key: cord-0850496-fvh3a5pv
authors: Chen, Peter Xin; Wang, Sunan; Nie, Shaoping; Marcone, Massimo
title: Properties of Cordyceps Sinensis: A review
date: 2013-03-21
journal: J Funct Foods
DOI: 10.1016/j.jff.2013.01.034
sha: 65030a7a3a7b0a16eeddf57acdc055bf2e50f774
doc_id: 850496
cord_uid: fvh3a5pv

A great mystique and aura surrounds Cordyceps sinensis (syn.: Cephalosporium sinensis), an endoparasitic fungus which has claims of anti-cancer and anti-aging properties. Much research has been conducted over the years on crude extracts and its bioactivity. More research is now focused on culturing C. sinensis and on isolating and identifying pure compounds novel to C. sinensis in an attempt to alleviate strain on demand for the natural fungi. Several polysaccharides, nucleosides and sterols all have had reports of promoting health both in vitro and in vivo. Specific and novel compounds which are characteristic to C. sinensis are emerging with reports of two new epipolythiodioxopiperazines, gliocladicillins A and B capable of inhibiting growth of HeLa, HepG2 and MCF-7 tumor cells. Exclusive to natural C. sinensis, five constituents of cordysinin (A–E) has also been reported for the first time and has been linked to anti-inflammatory properties. Although it may still be premature to believe these results should translate into pharmaceutical use, there is sufficient evidence to warrant further research.

A paras itic fungus kno wn as Cor dyce ps has long been co ve ted as an exotic tr aditional Chinese medicine (TCM) with an ove rwhelming list of pharmacolo gical pro perties. Cor dyce ps are the most div erse gen us of the Cla vicipitaceae famil y in the Order Hypocr eales. Ov er 400 species ha ve been rep orted (Sung et al., 2007) making classification a rather tedious task. Eac h indi vidual species is specific to a singular host species, insect caterpillars . The caterpillar fungus kno wn as Cor dyce ps sinensis (Berk) Sacc. stands out as the most documente d species of Cor dyc eps with re ports fro m Tibetan doctors dating as far bac k as the late 1400 (Winkler , 2008a ; Winkler , 2008b ) . The natur al prod uction of Cor dyce ps undoub tedly summoned the inter est of first disco ve rers. The fungal spore s infect and take ove r the host or ganism causing its eve ntual demise . The fungus contin ues to gro w and emer ges fro m the corpse of the host or ganism. Adding to the mystique is the location in whic h the relationship betw een the fungus and the larv a of the ghost moth occurs. The caterpillar fungus liv es on the Tibetan Plateau including parts of India and western China, otherw ise kno wn as the ''Roof of the World''. During the 1993 National Games in Beijing, China wher e thre e female runners set 5 world recor ds for the 1500, 3000 and 10,000 meter race s. After testing ne gati ve for ana bolic ster oids and other banned substance s, it was later re veale d by the coac h that the runners had taken C. sinensis extr acts. Anecdotal evidence of the potential performance enhancin g pro perties of C. sinensis ignited consumer inter est. In fact, the larv a-fungi complex has long been used in TCM to ''in vigor ate the lung and nourish the kidne y'' in China for hundr eds of ye ars (Dong & Yao , 2008 ) .

Demand for the fung i was further accentu ated by the num ero us scientific report s stating specific pro -health-r elated claims. C. sinensis tonic can be pur cha sed ove r the counter and is belie ved to pro vide the imm une system with a boost especial ly after serious illness (Bai, Ren, & Yu, 1997; Chen, Zhang, Shen, & Wang, 2010; Cheung et al., 2009 ). An eve r gro wing list of symptoms rem edied using C. sinensis include respira tory , renal, liv er , nervo us system, car dio va scular diseases, cancer ous tumors, decre ased libido and even str ess, fatigue and aging (Belo , Marc hbank, Fitz gera ld, Ghosh, & Pla yfor d, 2006; Beno witz, Goldber g, & Irwin, 2002; Chen et al., 2005; Dong & Yao , 2008; Ji et al., 2009; Koh, Kim, Chang, & Suh, 2003b; Koh, Kim, Kim, Song, & Suh, 2003c; Koh, Suh, & Ahn, 2003a; Liu, Li, Zhao , Tang, & Guo , 2010; Song, Ming, Peng, & Xia, 2010; Woo Bok, Lermer , Chilton, Kling eman, & Towers, 1999 ; Yama guch i, Kago ta, Nakam ur a, Shinozuka , & Kunitomo , 2000) . In fact, long befor e an y of these studies had been pub lished, C. sinensis has officially been classified as a drug in the Chinese Pharmac opoeia since 1964 (Committee of Pharmacopo eia, Chinese Ministr y of Health, 1964 Health, , 2005 .

The va lue of the fung i recei ve d a sharp incr ease follo wing the se ve re acute respira tory syndr ome (SARS) outbr eak in China in 2003. With the inflated value of the fung i comes incr eased demand leading to se ve re price hikes. Accor ding to 2008 prices, the price of natur al C. sinensis rang ed from 3000 to over 18,000 USD per kg dep ending on size of the larv ae (Winkler , 2008a ) . In a decade (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) (2008) , the price of C. sinensis has incre ased 900% (Winkler , 2008b ) . This cr eates a str ong socioecon omic stra in in the Tibetan Plateau wher e man y villa gers ha ve fought over the desecr ation of grassla nds containing the sought -after fung i. The ava ila bility of the fung i is limited by its confined geogr aphic location. With the added effects of lar ge-scale harve sts, the fungi has been classified as an endangere d species by CITES (Convent ion on International Trade in Endanger ed Species ) Mana gement Authority of China and China Customer s. The scar city of natur al C. sinensis sparked no ve l appr oac hes such as artificial culti va tion of the pure my celium in liquid cultur e or on gra ins.

Man y subsequ ent studies were conducte d using cultur ed C. sinensis and ha ve yielded positi ve resul ts suggesting the culti vated fungi might possess the same health-pr omoting functio ns as the natur al counterpart Cheung et al., 2009; Dong & Yao , 2008) . On the backs of these reports, the USA passed the Dietar y Supplement Health and Educati on Act in 1994 givi ng wa y to a va st market consisting of websites selling capsule s and tonics made fro m inde pendentl y gro wn Cor dy ceps. Fueled by an incr easing wave of health-c onscious consum ers and countless supportin g scientific liter ature to boot, demand for Cor dyce ps pro ducts are at an all-time high. Yet with all this, no major pharma ceutical compan y has de veloped a supplement to capital ize on the market. The lack of support fro m big name pharmace uticals puts into questions the va lidity of some rep orts and if the findings are substan tial or mer ely ove rblo wn. Dong and Yao (2008) called out a stud y on C. sinensis and its pro tecti ve effects on PC12 cells fro m hydr oge n-per oxide induced neur onal toxicity (Li et al., 2003 ) . Althou gh the claim in vo lve d Cor dy ceps, the actual str ain used was named Cephalospo rium sinensis Chen sp . no v. supplied by Wan Fong Pharmac eutical Factor y (Zhejiang, China). It wou ld appear as though the lac k of regulation and quality contr ol has affected factories and their distributio n of C. sinensis . Yama guch i et al. (2000) pur cha sed their artificially culti va ted fruiting-bodies of C. sinensis from the Xinhui Xinhan Artificial Cor dyce ps Factor y (Guangdong, China) and successful ly radi cal sca ve ng ing po wer of both the water and ethano l extr acts. Ho weve r, both Dong and Yao (2008) and Paterso n (2008) belie ve the fungal material to be unauthentic claiming that the man ufactur er is actuall y selling C. militaris. These findings can cause dama ge to the repu tation of the ove rall bod y of work in vo lving C. sinensis and its ther apeutic pro perties. Asian countries ha ve a marked ad vanta ge in the Cor dyce ps market due to its inher ent oriental origin; these nations are also responsib le for deli veri ng the va st majority of liter ature reg ar ding the beneficial pro perties of Cor dyce ps .

The follo wing revie w focuses on the differ ent methods used to extr act and identify compoun ds deri ve d fro m C. sinensis and ho w the y ha ve been re ported to possess pharma ceutical pro perties.

An over whelming bod y of litera tur e in vo lving C. sinensis has been conducte d using solv ent extr acts of the fungus. Extr acting solv ents va ry based mainl y on polarity and ease of use. The lar ge vo lume of resear ch rela ted to C. sinensis extr acts (CSE) has whittled do wn the cho ice of solv ents to a critical fe w that ha ve been sho wn to be effecti ve in extr acting the desir ed compoun ds. In or der of incre asing polarity , eth yl acetate , ethanol, methanol and water figure amongs t the most commo nly used solv ents for extr action althoug h combinati on and sequent ial extr actions are also common practic e.

Water , a polar solv ent, has long been used in the field of food chemistr y in part due to its nontoxic ity and in va siv eness (Cheung et al., 2009 ). Its molecular structur e contains dipoles and can solv ate ions by orienting itself accor ding to electr ostatic attracti on betw een the ion and the water molecule . A hydrat ion shell is formed and sta bility is achi eve d. Yama guch i et al. (2000) performed a simple hot water extr action using 10 g of dried fruiting bodies of culture d C. sinensis blanch ed in 200 mL of hot wate r at 70 °C for 5 min. This pro duced a br own-color ed extr act whic h was then filtered and ly ophilized into a gra y po wder whic h was administ ered to rats to determin e its effects on cholester ol le vel s (Yamag uc hi et al., 2000) . Ji et al. (2009) seemingl y follo wed the same extr action pro tocol ho weve r, at a higher temper atur e and longer extr ac-tion time in or der to pro ve antia ging effects of C. sinensis extract . In brief summar y, C. sinensis po wder was added to wate r at a 1:20 ratio (w/v) and heated by autocla ving for 20 min to a temper atur e of 120 °C. The extr acted po wder was then filtered and ly ophiliz ed and a yield of 30% was ascertained. This simple extr action tec hnique was also emplo ye d by Song et al. (2010) to test repa rati ve effects of the extr act on kidne y functio n and its potent ial as a ther apeutic drug for kidne y diseases (Song et al., 2010 ) . Slight modifications included the 1:5 (w/v) po wder to water ratio and extr action tempera tur e and time set to 90 °C and 2 h, respe cti vel y. Koh et al. (2003c) performed a series experim ents in vo lving hot-w ater extrac t fro m my celia of C. sinensis in 2003 and effecti ve ly demonstr ated ph ysiological functio n as antibiotic gro wth promo ter (Koh et al., 2003a ) , antistr ess and antifatig ue effect (Koh et al., 2003c ) as well as hypoc holester olemic effect (Koh et al., 2003b ) of the hot water extr act. Extr act pre par ation proto col for the antibio tic gr owth pro moting follo wed that of Ji et al. (2009) and Yama guc hi et al. (2000) ho wever , at 100 °C (Koh et al., 2003a ) . A mor e thor ough and ela bora te extr action proce ss was emplo ye d for the subsequent experiments in vo lving antistr ess, antifatigu e and hypoc holester olemic effects of C. sinensis extr act wher eb y 50 g of freez e dried C. sinensis po wder was boiled quick ly at 100 °C for 5 min, homogenized and centrifuged. A sequent ial fracti onation of the resultin g residu e ensued using solv ents of incr easing polarity be ginning with eth ylacet ate, methanol and hotwate r. The final hot-w ater extr act was centrifu ged and the supernatant fre eze-dried. This mor e thor ough hot-w ater extrac tion proce ss did not pro vide a gre ater yield (29.3%) compare d to that of Ji et al. (2009) . Liu et al. (2010) pre pare d separate hot water and ethanol extr act, which were later combined for their experim ents in vo lving the ther apeutic effects of the extr act on br ain dama ge (Liu et al., 2010 ) . Once again , a str aight forw ar d extrac tion pro cedur e was follo wed with temper atur e set to 100 °C and extr action time of 3 h. An aliquot was rem oved and ly ophiliz ed and yield of 29% was determine d prior to the subsequ ent ethanol extrac tion. It wou ld appear that based on the ab ove menti oned extr act pre parat ion pro tocols, time of extr action appears to be of minimal value seeing as a 5-min extr action pro ve d just as effecti ve as a 3 h extrac tion. Temper ature ma y be a mor e critical param eter .

