Arch. Biol. Sci., Belgrade, 66 (4), 1411-1421, 2014 DOI:10.2298/ABS1404411M

1411

CORDYCEPS PRUINOSA PRODUCES CORDYCEPIN AND N6-(2-HYDROXYETHYL)- ADENOSINE IN CULTURE

ZEBIN MENG1,2, TINGCHI WEN1, JICHUAN KANG1,*, BANGXING LEI1 and KEVIN D.HYDE3

1 Engineering Research Center of Southwest Bio-Pharmaceutical Resources, Ministry of Education, Guizhou University, Guiyang 550025, China

2 Guizhou Bioresource Development and Utilization Key Laboratory, Guizhou Normal College, Guiyang 550018, China 3 Institute of Excellence in Fungal Research, School of Science, Mae FahLuang University, Chiang Rai 57100, Thailand

*Corresponding author: bcec.jckang@gzu.edu.cn

Abstract - Cordyceps species are entomophagous pathogens with medicinal properties, mostly linked to cordycepin and N6-(2-hydroxyethyl)-adenosine (HEA). An isolate of Cordyceps pruinosa (GZUCC 8552) was obtained from a fruiting body formed on the cocoon a Limacodidae insect collected in Guizhou Province, China. Morphological and molecular analysis (combined 5.8S ITS, RPB1 and 18S RNA) confirmed the species to be Cordyceps pruinosa. Metabolites of the isolate grown in liquid static and solid-state media were established by HPLC-MS. Cordycepin (5.311 mg/g) and HEA (0.558 mg/g) were produced by this strain. This is the first record of cordycepin from an isolate of Cordyceps pruinosa. As Cordyceps pruinosa is a good source of cordycepin and HEA, it could be used as an alternative to the over-collected Cordyceps sinensis.

Key words: Cordyceps pruinosa; characteristics; cordycepin; cultivation; N6-(2-hydroxyethyl)-adenosine

INTRODUCTION

Cordyceps species are entomopathogenic taxa living mainly on arthropods, they have a long history of medicinal use throughout Asia (Sung et al., 2007a; Tuli et al., 2013) and have worldwide occurrence (Tuli et al., 2013). Cordyceps (Clavicipitaceae, Hypo-creales, Ascomycota) species have recently been re-organized following molecular analysis and placed in Metacordyceps (Clavicipitaceae), Elaphocordyceps (Ophiocordycipitaceae), Ophiocordyceps (Ophiocordy-cipitaceae) and Cordyceps (Cordycipitaceae) (Sung et al., 2007a).

Cordyceps sensu lato species have long been used to promote longevity, relieve fatigue and treat numer-

ous diseases in traditional Chinese medicine (Russell and Paterson 2008). Recent studies suggest that sev-eral species in this genus possess wide-ranging phar-macological properties, such as immunomodulating, antioxidant, antitumor, hepatoprotective, nephro-protective, hypoglycemic and hypocholesterolemic activities, and effects on apoptotic homeostasis (Yue et al., 2013; Zhou et al., 2009; De Silva et al., 2012a, b).

Cordyceps sensu lato species are thought to have these broad pharmacological properties because they have a variety of active chemical constituents, which include cordycepin, adenosine, polysaccharides er-gosterol, protein and amino acids (Zhou et al., 2009; De Silva et al., 2012a, b). Cordycepin plays a partic-

1412 ZEBIN MENG ET AL.

ularly important role in pharmacological properties. Significant pharmacological benefits of cordycepin have been revealed, and include antitumor, immu-nomodulatory, anti-inflammatory, antioxidant, hy-perlipidemia regulation, anti-aging, neuroprotective function, and renoprotection activity (Ramesh et al., 2012), antibacterial activity (Ahn et al., 2000), anti-viral activity (Lovinger et al., 1973), promotion of learning and memory (Cai et al., 2013), apoptosis (Choi et al., 2011) and decreasing rheumatoid ar-thritis (Noh et al., 2009). Studies have also targeted cordycepin as a therapeutic agent against leukemia (Clinical Trials Gov, verified by Onco Vista, Inc., 2009) (Jeong et al., 2011). In addition, preclinical as-sessment of cordycepin and deoxycoformycin (pen-tostatin) in the treatment of second-stage African trypanosomiasis has been carried out (Vodnala et al., 2009). N6-(2-hydroxyethyl)-adenosine (HEA) is also an important bioactive compound produced by this genus; HEA behaves as a Ca2+ antagonist, an inotropic agent and radio protectant (Furuya and Hirotani, 1983) and as an analgesic substance (Chai et al., 2004).

