Supercritical fluid extraction of cordycepin and adenosine from Cordyceps kyushuensis and purification by high-speed counter-current chromatography

Separation and Purification Technology 66 (2009) 625–629

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Supercritical fluid extraction of cordycepin and adenosine from Cordyceps kyushuensis and purification by high-speed counter-current chromatography Jian Ya Ling a,∗ , Guo Ying Zhang b,c,1 , Jian Qun Lin a,2 , Zhao Jie Cui b,3 , Chang Kai Zhang a,4 a b c

27# Shanda Nan Lu, State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China 27# Shanda Nan Lu, School of Environmental Science and Engineering, Shandong University, Jinan 250100, China 27# Shanda Nan Lu, Shandong University of Traditional Chinese Medicine, Jinan 250014, China

a r t i c l e

i n f o

Article history: Received 17 August 2008 Received in revised form 30 November 2008 Accepted 20 December 2008 Keywords: Supercritical fluid extraction High-speed counter-current chromatography Cordycepin Adenosine Cordyceps kyushuensis

a b s t r a c t Supercritical fluid extraction (SFE) was used to extract cordycepin and adenosine from Cordyceps kyushuensis. An orthogonal array design (OAD) test, L9 (3)4 , including pressure, temperature, the mount of modifier and the flow rate of CO2 was performed to get the optimal conditions. The process was then scaled up by 30 times with a preparative SFE system under 40 MPa, 40 ◦ C, the mount of modifier (0.04 ml min−1 ) and a flow rate of CO2 (2.0 l min−1 ). The preparative SFE extracts were separated and purified by highspeed counter-current chromatography (HSCCC) with a selected two-phase solvent system composed of ethyl acetate–n-butyl alcohol–water at an optimized volume ratio of 1:4:5, and the collected fractions were analyzed by high-performance liquid chromatography (HPLC). Two nucleosides were obtained and yielded 8.92 mg of cordycepin, 5.94 mg of adenosine with purities of 98.5 and 99.2% from 400 mg SFE crude extraction, in one-step separation, respectively. © 2008 Elsevier B.V. All rights reserved.

1. Introduction The fungi of genus Cordyceps belonging to Clavicipitaceae, Ascomycotina [1,2], have received significant attention from medical and pharmacological researchers [3–5]. There are more than 300 species in this genus, Cordyceps are generally regarded as rich sources of biologically active compounds, which play important roles due to their biomedical functions. Cordyceps sinensis and Cordyceps militaris, two well-known Cordyceps sp., are commonly used for treatment of hyperglycemia, respiratory liver diseases, renal dysfunction and renal failure [6–8]. Modern studies recently demonstrated that various species possess multiple pharmacological actions, to be specific, anti-tumor, anti-microbial, anti-inflammatory and immunoregulatory effects [9,10]. Furthermore, a variety of the effective chemical constituents including cordycepin, adenosine, ergosterol, myriocin and polysaccharides have been isolated from various species [11–16].

∗ Corresponding author. Tel.: +86 531 88364427; fax: +86 531 88565610. E-mail addresses: [email protected], [email protected] (J.Y. Ling), [email protected] (G.Y. Zhang), [email protected] (J.Q. Lin), [email protected] (Z.J. Cui), [email protected] (C.K. Zhang). 1 Co-first author. Tel.: +86 531 88362128. 2 Tel.: +86 531 88364429. 3 Tel.: +86 531 88361176. 4 Tel.: +86 531 88364427. 1383-5866/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2008.12.022

Cordycepin, a nucleoside analogue 3 -deoxyadenosine (structure shown in Fig. 1) with a broad spectrum of biological activity, was first extracted from Cordyceps militaris, and also found in Cordyceps kyushuensis [14]. The combination of cordycepin with an adenosine deaminase (ADA) inhibitor had significant antifungal activity [17], although pharmacological investigations had proved that cordycepin has antibacterial compound against Clostridium spp. [18], antiviral activity against HIV-l in vitro [19], anti-tumor activities [20], antimalarial activity [21], and the selective interruption of nucleolus and synthesis in Hela cell [22]. Adenosine shown in Fig. 1 is recognized as an important modulator of neurotransmission and has been implicated in many physiological functions such as regulation of arousal and sleep, anxiety, cognition and memory [23]. The anti-immobility effect of adenosine in the mouse forced swimming test, via adenosine A1 and A2 A receptors, is mediated by an interaction with the opioid system, likely dependent on an activation of ␮- and ␦-opioid receptors and an inhibition of ␬-opioid receptors [24]. In C6 glioma cells, adenosine inhibited cell proliferation in time- and concentration-dependent manners [25]. Due to the high pharmacological activities, cordycepin and adenosine have recently drawn great attention in natural medication researches. A large quantity of pure materials is urgently needed for further studies. Two new techniques, supercritical fluid extraction (SFE) and high-speed counter-current chromatography (HSCCC) are particularly suitable methods for the extraction and separation of natural products [26,27]. As an ideal solvent, carbon dioxide is generally

