Studies on the factors affecting growth of mycelium and fruiting body formation, and antioxidant activities of the extracts of cordyceps militaris l ex st aman

militaris two strains; 2 evaluate the effect of different liquid culture method shake and static culture on the mycelial production; 3 evaluate the effects of different mycelium drying

Introdution (general introduction)

Cordyceps, a well-known conventional Chinese medicinal mushroom, belongs to the elegance Ascomycetes and Hypocreales (Kuo et al., 1996; Mao et al., 2005) The Cordyceps species is an abundant source of useful natural products with various biological activities (Das et al., 2010) Some Cordyceps species have long been used for medicinal purposes in China, Japan and Korea and other East Asian countries because of their various biological and pharmacological activities that were generally at tributed to the presence of important bioactive ingredients such as adenosine, cordycepin and exopolysaccharides (EPS) (Gu et al., 2007; Song et al., 1998) Cordyceps species for the conventional Chinese medicinal functions in particular include

The best-known species of the genus is C sinensis It has been employed medicinally for over 2000 years in China (Li et al., 2006; Liu et al, 2001) C sinensis can produce many kinds of physiologically active substances (such as adenosine, cordycepin and polysaccharides) (Gu et al., 2007; Khan et al., 2010) These materials are useful too many structures, such as the circulatory, immune, hematogenic, cardiovascular, respiratory, and glandular structures inside the human body Compared to C sinensis, C militaris is without difficulty cultured in each stable, solid, and liquid media liquid media with a lot of carbon and nitrogen sources C militaris has additionally been used inside the conventional Chinese medicinal drug for a protracted time Recently, C militaris has been an increasing number considered as an alternative for C sinensis because of their comparable chemical capacities and medicinal properties (Dong et al., 2012; Zheng et al., 2011) The latest studies have additionally established that

C militaris comprises many varieties of lively additives including cordycepin, ergosterol, mannitol, and polysaccharides, and reveals pharmacological functions It is now used for over one medicinal functions because of its many physiological activities (Das et al., 2010) Generally, the wild C militaris parasitizes larva or pupa of lepidopteran insects Although this genus has a worldwide distribution, C militarisis mainly distributed in East Asian countries, such as China, Japan and the Korean Peninsula (Fig 1) In China, C militarisis named ‘‘Dong Chong Xia Cao’’ or ‘‘Chong Cao’’, and is now used as a traditional Chinese medicine and health food in Chinese herbs The wild C militarys is native to remote, high elevations of Tibet, Sinkiang, Sichuang and northeast China As parasites, C militaris often exhibits a high degree of host specificity of insects (Zhu et al., 1998) Generally, spores of C militaris attack the larvae (or pupae) of butterflies and moths, and invade the body of the larvae The fungus lives inside the larva and grows The mycelium keeps the host alive, until the larva’s life dies the fungus then produces the fruit-bearing bodies or stroma The stroma is club shaped and orange with grainy surfaces These elongated fruiting bodies grow to a length of around 2 to 8 cm and have a width of about 0.5 cm (Baral 2017) Nowadays, the organic product assemblages of wild C militaris are costly a result of host particularity and extraordinariness in nature They grow extremely slowly in nature; their growth is limited to specific areas, and the fruit bodies are small and difficult to find In particular, the resources of wild fish are in danger due to overexploitation In addition, traditional Chinese medicine usually involves the consumption of fruiting bodies and parasitic larvae As a result, there are concerns regarding microbial contamination when consuming dead carcasses of larvae Therefore, it is forbidden to fully collect wild C militaris for widespread use as medicine To control these difficulties, artificial cultivation has been developed to produce C militaris fruit bodies on a large scale

Nevertheless, different grades of C militaris strains isolated from different places may have different cultural characteristics and pharmacological effects For example, the size, shape, and color of the fruiting bodies of C militaris produced by different strains are sometimes completely different In addition, it is also important to study the genetic variation between C militaris strains In the past decade, people have been paying attention to the genetic characteristics of C militaris Randomly amplified polymorphic DNA was used to investigate the genetic variation of 11 locations in Korea, and it was found that there was no correlation between genetic variation and geographic area (Sung Kyoung Choi et al., (2015) ) In addition, genetic variations of C militaris from different regions (including wild C militaris strains) were used in industrial production, and artificially produced fruit bodies were also studied through sequence analysis in the ITS area The results showed that the genetic variation of C militaris from the United Kingdom, China, Japan, South Korea and Norway is very small and has nothing to do with geographic origin Large-scale production does not affect the genetic stability of insects (Berch S.M et al., 2007) Though, compared with other fungi, many protein families in C militaris are reduced, which shows that its ecology is more restricted In addition, the Cordyceps genome does not contain genes for known human mycotoxins Studies showed these results are consistent with their long- term safe use of drugs These results provide a better understanding of the biology of Cordyceps and improve the development of medicinal compounds produced by the fungus Recently, through transcriptome and proteome analysis, the developmental stage of cultured C militaris at the transcription and translation level has been determined (Yin et al., 2012) Gene expression analysis showed that 2113 genes were up-regulated in the mycelium and 599 genes were up-regulated in fruiting bodies Functional annotations show that intracellular nucleotide-binding and metabolism, transcription regulation, and translation are more active in the mycelium, while carbohydrate metabolism and signal transduction are more active in fruiting bodies This research will promote the development of insects and pharmacological research (NANCY KELLER et al., 2006) a transformation system was established in C militaris by using an integrated vector with a benomyl resistance gene Their results showed that, compared with wild-type strains, C militaris carrying the heterogeneous laeA increased the productivity of secondary metabolites.

Fundamentals of medicinal mushrooms

Medicinal mushrooms including Cordyceps are a rich source of useful natural products with various biological activities (Gu et al., 2007) C militaris

(an insect pathogenic fungus; Fig 1), belongs to the Ascomycetes category, and has been widely used in East Asia as raw materials and folk supplements (Ying et al., 1987) It contains a variety of active ingredients (such as cordycepin, ergosterol, mannitol, and polysaccharides), and because of its multiple physiological activities, now it can be used for multiple medical purposes (Mizuno 1999; Nag and Wang, 2005) So far, more than 350 types have been discovered in the broad meaning of insect parasitic fungi Currently, its ingredients are being studied in detail in universities and other research institutions This greatly improves its medicinal value There are more than 2.500 varieties of mushrooms in the world today It is estimated that countries produced more than 10 million tons of edible and medicinal fungi 2004 year (Nag and Wang ,2005) This is why mushrooms as a rich source of biologically active compounds have recently been highly valued by medical and pharmacological researchers (Wasser 2002) The taxonomical data of C militaris and popular names of Cordyceps are given below:

Fig 1 Photograph of C militaris (Ying et al 1987).

Applications of medicinal mushrooms

C militaris are potential biological metabolites of many new drugs Since ancient times, there has been evidence that it can be used to rejuvenate various human systems WHO estimates that about three-quarters of the world’s population currently uses natural medicines and other forms of traditional medicines (such as medicinal mushrooms) to treat diseases (Inamdar et al.,

2008) including asthma (24%) (Blanc 2001) breast cancer (12%) (Burstein et al., 1999), HIV (22%) (Kassler et al., 1999), liver disease (21%) (Strader et al.,

2002), and rheumatological disorders (26%) (Rao et al., 1999) At the same time, the development of modern chemistry has allowed people to separate chemicals from medicinal mushrooms, which have become drugs or raw materials for many important drugs today Many modern drugs have been synthesized based on the structure and action mechanism of chemical substances isolated from medicinal mushrooms (Wasser 2002) Therefore, medicinal mushrooms have played a major role in the development of modern medicine and continue to be widely used in their original form (Mathews et al.,1999)

Fig 2 Chemical structures of cordycepin and adenosine

The main active ingredient of insect fruit bodies is cordycepin, which is first extracted from extracted insects and then found in Cordyceps sinensis

(Cunningham et al., 1951) and Cordyceps kyushuensis (Ling et al., 2002) The cordycepin (3′-deoxyadenosine, C10H13N5O3, m.p 225 °C, [α] D-47°) (Fig

