Preparative isolation of cordycepin, N6-(2-hydroxyethyl)-adenosine and adenosine from Cordyceps militaris by macroporous resin and purification by recycling high-speed counter-current chromatography

Accepted Manuscript Title: Preparative isolation of cordycepin, N6 -(2-hydroxyethyl)-adenosine and adenosine from Cordyceps militaris by macroporous resin and purification by recycling high-speed counter-current chromatography Author: Zhong Zhang Tuernisan Tudi Yanfang Liu Shuai Zhou Na Feng Yan Yang Chuanhong Tang Qingjiu Tang Jingsong Zhanga PII: DOI: Reference:

S1570-0232(16)30626-2 http://dx.doi.org/doi:10.1016/j.jchromb.2016.08.025 CHROMB 20216

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

7-4-2016 21-7-2016 15-8-2016

Please cite this article as: Zhong Zhang, Tuernisan Tudi, Yanfang Liu, Shuai Zhou, Na Feng, Yan Yang, Chuanhong Tang, Qingjiu Tang, Jingsong Zhanga, Preparative isolation of cordycepin, N6-(2-hydroxyethyl)-adenosine and adenosine from Cordyceps militaris by macroporous resin and purification by recycling high-speed counter-current chromatography, Journal of Chromatography B http://dx.doi.org/10.1016/j.jchromb.2016.08.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Preparative isolation of cordycepin, N6-(2-hydroxyethyl)-adenosine and adenosine from Cordyceps militaris by macroporous resin and purification by recycling high-speed counter-current chromatography Zhong Zhang a, Tuernisan Tudi b, Yanfang Liu a, Shuai Zhou a, Na Feng a, Yan Yang a, Chuanhong Tang a, Qingjiu Tang a,, Jingsong Zhang a, a

Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences, Key Laboratory of Edible Fungi Resources and Utilization (South)，Ministry of Agriculture, P. R. China, National Engineering Research Center of Edible Fungi,Shanghai 201403，China b College of Pharmacognosy, China Pharmaceutical University, Nanjing 210038, China

 Corresponding author: Qingjiu Tang; Tel.: +86-21-62205130; fax: +86-21-62205130. Corresponding author: Jingsong Zhang; Tel.: +86-21-62201754; fax: +86-21-62201754. E-mail addresses: [email protected] (Q.-J. Tang); [email protected] (J.-S. Zhang).

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Highlights ► A convenient method for preparing three nucleosides from Cordyceps militaris. ► Three nucleosides are cordycepin, N6-(2-hydroxyethyl)-adenosine and adenosine. ► Three nucleosides were isolated by macroporous resin NKA-Ⅱ. ► Three nucleosides were purified by recycling HSCCC at one time. ABSTRACT In this study, cordycepin, N6-(2-hydroxyethyl)-adenosine (HEA) and adenosine from the fruiting bodies of Cordyceps militaris were separated by using macroporous resin NKA-Ⅱ adsorption. The parameters of static adsorption were tested and the optimized conditions were as follow: the total adsorption time was 12 h, 50% ethanol was used for desorption and the desorption time was 9 h. The crude sample that was prepared by macroporous resin NKA-Ⅱcontained 3.4% cordycepin, 3.7% HEA and 4.9% adenosine. Then the crude sample was further purified by recycling high-speed counter-current chromatography (HSCCC) with ethyl acetate, n-butanol, 1.5% aqueous ammonium hydroxide (1:4:5, v/v/v) as the optimized two-phase solvent system. Three nucleosides including 15.6 mg of cordycepin, 16.9 mg of HEA and 23.2 mg of adenosine were obtained from 500 mg of crude sample in one-step separation. The purities of three compounds were 98.5, 98.3 and 98.0%, respectively, as determined by high performance liquid chromatography. Keywords: Cordyceps militaris; Macroporous resin; High-speed counter-current chromatography; Cordycepin; N6-(2-hydroxyethyl)-adenosine; Adenosine;

1. Introduction Cordyceps militaris, a famous edible and medicinal mushroom, has been used as a traditional Chinese medicinal fungus for a long time. Accumulating data suggests that C. militaris possesses multiple pharmacological activities, including anti-cancer, anti-bacterial, anti-inflammatory and antioxidant properties [1-3]. Recent research has demonstrated that both the fungal mycelia and the fruiting bodies of C. militaris contain many kinds of bioactive components such as polysaccharide, nucleoside (including cordycepin, N6-(2-hydroxyethyl)-adenosine, adenosine, uridine and guanosine), mannitol, ergosterol, lectin and so on [4-7].