Fr actionatin g C. sinensis extr act with va rious solv ents is a rela tiv el y no vel appr oac h and certainl y critical for thor ough in ve stigation of activ e compon ents dire ctly responsib le for the man y ph ysiological roles associate d with C. sinensis . One such stud y in vo lve d structur al analy sis of pol ysacc harides in culture d my celia of C. sinensis responsib le for monoc yte activ ation and impr ove d innate imm unity (Akaki et al., 2009) . Cultur ed mycelia wer e defatted by incubating for 6 h in ethanol three times. The extr action was then performed on the residue using distilled wate r at 100 °C for another 6 h. Follo wing centrifugati on, the supernat ant was reco ve red and concent rated do wn to half its initial vo lume and ethanol was added to make up the differ ence . The resulting preci pitate was freez e-dried and re pre sented the crude pol ysacc haride fracti on with a yield of 4.2%. A portion of this crude extrac t was dissolv ed in wate r and dial ysis was performed to rem ove lo w-molecula r-w eight constituen ts. After 3 da ys of dial ysis, the liquid within the membr ane was rem ove d and centrifuged resulti ng in a water soluble polys acch aride fra ction (2.6%) and a water insolub le polys acch arides fractio n (0.2%). All three fra ctions were then subjecte d to further analysis after suspensio n in distilled water at a concent ratio n of 1 mg/mL . Sugar content , cytokine pro duction, Fourier tran sform infr are d spectr oscop y (FTIR), nucl ear ma gnetic resonance (NMR) and meth ylation analy ses were performed on all thr ee fra ctions in or der to pinpoint whic h fracti on was responsib le for the imm unostim ulation and subsequentl y inve st efforts into one particula r fra ction.

In 2011, a gro up of resear chers set out to explor e the antiinflammator y pro perties of extr acts of C. sinensis my celia (Yang, Kuo , Hwa ng, & Wu, 2011 ) . The intensi ve extr action protocol was based on a crude ethanol extrac t subsequentl y partitioned into n-hexane-solu ble and water -solub le fra ctions. The n-hexan e fra ction was further sepa rated into n-hexane/ n-hexane and n-hexane/Me OH-H 2 O fractio ns. The water -solub le fra ction was furthe r partitio ned with eth yl acetate to giv e eth yl acetate and water -solub le fra ctions. The crude and the partitio ned fra ctions wer e tested for inhibitio n of super oxide anion genera tion and elastase activ ity with the n-hexane/n-hexane , hexan e/MeOH-H 2 O and eth yl acetate fra ctions containing the most inhibitio n and elastase acti vity . Further purification of these fractio ns led to the disco ve ry of five no ve l compoun ds and 45 kno wn compoun ds. NMR and mass spectr oscop y was utilized to anal yze structur es. The five no vel compou nds comprise cor dysinin s and its va rious constituen ts simpl y defined as compou nds 1 thro ugh 5 ( Fig. 1 ).

In an attem pt to elucidate specific bioacti ve compoun ds fro m my celia of C. sinensis with antitumo r activ ity , an 'acti vity guided chem ical fra ctionation ' of the methanol extr act of C. sinensis was performed and led to the isolation of tw o antitumor compound s (Woo Bok et al., 1999; Gonza ´lez-Sarrías, Li, & Seer am, 2012) . A crude methanol extr act was pre par ed using 150 g of dried my celium and 500 mL of MeOH. The pro cess was re peated three times and the supernat ants combin ed and the solv ent was evapora ted under pre ssur e. The crude extr act was used in an antipr olifer ation assa y using a K562 ery thro leukemia cell line and was sho wn to ha ve the ability to inhibit pro lifer ation by 36%. No further testing was performed on the crude extr act and instead extr action focused on the rem aining residue from the crude MeOH extr action. The resid ue was redissolv ed in a 1:1 (v/v) mixtur e of MeOH/ H 2 O and partitioned using hexanes. The aqueous la yer was then extr acted with eth yl acetate . Eth yl acetate extr acts were concentr ated under press ure to rem ove the solv ent and a bro wn oil remained as a result. Silica gel-flash colum n chromatogr aph y elution was performed using CHCl 3 with ste pwise incre ase of the solv ent polarity . In all, 14 fractio ns were collecte d and tested for antitumo r activ ity . Fr action s that tested positi ve wer e further chroma togr aphed. This led to tw o fra ction FB1 and FB2. FB1 was report ed as a white solid and was recr ystallized into a white po wder and subsequentl y la belled compound 2. Further sep ara tion of FB2 using silica gel chr omatogr aph y resulted in the isolation of compound 1a and a fra ction FC1 which was injected into HPLC and ga ve compoun ds 3 and 4. The structur e of these compoun ds were resolve d using NMR and in sum, a ster oidal gly coside 5a,8aep i-dioxy-24(R)-methylch olesta-6,22-d ien-3 b-D-gluco pyra -noside (compound 1a) and 5a,6a-epo xy-24(R)-methylch olesta-7,22-dien-3b-ol (compound 2) were isolated and identified for the first time as anti-tumor compoun ds. Er goster yl-3-Ob-D-glucop yr anoside (compound 3) and 22,23-dih ydr oer gostery l-3-Ob-D-glucop yra noside (compound 4) were also isolated follo wing this extr action pro tocol but ha ve pre viousl y been isolated (Shiao , Lin, Lien, Tzean, & Lee, 1989 ).

The va st majority of resea rch conducted on the extr act of C. sinensis utilized ethanol, methanol or hot water as extrac tion solv ents mainl y due to common pra ctice and their ab ility to extr act polar components (Wu, Zhang, & Leung, 2007 ) . Eth yl acetate , although the least polar of the thre e aformenti oned solv ents, has garneshe d mor e attention of late and has gained in populari ty . Studies condu cted by Zhang, Wu, Hu, and Li (2004) and Zhang and Wu, (2007) ha ve effecti ve ly sho wn that eth yl acetate extr acted C. sinensis my celium to be a potent inhibitor of pro lifer ation of cancer cells Zhang et al., 2004) . Pre par ation of the extr acts follo wed a sequent ial or der starting fro m non-polar to polar solv ents. The dried my celium po wder was first extr acted using petr oleum ether , eth yl acetate , ethanol and water at a 1:10 (w/v) for eac h solv ent. The extr action pro cess in vo lv ed the use of an orbital shaker at room temper atur e for 24 h exce pt in the case of the hot water extr action wher e a hot plate and stir bar were used for a period of 3 h. After eac h roun d of extr action, the extr act was filtered and the solv ent was roto va pped to dry ness to affor d the crude extr act. In a mor e recent stud y aimed at identifying no ve l anti-cancer agents fro m C. sinensis , Chen et al. (2009) extensi ve ly extr acted the fung i with eth yl acetate six times ove r. After va cuum evapora tion of the solv ent, 40 g of the crude extr act was obtained and further fra ctioned by silica gel chr omatograph y using a CH 2 Cl 2 -MeOH elution gr adient. At a ratio of 99: 1 (v/v) CH 2 Cl 2 -MeOH, a 1 g activ e fracti on was collected and sep ara ted once mor e on a smaller silica gel column using 99.5: 0.5 (v/v) CH 2 Cl 2 -MeOH as eluents . Purification of a 150 mg activ e fra ction using re ve rsed-phase high-performance liquid chr omatogr aph y (RP HPLC) yielded 30 mg of glicladicillin A. Purification of a 120 mg activ e fractio n using RP HPLC resulte d in 25 mg of glicladic illin B. A thir d compound was isolated fro m a 100 mg activ e fra ction ag ain using RP HPLC and was identified as 11,11 0 -dideoxyv erticillin (18 mg).

Pr essurized liquid extr action (PLE) PLE pro motes the high extr action pro perties of the solv ent used by sustaining high temper atur e and press ure within the extr action cartridge or cell. The high temper atur e ena ble s high solubility and diffusion while high pre ssur e ensur es the solv ent is belo w boiling point. Both par ameters combine d, this allo ws the solv ent to penetr ate the sample with mu ch mor e efficiency resulting in fast extr action time using less solv ent (Luthria 2012). Li et al. (2004) performed PLE on po wder ed C. sinensis in a stud y in vestigating the prese nce of er goster ols and nu cleosides by HPLC. The extr action pro cess in vo lve d mixing the Cor dyc eps po wder with diatomaceo us earth in a 1:1 pro portion and subsequentl y placed in a stainless steel extr action cell. Optimiza tion trials scrutin ized par ameters including type of solv ent, temper atur e, static extr action time and static cycles. Temper atur e and extr action time pro ved to be most influential to extr action efficiency. Taking every thing into account , the pro posed optimal PLE condition s were : solv ent, methanol; temper ature , 160 °C; static extr action time , 5 min; press ure , 1500 psi; static cycle , 1 . This extr action tec hniqu e successful ly reco vered a plethor a of compoun ds most notab ly cor dyce pin whic h had pre viousl y not been found in C. sinensis follo wing HPLC detection (Shiao , Wang, Lin, Lien, & Wang, 1994 ) . Cor dyce pin is largely pre val ent in C. milita ris and rar ely detected in C. sinensis althoug h this stud y using PLE confirmed its pre sence in both cultur ed and natur al C. sinensis .

Nucleo sides are consider ed the main bioacti ve componen t in C. sinensis (Li, Yang, & Tsim, 2006 ) . Various ph ysiological processes ar e mediate d via nu cleoside rece ptors includin g nucleic acid synthe sis, stim ulating imm une respo nse (Cheung et al., 2009) , influence fatty acid meta bolism (Koh et al., 2003a) , assisting in iro n ab sorption in the gut and contribute repa rativ e pro perties in dama ged gastr ointestin al tract (Marc hbank, Ojobo , Pla yfor d, & Pla yfor d, 2011 ; Yang, Li, Feng, Hu, & Li, 2010) . Solub le nucl eosides are conside red by some the main bioacti ve componen ts in C. sinensis (Xie , Huang, Hu, He , & Wong, 2010) . To date , mor e than 10 nu cleosides, nu cleobases and related compoun ds ha ve been isolated and identified in Cor dyc eps (Fan et al., 2006 ) . These include adenine , adenosi ne, cytosine , cytidine , uridine , guanine , guanosine , hypoxanth ine, inosine , th ymine , th ymidine , 2 0deoxyurid ine and cor dyce pin (Yang, Ge , Yong, Tan, & Li, 2009 ). Adenosine has been sho wn to ha ve re gulator y function on the centr al nervo us system (CNS) and has anticon vulsant acti vity as evidenced in vivo in rats (Ballarin, Herr era -Marschitz, Casas, & Unge rstedt, 1987) . Oxidati ve deamination by the enzyme adenosine deaminase substitutes the amino gr oup on adenosin e with a hydr oxyl gro up resul ting in the related nu cleoside inosine which has pro ve n to mediate axonal gr owth and could help impr ove the condition of patient s suffering fro m CNS injur y (Beno witz et al., 2002 ) . Cor dyce pin (3 0 -deoxy adenosin e) is pur e compound certainl y worth y of attention. The pre sence of cor dycep in has been firmly esta blish ed in C. militar is using a highspee d counter -curr ent chro matogr aph y tec hnique yielding cor dyce pin with purity of 98.9 and 91.7% reco ve ry (Zhu, Liang, Lao , Zhang, & Ito , 2011) . Ho wever , contro ve rsy lingers reg ar ding its ava ila bility in C. sinensis (Fan et al., 2006 ) . Cor dyce pin has been classified as an anti-canc er compound stemmi ng fro m its ab ility to substit ute adenosin e ther eb y terminating syn-thesis of RN A molecule s (Paterson, 2008 ) . Extr action and detection of nucl eosides enab les differe ntiation betw een va rious stra ins of Cor dyce ps as well as to ensur e identification of counterfeits that ha ve become mor e wide spr ead in rece nt years due to high va lue dri ven by scar city of the natur al prod uct.