2), a nucleoside analogue (Ahn et al., 2002; Cunningham et al., 1951), It is considered being a nucleic acid antibiotic, which may inhibit the cancerization of cells, helping to normalize cancer cells as a component of genetic DNA (Cunningham et al., 1951) In addition, it is reported that cordycepin is converted into 5'-monophosphate triphosphate in the cell, inhibiting the activity of several enzymes in the purine biosynthesis pathway (Fig 3) (Masuda et al., 2006) Recent studies have shown that C militaris extracts have a variety of pharmacological effects, such as inhibiting the proliferation of human glomerular mesangial cells (Zhao Long et al., 2000), anti-fibrotic (Nan et al., 2001), anti-angiogenetic, improvement of insulin resistance and insulin secretion (Choi et al., 2004b In addition, it is reported that the cordycepin itself also has anti-tumor, anti-proliferation, anti-metastasis, insecticidal and antibacterial effects (Song et al., 1998) Therefore, the medicinal mushroom C militaris is one of the most important drug candidates and may be used as the medicinal base in the future

Fig 3 Inhibition of purine and polynucleotide biosynthesis by cordycepin

1.4 Antioxidant effect of renal herbs

The antioxidant activity in the xanthine oxidase, hemolysis and lipid peroxidation assay system was demonstrated from the polysaccharide fraction of cultured C sinensis mycelium (Li et al., 2001) Pheochromocytoma PC12 cells were protected against H2O2-induced injury by a 210-kDa polysaccharide from C sinensis mycelia (Li et al., 2003) Before exposure to H2O2, treatment of cells with 100 mg/ml polysaccharides can significantly increase the survival rate of PC12 cells in culture by more than 60% In parallel, treatment with polysaccharides significantly reduced the production of malondialdehyde in cultured cells induced by H2O2, and pretreatment of polysaccharides significantly reduced glutathione in H2O2 treated cells in a dose-dependent manner Changes in peroxidase and superoxide dismutase activity The biological activity of Clostridium sinensis polysaccharide and its antioxidant activity on H22 tumor-bearing mice (Chen et al., 2006) The growth of H22 tumor was inhibited, the activity of superoxide dismutase in liver, brain and serum of tumor-bearing mice was enhanced, and the activity of glutathione peroxidase in liver and brain was enhanced It was found that the water and ethanol extracts of C sinensis fruit bodies have effective antioxidant activity (Yamaguchi et al., 2000)

1.5 Advances in research of polysaccharides in Cordyceps s pecies

Fungi belonging to the genus Cordyceps belong to the clavicle family of the Hypocreales family There are more than 350 species called Cordyceps in the world, of which about 120 species have been reported in China (Jiang and Yao 2004) Cordyceps sinensis (Berk.) Sacc What are the fungal parasites on Lepidoptera larvae? In late autumn, the fungus infects the caterpillar and devours its host In the early summer of the second year, the grass-like fruit bodies protrude from the "head" of the dead host Because of this special life cycle, it is called ’winter-worm and summer-grass’ or ’worm-grass’ in China (Chen et al., 2005) Certain Cordyceps strains are precious medicinal fungi in traditional Chinese medicine and have long been used as general supplements and aphrodisiacs However, they grow slowly and only in high altitude areas, so the supply is insufficient to meet demand In addition, due to improper development, the resources of Cordyceps are in serious danger Cultivate mycelium and use fruit body instead of steroid production, although the key mechanism of fungal infection is not yet clear (Chen et al., 2005) Recent studies have shown that Cordyceps and its anamorph have a variety of biologically active substances, such as polysaccharides, cordycepin, and ergosterol, which have a wide range of pharmacological effects (Buenz et al.,

2005) Cordyceps polysaccharide (CP) is the most active and important one, with great potential, and is regarded as an important tool for researching

Cordyceps product development Compared with other medicinal fungi, Cordyceps sinensis and its anamorphs, as a luxurious traditional Chinese medicine, have a variety of unique pharmacological effects, which are reflected in the biological types and pharmacodynamics of Cordyceps polysaccharides

Although the pharmacologically active components of Cordyceps are still unresolved, at least two chemical components of cordycepin and cordycepic acid have been identified and have been proposed as important active components (Fig 4) (Huang et al., 2003) It is generally believed that cordycepin is the main biologically active component of Cordyceps sinensis

The cordycepin was originally extracted from insects and its structural formula was confirmed to be 3'-deoxyadenosine (Cunningham et al., 1950) Cordycepic acid is an isomer of quinic acid and is one of the main active pharmaceutical ingredients This structure was first inaccurately inferred as 1,3,4,5- tetrahydroxycyclohexane-1-carboxylic acid in 1957(Chatterjee 1957) Subsequently, the structure of the crystalline substance was identified as d- mannitol (Sprecher and Sprinson 1963) The content of cordycepic acid in

Cordyceps sinensis is usually 7–29%, which varies in each growth period of the fruit body (Jiang 1987) Now, it has been used as a raw material for injections and a supplement for other drugs

Fig 4 Chemical structure of cordycepin and cordycepic acid

Cordyceps strains are insect pathogenic fungi, mainly living on arthropods, and have a long history of medicinal use throughout Asia (Sung et al., 2007; Tuli et al., 2013) and have worldwide occurrence (Tuli et al., 2013)

Cordyceps species have recently been reorganized following molecular analysis and placed in Meta Cordyceps (Clavicipitaceae), Elapho Cordyceps

Cordyceps (Cordycipitaceae) (Sung et al., 2007)

Cordyceps sensu lato species have been widely used in traditional

Chinese medicine to promote longevity, relieve fatigue and treat various diseases (Russell and Paterson 2008) Recent studies have shown that several species in this genus have a wide range of pharmacological properties, such as immune regulation, antioxidants, anti-tumor, liver protection, kidney protection, hypoglycemic and cholesterol-lowering activities, and anti- apoptotic homeostasis influences (De Silva et al., 2012; De Silva et al., 2012; Yue et al., 2013; Zhou et al., 2009) Cordycepin plays an important role in pharmacological properties Cordycepin has significant pharmacological effects, including anti-tumor, immune regulation, anti-inflammatory, antioxidant, hyperlipidemia regulation, anti-aging, neuroprotective function, and renal protective activity Antibacterial activity (Ahn et al., 2000), antiviral activity (Lovinger et al 1973), promotion of learning and memory (Cai et al.,

2013), apoptosis (Choi et al 2011) and decreasing rheumatoid arthritis (Noh et al., 2009) Studies have also targeted cordycepin as a therapeutic agent against leukemia (Jeong et al., 2011) In addition, preclinical evaluation of cordycepin and deoxycofamycin (pentostatin) in the treatment of stage 2 trypanosomiasis in Africa 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 over 530 kinds of Cordyceps, only 12 species of Cordyceps are reported to produce cordycepin, although it is probably produced by most (Yang and Dong 2011)

1 To observe the appearancen and structure of fruiting body and mycelium with microscope

2 To evaluate effects of different factors on the mycelium growth of C militaris two strains (strains AG-1 and PSJ-1) for spawn production

3 To evaluate effect of different liquid culture, shake and static culture the mycelial growth of C militaris two strains (strains AG-1 and PSJ-1) for mycelial production

4 To evaluate effect of different liquid culture, shake and static, oven drying and free drying on the C militaris two strains (strains AG-1 and PSJ-1) for mycelial production

5 To evaluate effect of different the liquid culture on the C militaris two strains AG-1 and PSJ-1 fruiting body growth

6 To evaluate effect of different the temperatures culture on the C militaris two strains AG-1 and PSJ-1 fruiting body growth

7 To evaluate effect of pupa powder sources and concentration on the C militaris two strains AG-1 and PSJ-1 fruiting body growth

8 To evaluate effect of different liquid culture methods (shake, static, oven drying, and free drying) on the mycelium of antioxidant compound and activity of the C militaris two strains AG-1 and PSJ-1.

Advances in research of polysaccharides in Cordyceps Species

Fungi belonging to the genus Cordyceps belong to the clavicle family of the Hypocreales family There are more than 350 species called Cordyceps in the world, of which about 120 species have been reported in China (Jiang and Yao 2004) Cordyceps sinensis (Berk.) Sacc What are the fungal parasites on Lepidoptera larvae? In late autumn, the fungus infects the caterpillar and devours its host In the early summer of the second year, the grass-like fruit bodies protrude from the "head" of the dead host Because of this special life cycle, it is called ’winter-worm and summer-grass’ or ’worm-grass’ in China (Chen et al., 2005) Certain Cordyceps strains are precious medicinal fungi in traditional Chinese medicine and have long been used as general supplements and aphrodisiacs However, they grow slowly and only in high altitude areas, so the supply is insufficient to meet demand In addition, due to improper development, the resources of Cordyceps are in serious danger Cultivate mycelium and use fruit body instead of steroid production, although the key mechanism of fungal infection is not yet clear (Chen et al., 2005) Recent studies have shown that Cordyceps and its anamorph have a variety of biologically active substances, such as polysaccharides, cordycepin, and ergosterol, which have a wide range of pharmacological effects (Buenz et al.,

2005) Cordyceps polysaccharide (CP) is the most active and important one, with great potential, and is regarded as an important tool for researching

Cordyceps product development Compared with other medicinal fungi, Cordyceps sinensis and its anamorphs, as a luxurious traditional Chinese medicine, have a variety of unique pharmacological effects, which are reflected in the biological types and pharmacodynamics of Cordyceps polysaccharides.