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J.Y. Ling et al. / Separation and Purification Technology 66 (2009) 625–629 Table 1 L9 (3)4 experimental design.

1 2 3

Fig. 1. Chemical structures of cordycepin and adenosine.

A (pressure, MPa)

◦

B (temperature, C)

C (flow rate of modifier, ml min−1 )

D (flow rate of CO2 , l min−1 )

35 40 45

35 40 45

0.02 0.03 0.04

1.0 1.5 2.0

flow), the sample was subjected to dynamic extraction by flowing CO2 at a set rate for 2 h. 2.3. Scaling-up SFE

used in SFE instead of organic solvent because of the unusual properties such as high diffusivity, liquid-like density, low viscosity, non-toxicity, non-explosiveness, and easy removal from the extracts. In addition, SFE also has the ability to use low temperatures leading to less deterioration of the thermally labile components [28–33]. HSCCC is a unique liquid–liquid partition chromatography technique without solid support matrix [34]. So it eliminates the irreversible adsorptive loss of samples onto the conventional chromatographic column. This method has been successfully applied to the purification of several natural products with minimum sample preparation and clean-up procedures [35–39]. This work is the first to report on combination of SFE and HSCCC to isolate cordycepin and adenosine. We herein optimized experiment parameters by an analytical-scale SFE system using an L9 (3)4 test design. Then, the extraction was scaled up by 30 times by a prepared-scale SFE system. Subsequently, cordycepin and adenosine were purified from natural Cordyceps kyushuensis by HSCCC after the two-phase solvent system was selected. 2. Experimental

Under the optimized SFE conditions, SFE extraction was scaled up 30-fold using a 1000 ml vessel. A 195 g amount of sample was extracted statically for 1 h, and after dynamic extraction for 3 h by flowing liquid CO2 at a rate of 2 l min−1 , the extract was depressed directly into a collection vessel and stored in a refrigerator for subsequent HPLC analysis and HSCCC separation. 2.4. Selection of the two-phase solvent system The composition of the two-phase solvent system was selected according to the suitable partition coefficient (K) of cordycepin and adenosine. The K-values were determined by HPLC as follows: approximately 0.1 mg of standards were added to a 10-ml test tube with a cap, to which 2.5 ml of each phase of the pre-equilibrated two-phase solvent system was added. The sealed test tube was shaken vigorously for several minutes to equilibrate the standard thoroughly. An equal volume of the upper and lower phase was then analyzed by HPLC. The Kvalue was expressed as the peak area of standard in the upper phase divided by the peak area of the compound in the lower phase.

2.1. Reagents and materials 2.5. Preparation of two-phase solvent system and sample solution Carbon dioxide (99.9% purity) was obtained from Daxing Gas Company (Beijing, China). High performance liquid chromatography (HPLC)-grade methanol was obtained from TEDIA (Fairfield, OH, USA). All organic solvents used for HSCCC were of analytical grade and purchased from Guangcheng Chemical Factory (Tianjin, China). Cordycepin and adenosine were purchased from Sigma (St. Louis, MO, USA), and diluted to the desired concentration prior to use. Natural Cordyceps kyushuensis was collected from Mount Meng, Shandong Province of China in August 2006, and identified by Prof. Yinglan Guo (Institute of Microbiology, Chinese Academy of Science, Beijing, China). 2.2. Optimization of SFE extraction A Speed SFE system (Applied Separations, Allentown, PA, USA) fitted with a 10 ml stainless steel extraction vessel was used for optimizing the extraction conditions with a modifier added with a WellChrom K-501 HPLC pump (Knauer, Berlin, Germany). A micrometering valve is used as restrictor valve to control the flow rate of supercritical CO2 . Extraction temperatures were monitored using a thermocouple, and were found to be accurate to within ±1 K. The precision of the pressure measurement was ±1 Pa. An OAD L9 (3)4 test design was employed where pressure, temperature, the flow rate of methanol as a modifier and the flow rate of CO2 were considered to be the four major factors for effective extraction. Combinations of the three different levels of each factor were listed in Table 1. In each test, 6.500 g dried stroma of Cordyceps kyushuensis were shattered to powder (60–80 mesh) and placed into the extraction vessel. After 45 min of static extraction (no liquid