Although there are more than 530 species of Cordyceps sensu lato (Index Fungorum, 2013), cordycepin production had been reported in only 12 Cordyceps species (Yang and Dong, 2011), although it is probably produced by most. There are no reports of cordycepin production by Cordyceps pruinosa (C. pruinosa). In the present study, we isolated C. pru-inosa from the cocoon of a Limacodidae insect from Guizhou Province, China, and determined cordyc-epin and HEA production by its culture mycelium. C. pruinosa provides a new source of cordycepin and HEA.

MATERIALS AND METHODS

Strains separation

A strain of C. pruinosa (GZUCC 8552) was isolated from the cocoon of a Limacodidae insect collected in the Leigong Mountains in Guizhou Province, China. To obtain ascospores, the sclerotia of a fresh specimen were wrapped in moist tissue and its fertile

head was suspended over a sterile glass slide so that the ascospores were released onto the slide. The as-cospores were then transferred to PDA for germina-tion; a few single spore colonies were obtained and incubated at 23℃ for 10 d.

Morphological studies

The purified single colony was incubated on PDA in darkness for 10 days at 20O, then under 14 h:10 h light:dark (500 lux light) at 25O and 80-90% humidity for 10 days. Microcharacters of the fruiting bodies were examined under an Olympus CX31 compound microscope and photographed. The mycelia were collected for DNA extraction.

DNA extraction and reagents

Mycelia of strain GZUCC 8552 were collected for PCR. Taq enzyme and dNTPs was purchased from Shanghai Sangon. An Agarose Gel DNA Purification kit ver 2.0 was purchased from TRKARA Company. Fresh, sporulating cultures on PDA agar were used for DNA extraction following Tigano-Milani et al. (1995); the extracted DNA was stored at -20O.

PCR amplification and determination of DNA sequences

The PCR amplification and sequencing of ITS1-5.8S-ITS2 rDNA were conducted as described in Wen et al. (2012), and the primers ITS4 (5’-TCCTC-CGCTTATTGATATGC-3’) and ITS5 (5’-GGAAG-TAAAAGTCGTAACAAGG-3’) (White et al., 1990) were used. The PCR amplification and sequencing of nrSSU were conducted as described in Sung et al. (2007b), and the primers NS1 (5’-GTAGTCATAT-GCTTGTCTC-3’) and NS4 (5’-CTTCCGTCAAT-TCCTTTAAG-3’) (White et al., 1990) were used. In the amplification and sequencing of RPB1, we fol-lowed Castlebury et al. (2004); the primers CRPB1A (5’-CAYCCWGGYTTYATCAAGAA-3’) and RP-B1Cr (5’-CCNGCDATNTCRTTRTCCATRTA-3’) (Castlebury et al., 2004) were used. All PCR prod-ucts were sequenced by GenScript Biotechnology Co., Nanjing, China.

CORDYCEPS PRUINOSA PRODUCES CORDYCEPIN AND N6-(2-HYDROXYETHYL)-ADENOSINE IN CULTURE 1413

Sequence alignment and phylogenetic analysis

Blast searches were made to reveal the closest matches in GenBank for phylogenetic analysis. The taxon information and GenBank accession numbers used in the molecular analysis are listed in Table 1. DNA sequences were assembled using Tex-Edit Plus (Bender, TomBB@aol.com). The alignment of the sequence files was conducted using the CLUS-TAL W software (Thompson et al., 1994). Align-ments were manually adjusted to allow maximum sequence similarity. Gaps were treated as missing data. Phylogenetic analyses were performed with PAUP version 4.0b10 (Swofford, 2004). Then the phylogenetic tree was constructed using maximum parsimony (MP) methods. Confidence values for individual branches were determined by bootstrap analysis with 1 000 replications. Trees were viewed in Treeview and exported to graphics programs (Page, 1996).