Se ve ral detectio n methods ar e common ly availab le suc h as thin la ye r chr omatogr aph y (TLC) (Ma & Wang, 2008 ) , HPLC (Song, Liu, Li, & Jin, 2008 ) and capillar y electr ophor esis (Li, Feng, Ni, & Zhang, 2008) ho wever , limitations are encounter ed in the wa y of sensitiv ity , selecti vity and suita bility . Ion-exchan ge chro matogr aph y (IEC) is yet another method for separatio n of nucl eosides ho we ve r, the K + and Na + pre sent in the mobile phase pre vents further anal ysis and detectio n via mass spectr ometry (MS). Ion-pairin g re ve rsed-phase liquid chr omatogr aph y (IP-RP-LC-MS) has been commo nly used in other fields for the sep ara tion of nucl eotides . Reagents used her e ar e critical for optimal mass spectrom etry results. Another effecti ve method of sep ara tion and detection in vo lve s liquid chr omatogr aph y sep ara tion coupled with electr ospra y ionization -mass spectr ometry (LC/ESI-MS) (Xie et al., 2010 ) .

High perfor mance liquid chr omatogr aph y (HPLC)

One of the first attempts at genera ting a pro file of nucl eosides and nitro gen bases for C. sinensis was performed by Shiao et al. (1994) and emplo ye d the use of reve rsed-pha se HPLC. Gradi ent elution using tw o-solv ent system consisting of 2.5% MeOH and 20% MeOH in 0.01 M ammonium phosphate re veale d the pre sence of major nucl eosides and nitro gen bases, ura cil, guanin e, uridine , guanosine and adenosin e. Shiao et al. (1994) were successfu l in pro ducing RP-HPLC pro files of va rious Cor dy ceps species and Paecilom yces species pro viding the first ste p in distingui shing betw een the va rious species and the use of metab olites as markers for quality contro l. The use of a phospha te buffer impr oves chr omatograp hic performance ho we ve r can result in ion suppr ession for mass spectr ometry detectio n and poor sepa ration (Kla witter , Sch mitz, Kla witter , Leibfritz, & Christian s, 2007). RP HPLC was used by Chen et al. (2009) to identify three anti-cancer compoun ds fro m acti ve fra ctions of a crude eth yl acetate extr acted C. sinensis . Mobile phase par ameters emplo ye d in detectin g these compoun ds va ried and ther efor e three sepa rate runs were performed. In the isolation of gliocladicillin A, 50-55% acetonitril e in wate r ran for 5 min and the concentr ation of acetonitrile was held constant at 55% for the remainin g 25 min with peak retention time observ ed at 14.3 min. Gliocladic illin B was observ ed at a retention time of 19.5 min using 60-85% MeOH as mobile phase for 40 min. The last compoun d 11,11 0 -dideoxyv erticillin was eluted at 18.0 min using 60% acetonitrile in water for 5 min follo wed by 60-70% acetonitrile for 25 min.

Capillary electr ophor esis (CE)-mass-spectroscop y (MS) CE pro vides an attr activ e method for the identification of nucle osides and nu cleobases due to its simplicity of use, high sepa ration efficiency, small sample vo lume and lo w or ganic solv ent consumpt ion (Yang et al., 2009 ) . Optimize d CE-MS conditions were used in volving 100 mM formic acid containing 10% (v/v) methanol as CE electr olyt e. The sheath liquid assists the tran sfer of the anal yte fro m liquid phase to gas phase . Along with the CE electr ol yte , this ensur es sta bl e electr ospra y. Optimal sheath liquid conditions were 75% (v/v) methanol contai ning 0.3% formic acid. Follo wing optim al conditions, tw elve nucl eosides and nu cleobases were detected. This include s cytosine , adenine , guanine , cytidine , cor dyce pin, adenosine , hypoxanth ine , guanosin e, inosine , 2 0 -deoxyuridin e, uridine and th ymidine in both natur al and cultur ed C. sinensis (Yang et al., 2009 ) . Quantitati ve results indicate total content of nu cleosides is mu ch higher in the cultur ed fung i with the exce ption hypoxanthi ne and inosine . Cor dycep in, whic h is mostl y found in C. militaris , was onl y detected in min ute amounts in natur al C. sinensis and was not detected in culture d samples .

Selection of the mobile phase must take into conside ratio n of the sepa ration of the nucl eosides but also the ESI component wher eb y the analy te mu st be in a volatile state. For examp le, pre vious HPLC methods (Guo , Zhu, Zhang, & Zhang, 1998 ) used grad ient elution consisti ng of a KH 2 PO 4 buffer and methanol ho wever the lo w volatility and high salt content render ed this system incompati ble for LC/ESI-MS . An optimiz ed method was de ve loped and va lidated. Xie et al. (2010) suggests the use of ammonium acetate and methanol for the chr omatogr aphic compon ent. Optimiza tion of ESI-MS invo lve d testing in positiv e and nega tiv e ion mode and scanning betw een m/z 50-350 per second . Positiv e ion mode enab led the detection of th ymine , adenine , adenosine , cor dyce pin and 2-ch loroa denosine (internal standar d). Selecti ve ion monitorin g (SIM) mode was used to detect as well as to quantify the four main nu cleosides (thymine , adenine , adenosine and cor dyce pin). Xie et al. (2010) used [M+H] + at m/z 127, 136, 268, 252 and 302 for monito ring. The ionization temper atur e was set to 400 °C. The Cor dyce ps samples fro m differe nt sour ces were extr acted using distilled water and ultr asonicated. The va cuum dried filtrate was dissolv ed in methanol prior to chro matogr aphic sepa ratio n and subsequ ent ESI-MS detection. Follo wing this pro tocol, the four main nu cleosides were detected with 98.47-99.32% reco ve ry rates.

The limit of detectio n (LOD) is the lo west quanti ty of a detecta ble substance distinguish ab le fro m the blank sample while the limit of quantification (LOQ) encompa sses not onl y the lo west concentr ation of detectio n but also the limit at whic h one can reasona bl y distinguish differ ences betw een tw o differe nt val ues by ack no wledging a pred efined le ve l for bias and impr ecision (Armbrus ter & Terry , 2008 ). LOQ and LOD for th ymine , adenine , adenosine and cor dyce pin were respecti vel y 1.0 and 0.2, 1.8 and 0.6, 0.6 and 0.1 and 0.5 and 0.1 lg/mL (Xie et al., 2010 ) . Concentr ations of th ymine , adenine, adenosine and cor dycep in were 138.5-174.2, 20.4-38.1, 79.6-186. and 31.3-91.2 lg/g (Xie et al., 2010 ) . Fan et al. (2006) claim to ha ve de ve loped a method combini ng press urized liquid extr action (PLE) and HPLC-ESI-MS that ''significantly in-cr eases the selecti vity , sensiti vity and repe ata bility of assa y''. Similar to that of Xie et al. (2010) , ammonium acetate and methanol were used as the mobile phase with UV detection occurring at 254 nm. Positiv e ion mode was used ho wever; ne gati ve ion mode was used for uridine detectio n. The addition of PLE prior to LC/ESI-M S resulted in the disco ve ry of 43 compoun ds and the identification of 16 of those compoun ds. The identified compoun ds include cytosine , cytidine , ura cil, uridine , th ymine , th ymidine , adenine , adenosine , 2 0 -deoxy adenosine , cor dyce pin, hypoxanthin e, inosine , guanine , guanosine , 3 0 -amino-3 'deoxy adenosine and 6-h ydr oxy eth yladenosin e (Fan et al., 2006 ) . With the exclusion of 2 0 -deoxy adenosine , 3 0 -amino-3 0 deoxy adenosin e and 6-h ydrox yeth yladenosin e, the rem aining 13 compound s were further in ve stigated for linear ran ge and detectio n limits. For comparison sake , LOQ and LOD for th ymine , adenine , adenosin e and cor dyce pin wer e respe ctiv ely 45.21 and 11.32, 24.39 and 6.11, 3.44 and 1.72 and 4.64 and 1.16 ng/mL (Fan et al., 2006 ) . A mu ch lo wer detectio n limit was observ ed in rela tion to Xie et al. (2010) . In terms of concent ratio n of these four nu cleosides, compariso n betw een the tw o studies is rathe r cumbers ome since Xie et al. did not specify the natur e (culture or natur al) of the Cor dyce ps used in the stud y leading one to make assumpti ons based on the source of the Cor dyce ps . Th ymine and cor dyce pin were detecte d in muc h greate r concentr ations accor ding to Xie et al. (2010) while concent ratio ns of adenine and adenosin e meas ured by Fan et al. (2006) overw helmingl y trumpe d that of Xie et al. (2010) . The reco ve ry rate was mor e than 93.6% on aver age (Fan et al., 2006 ) . The 13 compoun ds were further quantified and compar ed amongst natur al and cultur ed C. sinensis fro m va rious regions in China along with data fro m cultur e C. militaris . Briefly, uridine , adenosin e and guanosin e wer e dominant in culture d ove r natur al C. sinensis . Ho wever , inosine figured mor e pro minentl y in natur al C. sinensis. Cor dyce pin was detected in small amounts in natur al C. sinensis and essentia lly not prese nt in the cultur ed fungus. The small amount of cor dyce pin in natur al C. sinensis pales in compariso n to that found in C. militaris .