Cordycepin (3'-deoxyadenosine) shown in Fig.1 is a nucleoside analogue compound. As an important bioactive compound in C. militaris, cordycepin possesses many pharmacological functions, such as anti-bacterial [8], anti-tumor [9], anti-inflammatory [10], anti-virus [11], anti-aging [12], anti-oxidant activity [13], immunomodulatory effect [14]. The combination of cordycepin with deoxycoformycin in the treatment of African trypanosomiasis is under the phaseⅡclinic trials in U.S.A [15]. N6-(2-hydroxyethyl)-adenosine (HEA) (Fig.1) is another kind of special nucleosides in C. militaris, which is the first calcium antagonists from biological sources and can be used as an inotropic agent [16]. HEA also possesses the activity of inhibiting proliferation of tumor cell and protection of the brain [17-18]. As an analgesic, HEA does not cause the addiction, which is different with other synthetic opioid analgesics commonly used. In addition, it was found that HEA can bind with human serum albumin (HSA) to form the HEA-HSA complex at room temperature by hydrophobic interaction [19]. Adenosine (Fig.1), a naturally and ubiquitous nucleoside, involved in a variety of physiological and pathophysiological regulatory mechanisms [20-21]. As signaling through adenosine membrane receptors, it was denominated four types as A1, A2a, A2b, and A3, which can modulate cell proliferation, differentiation, and apoptosis [22-23]. Owing to their pharmacological activities, these three compounds, as long as C. militaris have attracted more and more researchers’ attention in drug development of natural products. More pure compounds of them are required for further bioactive research in vivo. However, as these three nucleosides having similar chemical structures, yet there is no efficient technology to separate and purify them from extractions of C. militaris in large scale. Macroporous resin adsorption technology has recently drawn more attention in pharmaceutical applications and has also been used for separation and purification of compounds from natural resource [24-26]. As a unique liquid-liquid partition chromatography technique, high-speed counter-current chromatography (HSCCC) has also been used in separation and purification of active components from natural and synthetic products [27-28]. The well-known advantage of HSCCC is that it uses no solid support for the stationary phase and it allows preparative separation of solutes in a two-phase solvent system. It also has the advantages of high recovery, high efficiency and ease of scale-up. Macroporous resin adsorption technique and HSCCC are suitable methods of separation and purification of natural products in scale-up process [29]. At present, there is a report about the separation of cordycepin and adenosine by HSCCC [30].

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However, as HEA and adenosine always exist in C. militaris together and they are similar in many physical properties such as solubility, it is very difficult to completely separate them by HSCCC. It is reported that recycling mode of HSCCC, which can enhance the separation efficiency of the column and improve the resolution, has been used in the separation of components with similar structures when a single run cannot work in a separation[31]. The recycling mode of HSCCC is a simple and practical strategy for the increase in column length and it has the advantage that no increase pressure in the column inlet [32]. It solves the problems of higher column pressure and increased experiment cost associated with long columns by recycling the column output back into the column. The significant advantage of recycling mode of HSCCC lies in the fact that the available number of theoretical plates can be increased without increasing the actual length of the chromatographic column [37]. Besides that, no fresh solvent is required during the recycling periods and reduce the overall solvent consumption [37]. Currently, the recycling mode of HSCCC has been applied to the preparative isolation of natural products including echinacoside [33], (R, S)-naproxen [34], isoflavones [35], bovine serum albumin binders [36], tanshinones [37] and amlodipine besilate enantiomers [38]. To the best of our knowledge, no paper reported on the use of HSCCC for the separation and purification of cordycepin, N6-(2-hydroxyethyl)-adenosine and adenosine from the C. militaris at one time. In this study, a convenient and successful method combining the recycling HSCCC with macroporous resin adsorption technology was established for separation and purification of cordycepin, N6-(2-hydroxyethyl)-adenosine and adenosine from the fruiting bodies of C. militaris.