Cordycepin and Adenosine

Although the pharmacologically active components of Cordyceps are still unresolved, at least two chemical components of cordycepin and cordycepic acid have been identified and have been proposed as important active components (Fig 4) (Huang et al., 2003) It is generally believed that cordycepin is the main biologically active component of Cordyceps sinensis

The cordycepin was originally extracted from insects and its structural formula was confirmed to be 3'-deoxyadenosine (Cunningham et al., 1950) Cordycepic acid is an isomer of quinic acid and is one of the main active pharmaceutical ingredients This structure was first inaccurately inferred as 1,3,4,5- tetrahydroxycyclohexane-1-carboxylic acid in 1957(Chatterjee 1957) Subsequently, the structure of the crystalline substance was identified as d- mannitol (Sprecher and Sprinson 1963) The content of cordycepic acid in

Cordyceps sinensis is usually 7–29%, which varies in each growth period of the fruit body (Jiang 1987) Now, it has been used as a raw material for injections and a supplement for other drugs

Fig 4 Chemical structure of cordycepin and cordycepic acid

Cordyceps strains are insect pathogenic fungi, mainly living on arthropods, and have a long history of medicinal use throughout Asia (Sung et al., 2007; Tuli et al., 2013) and have worldwide occurrence (Tuli et al., 2013)

Cordyceps species have recently been reorganized following molecular analysis and placed in Meta Cordyceps (Clavicipitaceae), Elapho Cordyceps

Cordyceps (Cordycipitaceae) (Sung et al., 2007)

Cordyceps sensu lato species have been widely used in traditional

Chinese medicine to promote longevity, relieve fatigue and treat various diseases (Russell and Paterson 2008) Recent studies have shown that several species in this genus have a wide range of pharmacological properties, such as immune regulation, antioxidants, anti-tumor, liver protection, kidney protection, hypoglycemic and cholesterol-lowering activities, and anti- apoptotic homeostasis influences (De Silva et al., 2012; De Silva et al., 2012; Yue et al., 2013; Zhou et al., 2009) Cordycepin plays an important role in pharmacological properties Cordycepin has significant pharmacological effects, including anti-tumor, immune regulation, anti-inflammatory, antioxidant, hyperlipidemia regulation, anti-aging, neuroprotective function, and renal protective activity Antibacterial activity (Ahn et al., 2000), antiviral activity (Lovinger et al 1973), promotion of learning and memory (Cai et al.,

2013), apoptosis (Choi et al 2011) and decreasing rheumatoid arthritis (Noh et al., 2009) Studies have also targeted cordycepin as a therapeutic agent against leukemia (Jeong et al., 2011) In addition, preclinical evaluation of cordycepin and deoxycofamycin (pentostatin) in the treatment of stage 2 trypanosomiasis in Africa 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 over 530 kinds of Cordyceps, only 12 species of Cordyceps are reported to produce cordycepin, although it is probably produced by most (Yang and Dong 2011)

1 To observe the appearancen and structure of fruiting body and mycelium with microscope

2 To evaluate effects of different factors on the mycelium growth of C militaris two strains (strains AG-1 and PSJ-1) for spawn production

3 To evaluate effect of different liquid culture, shake and static culture the mycelial growth of C militaris two strains (strains AG-1 and PSJ-1) for mycelial production

4 To evaluate effect of different liquid culture, shake and static, oven drying and free drying on the C militaris two strains (strains AG-1 and PSJ-1) for mycelial production

5 To evaluate effect of different the liquid culture on the C militaris two strains AG-1 and PSJ-1 fruiting body growth

6 To evaluate effect of different the temperatures culture on the C militaris two strains AG-1 and PSJ-1 fruiting body growth

7 To evaluate effect of pupa powder sources and concentration on the C militaris two strains AG-1 and PSJ-1 fruiting body growth

8 To evaluate effect of different liquid culture methods (shake, static, oven drying, and free drying) on the mycelium of antioxidant compound and activity of the C militaris two strains AG-1 and PSJ-1.

Introduction Cordyceps

Since 2000 B.C, Cordyceps (the name of insect fungi) and its existence have been widely known Medicinal mushrooms (Cordyceps sinensis) are a rich source of useful natural products with various biological activities (Gu et al., 2007) One of the most important Chinese medicines is C militaris (an insect disease fungus), which belongs to the Ascomycetes category and has been widely used as raw materials and folk supplements in East Asia (Ying et al., 1987) It contains a variety of active ingredients (such as cordycepin, polysaccharides, ergosterol, mannitol, etc.) Due to its multiple physiological activities, it can now be used for a variety of pharmaceutical purposes (Mizuno 1999; Nag and Wang 2005) So far, more than 350 types have been discovered in the widespread influence of animal fungi that parasitize insects Currently, its elements are being studied in detail in universities and other research institutions This greatly improves its medicinal value There are more than

2500 varieties of mushrooms in the world today It is estimated that more than

10 million tons of edible and medicinal fungi were produced in various countries last year (Royse 2005) This is why mushrooms have recently received great attention from medical and pharmacological researchers due to their rich bioactive compounds (Wasser 2002b)

Currently, herbal medicine is becoming more and more popular and important in the public and scientific circles Contrary to the regulated status in China and other countries/regions, herbal medicines are regarded as dietary supplements in the United States, and in the United States, the use of herbal medicines has steadily increased, and it has aroused concerns about safety, efficacy, and its impact on safe patients (Lee 2000) Somewhere else, the increasing use of herbal products worldwide and the development of the herbal product industry have caused increasing concerns about their safety (Gogtay et al., 2002) WHO estimated that about three-quarters of the world's population currently use herbs and other forms of traditional medicines to treat their diseases (Nazma et al., 2008) including breast cancer (12%) (Burstein et al.,

1999), liver disease (21%) (Strader et al 2002), HIV (22%) (Kassler et al.,

1999), asthma (24%) (Blanc et al., 2001) and rheumatological disorders (26%) (Rao et al., 1999)

The acceptance and recognition of herbal medicines are partly because of the recognition of the value of traditional pharmacopoeias and local pharmacopoeias, and including certain drugs derived from these medicinal materials into medicines (DeSmet et al., 1992; Winslow and Kroll 1998), make everyone can afford the needs of medical care, and people's awareness of the prescription, price, and even danger of medicines Another important point that has sparked this interest is that natural medicines are somewhat safer, more effective, and have fewer side effects than drug-derived drugs At the same time, the development of modern chemistry has allowed people to separate chemical drugs from medicinal herbs, and these herbs have become the drugs or raw materials for the synthesis of many important drugs used today By studying the mechanism of action of chemical substances first isolated from herbs including herbal mushrooms, more modern drugs have been synthesized Therefore, herbal medicine has played a major role in the development of modern medicine and continues to be widely used in its original form (Mathews et al., 1999)

The main active constituent of C militaris fruiting bodies is cordycepin, which was first extracted from C militaris and then found to be present in

Cordyceps sinensis (Cunningham et al., 1951) and Cordyceps kyushuensis

(Ling et al., 2002a) The important bioactive compound cordycepin (3′- deoxyadenosine, m.p 225 °C, α D-47 °C), a nucleoside analogue (Ahn et al., 2002; Cunningham et al., 1951), is considered as a nucleic acid antibiotic that might inhibit canceration of cells contributing to the normalization of cancer cells as one of constituents of gene DNA (Cunningham et al., 1951) The recent studies have demonstrated that the extracts of C militaris have multiple pharmacological actions, such as, inhibition of human glomerular mesangial cell proliferation (Zhao-Long et al., 2000), anti-fibrotic (Nan et al., 2001), anti- angiogenetic (Yoo et al., 2004), improvement of insulin resistance and insulin secretion (Choi et al., 2004), anti-inflammatory (Won and Park 2005), and growth inhibition of U937 leukemia cells (Park et al., 2005) Besides, it is also reported that cordycepin itself acts as an anti-tumour, anti-proliferative, anti- metastatic, insecticidal and anti-bacterial compound (Song et al., 1998) Wherefore, the medicinal mushroom C militaris (Fig 1) is one of the important foundations of herbs for human well-being in the future Table 1 and Table 2 respectively give the mycological data of Cordyceps and the popular name of