Fruiting culture preparation

C. pruinosa (strain GZUCC 8552) was initially cul-tured on a PDA plate, and then used as seed cul-ture by punching out 5 mm agar discs with a self-designed sterilized cutter. The seed culture was placed in a 250 mL flask containing 50 mL of basal medium (20 g/L sucrose, 20 g/L peptone, 0.5 g/L MgSO4·7H2O and 1 g/L K2HPO4 with 1 000 mL dis-tilled water), and placed on a rotary shaking incu-bator at 130 rev/min at 26OC in darkness for 4 d. Two media were prepared. Solid-state media was prepared by mixing 20 g of rice and 30 mL of nu-trient solution (20 g/L sucrose, 20 g/L peptone, 0.5 g/L MgSO4·7H2O and 1 g/L K2HPO4 with 1 000 mL distilled water) in a cylindrical glass bottle. Liquid static culture media was prepared by mixing 200 mL of basic medium (10 g/L sucrose, 10 g/L glucose, 10 g/L peptone, 1 g/L MgSO4·7H2O, 1 g/L K2HPO4 and 0.5 g/L KH2PO4) with 1 000 mL distilled water in a cylindrical glass bottle. Media were autoclaved for 30 min at 121OC, and each glass bottle was in-oculated with 5 mL of liquid inoculum of GZUCC 8552. After inoculation, the solid-state media were incubated at 20OC in the dark for 10 days, then un-

der 14:10 L:D (500 lux light) at 25OC and 80-90% humidity for 50 days. The liquid static media were incubated for 120 days in the same conditions. Then mycelia from both were collected for drying to a constant weight at 55OC.

Identification of cordycepin and HEA by HPLC-MS

Water extracts of dry mycelia of this strain were pre-pared for compound analysis. Cordycepin and HEA in mycelium were analyzed by HPLC-MS (1100 se-ries, Hewlett-Packard Company, USA). HPLC was with an RP-C18 column (5 μm, 4.6 × 250 mm) (Up-elco, Bellefonte, PA, USA). The mobile phase con-sisted of water and methanol (90:10, v/v). Elution was performed at a flow rate of 1.0 mL min–1 with column temperature at 45℃, injection volume of 20 µL and UV wavelength of 254 nm.

MS was equipped with an electron ionization (ESI) interface. Peaks were detected in both scan and SIM mode. The selected values were as follows: nee-dle potential, 4 000 V; gas temperature, 350O; drying gas, 8.0 L/min; collision-induced dissociation voltage 70 eV; nebulizer pressure, 0.12 MPa. Data were col-lected on an HP ChemStation. Spectra were scanned over a mass range of m/z 50-800.

Standard cordycepin (from Sigma) and HEA (from the Dalian Institute of Chemical Physics, Chi-nese Academy of Sciences) were dissolved in distilled water for calibration.

Content of cordycepin and HEA in C. Pruinosa GZUCC 8552

The content of cordycepin and HEA in the mycelia strain was checked by HPLC under the same condi-tions as given above. Standard solutions of cordyc-epin were prepared into 90, 135, 180, 225, 270 mg/L by diluting with distilled water from 450 mg/L stock, and standard solutions of HEA were prepared into 7.7, 15.4, 22.1, 29.8, 38.5 mg/L by diluting with dis-tilled water from 77 mg/L stock. The standard curve was calibrated by plotting the peak area vs. concen-tration.

1414 ZEBIN MENG ET AL.

RESULTS

Morphological characters of Cordyceps pruinosa strain GZUCC 8552

The fruiting body forming on the cocoon of a Lima-codidae insect, wild strain GZUCC 8552, is shown in Fig. 1A. The liquid static culture and solid-state fermentation of strain GZUCC 8552 are shown in Figs. 1C and 1D, respectively. A colony 35-50 mm in diameter on PDA at 25℃ after 20 days was dense, white at fi rst, later light yellow, radial with a 3-4mm high region protruding in the center part of the colo-ny (Fig. 1B); reverse: center dark yellow, periphery light yellow. The mycelium was 1.5-2 μm wide. Co-nidiophores were 6-10 ×0.5-1 μm, hyaline, smooth, phialides were mainly slender conical. Conidia were 4.5-6 ×1.5-2.5 μm transparent, smooth-walled, cy-lindrical; conidia at the apex of chains were 7-10 ×2-2.5 μm, two-celled, chains with typical imbricate arrangement (Fig. 1E).

Note: Th e characters are typical of C. pruinosa (Liang et al., 2007).

Phylogenetic analyses

Th e combined datasets comprised 3 566 characters aft er alignment, of which 1 394 characters were par-simony-informative, 1 751 constant, and 421 parsi-mony-uninformative. Parsimony analysis generated 5 000 trees; SH test verifi ed that they were similar (tree length = 5,173 steps, CI = 0.567, RI = 0.742, RC = 0.420, HI = 0.443); the most parsimonious tree is shown in Fig. 2.