2. Experimental 2.1. Materials and reagents The fruiting bodies of C. militaris were purchased from Shun Taiyuan Biotechnology Co., Ltd (Jiangsu, China) and identified by Associate Professor Chuanhong Tang ((Shanghai Academy of Agricultural Sciences)). Macroporous resin NKA-Ⅱ(Table 1) (Tianjin Haiguang Chemical Co., Ltd, Tianjin, China) was used for absorbing active compounds. Macroporous resin NKA-II, formed by crosslinking polymerization of styrene, is a homogeneous red brown opaque sphere. The standard cordycepin, adenosine and N6-(2-hydroxyethyl)-adenosine were purchased from Sigma-Aldrich Company (USA). Methanol of HPLC-grade was bought from ANPEL Scientific Instrument Co., Ltd (Shanghai, China). All other solvents were analytical grade (Sinopharm Chemical Reagent Co., Ltd, Shanghai, China). 2.2. Apparatus The HSCCC apparatus is a TBE-300B HSCCC (Tauto Biotechnique Company, Shanghai, China) equipped with a set of three multilayer coils connected in series (internal diameter of the PTFE tube = 1.5 mm, total volume = 300 mL) and a 20 mL sample loop. Theβvalue of the multilayer coil ranged from 0.5 at the internal terminal to 0.8 at the external terminal (β= r/R, R = 5 cm, where r is the rotation radius or the distance from the coil to the holder shaft, and R, the revolution radius or the distance between the holder shaft and central axis of the centrifuge). The rotation speed of the column coils was in the range between 0 and 1000 rpm, where the optimum speed of 850 rpm was used. The system was also equipped with a TBP5002 constant flow pump (Tauto Biotechnique Company, Shanghai, China), a model of UV-500 monitor (Tauto Biotechnique Company, Shanghai, China) and an DC-0506 constant temperature circulator (Shanghai Shunyu henping Scientific Instruments Company, Shanghai, China). N2010 chromatography workstation (Zhejiang University, Hangzhou, China) was employed to record the chromatograms. 1H NMR and 13C NMR were recorded on a Bruker Avance 500 NMR spectrometer operating at 500 MHz (1H) and 125 MHz (13C) respectively. Chemical shifts were given in ppm and tetramethylsilane (TMS) was used as an internal standard. Analysis high-performance liquid chromatography (HPLC) was performed using a Waters 2695 HPLC system (Waters, Milford, USA) equipped with a quaternary solvent delivery system, an autosampler, an ultraviolet detector and a Parallax detector. 2.3. Studies of static adsorption and desorption 2.3.1. Adsorption amount at equilibrium

Dried and powdered fruiting bodies (0.5 kg) of C. militaris was suspended in boiling water (10 L) and stirred for 2 hours. After cooling down to the room temperature, the solution was centrifuged and concentrated to get the solution of crude extract (2 L) and 30 g of pretreated dry macroporous resin NKA-Ⅱ was introduced into 100 mL Erlenmeyer flasks (each Erlenmeyer flask contains 2 g of macroporous resin). 10 mL, 20 mL, 30 mL, 40 mL and 50 mL of crude extract solutions (cordycepin, 262 μg/mL; HEA, 342 μg/mL; adenosine, 383 μg/mL) were added to each flask (the solution of each volume was added to three flasks). The flasks were shaken (150 rpm) at 25℃ for 12 h. After adsorption equilibrium, the solutions were analyzed by HPLC. The adsorption capacity of the resin was evaluated by the following equation:

Qe = (𝐶0 − 𝐶𝑒 )𝑉0 /𝑊

(1)

4 where Qe is the adsorption amount at equilibrium per 1 g absorbent; C0 and Ce are the initial and equilibrium concentrations of solution, respectively (mg/mL); V0 is the initial volume (mL) and W is dry weight of the adsorbent (g).

2.3.2. The time of adsorbed equilibrium

Three aliquots of 50 mL solutions of crude extracts (cordycepin, 262 μg/mL; HEA, 342 μg/mL; adenosine, 383 μg/mL) were added to each 100 mL Erlenmeyer flask containing 2.0 g pretreated dry macroporous resin NKA-Ⅱ. The flasks were shaken (150 rpm) at 25℃. Adsorption capacities (Qe) of resins were analyzed by HPLC after 1, 2, 3, 6, 9, 12, 24 h adsorption. The adsorbed ratio (%) of the resin at different adsorbed time was evaluated by the following equation.