Table 1 Mycological data of C militaris (Shonkor Kumar Das et al., 2010)

Sub-phylum Ascomycotina Class Ascomycetes/ Pyrenomycetes Order Hypocreales

Table 2 Popular names of Cordyceps

Common name Caterpillar fungus, Cordyceps , Cetepiller mushroom

Latin/English name C militaris, Cordyceps mushroom

Chinese name Dong Chong Xia Cao, Summer grass-winter worm, Hia tsao tong tchong

Japanese name Tochukaso/Tochukasu, Totsu kasu

Korean name Tong ch'ug ha ch'o

Nepali name Yarsagumba, Jeebanbuti, Sanjivani, Kiraghans

Other names Chong cao, Dong chong cao, Aweto

Source: (Shonkor Kumar Das et al., 2010)

The description and distribution of Cordyceps samples

The stock culture is usually stored on a 39-g/l PDA media slant at 5 °C (Das et al., 2010)

2.2.1.1.2 Pre-culture (slant and plate culture)

Both of the slant andplate culture are preparedon 39-g/l PDA and, usually stored at 25 °C

There are many indigenous/popular cultural technologies, and their location, environment, and cultural goals considered vary These are discussed below

Glass beads are added to potato dextrose medium and then sterilized in an autoclave at 121°C for 20 minutes After cooling, the medium was inoculated with several stock cultures After standing for one week at 25 °C, shake the medium once a day to disperse the hyphae When the fungus proliferated, it was inoculated into a bead-free potato dextrose medium and cultured in it for 5 days to produce a seed culture In the case of a solid medium, a small amount of actively growing hyphae from the slope is used for seed culture Subsequently, a certain amount of sawdust can be added to the culture medium to expand the culture scale (Mizuno 1999)

Inoculate the hulled rice medium or wheat medium with liquid spawning Vaccination helps to replenish water and add auxiliary nutrients After culturing at 25°C for about 20 days, the fungus proliferated in the entire medium and then aged for about 1 month (Mizuno 1999)

The pre-cultivation is the same as above, and the worm mycelium is transferred to the seed culture medium by punching out a plate of about 5 mm with a sterile cutter The seed culture is grown in 250 or 500 ml shake flasks containing 50–100 ml liquid medium and incubated on a rotating shaker (50–

150 rpm, depending on the purpose of cultivation) at 25°C for 5–7 days, or The day when the carbon source becomes zero Shaking culture is also a submerged culture All the conditions of this technique depend on the research purpose, the time allocated, and the required environment (Mizuno 1999)

Before the surface liquid culture, the active PDA slant of mycelium was prepared by culturing at 25°C for 8 days, and then the seed culture transferred from the active slant was grown on the PDA medium on the plate for 13-20 Place it at 25 °C for 2 days (depending on the type of strain, 13 days for the control, but usually 20 days for the mutant) The inoculum was prepared by punching out a 1 cm disc of the PDA plate culture using a sterile cylindrical cutter The surface liquid culture is started by inoculating the inoculum (seed plate) into a 500 ml culture flask (the size varies with the purpose of the research), the flask has a diameter of 8.5 cm, a height of 14.0 cm, and a bottleneck diameter and height of 4.5 And 4.0 cm The working volume of the liquid culture medium on the surface of each bottle is 100 ml, and a cotton is installed at the bottleneck during the culture process Place the prepared bottles in an incubator kept at 25±1°C The sampling port can be sampled at various intervals for analysis Before sampling, it is best to stir for 5 seconds with a magnetic stirrer Then, the target substance in the filtrate can be analyzed (such as cordycepin, glucose, pH, etc.) (Masuda et al., 2006)

2.2.1.1.3.5 Continuous culture or repeated batch culture

It is well known that the vicious culture system rather than the single-cycle cell culture technology can maximize productivity, because, in the shuttle culture system, the growth of fungal/mushroom cells is significantly slower due to the lack of required nutrient supply, the end of each cultural cycle Therefore, the conventional culture system is limited to the duration accompanied by low productivity, and the repeated batch culture is independent of the duration and can provide better productivity Repeated batch culture technology is a method of removing useless medium at the end of each batch and replenishing the replaced medium with a fresh medium (Fig 5) The constant addition of fresh media and the elimination of useless media provide the environment that cells actually need to achieve higher productivity Therefore, the repeated batch technology using surface liquid culture is considered to be the best method to obtain the highest productivity (Das et al., 2010)

Fig 5 Replenishment of medium in a repeated batch culture

Many solid and liquid media are used to cultivate C militaris The solid medium can be beech wood flour, rice bran, wheat bran, brown rice, wheat grain, and culture bags or bottles can be used for this purpose According to the purpose of culture, several kinds of liquid media can also be used, and according to the concentration of the medium components, it can be a basic, enriched and enriched medium A basal medium with a minimum of medium components aimed at the growth of fungi and an enriched medium with a higher/optimal medium concentration aim to achieve peak yields For this purpose, suitable additives that may affect its growth and increase yield are usually used As far as we know, the yield of the basal medium is lower, while the best medium for C militaris mutant and control show higher yields, 6.84 and 2.45 g/l, respectively (Das et al., 2010) A further higher production was also attained using C militaris mutant using adenosine (6 g/l) as an additive as 8.57 g/l and, this is the highest production of cordycepin reported until today (Das et al., 2009) A repeated batch culture also showed a higher productivity than those of the control, prospective for pharmaceutical uses A table having a basal, optimized and optimized medium with additive is given below (Table

Table 3 Composition of different types of media for the mutant and control of C militaris

IV Others (diluted to 1/10 concentration of Vogel's medium) (Same for both mutant and the control)

Source : (Shonkor Kumar Das et al., 2010)

A variety of compounds have been purified and their structures were elucidated Cordycepin (3′-deoxyadenosine, C10H13N5O3), 3′-amino-3′- deoxyadenosine (C10H14N6O3), homocitrullyl aminoadenosine (C17H27N9O5), adenine (C5H5N5), cordycepic acid (C6H14O6) (D-mannitol) have been reported from Cordyceps species (Cunningham et al., 1950; Liu et al., 1989) Nucleosides and their bases are determined in Cordyceps (Li et al., 2001b; Li et al., 2004) Determination of adenosine (C10H13N5O4) and 3′- deoxyadenosine (cordycepin) using HPLC method also reported by some researchers (Huang et al., 2003; Li et al., 2004) Besides these, ergos- terol (C28H44O) in Cordyceps can be determined by HPLC method (Li et al., 2004; Li and Li 1991) Ophiocordin (C28H26N2O10), an anti-fungal antibiotic, is found in this

Cordyceps (Boros et al., 1994; Kneifel et al., 1977) Bioxanthracenes (C34H36O10) were also isolated from Cordyceps (Isaka and Kongsaeree 2001; Jaturapat et al., 2001).In addition, cordyheptapeptide (C49H65N7O8), a novel cycloheptapeptide, was isolated from a strain of Cordyceps together with four known bioxanthracenes There were only two previous reports on the isolation of cyclic peptides from this genus and these were C militaris and C sinensis (Rukachaisirikul et al., 2006) Hypoxanthine (C5H4N4O) was also separated along with adenine and cordycepin from Cordyceps (Huang et al., 2003) Water-soluble crude polysaccharides were obtained from the fruiting bodies of cultured C militaris by hot-water extraction followed by ethanol precipitation These polysaccharides were successively purified by chromatography giving several fractions (Yu et al., 2007) Finally, four exopolysaccharides with different molecular masses ranging from 50 kDa to 2260 kDa were also reported from C militaris by several researchers (Kim et al., 2003a; Yu et al.,

2004) Ten-membered macrolides (C10H14O4), cephalosporolides C, E and F, cordycepin, pyridine-2, 6-dicarboxylic acid (C7H5NO4) and 2-carboxymethyl- 4-(3′-hydroxybutyl) furan were also reported from C militaris (Rukachaisirikul et al., 2004) In addition, a method for concurrent determination adenosine and cordycepin from C sinensis and C militaris also described (Huang et al.,