Ion-pairing-r ever se phase liquid chr omatog raph y-MS (IP-RP-LC-MS) Ion-pairi ng chro matogr aph y requ ires an additiona l rea gent in the mobile phase char ged for inter action with the incoming anal yte. The reagent used her e can influence the quality of the ensuing MS by clouding and polluting the resulting spectr a. The vo latility of the rea gent is ther efor e trem endousl y importan t to incr ease the sensiti vity of the MS for the sampled anal ytes. Some commonl y used rea gents include N,Ndimeth ylhexylamin e (DMHA) (Vinas et al., 2010 ) , 1,5-dim ethylhexyla mine (1,5-DMHA) and hexylam ine (HMA) (Fr omentin , Ga ve gnano , Obikhod, & Schi nazi, 2010 ). The amine inter acts with the positi ve ly char ged nucle otide phosphate s. Although capa ble of anal ysis nu cleotides , these solv ent systems requ ired high concentr ations (5-20 mM) of the ion-pairing rea gent often resulti ng bac kgr ound signals and decr eased sensiti vity . Pentadecafluorooc tanoic acid (PDFOA), a perfluorinated carboxylic acid, had pre viousl y been used to detect 10 underi va tized amino acids (Asp, Asn, Ser , Gly , Gln, Cys, Glu, Thr , Ala, Pro ) which were less common using pre vious detectio n methods (Petritis, Chaimbau lt, Elfakir , & Dre ux, 1999) . Compar ed with other perfluorinated carboxylic acids, the long n-alk yl chain PDFO A pro ve d best at sep ara tion and was emplo yed for the detectio n nucle otides and nucl eobases along with deriv ati ve compou nds in Cor dyceps . Yang et al. (2010) performed sepa ration of the extr act using a gra dient mobile phase consist ing of 0.25 mM PDFO A in solution and acetonitrile with peaks detectin g at 260 nM. Validation of this solv ent system comes via results indicatin g lo w limits of detection. The overal l LOD and LOQ for all 16 in vestig ated compound s wer e less than 0.16 and 0.41 lg/mL, respecti vel y . The va lues are not sta gge ring and ar e in fact compar ab le to pre viousl y conducte d MS (Fan et al., 2006; Xie et al., 2010 ) . The ad va ntage to using PDFO A in ion-pairing chro matogr aph y is attributed to highl y resolve d peaks due to decr eased peak tailing whic h occurs when compoun ds ar e str ongly inter acting with the stationar y phase as well as the stain-less steel har dware . This is effecti ve ly reduced with the addition of an ion-pairing rea gent in the mobile phase facilita ting identification of peaks.

Pol ysacch arides are water solubl e and are obtained by ethano l pre cipitation. Polys accha rides comprise a major chem ical constit uent found in C. sinensis and the y are responsib le for man y of its pharmacolo gical prop erties. Based on a 2008 revie w, polys acch arides ha ve been sho wn both in vitr o and in vivo to possess anti-inflammator y, antioxid ant, anti-tum or , anti-m etastic, imm unomodula tory , hypogl ycemic, ster oidogenic and hypolipidae mic (Paterson, 2008 ) .

Ethano l has pre viousl y been used to pre cipitate pol ysacc harides from water Wang et al., 2011 ) . Chen et al. (2010) examined effects of the extr act while Wang et al. (2011) focused primaril y on structur al char acterization of the extr act. Pre par ation of the pol ysacc haride fracti on differ ed in appr oac hes. Chen et al. (2010) isolated the my celia fro m the liquid medium by centrifu gation prior to boiling with distilled water and then pre cipitating with ethano l. After washing with ethanol to get rid of unw anted polar compou nds, the pre cipitate was dried and redi ssolve d in water . DEAE-32 cellulos e column was used to elute the neutr al and acid pol ysacch aride fra ctions. Water and a gra dient NaCl solutio n (0-2 M) was used for elution with monitorin g of the eluate via phenol-sulfuric monitor ing. The supernat ant was freez e-dried after dial ysis thus compl eting the extr action proce ss. DEAE elution pro file sho wed two peaks; first peak was that of neutr al pol ysacc harides eluted with water while the second peak is that of the acid pol ysacc harides eluted from the NaCl grad ient. A mor e thor ough in ve stigation of the acidic pol ysacc harides fra ction was perform ed by Wang et al. (2011) . In their extr action pro tocol, the boiling of the my celia ste p was bypasse d and instead used centrifugati on to collect the liquid supernat ant fro m the liquid culture medium used to ferment the fungus. Ethanol preci pitation was then used to collect the exopol ysacc ha-ride whic h was subsequ ently freez e-dried and report ed as a 'whitish and fibrous solid' (Wang et al., 2011 ) .

To get rid of prote in content, the crude exopol ysacch aride extrac t was tr eated with Se vag rea gent (1-butanol/chlor oform 1:4, v/v). Dial ysis and fre eze-dr ying completed the exopol ysacc haride pre par ation ste p. Further purification was require d in or der to obtain acidic pol ysacc harides. The dried exopoly sacc haride was dissolv ed in a Tris-HCl solution ove rnight and the supernatant collected and loaded onto a DEAE-52 cellulos e column for ion-exc hange chr omatogr aph y. Some what similarl y to Chen et al. (2010) , a NaCl gr adient (0.2 and 0.4 M in Tris-HCl) was used for elution with monitorin g for the prese nce of pol ysacc harides by the anthr one-su lfuric acid method. Elution pro file sho wed a total of four distinct peaks. The first peak was eluted with the buffer solution at 0 M NaCl and the three later peaks were eluted at 0.2 M NaCl. The combined eluates repr esenting the thre e latter peaks wer e collection and further purified by gel-permeatio n chr omatogr aph y on a Se phadex G-75 column with eac h fra ction eluted with 0.2 M NaCl. The collected eluates fro m this ste p wer e dial ysed ag ainst water and fre eze-dried to pro duce the acidic pol ysacch aride fractio n repr esenting 52% of the original crude exopol ysacch aride (Wang et al., 2011 ) . Agar ose gel electro phor esis stained with the positi ve ly char ged toluidine blue displa ye d a single red dot upon being subjected to the acidic pol ysacc haride fra ction. This chan ge in color indicates the pre sence of pol ya nions within the fracti on ther eb y confirming the acidic pro perty as well as its homogeneity as evidenced by the prese nce of a singular point. A single peak was also observ ed when subjecte d to high performance gel permeation chr omatogr aph y (HPGPC). A retention time of 25 min would suggest a molecular weig ht of 36.3 kDa.

In a stud y focused on pol ysacch aride fro m C. sinensis and its ab ility to combat renal failure , a crude pol ysacch aride extract (21 g; 7% yield) was obtained and subjecte d to a DEAE-52 cellulose anion-e xch ang e colum n eluting with 0.05 M NaCl solution . Gel-perme ation purification was performed on a Se phadex G-100 colum n and eluted with distilled water . The collected eluate was ly ophilized resul ting in a white po wder (1.6 g) repr esenting 7.6% of the crude pol ysacchari de. Purity of the final fra ction (CPS2) was ve rified with the Bra dfor d assa y displa ying ne gati ve results indicating the lack of pro teins. Spectr ophotome ter anal ysis at 260 and 280 nm sa w no ab sorption indicating the ab sence of pro tein and nucl eic acid. HPGPC anal ysis using 0.1 M NaNO 3 displa yed a singular sharp peak at rou ghly 16 min thus suggests fractio n homogeneity . A dextr an standar d was used to determine a molecular weight of 43.9 kDa. Wang et al. (2009) extract ed a polys acch aride fracti on called CPS1 fro m fruiting bodies of cultur ed C. sinensis , whic h was sho wn to possess antioxidant acti vity and was subjecte d to further charac terization. The impr essi ve health claims pro mised by both fra ctions were intriguing and pro mpted the rese arc hers to dig dee per and attempt to char acterize their respe ctiv e structur es.

Str uctur al analysi s A va riety of anal ysis methods are curr entl y avai lab le to resea rchers. In almost all cases, multiple methods are used to pro vide a thor ough and mor e conclusi ve structur e determina tion.

Monosac cha ride composit ion of a polys acch aride fra ction can be determine d using a 1-phen yl-3-meth yl-5-p yra zolone (PMP) pre -column deri va tion Wang et al., 2010 ) . Wang et al. (2009 ha ve been demonstr ating the positi ve health effects of water -solub le polys acch arides isolated fro m C. sinensis follo wing hot-w ater extr action and ethanol precip itation, as discussed pre viously . These health claims included prote ction against kidne y dama ge and antioxidan t activ ity in vitr o . Monosac char ide composition of both pol ysacc haride fra ctions wer e in vestig ated using PMP labe lling and was determine d that CPS1 was composed of glucose , mannose and galact ose at a molar ratio of 2.8:2.9:1, respecti ve ly . CPS2 was composed of mannose , glucose and galactose at a ratio of 4:11:1, respecti ve ly . The same pro tocol was follo wed in both cases. Briefly, 2 M trifluoroacetic acid (TFA) hydr olyz ed the pol ysacc haride with the hydr olys ate la beled with PMP by incubating with 0.6 M NaOH and 0.4 M PMP . Aqueous phase fro m a chlo rof orm extr action was then anal yzed via HPLC with all standa rds label ed follo wing the same pro cedur es. CPS1 alone was subjecte d to a partial acid hydr olys is experiment in volving 0.05, 0.2, 0.5 M TF A and follo wing dial ysis with a membr ane size cut-off at 1000 Da, four fracti ons (f1, f2, f3, and f4) were obtained and underw ent HPLC anal ysis. The fra ction with the lar gest molecular weight was collecte d in f1 with fra ction resulting from 0.5, 0.2 and 0.05 M TF A hydr olysis ensuing in decre asing or der of molecula r weight. The significant pre sence of manno se and glucose in f1 is indicati ve of the bac kbone structur e composit ion of CPS1. Monosaccha rides of f2, f3 and f4 sho wed an ab undance of glucose and galactose sugge sting their pre sence in the br anch structur e of CPS1.

Periodate oxidation aims at deduci ng structur al informa tion relating to bra nchin g and linka ge of a polys acch aride based on the uptake of periodate (Stoddart, 1984 ) . Smith de grad ation is a useful extensio n of periodate oxidation wher eb y the pre viously oxidized pro duct is reduced to a poly alcohol with boro hydride and subsequ ently hydr olys ed with a mild acid. Wang et al. (2009 performed this oxidation -de gra dation pro cedur e with the componen ts of the pol ya lcohol determin ed using HPLC. The pol ysacc haride of CPS1 and CPS2 sho wed lar ge perioda te consumpti on indicating the existenc e of a 1,2-gl ycosidi c linka ge or 1,4-gl ycosidic linka ge or both. Trace amounts of formic acid was also detected impl ying the existenc e of a 1,6-gl ycosidic linka ge. HPLC detected the prese nce of mannose , glucose , galactose , gly cero l and erithrito l in the hydrol yzed pro duct follo wing periodate-oxid ation and Smith deg radation of CPS1. Mannos e, gly cero l and erithrito l were detected in the hydr olyz ed product of CPS2. The pre sence of manno se, galactose and glucose indicate s a 1,3-, 1,2,3-, 1,2,4-, 1,3,4-, 1,3,6-or 1,2,3,4-link age whic h was not oxidized by perioda te. Gly cero l stems fro m 1,2-gl ycosidic linka ge or 1-linked gly cosidic linka ge and erithrito l fro m 1,4 or 1,4,6-link ed glycos idic linka ge. Pre dictions were made in re gar ds to the bac kbone structur e of CPS1 and was hypothesize d to ha ve 1 ! 2 and 1 ! 4-linka ge of manno se, 1 ! 3,6 and 1 ! -linked glucose and 1 ! 3-linked galactose . NMR spectr oscop y was used later on to complete the structur al charac terizatio n of CPS2.