The selected solvent system, ethyl acetate–n-butyl alcohol– water (1:4:5, v/v/v), was prepared by adding all the solvents to a separation funnel according to the volume ratios and thoroughly equilibrated by shaking separately. After thoroughly equilibrating, the upper phase and lower phase were separated and degassed by ultrasound for 30 min prior to use. The sample solution was prepared by dissolving the crude sample in 5 ml upper phase of the solvent system. 2.6. HSCCC separation The SFE extract of Cordyceps kyushuensis was separated by a TBE300A high-speed counter-current chromatograph (Tauto biotech., Shanghai, China) with three multilayer coil separation column connected in series (I.D. of the tubing = 1.6 mm, total volume = 260 ml) and a 20 ml sample loop. The revolution radius was 5 cm, and the ˇ values of the multilayer coil varied from 0.5 at internal terminal to 0.8 at the external terminal. The upper phase of ethyl acetate–n-butyl alcohol–water (1:4:5, v/v/v) was pumped into the multilayer-coiled column with a ÄKTA prime plus system (GE Healthcare, Piscataway, NJ, USA). After the column was totally filled with the two phases, only the lower phase was pumped at a flow-rate of 1.5 ml min−1 ; and at the same time, the HSCCC apparatus was rotated at a revolution speed of 850 rpm. After hydrodynamic equilibrium was reached, 5 ml of the sample solution containing 400 mg of the crude extract was introduced into the column through the injection valve. All through the experiment the separation temperature was controlled at 25 ◦ C by a Multi Temp

J.Y. Ling et al. / Separation and Purification Technology 66 (2009) 625–629

Table 4 The K (partition coefficient) values of cordycepin and adenosine in different solvent systems.

Table 2 L9 (3)4 test results. Test no.

A

B

C

Yield (%)a

D

Cordycepin 1 2 3 4 5 6 7 8 9

1 1 1 2 2 2 3 3 3

1 2 3 1 2 3 1 2 3

1 2 3 2 3 1 3 1 2

1 2 3 3 1 2 2 3 1

627

Adenosine

0.425 0.558 0.700 0.711 0.732 0.630 0.696 0.658 0.585

0.432 0.474 0.538 0.547 0.510 0.464 0.481 0.532 0.492

a Extraction yield (%) = (the amount of cordycepin or adenosine in extract/sample mass) × 100.

III Thermostatic Circulator 230 VAC (GE Healthcare, Piscataway, NJ, USA). The effluent from the tail end of the column was continuously monitored with ÄKTA prime plus system at 254 nm and the chromatogram was recorded. Each peak fraction was collected manually according to the obtained chromatogram and each collection was treated by vacuum freeze-dry and dissolved by methanol for analysis by HPLC. 2.7. HPLC analysis and identification of HSCCC fractions The crude extract and each purified fraction from the preparative HSCCC separation were analyzed by Agilent 1100 HPLC system (Agilent, Santa Clara, CA, USA) with a Kromasil 100-C18 column (250 mm × 4.6 mm I.D., 5 ␮m) at 260 nm and at a column temperature of 30 ◦ C. The mobile phase, a solution of phosphate buffer (pH 6.5): methanol = 17:3 (v/v), was eluted at a flow rate of 1.0 ml min−1 . Identification of the target compounds cordycepin and adenosine was based on comparison with the standards. The identification of HSCCC peak fractions was carried out, respectively, by LC-ESI-MS on an Agilent 1100 HPLC-MSD SL ion trap mass spectrometer (Agilent, Santa Clara, CA, USA) and by 1 H Nuclear magnetic resonance (NMR) and 13 C NMR spectra on a Bruker Avance 400 MHz NMR spectrometer (Bruker BioSpin, Switzerland). Ultraviolet (UV) spectra were obtained on a Shimadzu 2450 spectrophotometer (Shimadzu, Kyoto, Japan), and Infrared spectrum (IR) data on a Nicolet 20sx spectrophotometer (Thermo, Waltham, MA, USA).

