus C. millitaris. We herein report a successful semi-preparative separation and purification of cordycepin from the crude extract of C. millitaris using two different solvent systems by HSCCC
EXPERIMENTAL
Reagents and Materials
All organic solvents used for HSCCC were of analytical grade and purchased from Shanghai Suran Chemical Factory, Shanghai, China. Methanol used for HPLC analysis was of chromatographic grade and purchased from Sinopharm Chemical Reagent Co., Ltd., Shanghai, China.
The cordycepin standard was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.
The dried fruiting bodies of C. millitaris were supplied by Key Laboratory of Biotechnology for Medicinal Plant of Jiangsu Province, Xuzhou Normal University, China.
Apparatus
The preparative HSCCC instrument employed in the present study is a Model HHS-400A miltilayer coil planet centrifuge (Shanghai Tonghong Machine Co., Ltd., Shanghai, China) equipped with a polytetrafluoroethylene multilayer coil of 130 m × 1.6 mm i.d., with a total capacity of 260 mL. The β value of the preparative column varied from 0.33 at internal to 0.86 at the external (β = r/R, where r is the rotation radius or the distance from the coil to the holder shaft, and R is the revolution radius or the distance between the holder axis and central axis of the centrifuge). The rotation speed is adjustable from 200 to 850 rpm, and 750 rpm was used in the present study.
The system was also equipped with one NS-1007 constant flow pump, a Model 8823B-UV monitor operating at 254 nm, a Yakogawa 3057 recorder and a manual injection valve with a 5 or 10 mL sample loop. The HPLC system used throughout this study consisted of a P3000 pump, a UV3000 detector (Beijing Chuang Xin Tong Heng Science & Technology Co., Ltd. Beijing, China), and a sample injector (Model: 7725) with a 20 µL loop. Evaluation and quantification were made on a CXTH-3000.
Preparation of Crude Extract
Dried fruiting bodies of C. millitaris (20 g) were comminuted and extracted with 400 mL of distilled water by using ultrasonic (40 KHz) for 20 min. The sample extraction procedure was repeated 3 times. The filtrates were combined together and concentrated by vacuum distillation, yielding 8.4 g of crude sample which was stored in a refrigerator for the subsequent HSCCC separation [10].
Preparation of the Two-Phase Solvent Systems and Sample Solution
Two-step separation strategy was used in the present study on HSCCC with two different types of two-phase solvent systems composed of ethyl acetate–1-butanol–water (3:2:5, v/v/v) and trichloromethane– methanol–1-butanol–water (2:1:0.25:1, v/v/v/v). Each solvent mixture was thoroughly equilibrated in a separatory funnel at room temperature and the two phases separated shortly before use. The upper aqueous phase was used as the stationary phase and the lower organic phase as the mobile phase.
The sample solution of the first step was prepared by dissolving 500 mg of the crude extract in 10 mL of each phase (1:1, v/v) of the first solvent system. The sample solution of the second step was prepared by dissolving 50mg of dried peak fraction of the first step separation in 5 mL of each phase (1:1, v/v) of the second solvent system.
The standard working solution of cordycepin (0.1 mg/mL) was prepared in mobile phase of HPLC.
HSCCC Separation Procedure
First, the multilayer coiled column was entirely filled with the upper aqueous phase as the stationary phase. Then, the lower organic mobile phase was pumped into the head end of the column at a suitable flow-rate of 2.0 mL/min while the apparatus was rotated at an optimum speed of 750 rpm. After hydrodynamic equilibrium was reached as indicated by a clear mobile phase eluting from the tail outlet, the sample solution was injected through the injection valve. The effluent from the tail end of the column was continuously monitored by a UV detector at 254 nm, and the peak fractions were collected according to the chromatogram.
HPLC Analyses and Identification of HSCCC Fractions
The crude sample and each purified fraction from the preparative HSCCC separation were analyzed by HPLC with an Alltima C18 column (250 mm×4.6 mm, 5 µm) and column temperature of 25°C. The mobile phase, a mixture of methanol and water (17:83, v/v), was set at a flow-rate of 0.8 mL/min. The effluent was monitored by a UV detector at 260 nm [11].
RESULTS AND DISCUSSION
A successful separation of the target compounds using HSCCC requires a careful search for a suitable two-phase solvent system to provide an ideal range of partition coefficients for the applied material.
After trying several solvent systems, we found that the solvent system composed of ethyl acetate–1-butanol –water (3:2:5, v/v/v) was most suitable for separation of the crude extract of C. millitaris by HSCCC. The chromatogram of HSCCC separation is shown in Fig. 1. Each fraction was analyzed by HPLC with the C18 column and the target fraction was eluted at the retention time of 1.9 h to 2.5 h with a purity of 85.3%. To further improve the purity of cordycepin, the solvent system composed of trichloromethane–methanol–1-butanol–water (2:1:0.25:1, v/v/v/v) was used for the second step HSCCC separation (Fig. 2). The target fraction was eluted at the retention time of 6 h to 7.5 h with a purity of 98.1% by HPLC analysis. The yield of cordycepin after two-step HSCCC separation was 80.1%.
Fig.1. Chromatogram of the crude extract by preparative HSCCC.
Conditions: revolution speed: 750 rpm; solvent system: ethyl acetate–1-butanol–water (3:2:5, v/v/v); stationary phase: upper aqueous phase; mobile phase: lower organic phase; flow-rate: 2.0 mL/min; detection: 254 nm; sample size: 500 mg; injection volume: 10 mL; retention of the stationary phase: 25.0%
For further confirmation, HSCCC fraction of supposed cordycepin was mixed with the solution of standard sample of cordycepin where HPLC separation of this mixture showed a single peak as shown in Fig.3.
CONCLUSION
Using the two-step HSCCC separation method, we were able to purify cordycepin efficiently at purity of over 98% with a relatively short separation time. The two-step HSCCC separation method with a suitable set of two-phase solvent systems yields high purity cordycepin directly from a crude extract of C. millitaris.
Fig.2. Chromatogram of the second step HSCCC Purification.
Conditions: revolution speed: 750 rpm; solvent system: trichloromethane–methanol–1-butanol–water (2:1:0.25:1, v/v); stationary phase: upper aqueous phase; mobile phase: lower organic phase; flow-rate: 2.0 mL/min; detection: 254 nm; sample size: 50 mg; injection volume: 5 mL; retention of the stationary phase: 73.1%
Fig.3. (A) HPLC chromatogram of the standard sample of cordycepin (B) HPLC chromatogram of the standard sample of cordycepin with HSCCC fraction of supposed cordycepin.
Conditions: an Alltima C18 column (250 mm×4.6 mm, 5 µm), column temperature: 25 °C; mobile phase: methanol and water (17:83, v/v); flow-rate of 0.8 mL/min; detection: 260 nm. The concentrations of standard working solution was 0.1 mg/mL
ACKNOWLEDGMENTS
This research is partially supported by the China National 863 Hi-Tech Program under Grant 2007 AA021506.
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