Er = (𝐶0 − 𝐶𝑒 )/𝐶0 × 100%

(2)

where C0 and Ce are the same as defined above; Er is the adsorbed ratio (%).

2.3.3. Ethanol concentration of desorption

Pretreated dry macroporous resin NKA-Ⅱ(30 g) was introduced into 100 mL Erlenmeyer flasks (2 g of macroporous resin per flask). 50 mL of crude extract solutions (cordycepin, 262 μg/mL; HEA, 342 μg/mL; adenosine, 383 μg/mL) were added to each flask. The flasks were shaken (150 rpm) at 25℃ for 12 h. After the resins attained saturation, they were washed with de-ionized water to remove un-adsorbed components. Ethanol-water solution (40 mL) with series concentration of 10%, 30%, 50%, 70%, and 95% were added to each flask. The flasks were shaken (150 rpm) at 25 ℃ for 9 h, and the solutions were analyzed by HPLC. The desorption (%) ratio of the resin was evaluated by the following equation.

Dr = 𝐶𝑑 𝑉𝑑 /[(𝐶0 − 𝐶𝑒 )𝑉0 ] × 100%

(3)

where C0, Ce and V0 are the same as defined above; Dr is the desorbed ratio (%); Cd is the concentrations of the desorbed solutions (mg/mL); Vd is the volume of the desorbed solutions (mL).

2.3.4. Ethanol volume of desorption

Pretreated dry macroporous resin NKA-Ⅱ(30 g) was introduced into fifteen 100 mL Erlenmeyer flasks (2 g of macroporous resin per flask). 50 mL of crude extract solutions (cordycepin, 262 μg/mL; HEA, 342 μg/mL; adenosine, 383 μg/mL) were added to each flask. The flasks were shaken (150 rpm) at 25 ℃ for 12 h. After adsorption equilibrium, the resins were washed with de-ionized water to remove un-adsorbed components. 10 mL, 20 mL, 30 mL, 40 mL and 50 mL of ethanol in water (50%, V/V) were added to each flask (the ethanol of each volume was added to three flasks). The flasks were shaken (150 rpm) at 25 ℃ for 9 h, and the solutions were analyzed by HPLC. 2.3.5. The time of desorption

Three aliquots of 50 mL solutions of crude extracts (cordycepin, 262 ug/mL; HEA, 342 ug/mL; adenosine, 383 ug/mL) were added to 100 ml Erlenmeyer flask separately, containing 2.0 g pretreated dry macroporous resin NKA-Ⅱeach. The flasks were shaken (150 rpm) at 25 ℃. After adsorption equilibrium, the resins were washed with de-ionized water to remove un-adsorbed components. 40 mL 50% ethanol-water solution was added to each flask. The flasks were shaken (150 rpm) at 25 ℃ for 12 h, and the solutions were analyzed by HPLC after 0.5, 1, 2, 3, 4, 6, 9, 12h desorption. 2.4. Preparation of crude sample A total of 2.0 kg of dried and powdered fruit bodies of C. militaris was suspended in boiling water (30 L) and stirred for 2 hours. After cooled to the room temperature, the precipitation was filtrated out. 1 kg of macroporous resin NKA-Ⅱ was added in supernatant and stirred at 25℃ for 12 hours. Then the macroporous resin NKA-Ⅱ was washed with 15 L water and then 50% ethanol was added, After stirred at 25℃ for 9 hours, the ethanol elution were evaporated to obtain the crude sample (60.5 g). 2.5. Selection of two-phase solvent system The two-phase solvent system was selected according to the suitable partition coefficient (K) of each target compound. The K-value was expressed as peak area ratio of the compound in the upper phase (A U) divided by that in the lower phase (AL) (K = AU /AL). The K values of target compounds were determined by HPLC analysis as follows: 5 mg of crude samples were added to a test tube, to which 5 mL of each phase of the pre-equilibrated two-phase solvent system was added. The test tube was rigorously shaken for several minutes until equilibrium was established. Both the solutions of the upper and lower phases were analyzed by HPLC to obtain the K-value of target compounds. 2.6. Preparation of two-phase solvent system and sample solution The selected solvent system that consisted of ethyl acetate, n-butanol and 1.5 % ammonia water (1.5 % of ammonnnia in water) with the volume ratio of 1:4:5 was prepared by adding the solvents to a separation