Th e data set comprises 32 species (Fig. 2), in-cluding the isolate C. pruinosa strain GZUCC 8552 and other strains from GenBank. C. pruinosa formed a separate clade from other species of Cordyceps with strong bootstrap support (100%).

Identifi cation and content of cordycepin and HEA in Cordyceps pruinosa strain GZUCC 8552

by HPLC-MS

Th e HPLC analysis of water extract of the C. pruinosa strain GZUCC 8552 by liquid static culture showed two peaks at retention times of 8.794 min and 10.796

Fig. 1. Strain GZUCC 8552. A – Fruiting body forming on the cocoon of a Limacodidae insect. B – Culture on PDA aft er 20 days. C – Liquid static culture. D – Solid-state fermentation. E – Conidia. Scale bars: E = 10 μm.

CORDYCEPS PRUINOSA PRODUCES CORDYCEPIN AND N6-(2-HYDROXYETHYL)-ADENOSINE IN CULTURE 1415

Table 1. Cordyceps pruinosa and its related species and their NCBI accession numbers used in this study.

Species Voucher 1NCBI accession number

ReferenceITS 18S RPB1

Cordyceps pruinosa GZUCC 8552 KF359948 KF359949 KF359950 This study

Beauveria bassiana IFO 4848 AB027382 AB027336 — Sung and Spatafora (2004)

Beauveria bassiana ARSEF 300 AY532015 — HQ880831 Morar-Bhana et al. (2011)

Beauveria bassiana ARSEF 751 AY532045 — HQ880835 Shin et al. (2011)

Cordyceps bifusispora EFCC 8260 — EF468953 EF468855 Sung et al. (2007a)

Cordyceps brongniartii NBRC 101395 JN943298 JN941759 JN992493 Schoch et al. (2012)

Cordyceps brongniartii BCC 16585 JN049867 JF415951 JN049885 Kepler et al. (2013)

Cordyceps cardinalis OSC 93609 — AY184973 DQ522370 Sung et al. (2007a)

Cordyceps cardinalis OSC 93610 JN049843 AY184974 EF469088 Kepler et al. (2013)

Cordyceps cf. ochraceostromata ARSEF 5691 JN049849 EF468964 EF468867 Sung et al. (2007a)

Cordyceps coccidiicola AB031196 AB031195 — Sung et al. (2007a)

Cordyceps coccidioperitheciata N.H.J. 6709 JN049865 EU369110 EU369067 Kepleret al. (2013)

Cordyceps cochlidiicola AB027377 AB027331 — Sung et al. (2007a)

Cordyceps confragosa CBS 101247 JN049836 AF339604 DQ522407 Sung et al. (2007a)

Cordyceps elongata OSC 110989 — — EF468856 Sung et al. (2007a)

Cordyceps gunnii OSC 76404 JN049822 AF339572 AY489650 Sung et al. (2007a)

Cordyceps gunnii ARSEF 6828 HM140630 — — Chan et al. (2011)

Cordyceps kanzashiana AB027371 AB027325 — Nikoh and Fukatsu (2001)

Cordyceps konnoana EFCC 7315 — EF468959 EF468861 Sung et al. (2007a)

Cordyceps kyusyuënsis EFCC 5886 — EF468960 EF468863 Sung et al. (2007a)

Cordyceps longissima EFCC 6814 — — EF468865 Sung et al. (2007a)

Cordyceps melolonthae OSC 110993 — DQ522548 DQ522376 Sung et al. (2007a)

Cordyceps militaris OSC 93623 JN049825 AY184977 DQ522377 Sung et al. (2007a)

Cordyceps militaris NBRC 100741 JN943437 JN941755 JN992489 Schoch et al. (2012)

Cordyceps militaris ARSEF 5050 HQ880829 — HQ880901 Rehner et al. (2012)

Cordyceps nigrella EFCC 9247 JN049853 EF468963 EF468866 Sung et al. (2007a)

Cordyceps nipponica NBRC 101407 JN943302 JN941752 JN992486 Schoch et al. (2012)

Cordyceps nipponica NBRC 101408 JN943303 JN941751 JN992485 Schoch et al. (2012)

Cordyceps nutans OSC 110994 — DQ522549 DQ522378 Sung et al. (2007a)

Cordyceps pluricapitata NBRC 100745 JN943304 JN941750 JN992484 Schoch et al. (2012)

Cordyceps pluricapitata NBRC 100746 JN943306 JN941749 JN992483 Schoch et al. (2012)

Cordyceps pseudomilitaris NBRC 101411 JN943308 JN941746 JN992480 Schoch et al. (2012)