2004) In Cordyceps species, adenine, adenosine, guanosine (C10H13N5O5), uracil (C4H4N2O2), uridine (C9H12N2O6) and inosine (C10H12N4O5) can be determined by a capillary electrophoresis (Gong et al., 2004) Cicadapeptins I and II (peptides containing α-aminoisobutyric acid, C50H90N10O11) and myriocin (C21H39NO6, a fungicide) were also reported in this species (Krasnoff et al., 2005) A glycoprotein with N-acetylgalactosamine (C8H15NO6) was also isolated from Cordyceps (Kawaguchi et al., 1986) An inhibitor of the prophenoloxidase activation was isolated from the culture filtrate of C militaris and identified as pyridine-2, 6- dicarboxylic acid, also known as dipicolinic acid (DPA, C7H5NO4) (Watanabe et al., 2006) A lectin from C militaris exhibited hemagglutination activity in mouse and rat erythrocytes, but not in human ABO erythrocytes (Jung et al., 2007) The nucleic acid-related compounds obtained from C militaris are shown in Table 4

Table 4 Nucleic acid-related compounds obtained from C militaris

No Contents Extract from mycelia (mg)

(Source: (Shonkor Kumar Das et al., 2010) All values were estimated on the basis of a working volume of 1 L)

A number of valuable biological activities have been encountered for C militaris by several authors (Table 5) It was revealed that most of biological activities more or less similar to those of C sinensis (Nag and Wang 2005)

Table 5 Biological activities of C militaris

No Biological activity References Biological activity

1 Pro-sexual (Yu et al., 2007)

2 Anti-inflammatory (Yu et al., 2004) Larvicidal (Kim et al, 2002)

(Won et al., 2005) Anti-fibrotic (Nan et al., 2001)

(Yu et al., 2007) Steroidogenic (Shih et al., 2007) (Chen et al., 2004) Hypoglacaemic (Choi et al., 2004)

Anti-tumour/anti- acncer/anti- leukemic

(Liu et al, 1997) (Yu et al., 2007)

Hypolipidaemic (Yu et al., 2004) (Muller et al., 1977) (Shen et al, 2001) (Kodama et al., 2000) Anti-angiogenic (Yoo et al., 2004) (Penman et al., 1970) Anti-diabetic (Choi et al., 2004)

Anti-proliferative (Liu et al., 1997)

Anti-HIV (Mueller et al.,

Anti-fatigue (Jung et al, 2004)

Neuroprotective (Ribeizo., 1995) (Mao et al., 2006) (Gu et al., 2007) (Shih et al., 2007)

7 Anti-microbial (Park, 1996) (Yu et al., 2007)

Anti-bacterial (Ahn et al., 2000) (Won et al., 2005)

(Lin et al., 2008) (Yu et al., 2004) (de Julian-Ortiz et al.,

2000) (Mueller et al., 1991) (Wu et al., 2000) (Mao et al., 2006) (Yu et al., 2007)

9 Anti-fungal (Mao et al., 2006)

(Yu et al., 2007) (Shih et al., 2007)

10 Anti-protozoal (Trigg et al., 1971)

Source: (Shonkor Kumar Das et al., 2010)

Medicinal uses/clinical applications of Cordyceps including C militaris

C sinensis is more widely used than C militaris, and its clinical applications are more or less similar Although it is well known as sexual intercourse, anti-inflammatory, and anti-cancer drug, it is currently used in many clinical cases such as lung function, cough, sputum, dizziness, memory loss, muscle amblyopia, visual failure, cold virus loss of appetite, night sweats, Pale complexion, pale lips, buzzing ears, toothache and lose teeth, insomnia and thirst, cold or warm limbs, knee pain or pain, neurasthenia, diabetes, nocturia, impotence, slow recovery from anemia (Hui et al., 2006; Mizuno

Current state, limitations and remedies

The natural C militaris is expensive in the local market In the present state, the production of cordycepin from the fruiting body of C militaris is not likely to reach commercial levels due to some practical limitations (Das et al.,

2008) For example, C militaris is very scarce in nature due to the requirements of specific hosts and strict growth environments (Das et al., 2010) On the other hand, it is necessary to note that the chemical synthesis of cordycepin requires a complicated process resulting a lower productivity, moreover, a large volume of organic solvents those are harmful substances to the environment may be discharged (Aman et al., 2000; Hansske and Robins 1985) The friendly production of cordycepin from the cultured mycelia of C militaris in a large scale is currently an acute issue Some experiments have already proven that the chemical components of natural and cultured C militaris are similar (Jiang and Sun 1999; Tong et al., 1997) It is well known that since its discovery as an ancient medicinal tonic, it was facing several limitations until today Negligence, lack of adequate awareness (lack of extension technology), systematic data, and insufficient indepth research are the major limitations regarding the medicinal fungus C militaris Once it was used only by the elite society as a secret of life, but time has changed allowing universal access Studies on such novel components of the mushroom C militaris with respect to their efficacy, safety profile, adverse interaction, proper standardization, etc should be conducted with utmost priority not only by the respective manufacturers but also by the pharmacy, pharmacognosy and medicinal enterprises (Shonkor Kumar Das et al., 2010)

Future prospects nature is the source of all the raw materials that we need

About 2 to 30 years ago, most medicines came from herbal medicine People like to use natural medicines for many reasons because patients will be more distressed after using chemically synthesized medicines, not medicines like natural mushrooms that can conquer life but have no side effects on human health In order to maintain appropriate growth, the pharmaceutical industry needs to innovate and obtain high output rates of low-cost materials with reasonable safety The combination of modern chemistry and bio-based materials (such as biological metabolites) provides room for innovation in the mushroom-based pharmaceutical industry In the near future, biological metabolites extracted from medicinal mushrooms (for example, cordycepin, polysaccharides, etc.), such as C militaris, will have a function comparable to today's oil and gas crackers It is mentionable that the highest cordycepin production was recently obtained in surface liquid culture using C militaris mutant (Das et al., 2009) Therefore, bio-metabolites, such as cordycepin, polysaccharides and alike materials of C militaris will be the key future driving force in the realm of green pharmacology and pharmacognosy.

Polysaccharide content in the cultured Cordyceps

In nature, in Chinese C sinensis is produced on the Qinghai-Tibet Plateau above 3000 m above sea level Due to geographical constraints, natural resources are limited and very expensive Since it is difficult to form a substrate, cultivation is difficult However, after a period, fermentation technology has been successfully developed for the production of Cordyceps, which provides an extensive amount of mycelium for the study of Cordyceps polysaccharide (CP) The polysaccharide content of different Cordyceps strains Changes, including monosaccharide composition, physical and chemical properties, and biological activity (Zhong et al., 2009)

2.6.2 Differences in polysaccharide CP content

As mentioned above, there is variation in the polysaccharide content of

Cordyceps spp (Wang et al., 2007) found that polysaccharide content in the mycelia of C sinensis was 157.3 mg/g Although, by comparison, the content of total sugars between the corpus (part of the insect) and fruiting body (ascoma) was slightly different (24.2 and 24.9 %, respectively), the content in mycelium was higher (39.4 %) By colorimetric assay using sulphuric acid anthrone (Bai et al., 2001) found that the content of polysaccharides in C sinensis and C hawkesii (Gray) Cooke was 3.5 and 0.7 %, respectively, which was much lower than in C sinensis

C militaris (L ex St Amans) Link is considered as the ideal Cordyceps sp and can be cultivated well in solid or liquid culture (Huang et al., 2006) studied the nonvolatile components in the fruiting bodies and mycelia of C militaris and found that total sugars were 260.64 and 389.47 mg/g, respectively, but reducing sugar content of the fruiting body was higher than that of mycelia (Wen et al., 2006) compared the polysaccharide content of the silkworm

Cordyceps fruiting body, stroma, sclerotium and mycelium and found it the highest (86.49 mg/g) in the fruiting body, followed by stroma and sclerotium

In the artificial medium, the Cordyceps polysaccharide (CP) content in dry matter was 0.14 % (Zhang 2003)