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funnel and thoroughly equilibrated by vigorously shaking at room temperature. The solvents were left overnight and the upper and lower phase were separated and degassed by ultrasound for 30 min prior to use. The sample solution was prepared by dissolving 500 mg crude sample in 20 ml solvent of lower phase of the solvent system. 2.7. Recycling HSCCC separation The multilayer-coiled column was entirely filled with the upper phase (stationary phase) of the solvent system. Then the apparatus was rotated 850 rpm, while the lower phase (mobile phase) was pumped into the column from head to tail at 1.5 mL min-1. The effluent from the outlet of the column was continuously monitored at 254 nm and the separation temperature was controlled at 25 ℃. After the mobile phase front emerged and hydrodynamic equilibrium was established in the column, 20 mL of sample solution containing 500 mg of crude sample was introduced into the column through the injector. The recycling HSCCC was employed by channeling the outlet of the detector to the inlet of the pump. When the target components were about to emerge, the switching valve was changed to the recycling mode. While the target components were completely separated after several HSCCC recycles, recycling elution mode was stopped to collect the peak fractions according to the chromatogram. 2.8. HPLC analysis and identification of HSCCC fractions Qualitative and quantitative analyses of cordycepin, N6-(2-hydroxyethyl)-adenosine and adenosine in the collected fractions were performed by HPLC on a reverse phase Ultimate AQ-C18 column (Welch Materials Inc, Shanghai, China). The mobile phase consisted of ultrapure water (A) and methanol (B) in gradient mode: 0-5 min, 5% B; 5-15 min, 5-15% B; 15-20 min, 15-25%B; 20-25 min, 25%B; 25-26 min, 25-5%B; 26-35 min, 5%B. The effluent was monitored at 254 nm and the flow rate was kept at 1.0 mL/min. The structural identification was elucidated by 1H NMR and 13C NMR.

3. Results and discussion 3.1. Optimization of parameters of static adsorption and desorption As shown Fig. 2A, macroporous resin NKA-Ⅱ absorbed more than 3.8 mg cordycepin, 4.7 mg HEA and 4.7 mg adenosine on per g resin. The adsorbed amount increased with the extension of adsorption time, and reached equilibrium at 12 h (Fig. 2B). The three nucleosides absorbed on the resin NKA-Ⅱ could be easily desorbed by 50%, 70% and 95% ethanol-water solution (Fig. 2C). 50% ethanol-water solution is the most suitable for desorption of the nucleosides, as the purity of target compounds in elution decreased when increasing the ethanol concentration. In addition, as shown in Fig. 2D and Fig. 2E, the nucleosides absorbed on the resin desorbed completely when the ethanol volume of desorption increased 30 mL (ratio of the resin and ethanol volume is 1:15 (g/mL) ) and time of desorption reached in 9 h. 3.2. Preparation of crude fraction by macroporous resin Through adsorption and desorption on the macroporous resin NKA-Ⅱ, three nucleosides including cordycepin, HEA and adenosine were successfully enriched. Their content increased from 0.1%, 0.1%, and 0.2% to 3.4%, 3.7% and 4.9%, respectively (Fig. 3). The recovery of three nucleosides was 95.1%, 94.3% and 94.8%, respectively. 3.3. Selection of two-phase solvent system Previous studies indicated that the nucleosides are a series of polar compounds and the higher polarity systems such as ethyl acetate-ethanol-water, ethyl acetate-n-butanol-water and n-butanol-ethanol-50% saturated ammonium sulfate have been used for separation of these compounds [39]. Therefore, these three kinds of polar solvent systems were selected to study the partition coefficient (K) of three nucleosides. As shown in table 2, we have found that three nucleosides have small K values in solvent system composed of ethyl acetate-ethanol-water (5:2:5, v/v) and small separation factor α (α = Kadenosine/KHEA) in the solvent system composed of ethyl acetate-ethanol-water at the volume ratios of 5:3:5. We also investigated solvent systems composed of ethyl acetate-n-butanol-water (2:3:5, 2:4:5, 1:4:5, v/v), the results showed that the best K value was obtained in the solvent system composed of ethyl acetate-n-butanol -water at the volume ratios of 1:4:5 (Table 2). But the K value of HEA and α value (α = Kadenosine/KHEA) were small (K < 0.5, α < 1.5). According to property of weak base of three nucleosides, ammonium hydroxide and ammonium sulfate were added to the solvent systems to increase the solubility of target compounds in the upper phase. The results indicated that the compounds in ethyl acetate-n-butanol-1.5% aqueous ammonium hydroxide (1:4:5, v/v) and ethyl acetate-n-butanol-1% aqueous ammonium sulfate (1:4:5, v/v) have suitable K value, but low retention of the stationary phase (<40%) was obtained in the second solvent system. Based on the above results,