Cordyceps pseudomilitaris NBRC 101413 JN943310 JN941745 JN992479 Schocha et al. (2012)

Cordyceps ramosopulvinata AB027372 AB027326 — Nikoh and Fukatsu (2001)

Cordyceps ramosopulvinata SU-65 — — DQ127244 Kepler (2010)

Cordyceps scarabaeicola ARSEF 5689 JN049827 AF339574 DQ522380 Sung et al. (2007a)

Cordyceps superficialis MICH 36253 — EF468983 EF468883 Sung et al. (2007a)

Cordyceps takaomontana OSC 111007 — DQ522559 DQ522395 Sung et al. (2007a)

Cordyceps takaomontana ARSEF 5135 AY624196 — JN049896 Kepler et al. (2013)

1416 ZEBIN MENG ET AL.

min, respectively (Fig. 3B), which were consistent with the retention times of standard cordycepin (8.655 min, Fig. 3A) and standard HEA (10.606 min, Fig. 3C). Fig. 4 provides mass fragmentation patterns of the peak at retention times of 8.794 min by LC-MS, the m/z spectrum of the peak showing domi-nant ions [M + H]+ at 252.1 under positive ionization conditions. Thus, the measured molecular weight was deduced to be 251.1, which is in good agree-ment with the value of standard cordycepin. Fig. 5 provides mass fragmentation patterns of peak at re-tention times of 10.796 min, and the m/z spectrum of this peak showed dominant ions [M + H]+ at 312.1 under positive ionization conditions. The measured molecular weight was deduced to be 311.1, which is consistent with that of standard HEA.

The standard curve for cordycepin was y = 3087.2x + 3024 (R² = 0.9997) and that of HEA was y = 395.2x - 9.8 (R² = 0.9996). According to the HPLC data of C. pruinosa strain GZUCC 8552 and the standard curves of cordycepin and HEA, the con-tents of cordycepin and HEA in C. pruinosa strain GZUCC 8552 mycelia by liquid static culture were

5.311 mg/g and 0.558 mg/g, respectively. However, cordycepin of the mycelium by solid-state fermenta-tion was not found; HEA content was 0.198 mg/g.

DISCUSSION

In this study, an isolate of C. pruinosa (strain GZUCC 8552) was obtained from a fruiting body forming on the cocoon of a species in Limacodidae from Guizhou Province, China. Combined gene analysis of 5.8S ITS, RPB1 and 18S gene sequence data and morphological characteristics were used to identify the isolate as C. pruinosa. Cordycepin and HEA were confirmed as being produced by this fungal strain by HPLC-MS. The cordycepin and HEA contents of the strain produced by mycelia in liquid static cul-ture were 5.311 mg/g and 0.558 mg/g, respectively. In solid-state fermentation, the strain produced 0 mg/g of cordycepin and 0.198mg/g of HEA. This is the first report of cordycepin from C. pruinosa. Thus, C. pruinosa provides a new source of cordycepin and HEA.

Species Voucher 1NCBI accession number

ReferenceITS 18S RPB1

Cordyceps tricentri AB027376 AB027330 — Sung et al. (2007a)

Cordyceps tuberculata OSC 111002 JN049830 DQ522553 DQ522384 Sung et al. (2007a)

Cordyceps tuberculata NBRC 106949 JN943318 JN941741 JN992475 Schoch et al. (2012)

Cordyceps variabilis ARSEF 5365 — DQ522555 DQ522386 Sung et al. (2007a)

Cordyceps variabilis OSC 111003 — EF468985 EF468885 Sung et al. (2007a)

Cordyceps yakusimensis — AB044632 — Sung et al. (2007a)

Cordyceps pruinosa ARSEF 5413AUT JN049826 AY184979 DQ522397 Sung et al. (2007a)

Ophiocordyceps cf. pruinosa EFCC 5197 — EF468965 EF468868 Sung et al. (2007a)

Ophiocordyceps cf. pruinosa N.H.J. 10627 — EF468967 EF468870 Sung et al. (2007a)

Aschersonia placenta BCC 7869 JN049842 EF469121 EF469085 Sung et al. (2007a)

1 ARSEF, USDA-ARS Collection of Entomopathogenic Fungal cultures, Ithaca, NY; BCC, BIOTEC Culture Collection, KlongLuang, Thailand; CBS, Central Bureau voor Schimmelcultures, Utrecht, the Netherlands; EFCC, Entomopathogenic Fungal Culture Collection, Chuncheon, Korea; IFO, Institute for Fermentation, Osaka, Japan; KEW, Mycology collection of Royal Botanical Garden, KEW, Surrey, UK; MICH, University of Michigan Herbarium, Ann Arbor, MI; NBRC, NITE Biological Resource Center, Japan ; N.H.J., Nigel Hywel-Jones personal collection; OSC, Oregon State University Herbarium, Corvallis, OR; Guizhou University Culture Collection, Guiyang, Guizhou, China, GZUCC.2. AUT Authentic material.