GC is a common ly used analy tical tool for sepa rating and analy zing va porized compoun ds. As the name would suggest, the compoun d must be first con verted into a vo latile deri vati ve . Se vera l deri vati zation pro cedur es exists suc h as trimeth ylsil yl ether , deuterioaldito l acetate , per acetylatio n and aldono nitrile acetate (Price , 2004 ) . Deri vati zation to aldononitri le acetate allo ws for short sample pre par ation time and pro duces well resolve d peaks and sta bl e compoun ds for quanti tativ e and qualitati ve analy sis (Guan, Yang, & Li, 2010 ) . Cultur ed and natur al C. sinensis extr acts were pre pare d using a pre ssurized liquid extr action (PLE) tec hnique and anal yzed using GC/MS in a rece nt stud y conducte d by Guan et al. (2010) . Under optimized condition s, pol ysacch arides fro m a water extrac t were hydrol yzed at 100 °C for 2 h using 2 M of TF A in or der to release sacc harides . The hydr olyz ed pro duct was then deri vatized. Deri va tization in volves firstly, con ve rsion of the anal yte into an oxime by reacting with hydr oxylamin e hydr ochloride follo wed by an ac ylation reaction in vo lving acetic anh ydride . Optima l deri va tization condition s were used based on kno wledge fro m a pre vious stud y suggesting amount of reagent, temper atur e and dura tion of oximati on and ac ylation can influence deriv atization (Li, Wu, & Zhang, 1981) . Based on their own prel iminar y optimiz ation experiments, Guan et al. (2010) found that oximation and ac ylation reactions performed best at a temper atur e of 90 °C and a rea ction time of 30 min. The ratio of reagent to anal yte was optimal at 0.5-2.0 (mg/mg). Column sep ara tion was perform ed using 5% phen yl meth yl siloxane at a temper atur e of 175 °C with gra dual incre ase to 185 °C and finally to 230 °C. MC par ameters include s ionization ener gy of 70 eV and a scan rang e of 40-550 am u. Fr om these experim ental par ameters, ten monosacc harides (rhamnose, ribose , ara binose , xylose , manno se, glucose , galactose , mannito l, fructose and sorbose) were detected in both culture d and natur al C. sinensis samples. Contr ast betw een the carboh ydr ate pro file of cultur ed and natur al C. sinensis was esta bli shed and sho wed that natura l C. sinensis contained mor e than 7.99% fre e mannitol compar ed to that fro m the cultur ed samples .

NMR spectr oscop y is a popular method used for structur e determina tion genera ting highly informa ti ve spectr a all the while pro viding high-r esolution and quantitati ve resul ts. This non-destr ucti ve method , prese rve s the ingr edient of the original sample . One-dimen sional (1D) carbon or pro ton NMR exploits the ma gnetic pro perties of NMR activ e nu clei ( 1 H or 13 C) and can be analy zed further to determin e ph ysical and chemical charac teristics of the atom and, in a lar ger sense , the molecule itself. Although 1D NMR can pro vide the user with adequa te informa tion pertainin g to the structur e of simple unkno wn compoun ds, lar ge compl ex compoun ds will usuall y genera te cro wded spectr a resulti ng in the ove rwhelming of small NMR peaks and hea vy signal overl ap . This prob lem is alle viated by incorpor ating an additiona l par ameter or dimension. Multidimens ional spectr a is conside red as a major ach ie ve ment in NMR spectr oscop y capa ble of resolving the structur es of lar ge pro teins and nucl eic acids. For bioacti ve compou nds determina tion within C. sinensis extrac ts, 1D and 2D NMR spectr oscop y are most fre quentl y emplo ye d. Homo-and heter onuc lear NMR experim ents suc h as corre lation spectr oscop y (COSY), total corr elation spectr oscop y (TOCSY), heter on uclear single quantum corr elation (HSQC), heter onuc lear multiple quantum corre lation (HMQC) and heter onuc lear multi ple-bond corr elation (HMBC) can be used for structur e elucidation .

Pol ysacch arides extr acted fro m cultur ed and natur al C. sinensis ha ve dra wn conflicting conclusio ns as to the structur e of the activ e compou nds. Recentl y, a hydr ophilic pol ysacch aride fra ction isolated fro m cultur ed C. sinensis , was examined after recei ving positiv e resul ts suggesting antifibrotic effect in renal fibrosis (Zhang, Liu, Al-Assa f, Phillips , & Phillips, in press ). Using this bioacti ve fra ction, monosacc haride composition and structur al analy sis was studied by way of meth ylation anal ysis and 1D and 2D NMR spectr oscop y (Nie et al., 2011 ) . Meth ylation anal ysis pro duced partiall y meth ylated alditol acetates (PMAA) which were identified via mass spectr ometr y. This type of analy sis determine s the mode of linka ge and based on this stud y, would suggest that the hydr ophilic pol ysacc haride fra ction is compose d mainl y of a glucop yr anosyl bac kbone 1 ! 4 linked (65.7%). Non-r educing termin al-Dglucop yra nosyl residue had a peak ar ea per centa ge of 20.7% (Nie et al., 2011 ) . At this junctur e, discr epa ncies in the assigned linka ge ha ve alre ad y emer ged. Akaki et al. (2009) reported 1 ! 3 linka ge with suppor ting evidence by Bai et al. (1997) . Discr epa ncies in liter atur e can be attributed to man y factors ho weve r most likel y due to differe nce in species and extr action methods. Akaki et al. (2009) ho wever , failed to perform furthe r structur al in vesti gation using NMR spectr oscop y in or der to confirm their meth ylation analy sis conclusio ns. Nie et al. (2011) further elucidat ed structur al charac teristics of the pol ysacc haride fra ction. Based on meth ylation analysis, 1 H and 13 C NMR, HMQC, HMBC, COSY , and TOCSY , a conclusion was made as to the hypothesize d structur e of the bioacti ve fra ction compr ising a glucop yra nosyl a-linked 1 ! 4 bac kbone with 1 ! 3 linked br anch ing at the O-2 or O-6 glucop yr anosyl and an a-terminal-D-glucop yra nosyl in the side chain .

FTIR anal ysis compl emented with 13 C NMR analy sis determined the main compoun d in CS-Pp was 1,3b-D-glucan (Akaki et al., 2009). Alditol acetates obtained fro m hydr olys is of meth ylated CS-Pp was in vesti gated using GC and GC/MS . Thr ee peaks emer ged corr esponding to 2,3,4,6 -tetr a-O-methylglucit ol, 2,4,6-tri-O-me th ylglucit ol and 2,4-di-O-m eth ylglucitol. These conclusio ns wer e enough for the authors to deem the acti ve compou nd found in CS-Pp is a 1,3b-D-glucan with 1,6-br anch ed chains. Similar imm unostim ulating results ha ve pre viousl y been determin ed and credi ted to the insoluble 1,6-br anch ed, 1,3b-D-glucan, Letinan (Bai et al., 1997 ) . Ho we ve r, the compound Akaki et al. (2009) isolated, had a muc h smaller particle size and could stim ulate an imm une respo nse follo wing ora l administ ratio n as pre viousl y demonstra ted (Koh et al., 2003a ) .

As summarized in Tab le 1 and Tab le 2, the follo wing section described major pharmace utical compoun ds in Cor dyc eps extract s and their thera peutic pro perties (i.e. antiaging, repa rativ e prop erties, anti-canc er/anti-tum or , imm uno-stim ulation and antioxid ant) (Akaki et al., 2009; Chen et al., 2009; Dong & Yao , 2008; Ji et al., 2009; Koh et al., 2003c; Woo Bok et al., 1999; Yang et al., 2011) .

Solv ation pro perties of water ha ve been exploited and used to determine va rious health benefits of C. sinensis . Antia ging effect of C. sinensis is of particula r inter est (Ji et al., 2009 ) . Hot wate r extr act was pre pare d and administ ere d to male and female rats. Under D-galactose induced aging, accum ulation of metab olites occurs in nerve cells leading to symptoms associated with aging (Song, Bao , Li, & Li, 1999 ). This aging model was effecti vel y emplo ye d by Ji et al. (2009) to demonstr ate common aging symptom s such as memor y dysfunction, decline in sexual function and decr eased activ ity of antioxid ant enzymes. Water extr act of C. sinensis (WECS) fed rats sho wed impr ove d resistance to the symptom s of induced aging in a dose-de pendent manner . Although pur e compou nds within WECS were not identified nor isolated, a thor ough in ve stigation of the effects of the extr act was conducted. The rats were subjected to learning and memor y tests and sexual functio n test. The ultra structur e of the hippocam pus was observ ed with an electr on micr oscope and visuall y demonstr ated the prese rva tiv e effects of WECS on the cellular structur e of the hippocamp us. The bra ins and liv ers were subsequ entl y remo ve d in or der to perform age-r elated enzyme assa ys, which once again demonstr ated WECS ab ility to impr ove the activ ity of antioxidati ve enzyme s suc h as super oxide dism utase , catalase, GSH-Px all the while lo wering the activ ity of lipid peroxidation and the activ ity of monoami ne oxidase B, both indicators of aging.

More rece ntly , the repa rati ve pro perties of C. sinensis extr acts wer e in vestigated using HT29 cells, a human colon cancer cell line (Marc hbank et al., 2011 ) . Effect of C. sinensis extr acts on gastric dama ge was explor ed in vivo on rats that had been administer ed indomethacin , a non-ste roid al anti-inflammatory drug. C. sinensis gro wn on rice and potato medium along with a po wder ed dried extr act fro m my celia gr own in liquid culture were further extr acted using hot-w ater . Whole and finely gro und up fruiting bodies of C. sinensis gro wn on rice and potato medium were extr acted for comparison purposes and concluded that the suppleme ntal ste p of grinding the fruiting bodies only impr ove d pro-proli ferat iv e activ ity . Ethanol -soluble and ethanol -insolub le sub-fr actions were also collected from gro und-rice and potato hot water extr act in or der to fur -ther examine possib le acti ve componen ts. Wound healing invo lve s the migr ation, in va sion and prol iferat ion of cells. On aver age, C. sinensis extr act stimul ated cell proli ferat ion thre efold, cell migr ation increa sed by 69% and in va sion by 17%. In compariso n, the liquid-p hase-gr own pro duct was less effecti ve ho weve r yielded similar pro-proli ferat iv e effects. Of interest, 93% of the pro-p roli ferat iv e activ ity was found in the ethanol-so lub le sub-fr action and pro -migr ator y activ ity was split 61:39 betw een ethanol-so lub le and ethanol -insolub le sub-fr actions. Pro tecti ve effects of C. sinensis extr acts were also seen in vivo on rats with gastric dama ges induced by indometh acin. Based on in vitr o studies, C. sinensis extr acts displa yed no anti-a poptosis activ ity as it sho wed no influence on caspase-3 acti vity ther eb y suggesting its gastr opr otecti ve effects ma y stem fro m its ab ility to stim ulate cell migr ation and prolifer ation. Pre vious resear ch by the same gro up sho wed that pure adenosin e compoun ds had pro -pr olifer ativ e, pro-migr atory and gastric injur y pro tecti ve effects ther efore rele va nce can be made in re gar ds to the ethanol solub le sub-fr action whic h contains nucl eotides and nucl eosides (Belo et al., 2006 ) .