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solvent systems composed of ethyl acetate-n-butanol-10% aqueous ammonium sulfate (1:4:5, v/v), n-Butanol-ethanol-10% aqueous ammonium sulfate (5:1:5, v/v) and n-Butanol-ethanol-50% saturated aqueous ammonium sulfate (1:1:2, v/v) were also tested to obtain the optimum partition coefficient (Table 2). The results showed that three nucleosides in the first and second solvent system have suitable K value and three nucleosides in the third solvent system have too large K value. But when the salt was added to solvent system, it was easy to block up the multilayer coil separation column and difficult to eliminate the salt from the collected fractions. To sum up, the solvent system composed of ethyl acetate-n-butanol-1.5% aqueous ammonium hydroxide at the volume ratio of 1:4:5 was selected to separate these three nucleosides in the crude sample. 3.4. Recycling HSCCC separation As shown in Fig. 4, the solvent system of ethyl acetate-n-butanol-1.5% aqueous ammonium hydroxide (1:4:5, v/v) was used in the HSCCC. The flow rate of the mobile phase, the temperature and the revolution speed of the separation column were optimized according to the methods by Ito [40]. The results indicated that cordycepin could be purified from the crude fraction, but HEA and adenosine could not separate effectively because of their similar polarity. The recycling HSCCC have shown its unique advantage in achieving effective separations through preventing stationary phase loss when effective separation could not be achieved in a single run [31]. Therefore, the recycling HSCCC was proposed for the further separation of HEA and adenosine and the operation procedure was as follow: After the peak 1 was collected completely, the switching valve was turned to form a recycling mode. When peak 3 (cordycepin) was emerged, the switching valve was immediately returned to its original position to collect the peak 3 (cordycepin). After the peak 3 was collected completely, the switching valve was turned to form a recycling mode again. Then a fork-like peak was formed in the second cycle, which suggested the trend to separation of HEA and adenosine (Fig. 4). In Fig. 4，peak 1 are unknown mixture. Peak 2 are the mixture of HEA and adenosine that needs recycling HSCCC. Peak 3 is the compound cordycepin. After seven cycles, the peak 2 was divided into two peaks completely and two compounds (HEA and adenosine) were successfully separated by recycling HSCCC. Through adsorption and desorption on the macroporous resin NKA-Ⅱ, ammonium hydroxide was removed and three nucleosides including cordycepin, HEA and adenosine were purified from HSCCC factions. The recovery of three nucleosides was 99.1%, 98.9% and 98.4%, respectively. Finally, total of 15.6 mg cordycepin, 16.9 mg HEA and 23.2 mg adenosine were produced from 500 mg crude sample by recycling HSCCC separation with the purities of 98.5 %, 98.3% and 98.0% respectively. 3.5. Structure identification of compounds The HPLC chromatograms of HSCCC fractions were shown in Fig. 5 and HPLC analysis conditions were the same shown in Fig. 3. The chemical structures of isolated compounds were identified by 1H NMR and 13

C NMR and comparison with published data.