C sobolifera, also known as chanhua, chanyongcao, chanrong, etc in

China, has a refreshing and cooling effect, and is used as a relatively rare drug for improving acuity of vision, fever reduction, detoxification, relieving convulsion, and other purposes However, little is known about it To find out the main active components and to facilitate further development and utilization (Wen et al., 2006) studied the content of CP in C sobolifera and found it to be 94.88 mg/g, which was significantly higher than in C militaris (32.30 mg/g), but slightly lower than in C sinensis (125.68 mg/g)

C gunnii, another species of the genus, is a large Cordyceps hosted on Hepialidae larvae and was first reported in 1983 by (Liang 1983) C gunnii and its anamorphs have rich bioactive substances The polysaccharide has unique pharmacological effects, and has good development prospects and utilization value (Liang 1983) Cultivation of C gunnii for different growth periods showed that the accumulation of polysaccharide in the mycelia was slow, which increased in the logarithmic growth phase and was maximum at 28.2 mg/g CP content evaluation in stroma, parasited worm, whole teleomorph and mycelia showed it to be maximum in mycelia, followed by stroma (Li et al., 199; Meng et al., 2002)

2.6.3 Differences in Cordyceps polysaccharide (CP) under different cultural conditions

Generally, the CP content in the fruiting bodies is low in any basic culture medium, which could be in creased through the optimization of medium composition and cultural conditions This not only reduces the cost, but also improves the economic efficiency Studies have also been carried out on the influence of microelements on the biomass and polysaccharide content in the fruiting body studied the effect of different concentrations of lanthanum nitrate (La(NO3)3 on C brasiliensis mycelium growth and polysaccharide content

(Yang et al., 2006) The results showed that a certain concentration of lanthanum nitrate (La(NO3)3 could increase polysaccharide content When the concentration of lanthanum nitrate (La(NO3)3 was 0.3 g/L, the polysaccharide content was the highest (24.21 mg/g, 1.59 times more than the control) However, with the increase of (La(NO3)3 concentration, the polysaccharide content was gradually reduced When the concentration of (La(NO3)3 was 0.5 g/L, the polysaccharide content was 15.01 mg/g only (Yu and Qian 2006) studied the effect of selenium on the CP content in C militaris and found that the supplementation of Se resulted in 51.03 mg/g of CP, almost twice more than that of control, i.e without Se (26.71 mg/g) (Xiao et al., 2004) investigated the optimal culture requirements for mycelial growth and exopolysaccharide production by C jiangxiensis JXPJ 0109 in submerged culture

The optimal temperature, initial pH and incubation period for exopolysaccharide production were 28 °C, 7.0 and 10 days, respectively The effect of different medium ingredients on the exopolysaccharide production was in the order of yeast extract > maltose > tryptone > glycerol > KH2PO4 > CaCl2 > MgSO4, and the optimal medium contained (in g/L of distilled water): maltose (food-grade) 20, glycerol 8, tryptone 5, yeast extract 10, KH2PO4 1, and CaCl2 0.5 Under the optimal conditions, the maximum exopolysaccharide and biomass production were 3.5 and 14.5 g/L after 10 and 8 days of fermentation, respectively (Xiao et al., 2004) (Lu 2000) optimized the CP production by single factor and multifactor design experiments and reported the best production (10.11 mg/g) in a medium containing (in %): sucrose 3, peptone 10, MgSO4ã7H2O, KH2PO4 1.0, pH=7.5, fermented at 25 °C and 125 rpm for 5 days By the orthogonal experimental study of temperature, pH, incubation time and medium composition on the poly- saccharide production, the optimal conditions were 22 °C, pH=6.0 and incubation time for 5 days with a medium containing (in %): cornmeal 2, sucrose 5, yeast extract 0.03, NH4NO3

0.04, KNO3 0.08, MgSO4ã7H2O 0.02, K2HPO4 0.04, and FeSO4ã7H2O 0.02, which yielded 170.3 mg of polysaccharide per 100 mL of broth (Chen et al., 2005; Xiao et al., 2004) Studied submerged cultural conditions for the production of polysaccharide by C pruinosa and found that the molasses mixed with sugar, peanuts and vitamin B instead of sucrose, beef extract and yeast extract, in 25-litre fermentation tank for 54 h, shortened the fermentation time, which would not only reduce the cost, but also reach high polysaccharide production level (9.51 g/L) (Hsieh et al., 2005) studied the production of polysaccharides by C sinensis by using response surface methodology The composition of optimized medium for polysaccharide production calculated from the regression model of RSM was (in %): sucrose 6.17, corn steep powder 0.53, (NH4)2HPO4 0.5, and K2HPO4 0.15 at pH= 4.44, with a predicted maximum polysaccharide production of 3.17 g/L Under the experimental conditions with this medium, the maximum polysaccharide production was 3.05 and 3.21 g/L in a shake flask and a 5-litre jar fermentor, respectively When the pH was controlled at a higher level such as pH= 5.0, both the cell growth and polysaccharide production were inhibited A low pH (2.85) was required for maximum production of polysaccharides

2.6.4 Isolation and purification of the polysaccharide

Several studies have been carried out on the isolation and purification of polysaccharides from the fungal mycelia (Sun et al., 2003) studied the extraction conditions of C sinensis mycelia and found that the ratio of raw material powder in a 300-mesh size and water of 1:2.5, the extraction time of

24 h and pH of 7.5–8.0 were the best (Ruan 2005) found that the temperature was the most important factor for the polysaccharide extraction, followed by the pH The best results were obtained when extraction was carried out twice with 0.1 g of mycelium powder in 20 mL of distilled water (pH=4.2) at 100 °C for 70 min (Che et al., 2004) also obtained similar results, but with alcohol; the polysaccharide obtained was of better quality and purity (Che et al., 2004) studied the differences in extraction methods from C militaris, such as ultrasonic and water, ultrasonic and alcohol, and hot water and reflux methods

An orthogonal experimental design was used to study the effects of extraction temperature and duration, number of extraction cycles and water concentration The results showed that a water extraction temperature of 80 °C with three extraction cycles, each for 90 min, was found to be the best (Shi et al., 2006), through the single factor and orthogonal experiment, studied the effect of different extraction times, the ratio of material and water, and the microwave power The results showed that C militaris crude polysaccharide reached 10.97

% when extraction was done with water and 80 % microwave power for 20 min

In the traditional polysaccharide purification method, alcohol is used for precipitation, which, however, also precipitates the protein In order to get rid of the protein, (Liu et al., 2002) used orthogonal experiments optimizing the factors such as the ratio of sample and the mixture of chloroform and butanol, and the reaction time The results showed that the ratio of sample and the mixture of chloroform and butanol 1:2.4 (by mass per volume); chloroform and butanol 1:0.2 (by volume), and the reaction time of 5 min were the optimum conditions This resulted in the removal of 97 % of protein Combination of trifluoromethyl, trichloroethane and Sevag method for protein removal resulted in the removal of 96.2 % of protein (Pan et al., 2002) Another study on polysaccharide extraction from C gunnii by orthogonal design showed that the optimum extraction conditions were water at 70 °C for 2 hand three extraction cycles After concentration to 1/6 of the volume, ethanol was added to reach the final concentration of 85 % and the precipitate was recovered by centrifugation This was washed three times with 95 % ethanol, dried in vacuum and subjected to further purification by column chromatography on DEAE-Sephadex A-25 and Sephadex G-200, which resulted in four fractions The polysaccharide content was 13.9 % and at least one fraction was a protein- bound polysaccharide (Xiao et al., 2003) When C pruinosa mycelia were extracted with hot water, followed by precipitation of polysaccharide with alcohol, dialysis and subjected to Sevag method, followed by cellulose and Sephadex G-100 column chromatography, two pure polysaccharides, PS1 and PS2, were obtained (Huang et al., 1998) There are some other methods such as ion exchange and gel chromatography described for the purification of CP (Li et al., 2003; Yu et al., 2004) Under normal circumstances, the more purified

CP could be expected by combining a variety of methods with the repeated purification

2.6.4.2 Monosaccharide composition and structure analysis

Isolated one polysaccharide from C.sinensis, which contained D-Glc, D- Man, L-Ara and D-Gal in a molar ratio of 8:90:1:1 (Wu et al., 2006) The average molecular mass was approx 8.3ã104 Da (Li et al., 2003) purified one water soluble polysaccharide from C sinensis mycelia with molecular mass of