Solvent system

K

n-Hexane–ethyl acetate–methanol–water (1:5:1:5,v/v) n-Hexane–ethyl acetate–methanol–water (1:9:1:9,v/v) Ethyl acetate–n-butanol–water (4:1:5,v/v) Ethyl acetate–n-butanol–water (3:2:5,v/v) Ethyl acetate–n-butanol–water (2:3:5,v/v) Ethyl acetate–n-butanol–water (1:4:5,v/v) n-Butanol–water Ethyl acetate–water

Cordycepin

Adenosine

0.37

0.44

0.45

0.42

0.60 0.62 0.99 1.05 0.90 0.19

0.57 0.82 0.84 0.90 0.67 0.35

sets of temperature, pressure, the flow rate of modifier and the flow rate of CO2 were examined under L9 (3)4 test design, and the results were shown in Table 2, which indicate that there are great yield differences among each set of SFE conditions as a control index. Table 3 showed the results of orthogonal analysis. The flow rate of modifier was found to be the most important determinant of the yield. Pressure, the flow rate of CO2 and temperature also had significant influence on the yields. The optimal conditions for extraction of cordycepin and adenosine by SFE were 40 MPa of pressure, 40 ◦ C of temperature, 0.04 ml min−1 of flow rate of methanol and 2.0 l min−1 of flow rate of CO2 . 3.2. Optimization of HSCCC conditions In order to determine the optimal two-phase solvent system for the HSCCC separation, a series of experiments were performed in the present study. n-Hexane–ethyl acetate–methanol–water, ethyl acetate–n-butanol–water, n-butanol–water, ethyl acetate–water were used as the two-phase solvent system. The partition coefficients of the compounds of crude sample in these twophase solvent systems were given in Table 4. It can be seen from Table 4 that ethyl acetate–n-butanol–water with the volume ratio of 1:4:5 may be suitable for HSCCC separation because of good separation results and acceptable separation time. 3.3. Preparative-scale SFE

3. Results and discussion 3.1. Optimization of SFE conditions The products obtained from each L9 (3)4 test of the analytical SFE were quantitatively analyzed, extraction efficiencies at different

Under the above optimized SFE conditions, 195 g of sample Cordyceps kyushuensis powder was extracted, yielding about 0.75% cordycepin and 0.55% adenosine. HPLC analysis in Fig. 2 showed that the total 16.35 g of SFE extract contained approximately 8.94% cordycepin and 6.56% adenosine.

Table 3 Analysis of L9 (3)4 test results. Cordycepin yield (%)

K1 K2 K3 k1 k2 k3 R Optimal level



B

C

D

A

B

C

D

1.683a 2.073 1.939 0.561b 0.691 0.636 0.130c A2

1.832 1.948 1.915 0.611 0.649 0.638 0.038 B2

1.713 1.854 2.128 0.571 0.618 0.709 0.138 C3

1.742 1.884 2.069 0.581 0.628 0.690 0.109 D3

1.444 1.521 1.505 0.481 0.507 0.501 0.026 A2

1.460 1.516 1.494 0.487 0.505 0.498 0.018 B2

1.428 1.513 1.529 0.476 0.504 0.510 0.034 C3

1.434 0.938 1.617 0.478 0.313 0.539 0.226 D3

a

KiA =

b

kiA = KiA /3. RiA = max{kiA } − min{kiA }.

c

Adenosine yield (%)

A

extraction yield at Ai .

628

J.Y. Ling et al. / Separation and Purification Technology 66 (2009) 625–629

Fig. 2. HPLC chromatogram of the extracts from preparative SFE. Sample: methanol solution of preparative SFE extraction without any further treatment. Column: kromasil 100-C18 column (250 mm × 4.6 mm I.D., 5 ␮m); mobile phase: phosphate buffer (pH 6.5): methanol = 17:3 (v/v); flow rate: 1.0 ml min−1 ; detection wavelength: 260 nm.