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Compound A (HSCCC fraction A in Fig. 4): 1H-NMR (DMSO-d6, 500 MHz): δ 8. 35 (1H, s, H-8), 8.15 (1H, s, H-2), 7.29 (2H, br s, N6H), 5.88 (1H, d, J = 6.3 Hz, H-1′ ), 5.66 (1H, d, J = 4.4 Hz, OH-2′ ), 5.16 (1H, t, J = 4.4 Hz, OH-5′ ), 4.56 (1H, m, H-2′ ), 4.34 (1H, m, H-4′ ), 3.65 (1H, m, H-5′a ), 3.51 (1H, m, H-5′b ), 2.22 (1H, m, H-3′a ), 1.90 (1H, m, H-3′b )。13C-NMR (DMSO-d6, 125 MHz): δ 155.9 (C-6), 152.3 (C-2), 148.8 (C-4), 139.0 (C-8), 119.0 (C-5), 90.7 (C-1′ ), 80.6 (C-4′ ), 74.6 (C-2′ ), 62.6 (C-5′ ), 34.0 (C-3′ ). Compared with published data given in ref.[30, 41], compound A was in agreement with cordycepin. Compound B (HSCCC fraction B in Fig. 4): 1H-NMR (DMSO-d6, 500 MHz): δ 8. 35 (1H, s, H-8), 8.21 (1H, s, H-2), 7.74 (1H, br s, N6H), 5.88 (1H, d, J = 6.3 Hz, H-1′ ), 5.42 (1H, d, J = 6.3 Hz, OH-2′ ), 5.40 (1H, dd, J = 6.9, 4.7 Hz, OH-5′ ), 5.17 (1H, d, J = 4.7 Hz, OH-3′ ), 4.76 (1H, br s, OH-2′′ ), 4.61 (1H, dd, J = 11.3, 6.0 Hz, H-2′ ), 4.14 (1H, m, H-3′ ), 3.96 (1H, dd, J = 6.6, 3.5 Hz, H-4′ ), 3.67 (1H, dt, J = 12.1, 3.9 Hz, H-5′a ), 3.55 (1H, dt, J = 12.1, 3.9 Hz, H-5′b ), 3.56 (2H, m, H-1′′ ), 3.54 (2H, m, H-2′′ )。13C-NMR (DMSO-d6, 125 MHz): δ 154.6 (C-6), 152.2 (C-2), 148.2 (C-4), 139.7 (C-8), 119.7 (C-5), 87.8 (C-1′ ), 85.8 (C-4′ ), 73.4 (C-2′ ), 70.6 (C-3′ ), 61.6 (C-5′ ), 59.6 (C-2′′ ), 42.4 (C-1′′ ), Compared with published data given in ref. [18], compound B was in agreement with N6-(2-hydroxyethyl)-adenosine. Compound C (HSCCC fraction C in Fig. 4): 1H-NMR (DMSO-d6, 500 MHz): δ 8. 35 (1H, s, H-8), 8.12 (1H, s, H-2), 7.30 (2H, br s, N6H), 5.88 (1H, d, J = 6.3 Hz, H-1′ ), 5.63 (1H, d, J = 4.4 Hz, OH-2′ ), 5.17 (1H, d, J = 4.7 Hz, OH-3′ ), 5.14 (1H, t, J = 4.4 Hz, OH-5′ ), 4.60(1H, m, H-2′ ), 4.12 (1H, m, H-3′ ), 3.95 (1H, dd, J = 6.6, 3.5 Hz, H-4′ ), 3.64 (1H, m, H-5′a ), 3.53 (1H, m, H-5′b )。13C-NMR (DMSO-d6, 125 MHz): δ 155.9 (C-6), 152.3 (C-2), 148.8 (C-4), 139.0 (C-8), 119.0 (C-5), 90.7 (C-1′ ), 85.6 (C-4′ ), 72.9 (C-2′ ), 69.9 (C-3′ ), 59.8 (C-5′ ). Compared with published data given in ref.[42], compound C was in agreement with adenosine.

4. Conclusion Cordycepin, N6-(2-hydroxyethyl)-adenosine and adenosine, the three nucleosides from traditional Chinese medicine C. militaris, were separated and purified by macroporous resin adsorption and recycling HSCCC. After adsorption and desorption of the macroporous resin NKA-Ⅱ, the contents of cordycepin, N6-(2-hydroxyethyl)-adenosine and adenosine in the crude sample increased to 3.4%, 3.7% and 4.9%, respectively. After that, three nucleosides with the purities of over 98% were obtained by recycling HSCCC with a two-phase solvent system composed of ethyl acetate-n-butanol-1.5% aqueous ammonium hydroxide (1:4:5, v/v). These results indicated that the combination of macroporous resin and recycling HSCCC is a very efficient strategy for separating and purifying cordycepin, N6-(2-hydroxyethyl)-adenosine and adenosine from C. militaris. Furthermore, the technique of combining two methods may be widely used in the separation of other highly polar compounds in natural and synthetic products with similar structures.