210 kDa and its monosaccharide composition was glucose/ mannose/ galactose=1:0.6:0.75 (Wu 2006) isolated a polysaccharide with molecular mass of about 7.7ã103 Da from Cordyceps mycelia, with mannose/ galactose 1:9 After chemical analysis, IR, NMR spectroscopy and a-D-amylase digestion, it was identified as a-D-glucan with a skeleton of 1-4 and 1-3 connections, a-D-(1-6)-mannose skeleton through a-(1-3)-galactose residues of O-6 bond linking In addition, the mycelium of C sinensis contained a D-glucan

Cordyceps – A medicine mushroom and another fungal therapeutic

Too much about Cordyceps is unsubstantiated This literature is written to sell so-called medicines to potentially vulnerable people with serious diseases

On the other hand, there is convincing scientific information that indicates sig- nificant pharmacological properties, which are worth assessing (see Table 6) The present large review undertakes this task and deals with papers that use the name Cordyceps sometimes in its most general sense, especially when the revision of (Gi-Ho Sung et al., 2007) is considered There follows an extended introduction to the topic

Table 6 Medically related purported effects of various Cordyceps taxa

(or preparations) as described by various authors

Reputed for broad biological activities; tonic to replenish vital function (Shim et al., 2000) Prevents disease and onset of senility (Leung et al.,

2005) Reputed for broad biological activities; tonic to replenish vital function (Shim et al., 2000)

Treats a wide range of disorders; used for centuries; cultivated has same properties as natural (Yang et al,

2005) Treatment for wide range of diseases; anti- oxidant/anti-apoptotic properties (Buenz et al., 2004) Wide range of diseases (Hui et al., 2006)

Prized traditional medical materials (Ng et al., 2005) Immunomodulator (Kuo et al., 2005)

Highly valued for properties (Park et al., 2005) Benefits to the kidneys (Wojcikowski et al., 2004)

Wide range of diseases (Hui et al., 2006) Prized traditional medical materials (Ng et al., 2005) Popular nutraceutical and TCM (Jung et al., 2007)

4 C pruinosa Prized traditional medical material (Ng et al., 2005)

5 C ophioglossoides Prized traditional medical material (Ng et al., 2005)

The title relates to another in this journal concerning the fungal traditional Chinese medicine (FTCM), Ganoderma (Jang et al., 2006) In that case, the fungus was indeed a biofactory in the sense that numerous compounds have been reported from the fungus What is the situation with another FTCM,

Cordyceps? The immediate answer is that the state of the art is considerably less developed (see Table 7 for a list of secondary metabolites) There is a general impression that this fungus is being used in a modern con- text, before the benefits, and even what is being used, have been determined scientifically

Cordyceps is one of a growing number of FTCM being considered as cures for modern human diseases Many commercial products are available in the market (e.g Didanosine from Cordyceps militaris) These nutraceuticals are considered to relieve the ‘‘stress for humans of living in technologically developed societies” by stimulating basic and secondary responses of the immune system (Lakhanpal et al., 2005)

Table 7 Example of the range of species and some of the low molecular- weight secondary metabolites from Cordyceps

Cyclic peptides, cordycepin, 10-membered macrolides, cepharosporolides C, E and F, pyridine-2,6-dicarboxylic acid and 2- carboxymethyl-4-(30-hydroxybutyl) furan, dipicolinic acid

5 C sinclairii (2S,3S,4R)-(E)-2-Amino-3,4-dihydroxy-2- hydroxymethyl-14-oxoeicos-6-enoic acid

8 C ophioglossoides Ophiocordin, glycoprotein containing N- acetylgalactosamine

9 C heteropoda Cicadapeptins I and II, myriocin

The fungus represents a genus of perithecial ascomycetes (Phylum Ascomycota) classified in the Clavicipitaceae; a monophyletic group included in the order Hypocreales The genus contains over 400 species and the anamorphs of most are unknown Paecilomyces is considered traditionally to host the anamorphs but this has been disputed.(Sung et al., 2007) should be consulted for an up-to-date revision (and see later) Cordyceps are parasites of insects or fungi, often exhibiting a high degree of host specificity (Fig 6) However, the Cordyceps species associated with Lepidopteran hosts do not represent a monophyletic group There is even a high degree of genetic variation within Cordyceps sinensis, which creates difficulties in verifying samples A taxonomic review of the fungus is now available (see later section) a similar review is required for Ganoderma (Paterson, 2006) Larval infection via meiotic and/or mitotic spores/conidia and multiplication within the insect is from yeast-like budding However, the fungus grows through the insect by hyphae The accumulation of the biomass eventually kills the host (and/or a toxin(s) may be involved) It would be interesting to determine the biochemical parameters that cause these changes but this is not reported in the literature The fungus ruptures the host body following over wintering and forms the sexual perithecial stroma that are connected to the dead larva below ground which grow upward to emerge above the soil surface (Fig 6) The complete insect/fungus combination is used traditionally, but not exclusively, for medicinal purposes The present reviewer has seen no reports of the insect perse being given as a treatment

Fig 6 (a) C militaris on dead insect; (b) Cordyceps polycephala on dead insect host; (c) Cordyceps spp on Campanotus; (d) Cordyceps spp on an unidentified moth; (e) Cordyceps spp on a micropezid fly; (f) Cordyceps spp on another micropezid fly (Sung et al., 2007)

C sinensis (Berk.) Sacc., is one of the most famous traditional Chinese medicines (TCM) and health foods The fungus parasitises larvae of moths (Lepidoptera), especially Hepialus armoricanus (and Thitarodes), and converts each larva into a sclerotium, from which the stroma and fruit body grows The complex (including the larva body) has been used as a health food and traditional medicine to ‘‘invigorate the lung and nourish the kidney” in China for hundreds of years, and at least from the 17th century (Dong et al., 2008) Although what these preparations actually represented is impossible to determine given the difficulties in taxonomy of even modern times (Sung et al.,

2007) Understandably, conservation and sustainable harvest are important issues There is need for (a) research on biological screening, (b) a better understanding of the status in natural habitats, and (c) artificial cultivation of the fungus

Cordyceps and products are available in ‘‘Western” countries as over-the counter medicine/tonics which advertise them as Chinese herbs with anti-aging,

‘‘pro-sexual”, anti-cancer and immune boosting effects, although with poor supporting scientific evidence The believe is that C sinensis (CS) has various beneficial effects on humans, including those of a psychological nature The FTCM, is also called Dong Chong Xia Cao in Chinese (=winter worm summer grass) (Li et al., 2006) Primarily it is prescribed as a tonic for body strengthening after serious disease More recently, other treatments have been claimed such as for (a) respiratory, renal, liver, nervous system and cardiovascular diseases, and (b) tumours, aging, hypo- sexuality and hyperlipidemia (Kuo et al., 2006) It has been officially classified as a drug in the Chinese Pharmacopoeia since 1964 Furthermore, the outbreak of the Severe Acute Respiratory Syndrome (SARS) in China in 2003 has increased use considerably This would have been an excellent oppor- tunity to have determined how effective it was However, this does not appear to have been undertaken

The market demand for CS is growing sharply in many countries (Dong et al., 2008) They would surely be hailed as medical breakthroughs if the efficacy of any of these treatments were confirmed Nevertheless, the identities of active components have not been determined (in all cases) (Li et al., 2006) Research has shown that at least some of the traditional uses ‘‘may,” relate to pharmacological activities (Zhu et al., 1998) (if not pharmacological activities then what?) Herbs have been used throughout history to enhance physical performance, but scientific scrutiny with controlled clinical trials has only recently been used to study such effects (Bucci, 2000) The authors mention that Cordyceps remain untested which is surprising given the interest in the fungus The fungus is endemic to the alpine habitats of the Tibetan Plateau above 3000 m in south-western China, and there has been large-scale harvesting of the wild material from Nepal and India more recently It is agreed generally to have been over-harvested Furthermore, the price of natural products of CS is over US$ 12,000 kg/1 (2006 prices) for only ‘‘average quality” (how this is determined is not clear) in the market and increasing (Sharma et al., 2004) So one can understand the pressures on supply The socioeco- nomic implications of the FTCM are highly significant to the regions where it is harvested The fungus has officially been classified as an endangered species by CITES Management Authority of China and China Customers and this scarcity is of considerable concern to all Consequently, living strains have been isolated from natural CS and cultivated in large quantity by bioreactor technology which is a promising method to meet the needs of human consumption and to reduce the pressure on natural resources of the species (Dong and Yao 2008)