3.4. HSCCC purification and HPLC identification Fig. 3 showed the preparative HSCCC separation of 400 mg of the SFE crude sample using the optimized solvent system. The retention of the stationary phase was 78.6%, and the separation time was about 250 min in each separation run. Based on the HPLC analysis and the elution curve of the preparative HSCCC, all collected fractions were combined into different pooled fractions. Then, the target compounds were obtained and yielded 5.94 mg of A and 8.92 mg of B with the purity of 99.2 and 98.5% in onestep separation. The chromatograms of HPLC and UV spectra of the compounds were shown in Fig. 4. The structural identification was carried out by IR, UV, MS, 1 H NMR and 13 C NMR spectra as follows. HSCCC fraction A in Fig. 4(a): IR (KBr) 3332, 3140, 2922, 1679, 1606, 1577, 1479, 1421, 1384, 1339, 1297, 1205, 1101, 1070, 1038, 822, 721, 640 cm−1 ; UV max 260 nm; MS, C10 H13 N5 O4 , 267 [M]+ , 268 [M + H]+ ; 1 H NMR (DMSO-d6) ı: 8.36(1 H, s), 8.14(1 H, s), 7.36(2 H, s), 5.88(1 H, d), 4.60(1 H, dd), 4.14(1 H, dd), 3.97(1 H, m), 3.68(1 H, dd), 3.56(1 H, dd); 13 C NMR (CD3 OD) ı: 156.2, 152.5, 149.2, 140.1, 119.4, 89.0, 86.1, 73.6, 70.8, 61.9. Compared with the data given in ref. [40,41], peak A in Fig. 4 corresponded to adenosine. HSCCC fraction B in Fig. 4(b): IR (KBr) 3330, 3140, 1678, 1608, 1575, 1480, 1422, 1384, 1341, 1298, 1206, 1174, 1092, 831, 717 cm−1 ;

Fig. 3. HSCCC chromatogram of the SFE crude extract from Cordyceps kyushuensis. Two-phase solvent system: ethyl acetate–n-butyl alcohol–water (1:4:5, v/v/v); stationary phase: upper aqueous phase; mobile phase: lower organic phase; flow-rate: 1.5 ml min−1 ; revolution speed: 850 rpm; detection wavelength: 254 nm; sample size: 400 mg; injection volume: 5 ml; retention of stationary phase: 78.6%. (A) Adenosine; (B) Cordycepin.

Fig. 4. HPLC analyses and UV spectrum of cordycepin and adenosine purified from Cordyceps kyushuensis with HSCCC. HPLC conditions were the same as shown in Fig. 2. (a) HPLC analyses and UV spectrum of adenosine, fraction A purified by HSCCC; (b) HPLC analyses and UV spectrum of cordycepin, fraction B purified by HSCCC.

UV max 260 nm; MS, C10 H13 N5 O3 , 251 [M]+ , 252 [M + H]+ ; 1 H NMR (DMSO-d6) ı: 8.36 (1 H, s), 8.17(1 H, s), 7.32 (2 H, s), 5.87 (1 H, s), 5.67 (1 H, d), 4.67 (1 H, m), 4.56 (1 H, m), 3.72 (1 H, m), 3.55 (1 H, dd), 2.35 (1 H, m); 13 C NMR (CD3 OD) ı: 156.1, 152.6, 148.9, 139.2, 119.2, 90.9, 80.7, 74.8, 62.8, 34.2. Compared with the data given in ref. [30,42,43], peak B in Fig. 4 corresponded to cordycepin. 4. Conclusion Two nucleosides from the tradition Chinese medicine Cordyceps kyushuensis were extracted and purified by SFE and HSCCC. Under optimal SFE conditions, 40 MPa, 40 ◦ C, a mount of methanol (0.04 ml min−1 ) and a flow rate of CO2 (2.0 l min−1 ), the yields of cordycepin and adenosine were 8.94 and 6.56% in crude extract, respectively. At last, greater than 98% purity of cordycepin and adenosine were obtained by one-step HSCCC with a two-phase solvent system composed of ethyl acetate–n-butyl alcohol–water at an optimized volume ratio of 1:4:5. The combined use of the two methods has been developed successfully in this paper. It is a significantly simple purification procedure to a considerable production of nucleosides with a higher purity as compared with the conventional liquid–solid extraction methods. The results of the present paper demonstrated that SFE and HSCCC are sufficiently valuable techniques for the extraction, isolation and purification of cordycepin and adenosine from Cordyceps kyushuensis. Acknowledgements Financial supports from National Natural Science Foundation of China (Grant No. 30770041) and from the Science and Technology

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