Acknowledgment This project was supported financially by the National Science and Technology Support Program (No. 2013BAD16B08-02), the Special Fund for Agro-scientific Research in the Public Interest (No. 201303080).

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Figure captions Fig.1. Chemical structures of cordycepin, N6-(2-hydroxyethyl)-adenosine and adenosine Fig.2. Optimization of conditions of static adsorption and desorption: (A) max adsorbed amount; (B) time of adsorbed equilibrium; (C) ethanol concentration of desorption; (D) ethanol volume of desorption; (E) time of desorption Fig.3. HPLC chromatograms of cordycepin (a), HEA (b) and adenosine (c) in crude extraction (A) and crude fraction prepared by macroporous resin NKA-Ⅱ (B). Fig.4. Separations of cordycepin (A), N6-(2-hydroxyethyl)-adenosine (HEA) (B) and adenosine (C) by recycling HSCCC. Solvent system: ethyl acetate-n-butanol-1.5% aqueous ammonium hydroxide (1:4:5, v/v); sample solution: 500 mg of crude samples dissolved in 20 mL of the lower phase; flow rate: 1.5 mL /min in head-to-tail elution mode; revolution: 850 rpm; separation temperature: 25℃; detection wavelength: 254 nm; stationary phase retention: 43%. Recycling elution mode was firstly formed at retention time 160 min and was stopped at retention time 200 min, and recycling elution mode was secondly formed at retention time 300 min and was stopped at retention time 1670 min. Fig.5. HPLC chromatography of HSCCC fractions (A: cordycepin; B: N6-(2-hydroxyethyl)-adenosine (HEA); C: adenosine).

12

Fig.1

Fig.2

13

Fig.3 (1)

(2)

Fig.4

14

Fig.5 (1)

(2)

(3)

15

Table 1 Properties of macroporous resin NKA-Ⅱ

Resin

Specific surface area m2/g

Bore diameter nm

Skeletal density g/mL

NKA-Ⅱ

160-200

14.5-15.5

1.04-1.08

%

Particle size rang mm

Wet density g/mL

Property

42-52

0.3-1.25

1.02-1.08

polarity

Porosity

Table 2 Partition coefficients (K) of cordycepin, N 6-(2-hydroxyethyl)-adenosine (HEA) and adenosine in various solvent systems. Partition coefficients (K)

Separation factor(α)a

Solvent system HEA

adenosine

cordycepin

α1

α2

Ethyl acetate-ethanol-water (5:2:5, v/v)

0.15

0.21

0.34

1.40

1.62

Ethyl acetate-ethanol-water (5:3:5, v/v)

0.67

0.76

0.90

1.13

1.18

Ethyl acetate-n-butanol-water (2:3:5, v/v)

0.37

0.45

0.78

1.21

1.73

Ethyl acetate-n-butanol-water (2:4:5, v/v)

0.45

0.53

0.92

1.18

1.74

Ethyl acetate-n-butanol-water (1:4:5, v/v)

0.46

0.56

0.98

1.22

1.75

Ethyl acetate-n-butanol-1.5% aqueous ammonium hydroxide (1:4:5, v/v)

0.50

0.62

1.20

1.24

1.94

Ethyl acetate-n-butanol-1% aqueous ammonium sulfate (1:4:5, v/v)

0.54

0.66

1.25

1.22

1.89

Ethyl acetate-n-butanol-10% aqueous ammonium sulfate (1:4:5, v/v)

0.88

0.93

2.55

1.06

2.74

n-Butanol- ethanol -10% aqueous ammonium sulfate (5:1:5, v/v)

0.82

0.94

2.19

1.15

2.33

n-Butanol- ethanol -50% saturated aqueous ammonium sulfate (1:1:2, v/v)

2.29

2.58

6.28

0.22

2.43

a

α1= Kadenosine/KHEA, α2= Kcordycepin/Kadenosine