Table 1. Continued

CORDYCEPS PRUINOSA PRODUCES CORDYCEPIN AND N6-(2-HYDROXYETHYL)-ADENOSINE IN CULTURE 1417

Fig. 2. Phylogenetic relationships among the fungal isolate GZUCC 8552 and related species based on combined 5.8S-ITS rDNA, 18S, RPB1 genes. Bootstrap values (1 000 replicates) are indicated above the nodes. Type species are indicated with an asterisk. Th e tree is rooted to Glomerella cingulata.

1418 ZEBIN MENG ET AL.

Fig. 3. HPLC spectra of standard cordycepin A, extract of Cordyceps pruinosa strain GZUCC 8552 B and standard HEA C; retention time of standard cordycepin: 8.655 min, standard HEA: 10.606 min; retention time of cordycepin of Cordyceps pruinosa strain GZUCC 8552: 8.794 min, fungal HEA: 10.796 min.

Fig. 4. Electrospray mass spectra of cordycepin isolated from Cordyceps pruinosa strain GZUCC 8552, the molecular ion of cordycepin at m/z 252.1 [M + H+].

Fig. 5. Electrospray mass spectra of HEA isolated from Cordyceps pruinosa strain GZUCC 8552, the molecular ion of HEA at m/z 312.1 [M + H+].

CORDYCEPS PRUINOSA PRODUCES CORDYCEPIN AND N6-(2-HYDROXYETHYL)-ADENOSINE IN CULTURE 1419

C. pruinosa is important among Cordyceps spe-cies as it is used in traditional Chinese medicine. Recent studies have shown that polysaccharide from C. pruinosa can improve cellular immune function-ing (Liu and Fei, 2001), while a methanol extract can inhibit inflammatory mediators by suppressing NF-kB activation (Kim et al., 2003; Cui, 2009). A buta-nol fraction was shown to induce apoptosis in HeLa cells (Kim et al., 2010). However, as crude extracts of C. pruinosa and not pure compounds were used, the material basis of the functions is not clear. The discovery of cordycepin in C. pruinosa will help in research and establish the multiple pharmacological attributes of this traditional Chinese medicine.

Cordycepin is an important compound with broad significant pharmacological functions, with production only known in 12 Cordyceps species (Yang and Dong, 2011) and in Aspergillus nidulans (Kaczka et al., 1964). Cordyceps militaris has the highest known cordycepin yield of 8.57 g/L (Das et al., 2009). All other species have low cordycepin yields. In this study, the 5-mg/g cordycepin produc-tion of C. pruinosa is relatively high. It is believed that the capability of cordycepin production of this stain could be enhanced by optimizing both media and culture conditions. C. pruinosa is the best spe-cies for producing cordycepin after Cordyceps mili-taris.

Hao et al. (1999) reported that C. pruinosa could produce HEA at 3.1 mg/g. HEA production is only known to occur in C. pruinosa, Cordyceps militaris, Cordyceps sinensis, Paecilomyces cicadae, Cordyceps coccidiocola, Cordyceps takaomontana, Isaria japoni-ca and 3 Isaria sp. (H55, Is-l and H40)(Cleaver et al., 2011; Lei et al., 2013; Furuya and Hirotani, 1983). The medicinal properties of HEA are understudied. The strain of C. pruinosa studied is a good source of HEA, which can be increased after optimizing. Pro-duction of cordycepin and HEA was higher in liquid static culture than solid-state fermentation. One rea-son for this may be the length of culture, as longer periods could be helpful for cordycepin and HEA production. Liquid state culture is also more suitable for cordycepin and HEA production.

Acknowledgments - This work was supported by the National Natural Science Foundation of China (No. 31200016), the Agricultural Science and Technology Foundation of Guizhou Province (No. [2011]3054) and the Modernization of Tradi-tional Chinese Medicine Program of Guizhou Province (No. [2012]5008). We would like to thank the Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences for the use of the equipment employed in this study.

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