C. sinensis extr acts role as a prote ctiv e agent stre tch es beyo nd the stomac h and into the kidne ys, this accor ding to recent studies on extr acellular accum ulation of glomeru lar scler osis by Song et al. (2010) . A simple hot wate r extr action was performed using C. sinensis po wder and the filtrate was administ ere d to one of four gro ups of male rats . Adriam ycin was administ ered to thre e of four test gro ups and induce d kidne y dama ge. Urine prote in measur ement as well as biochemistr y index examina tion wer e conducted to quant ify the dama ge induced by adriam ycin and the pro tecti ve effects of the extr act. Hematoxy lin and eosin staining, and imm unologi cal histoc hemistr y examina tion were used to visual ly detect cha ng es in the renal or gan. In non-tr eated adriam ycin rats, otherwise referr ed to as the model gro up , urine prote in le ve l sa w consist ent incre ase over the 12-w eek period compar ed to the sham gro up subjecte d to isoton ic sodium chloride injections . This definitive ly sho ws adriam ycin effecti ve ness at inducing detecta ble kidne y dama ge. C. sinensis extr acts role as a prote cti ve agent was tested on rats fed C. sinensis extr act on a dail y basis (5 mL/kg). Another gr oup of rats were fed with 7.5 mg/kg of fosinopril sodium on a dail y basis as the positi ve contr ol gro up . Fosinopril sodium, or commonl y kno wn as Monopril, is a angiotensin con ve rting enzyme inhibitor and used for the pre ve ntion of kidne y failur es among other conditions. Urine prote in le ve ls in rats tr eated with either C. sinensis extr act or fosinopril sodium were muc h lo wer in compariso n to the model gro up . Furthermor e, bloo d urine nitr oge n (BUN) and serum cr eatinine (SCr) also sho wed decr eased expre ssion in rats tr eated with either C. sinensis extrac t or fosinopril sodium with the former demonstr ating mor e effecti ve ness than its counterpart. BUN is an -In rats fed CSE, stress symptoms wer e suppr essed by observing weight ch ange s of the adr enal gland, spleen, th ym us and th yro id Koh et al. (2003a Koh et al. ( , 2003b Koh et al. ( , 2003c -Hot Chen et al. (2009) end pro duct of pro tein meta bolism and excr eted by kidne ys and is an indicator of acute or chro nic renal failure . SCr is an amino acid deri ve d fro m mu scle tissue excr eted by glomerular filtration ther efore decre ased filtration rate caused by ren al dama ge incr eases SCr le ve ls. These observ ations were all further concr etized via visual inspectio ns. Based on this stud y, C. sinensis extr act can be consider ed an effecti ve ther apeutic agent in redu cing pro tein uria, impr ove renal functio n and inhibit glomeru lar scler osis adding ye t another benefit to the long list of health benefits associate d with water extr acted C. sinensis .

Eth yl acetate extrac ted C. sinensis has been pre viousl y sho wn to exhibit potent inhibitor y effects on va rious cancer cell lines and also in vivo anti-tum or activ ity has been detected in B16induce d melanom a C57BL/6J mice . Zhang et al. first rele ased a stud y on the effects of va rious C. sinensis extr acts ag ainst the pro lifer ation of human prem yel oc ytic leukem ia cell HL-60 in 2004 . Petro leum ether , eth yl acetate , ethanol and wate r extr acts were pre par ed and subjecte d to MTT (3-(4,5-Dimeth ylthiazol-2 -yl)-2,5-diphenyltetr azolium br omide) assa y to meas ure cell pro lifer ation as well as to determine cytotoxici ty potential of the va rious extr acts on HL-60 cells. Caspase-3 activ ity as well as clea va ge of pol y ADP-ribose pol ymera se (PARP) were also anal yzed; both are char acteristic of cell apoptosis. During apoptosis, acti vati on of caspase-3 enab les the clea vage of the nucl ear enzyme , PARP , and subsequ ently pre ve nts it fro m re pairing DN A. Although all four extrac ts exhibi ted significant inhibition of cell gro wth, the eth yl acetate extr act pro ved the overwhelmin g fa vo rite with an ED 50 (median effecti ve dose; a dose requir ed for quant al effect in 50% of the populatio n) of 625 lg/mL and became the focus for the rem ainder of the stud y. The eth yl acetate extr act contin ued to impr ess by activating caspase-3 in a dose-and time-de pendent manner . This had a cascading effect of PARP as evidenced by the clea va ge of the 113 kD prote in into 89 kD fra gments. These results confirms eth yl acetate extr acted mycelium's ab ility to induce apoptosis in HL-60 cells via caspase-3 mediate d PARP clea vage. Of significant inter est is the lo w ED 50 va lue obtained in this stud y which warra nts further scr eening in an attempt to identify the activ e compoun d in vo lv ed and its use as potential ther apeutic drug. The authors did sugge st the extr act contai ned er goster ols, gly cosides and polys acch arides. Ster ols ha ve pre viousl y been pro ve n to ha ve antitumor acti vity against Jurk at cells and K562 cells. Results fro m this stud y concluded that fra ction FB1 had 61% antitumor activ ity against Jur kat immortal ized T ly mphoc yte cells and 36.3% acti vity ag ainst K562 cells at 100 lg/mL. Fr action FB2 was found to ha ve 96% activ ity again st Jurk at cells and 77% activity against K562 cancer cells at 100 lg/mL (Woo Bok et al., 1999) . Sterols ar e ther efore the primar y suspect in vo lve d in gr owth inhibition and apoptosis inductio n. Based on this stud y, Zhang et al. (2005) contin ued their resear ch using four cancer cell lines (breast cancer MCF-7, mouse melanom a B16, human pre my eloc ytic HL-60 and human he patocellula r car cinoma Hep G2). The same extr action pro tocol was emplo yed as in the 2004 stud y and four extr acts were subjected to testing.

HPLC anal ysis was also incorpor ated in the latter stud y to determine the major chem ical constituen ts in eac h extr act. In vivo anti-tum or activ ity of the eth yl acetate extr act was tested on B16-indu ced melanoma in C57BL/ 6 mice . Initial cytotoxi city tests re ve aled nothing ne w to the resear chers as yet again eth yl acetate extr act pro ve d to be the most potent inhibitor in all the cancer cell lines based on IC 50 (concentration requir ed for 50% inhibitio n) va lues. In fact, the potenc y of the eth yl acetate is compar ab le to harrington ine, a positi ve contro l drug. Due to the ove rwhelming cytotoxic natur e of the eth yl acetate extr act, its potential dama ging effects on normal cells was put into question. Normal mouse bone marro w cells witness ed no mor e than 30% gr owth inhibitio n when tr eated with the extr act; mu ch lo wer than that ag ainst cancer cells at the same dose . The extrac t was tested in vivo on B16-induce d mice and was sho wn to reduce the weight and volume of the tumor by 48% and 62% in just 27 da ys. Cytoxan was used as a positi ve contr ol drug and althoug h it was able to suppr ess tumor gro wth almost completel y, damage to the spleen and str ong in vivo toxicity was observ ed; none of which was observ ed in the gr oup tr eated with the extract . HPLC analy sis pro vided insight into possib le chem ical constituents in volved in inhibiting cancer cell proli ferat ion. Pol ysacch aride and cor dyce pin were the least pre va lent constituents in all four extr acts except in water wher e wate r-soluble sugars contrib uted to the high concent ration of carboh ydr ates in the water extr act. Eth yl acetate extr act had the highest er goster ol content compar ed to the other extr acts. Pure compou nds found in C. sinenesis were assa ye d and er gosterol and b-sitoster ol displa ye d significant inhibitor y effect on cancer cell pro lifer ation. Although one ma y jump to conclusion and pro claim ster ols and its relati ve s as the main activ e anti-tumor compon ents in C. sinensis and its eth yl acetate extract , syner gy betw een va rious complexes is most likely in vivo and in vo lving vari ous mec hanisms.

A libr ary consist ing of 200 crude eth yl acetate extr act of C. sinensis was scre ened using a MTT assa y on HeLa cervical cancer cells with one str ain (Gliocladium sp .) emer ging as the most potent inhibitor of pro lifer ation with a GI 50 (dose requ ired for 50% gro wth inhibitio n) of 15 lg/mL. Further sep ara tion of the extrac t pro duced an even mor e potent fra ction with a GI 50 value of 2.0 lg/mL. The ste pwise 'bioassa y-dir ected' purification resulte d in the isolation of three activ e compoun ds with a respe ctiv e GI 50 val ue of 0.50, 0.10 and 0.25 lg/mL. The structur es of compoun ds 1, 2 and 3 were determine d using MS and NMR. Compo und 3 was first identified as 11,11 0 -dideoxyverti cillin and data fro m NMR and MS reve aled clues pertaining to the structur e of compound 1 and compariso ns compound 3 pro vided enough information to elucidat e a structur e for compound 1 and subsequentl y compoun d 2. Compound 3 (11,11 0 -dideox yvertic illin) had alre ad y been defined as a gro wth factor rece ptor tyro sine kinase with anti-tumor activ ity with two papers firmly esta bli shing this conclusion (Chen et al., 2009; Zhang et al., 2005 ) .The former two compoun ds are no vel ep ipol ythiodioxo piper azines and accor dingl y named gliocladicilli n A and B (Fig. 2) . Further eva luations revo lve d aro und these tw o no ve l compound s. Anti-pr olifera ti ve effects were substantiat ed using three human cancer cell lines; HeLa, He pG2 and MCF-7. The mec hanism by which prol iferat ion is inhibited was anal ysed using HeLa cells and the per centa ge of cells in eac h phase of the cell cycle was determine d using flow cytometr y. When trea ted with compoun d 1, an incre ase in the perce nta ge of cells in the G2/M phase fro m 22.7% to 41.8% was detecte d suggesting G2/M phase arr est was responsib le for inhibiting cell pro liferation. This apoptotic mec hanism was further confirmed by Annexin V staining and DN A fra gmentation anal ysis. Both time and dose were critical factors and dir ectly pro portiona l to the inhibitio n effect. The effects of compound 1 and 2 on the expr ession of the pro teins p21 and p53 were examined by Western blotting and sho wed significant increa se in expre ssion follo wing exposur e to compound s 1 and 2 leading to induction of cells to arre st in the G2/M phase . Mitotic exit requir es the de gra dation of the cyclin B subunit composing the pro tein kinase dimer CDK2/c yclin B. Western blots sho wed increa sed accum ulation of cylcin B suggesting compounds 1 and 2 effecti vel y pre ve nts the deg radat ion of cyclin B. The caspase pathw ay was also in ve stigated via imm unoblot s with resul ts indicatin g activ ation of caspase-8 , caspase-9, executio ner caspase-3 and its substr ate PARP-1 by clea vage. The ratio of Bax/Bcl-K l pro tein le vel s were examined and sho wn to modula te the activ ity of caspase-3.T he effects of these tw o no vel compoun ds in inhibiting cancer ous cell gro wth are firmly evidenced in vitr o ho wever , ho w would results tr anslate in vivo ? B16-indu ced C57BL/6J mice were injected with contr olled doses of compoun d 1 (0.25 and 0.50 mg/kg) or 2 (0.10 and 0.40 mg/kg) and examined after 21 da ys of treatm ent. When compar ed to tumors retrie ved fro m the contr ol gr oup , compou nd 1 was 69.8-87.2% inhibition while compound 2 was a similar 56.7-82.5% gro wth inhibition .

A stud y fro m Jap an in 2009 identified a potential polys acch aride that ma y be responsib le in inducing monoc yte activ ation (Akaki et al., 2009 ). The culture d C. sinensis was defatted using ethanol prior to extr action of the residu e with hot water . The supernat ant la ye r was furthe r concentr ated and ethanol was added to pre cipitate the crude pol ysacch aride (CS-P). A portion of the crude polys acch aride was further fra ctionated to yield soluble (CS-Ps) and insolub le pol ysacch arides (CS-Pp). Cytokine pro duction assa y measur ed tumor necr osis factor alpha (TNF-a) pro duction of mouse spleen cells C3H/HeJ as determine d by ELISA. CS-Pp seemingl y induced an ove rwhelming pro duction of cytokine compar ed to the other fractions. A mouse macr opha ge cell line RA W264.7 was used to determine the effect of CS-Pp , which was time and concentr ation-de pendent.

A mor e rece nt stud y focuses on the effect of pol ysacch aride fro m the fruiting bodies of cultur ed C. sinensis on splenic lym phoc ytes, macr opha ges, dela yed -type hypersensiti vity (DTH), anti-oxid ant activ ity and cytokine expre ssion in BALB/c mice exposed to 60 Co (Zhang et al., 2011a; Zhang et al., 2011b ). An imm unosuppr esion model was used in this stud y by subjecting the mice to the ionizing rad iation of 60 Co ther eb y stimul ating inflammation and causing suppr ession of the imm une system by way of re press ing ly mphoc yte pro lifer ation. Results fro m the ly mphoc yte pro lifer ation assa y would suggest the pol ysacch aride fed mice were mor e apt in stim ulating splenic lym phoc yte prol ifer ation and at 100 mg/ kg dose , irr adiated mice displa ye d enhanced imm unomo dulator y activ ity when compar ed to normal non-irr adiated mice . The bioacti ve pol ysacc haride ma y also affect cytokine regu lation and assist in imm uno-stim ulation in this manne r. Cytokines pla y a major role in all aspect of imm unity and oxidation. Cytokines act in a syner gistic manner with eac h cytokine amplifying the effects of eac h other . A bead-based mu ltiplexed method assa y was used to quantify IL-1 a, IL-2, IL-4, IL-5, IL-6, IL-10, IL-17, IFNc, TNFa and GM-CSF . This method allo ws for the quanti fication of se ve ral cytokines sim ultaneousl y ther eb y only requ iring a small vo lume of sample . This method also offers high precisio n and ove rall mor e rapid and cost-effec tiv e. Fi ve cytokines (IL-4, IL-5, IL-6, IL-10, IL-17) emer ged ab ove detectio n limits. When compar ed to the non-tr eated irr adiated contr ol gro up , IL-4 was significantl y decre ased as a result of trea tment with the pol ysacch aride fractio n. A 25.0% and 34.1% decr ease was observ ed in the 50 and 100 mg/kg bod y weight dose gro ups, respe ctiv el y. Meanwhile , IL-5 le ve ls were significantly incr eased by 38.6% and 49.1% in the 50 and 100 mg/kg bod y weig ht dose gro ups, respecti ve ly . IL-17 was the onl y other cytokine to ha ve exhibited note worth y resul ts. In the 50 and 100 mg/kg bod y weight dose gro ups, le vel s of IL-17 decre ased by 14.5% and 11.1%, respecti ve ly . IL-4 and IL-17 both displa y a va riety of pro inflammatory and imm une modulator y activ ities. The authors ther efore assumed the lo w le vel s of IL-4 and IL-17 ma y contribute to the inhibition of reacti ve oxygen species (ROS) production and ma y pro tect the or ganism again st inflammation and subsequ entl y pro tecting the imm une system. IL-5 is secr eted by T cells with mu rine IL-5 capa ble of inducing the prolifer ation and differ entiation of pre -activ ated B-cells. This enab les the secr etion of natur al antibodies suc h as IgM and IgA by matur e B-cells. The observ ed incre ase in IL-5, along with the decr ease in IL-4 and IL-17 le ve ls, can ther efore be suggested as a possib le mech anisms induced by the imm unostim ulating pol ysacc haride .

Ther e are incre asing evidence suggesting corre lations betw een the accum ulation of RO S and associate d degener ativ e diseases including senescence and cancer . Oxidati ve dama ge can affect DNA , pro teins and other macr omolecu les (Dong & Yao , 2008) . The role of antioxidants is to sca venge these fre e radicals and pre vent diseases caused by RO S. Se ver al pre vious report s successfu lly demonstr ate hydr oxyl and super oxide anion radica l sca veng ing activ ities of natur al and cultur ed C. sinensis extr acts in vitr o (Cai, Chen, Yin, & Zhang, 2004; Li, Su, Dong, & Tsim, 2002; Zhang, Pu, Yin, & Zhong, 2003) . Metal chelating and reducing po wer of C. sinensis extr acts had ye t to be observ ed. In vitr o, hot water extrac ts from natur al and cultur ed C. sinensis were subjecte d to determina tions of antioxidant activ ities includin g inhibitio n of linoleic acid per oxidation, sca venging ab ilities on DPPH, hydrox yl and super oxide anion radi cals and the reducing po wer and the chelating ab ility on ferr ous ions (Dong & Yao , 2008 ) . Re ports fro m this stud y would suggest that the natur al and cultur ed my celia of the fungus contains potent antioxi dant activ ities with the culture d my celia outperfo rming the natur al my celia in all in vitr o assa ys except for the linoleic acid per oxidation assa y. In terms of reducing po wer and metal chelating ab ilities, the hot-w ater extr acts pro ve d to ha ve a moder ate affect. Fr om this stud y, it is inter esting to note the stron g performance of the cultur ed my celia pro mpting its use over the depleted natur al fungus.

In vivo antioxid ant activ ities in the serum of 60 Co irr adiated BALB/c mice were in ve stigated using tw o assa ys in vo lving malond ialdeh yde (MDA) and super oxide dism utase (SOD) detectio n (Zhang et al., 2011a; Zhang et al., 2011b ) . MD A is a lipid perox idation indicator while SOD prote cts the cell fro m oxidati ve dama ge by con verting super oxide anions into hydrogen perox ide. In mice administ ere d lo w and interm ediate doses of C. sinensis extr act, MD A in serum was decr eased by 17% and 20% compar ed to the irr adiated untre ated contr ol gr oup . SOD le vels was enhance d by 11%, 2% and 15% in the 50, 100 and 200 mg/kg groups respecti ve ly . The authors claim the enhance d imm unity function of a pol ysacch aride extr acted fro m culture d C. sinensis ma y be attributed to the reduction of oxidati ve str ess and the stimu lating of antioxidant enzymes activ ity (Zhang et al., 2011a; Zhang et al., 2011b ) .

Althou gh Cor dyce ps is recognized as a dietar y supplemen t by the U.S . FD A in man y nations, Cor dyce ps is still regar ded as an unkno wn substan ce. This has shielded Cor dyce ps fro m the full scrutin y of modern science . Little informa tion is avai lable re gar ding detailed side effects and toxicity . In rare cases, dry mouth, nausea, or diarrhea has been report ed. An even rare r occurr ence is that of a systemic aller gic drug aller gy seen in a patient taking Cs-4. Rep orts of toxicity to war ds human ha ve not been pro ve n. In animal trials, mice ha ving recei ved intra peritoneal injectio ns of a Cor dyce ps extr act suffer ed no fatalities after 7 da ys at doses of up to 80 g/kg daily . When ora lly administ ered to rab bits for a period of 3 months at a dose of 10 g/kg dail y, no significant changes in kidne y or liv er function and bloo d tests re ve aled an y signs of toxicity . To put things into perspecti ve , clinical trials in vo lving C. sinensis normall y requir e 3-4.5 g of Cor dyce ps dail y and up to 3-5 daily , when tr eating se ve re liv er disease (Hollida y, Clea ver , & Wasser , 2005) . Anecdot al use of Cor dyce ps in the tr eatment of cancer patient s has pro duced pro mising results at 3-5 g of Cor dyce ps dail y with zero re ports of Cor dyceps related toxicity . Eth yl acetate extr act of C. sinensi s ha ve been sho wn to ha ve str ong anti-tum or acti vity both in vitr o and in vivo . It ha ve been re ported anti-pr olifer ativ e effect of the eth yl acetate extr act possess to war ds four cancer cells, includin g human br east cancer MCF-7 cells, mouse melanoma B16 cells, Human pro my eloc ytic leukemia HL-60 cells, and Human he patocellula r liv er car cinoma cell Hep G2 cells, At a dose of 200 lg/mL, 50% gro wth of ab ove four cells were inhibit after 2 da y tr eatment, and near complete inhibitio n by da y four . Inter estedl y, eth yl acetate extr act of C. sinensis was less toxic to normal mouse bone marr ow cells. Such selecti ve toxicity of eth yl acetate Cor dyce ps extr act permits the potential of C. sinensis as a pro mising chemother ap y drug for tr eatments certain type of cancers. In melanom a-induced mice , eth yl acetate extr act trea tment at a dose of 0.05 g/kg/ da y for 27 da ys displa ye d a significant ove rall decre ase in tumor weig ht and vo lume all the while causing no harm to the spleen, a major imm unity or gan. In contr ast, the positi ve control drug Cytoxan, sho wed strong in vivo toxicity as witnessed by the lo were d spleen index .

Imm unoglobu lin A nep hro path y (IgAN) is a chr onic disease wher eb y rest ing mesangial cells in the kidne y are stimulated promo ting release of cytokines and gro wth factors. The combined action of cytokines and gro wth factors enab les a vicious cycle of mesangial pro lifer ation which can lead to glomerular injur y and scler osis. Cultur ed human mesa ng ial cells (HMC) stimul ated with interleukin-1 (IL-1) and IL-6 caused an incre ase in mesangial cell prol iferat ion, incre ased prod uction of mediato rs and super oxide anion (Lin et al., 1999) . A fr action fro m the methanolic C. sinensis extr act was successful at inhibiting HMC activ ation by IL-1 and IL-6 in vitr o. This activ e fr action was fed to mice as part of their normal diet at a ratio of 2%. Acute toxicity test reve aled no chan ges in bod y weight and liv er function as a result of the 2% Cor dyce ps diet. Li ver to bod y weight ratio ho wever did increase and was speculate d to be the resul t of an incr ease in liv er cytoc hro me P450 content. The authors of the stud y point out that this incre ase in cytoch rome P450 does not cause an y adv erse effects and instead ma y eve n ha ve beneficial effects in IgAN (Lin et al., 1999 ) .

Although there is a lack of evidence suggesting that Cor dyceps ma y be toxic at high doses, mor e certainl y doesn't alw ays equal better . In a stud y focuse d on the pro tecti ve effects of C. sinensis extr act on isch emia-indu ced bra in infar ction, basal grip str ength, serum lactate deh ydr oge nase (LDH) le ve ls, hippocampus reduced glutathio ne (GSH), cerebr al cortex GSH, glutathione perox idase (GPx) acti vity , glutathione reductase (GR) activ ity and glutathio ne S tr ansfer ase (GST) activ ity were measur ed in a rat model at thr ee dosa ges (4, 8, 10 mg/kg /da y) (Liu et al., 2010 ) . Data fr om these indexes sho wed Cor dyce ps extrac t ha ving a rest ora ti ve pro perty when compar ed to the positiv e and ne gati ve contr ols. At a dose of 8 mg/kg /da y, greate st impr oveme nts wer e witnessed. At an incr eased dose of 10 mg/g/kg , a decre ase in health restoring pro perties was observ ed sugge sting that too muc h Cor dyce ps ma y not alw ays pro vide optimal results.

Of real concer n to all consumers of tr aditional Chinese medicine is the possibility of lead contaminat ion and adulterations of the materials. Lead wir e or solder is often inserted into the fungi to incre ase the weight of the pro duct. This pra ctice is widespr ead and the use of fillers is often times una vo ida ble . Lead wire is no w the filler of choi ce due to its hea vier mass compar ed to the less harmfu l twigs. Lead poisonings caused by Cor dyce ps ha ve been report ed with one patient ha ving a blood lead le vels of 130 lg/dl while another patient was asympt omatic with a bloo d lead le ve l of 46 lg/dl (Wu et al., 1996 ) .

Other well-kno wn fungi including mu shr ooms, truffles and mor els are consider ed rar e and exotic delicaci es and in some cases ha ve report ed imm unomodula ting and anti-tumor effects (Roupas, Ke ogh, Noakes, & Taylor , 2012 ). Althou gh Cor dyc eps are rar e and expensi ve , the y are by no means consider ed a delicac y due mainl y to their tough textur e. Instead , Cor dyc eps ar e belie ve d to possess medici nal prop erties mu ch like the Penicilliu m fung i fro m which the antibiotic penicil lin is synthesize d. Capsules are no w ava ilab le typicall y containing 600-750 mg of Cor dyce ps . Traditional ly , Cor dyce ps are cooked as part of ''medicinal dishes'' with various types of meats dep ending upon the tar geted illness (Zhu, Halpern, & Jon es, 1998) . Combination s of Cor dyce ps with certain foods ha ve been report ed to possess certain functio nalities. Rep resentati ve examples of traditi onal medicina l dishes include (1) a mixtur e of Cor dyce ps and old duc k meat could be used for the tr eatment of cancer , asthenia or reco very after se ver illness;

(2) a mixtur e of Cor dyce ps and hen yield hyposexuality ;

(3) a mixtur e of Cor dy ceps and blac k-bone hen could teat asthenia; (4) a mixtur e of Cor dyce ps and lean pork could release the symptom s of fatigue , male impotence and kidne y asthenia (5) a mixtur e of Cor dyce ps and sparr ow could use for antiaging/se nescence ; (6) a mixtur e of Cor dyce ps and quail could tr eat fatigue , poor appetite , kidne y asthenia and tuberculosis; (7) a mixtur e of Cor dyce ps and steamed turtle used for male/fem ale hyposexu ality , and finally a mixtur e of Cor dyce ps and ab alone could use for treat chr onic br onch itis, COPD , tuber culosis, arterioscler osis, catar acts, and for health y ove rall indi viduals (Zhu, Halpern, & Jo nes, 1998 ) .

Health-pr omoting prop erties stemming fro m Cor dyce ps was once consider ed mer e Chinese folklor e but has since been accepte d within the scientific comm unity . Ho we ve r, shortcomings in obtaining authentic fungi and loose taxonom ic regu lations are ke y detrim ents to the legitimac y of most papers. The papers that wer e discusse d in this revie w pro vide a small sampling of the plethor a of studies pra ising the health contribut ions of C. sinensis . The substan tial natur e of liter atur e suggesting positi ve outcome s deri ved fro m C. sinensis use is not met with an equal amount of litera tur e discussin g the bioc hemistr y behind the transf ormation of the liv ing insect into fung i. Furthermo re , as evidenced in Tab le 1, a limited va riety of solv ents are emplo yed in the extr action of C. sinensis. Non-polar solv ents deserv e mor e attention as the y are especiall y effecti ve for fungal compoun ds (Paterso n, 2008). Ne ve rtheless, the curr ent stoc k of scientific litera tur e, both mainstr eam and altern ativ e, contribut es to the evergro wing wea lth of kno wledge re vo lving Cor dy ceps and can pro vide leads for futur e resea rch.

Pr essurized liquid extr action (PLE)

555 3.4. Ion-pairi ng-re ve rse phase liquid chro matogr aph y-MS (IP-RP-LC-MS)

Structural analysis of monoc yte activ ation constituent s in cultured mycelia of Cor dy ceps sinensis. Fitoter apia

Limit of blank, limit of detection and limit of quantitation

Elemen ts determination and their relation to immunity

Striatal ad enosine le vels measured 'in viv o' by microdial ysis in rats with unilateral dop amine denervation

Gastropr ote ctiv e effects of oral nucleotide administration

Inosine stimulates axon gro wth in vitro and in the adult CNS

Sca venging effect of Cor dyceps gr owing in different en vironment on hydroxyl radical

Ecologybased screen identifies ne w metabolites from a cor dycepscolonizing fungus as cancer cell prolifer ation inhibitors and apoptosis inducers

Antiang ioge nic activity of 11,11 0 -dideoxy-verticillin, a natural product isolated from the fungus Shir aia bambusicola

Ef fects of the acid polysacc hari de fraction isolated from a cul tiv ated Cor dyceps sinensis on macropha ge in vitro

Cor dysinocan, a polysacc haride isolated from cultured Cor dyceps , activ ates immune responses in cultured Tlymphoc ytes and macropha ges: Signaling cascade and induction of cytokines

In vitro evaluation of antiox idant activities of aqueous extracts from natural and cul tured mycelia of Cor dyceps sinensis . Swiss society of Food Science and Tec hnology

Qualitativ e and qua ntitativ e determination of nucleosides, bases and their analogues in natural and cultured Cor dyceps by pressurized liquid extraction and high perf ormance liquid chr omatograph y-electrospray ionization tandem mass spectrometr

Simultaneous quantification of intracellular natural and antiretr oviral nucleosides and nucleotides by liquid chr omatograph y-tandem mass spectrometr

Anticancer effects of maple syrup phenolics and extracts on prolifer ation, apoptosis and cell cycle arr est of human colon cells

Evaluation of carboh ydrates in natural and cultured Cor dyceps by pressurized liquid extraction and gas chr omatograph y coupled with mass spectrometr

Determination of adenosine and 3-deoxuadenosine in Cor dyceps militaris (L.) link by HPLC

Cor dyceps

Antiaging effect of Cor dyceps sinensis extract

Dev elopmen t and validation of an assa y for the quantification of 11 nucleotides using LC/LC-electrospr ay ionization-MS

Hypocholester olem ic effect of hot water extract from mycelia of Cor dyceps sinensis

Antifatique and antistress effect of the hot-water fraction from mycelia of Cor dy ceps sinensis

Hot-water extract from mycelia of Cor dyceps sinensis as a subst itute for antibi otic gr owth promoters

Determinat ion of nucleosides of natural Cor dyceps sinensis in Qinghai pro vince by capillary electrophor esis

Simultaneous determination of er gos terol, nucleosides and their bases from natural and cultu re d Cor dy ceps by pressurised liquid extraction and highperformance liquid chr omato gr aphy

The fruiti ng bod y and its caterpillar host of Cor dyceps sinensis sho w close resembla nce in main constituent s and anti-oxidation activity

Studies of gas chr omatographic analysis of saccharides and alditols II. Some impro vements in the analysis of ace tylated aldononitril es by gas chr omatograph

Quality control of Cor dyceps sinensis, a va lued traditional Chinese medicine

A polysacc haride isolated from Cor dyceps sinensis, a traditional Chinese medicine, protects PC12 cells against hydroge n peroxide-induced injury

Inhibition of activ ated human mesang ial cell prolifer ation by the natural product of Co rdyceps sinensis (H1-A): An implication for treatment of IgA mesang ial nephr opat hy

Protecti ve effect of extract of Cor dyceps sinen sis in middle cerebr al artery occlusion-induced focal cerebra l ischemia in rats. Behavior al and Br ain Functions

Determinat ion of nucleosides in Cor dy ceps sinensis pre paration by dual-wa velength TLCscanning

Repar ative properties of the tr adit ional Chinese medici ne Cor dy ceps sinensis (Chinese cater pillar mushr oom) using HT29 cell cul ture and rat gastric damage models of injury

Elucidation of the structure of a bioactiv e hydrophilic polysacc haride from Cor dyceps sinensis by meth ylat ion analysis and NMR spectroscop y. Carbohydr ate Polymers

Cor dyceps -A traditional chinese medicinie and another fungal therapeutic biofactory? Phytoc hemistry

Ion-pair re versed-phase liquid chr omatograph y for deter mination of polar underiv atized amino acids using perfluorinated carbo xylic acids as ion pairing agent

Ac ylic sugar deriv ativ es for GC/MS ana lysis of 13

The role of edible mushr ooms in health: Ev aluation of the evidence

Natural products in Cor dyceps

Profiles of nucleosides and nitrogen bases in Chinese med icinal fungus Cor dyceps sinensis and related species

Adv anced glycation in Dgala ctose induce d mouse aging model. Mec hanism of Ageing and Development

Determi nation of cor dycepin from cul tured Cor dyceps sinensis by HPLC-D AD

The protecti ve effects of Cor dyceps sinensis extract on extracellular matrix accumulation of glom erular sclerosis in rats

The biosynthesis of polysacch arides

Ph ylog enetic classification of Cor dycep s and the Clavicipitaceous fung i. Studie s in Mycology

Ion-pair high-per formance liquid chr omatograph y with diode arr ay detection coupled to dua l electrospr ay atmospheric pressur e chemical ionization time-of -flight mass spectrometr y for the det ermination of nucleotides in bab y foods

Struct ural cha racterisation and im munomodulator y property of an acidic polysacc haride from mycelial culture of Cor dyceps sinensis fungus Cs-HK1

Struct ural determination and antioxidant activity of a polysacc haride from the fruiting bodies of cultured Cor dyceps sin ensis

Protection of chr onic renal failure by a polysacc haride from Cor dyceps sinen sis

The mushro oming fung i market in Tibet exemp lified by Cor dyceps sinensis and Tric holoma matsutake

Yartsa gunbu (Cor dyceps sinen sis ) and the fungal commodification of Tibet 's rural economy

Antiox idant capacity of 55 medici nal herbs traditionall y used to treat the urinary system: a comparison using a sequential three solvent extraction process

Antitumor sterols from the mycelia of Cor dyceps sinensis

Lead poisoning caused by contaminate d Cor dyceps, a Chinese herbal medicine: two case re por ts

Inhibitory effects of eth yl acetate extracts of Cor dyceps sinensis mycelium on va riou s cancer cells in culture and B16 melanoma in C57BL/6 mice

Analysis of the main nucleosides in Cor dyceps sinensis by LC/ ESI-MS . Molecule s

Inhi bitory effect s of water extr act s from fruiting bodies of cultured Cor dyceps sinensis on raised ser um lipid peroxide le vels and aortic cholester ol deposition in atheroscler otic mice

Deter mination of nucelosides and nucleobases in dif ferent speci es of Cor dyceps by capillary electrophore sis -mass spectrometry

Antiinflammatory principles from Cor dyceps sinensis

Determinat ion of nucleotides, nucleosides and their tr ansfo rmation products in Cor dyceps by ion-pairing re versed-phase liquid chr omatograph y-mass spectrometry

11,11 0 -Dideoxy-verticillin: A natural compound possessing gro wth factor rece ptor tyrosine kinase-inhibitory effect with anti-tumor act ivity . Anti-Cancer Drugs

Cor dyceps sinensis decreases TGF-B1 dependent epithelial to mesench ymal transdifferent iatio n and atte nuates renal fibrosis

Studies on the sca vengin g effect on superoxide anion free radical and hydroxyl free radical of Cor dyceps sinensis

Cor dyceps sinensis mycellium extract induces human prem yeloc ytic leukem ia cell apoptosis through mitochondrion pathwa

Induction of HL-60 apoptosis by eth yl acetate extract of Cor dyceps sinensis fungal mycelium

Cor dyceps sin ensis extracts attenuate aortic transplant arterios clerosis in rats

Effec t of polysacc haride from cultured Cor dyceps sinensis on immune function and anti-oxid ation activity of mice exposed to 60Co

The scientific redisco ver y of a pre vio us ancient Chinese herbal re gimen: Cor dceps sinensis: part II

Pre parati ve separ ation of high-purity cor dycepin from Cor dyceps milita ris (L.) link by high-speed countercurr ent chr omatograph