In vitro culture of the fungus has been employed increasingly and (Yang et al., 2005) state, ‘‘It is generally accepted that its cultivated Cephalosporium sinensis CS fungi possess the same functions as CS natural ‘‘herbs” (sic)”

Some other issues that require addressing are that natural C militaris is not readily available and is costly Thus, a growing number of so called Cordyceps products that derive from mycelial cultures of the asexual forms of these fungi have become commercially available (Hamburger, 2007) Mycelia cultivation has resulted in establishing a number of cultures derived from the holomorphic

CS These are referred to by the anamorphic names Paecilomyces hepiali and

Cephalosporium sinensis (CS), although the anamorph of CS appears to be

Hirsutella sinensis (and see later) However, the situation is confused with some taxonomist using outdated names To paraphrase (Buenz et al., 2005): while these strains undoubtedly support ecologically sustainable use of CS, the actual similarities between the wild fungus and the cultures are not clear

The consumption of complimentary medicine has increased dramatically, with over 42% of people in the United States of America reported as ‘‘users” Sales were US$ 3.3 billion in 1999 (Buenz et al., 2005) An important factor was the passage of the Dietary Supplement Health and Education Act in 1994 in the USA which opened the market for TCM (Cooper and Chang, 2001) One can appreciate how journals advocating these have increased concomitantly Why have they not been developed by big pharmaceutical companies and made available to the public in pure compound form? No doubt there could be many reasons why this has not happened (e.g not enough profit, ‘‘sticky” intellectual property rights issues see (Paterson et al., 2008) difficulty in mass production or synthesis, etc.) – apart from the possibility that they simply may not be effective Buenz et al mention that ‘‘one of the most inter)esting supplements is the not yet well-characterized C sinensis (Berk.) Sacc.”

C sinensis (Berk.) Sacc has attracted much research interest for antioxidant activity and there is considerable evidence of this from the fungus as a treatment of a wide range of diseases However, unauthenticated material has been used in some cases For example, a polysaccharide was isolated which can protect PC12 cells against hydrogen peroxide-induced neuronal cell toxicity, but the Cordyceps mycelia used was from the Wan Fong Pharmaceutical Factory (Zhejiang, China) and derived from Cordyceps sinensis Chen sp nov This is a nomenclaturally illegitimate fungal name, which raised doubts as to its relationship to CS In fact, it was later proved to be a different species (Dong and Yao, 2008) and this is a specific example of a general problem in the field An example of another problem is CordyMax Cs-

Preparations

The various pure compounds, extracts, whole fungus and other preparations as they relate to pharmacological activities are discussed next, as these divisions are considered most relevant to a biochemical/phytochemical perspectives of the topic

Data from pure compounds are the most revealing in terms of determining effects of the fungus/insect It is noted that these reports are scarce Some compounds from Cordyceps (as defined here) are not particularly unusual (Ng and Wang, 2005) review the chemical constituents and pharmacological properties The chemical constituents include (a) cordycepin (30- deoxyadenosine) and its derivatives, (b) ergosterol, (c) polysaccharides, (d) a glycoprotein and (e) peptides containing a-aminoisobutyric acid The activities ascribed to the fungus are anti-tumour, anti- metastatic, immunomodulatory, antioxidant, anti-inflammatory, insecticidal, anti-microbial, hypolipidaemic, hypoglycaemic, anti-aging, neuroprotective and renoprotective effects: So a vast a range of properties from a narrow spread of compounds

Polysaccharides account for the anti-inflammatory, antioxidant, anti- tumour, anti-metastatic, immunomodulatory, hypoglycaemic, steroidogenic and hypolipidaemic effects Effects Cordycepin contributes to the anti-tumour, insecticidal and anti-bacterial activity Ergosterol (a universal fungal compound) exhibits anti-tumour and immunomodulatory activity Finally, a DNase has been characterized These are not particularly novel compounds and one wonders why there are so many reports of the effects of crude extracts rather than much more work on the effects of novel pure compounds Cordycepin (Fig 7), 3-deoxyadenosine, is a derivative of the nucleoside adenosine differing from the latter by the absence of oxygen in the 30 position of its ribose entity As such is may be quite common Initially, it was extracted from Cordyceps; however, it is now produced synthetically Some enzymes do not discriminate between adenosine and so it can participate in certain reactions For example, it can be incorporated into RNA molecules causing premature termination of its synthesis It is classified as an anti- cancer compound

Fig 7 Chemical structure of cordycepin

Cordycepin inhibited the growth of Clostridium parapu-trificum and Clostridium perfringens, but had no effect on Bifidobacterium spp and Lactobacillus spp (Ahn et al., 2000) The presence of cordycepin in CS has been difficult to confirm, although it has been confirmed by NMR (Chen and Chu, 1996) However, other groups have not been able to detect this compound (Shiao et al., 1994) It is clearly important to confirm the presence of the compound in CS in terms of determining the active components of the fungus and ultimately for chemotaxonomic purposes (Cho et al., 2007) state that cordycepin is isolated from C militaris and is (claimed to be) an ingredient in TCM which is prescribed for various diseases, such as cancer and chronic inflammation (again note how vague this verbatim statement is) In this study, the novel effect of cordycepin inhibiting collagen-induced platelet aggregation was reported The data suggests that the inhibitory effect of cordycepin might be associated with the down-regulation of [Ca2 + ]i and the elevation of cAMP/cGMP production This result has obvious significance for prevention of thrombus formation Finally, cordycepin inhibited the growth of B16 melanoma cells inoculated subcutaneously into right murine footpads (Yoshikawa et al., 2004) Cordyheptapeptide A (Fig 7), a novel cycloheptapeptide, was isolated from a strain of Cordyceps together with four known bioxanthracenes There were only two previous reports on the isolation of cyclic peptides from this genus and these were from C militaris and CS The metabolite exhibited anti-malarial activity against Plasmodium falciparum and cytotoxicity to Vero cell lines In addition, the anti-malarial and cytotoxic activities of the bioxanthracenes were reported (Rukachaisirikul et al., 2006)

Fig 8 ORTEP view of cordyheptapeptide A (Rukachaisirikul et al., 2006)

In an extensive and impressive report, compounds desig-nated as ES-242s, were isolated from a Verticillium strain and identified as bioxanthracenes (Isaka et al., 2007) In addition, five novel ES-242 analogues were isolated with nine known compounds from a Cordyceps strain A closely related strain provided cordyheptapeptide A, cordyhepta- peptide B, and known ES-242s The structures of the novel bioxanthracenes were 60-O-desmethyl analogues of the compounds described Furthermore, cordyheptapeptide B has an N-Methyl- L-phenylalanine residue in place of the N-Methyl-L-tyrosine The isolation, structure elucidation, and anti-malarial activity of ES-242s and their analogues from the insect pathogenic fungus Cordyceps pseudomilitaris (from a Lepidoptera larva) were reported previously Cycloheptapeptide, cordyheptapeptide A, and some known ES-242s were isolated from a

Cordyceps strain from an elaterid larva In a continuing search for bioactive compounds from insect pathogenic fungi it was noticed that culture extracts of six Cordyceps strains, collected in the same location (from Coleoptera larvae, at Doi Innthanon National Park, Chiang Mai Province, Thailand), showed similar 1H NMR spectra This suggested the presence of bioxanthracenes (ES-242s) and cordyheptapeptide A as major constituent Two of these strains were subjected to mass fermentation (15 L) and chemical investigation As a result, five new ES-242 analogues and nine known compounds and cordy- heptapeptide A were isolated from an undefined strain

Cordyheptapeptide B, was isolated, together with other known compounds Some of these were tested for activity against P falciparum and cytotoxicity to KB cells (oral human epidermoid carcinoma), BC cells (human breast cancer), NCI-H187 cells (human small cell lung cancer), and noncancerous Vero cells (African green monkey kidney fibroblasts) Cordyheptapeptide A exhibited anti- malarial activity, while cordyheptapeptide

B was inactive and both cyclic peptides showed moderate cytotoxicity Furthermore, cordyformamide is a plausible biogenetic precursor of xanthocillin Y, and was isolated from a culture broth of Cordyceps brunnearubra BCC 1395 Cordy- formamide was found to exhibit activity against P falciparum, whereas it showed weak or no cytotoxicity (Isaka et al.,

2001) Production of the nonribosomal peptides cicapeptins I and II (Fig 9) were reported by (Krasnoff et al., 2005) which was the first report from fungi of consecutive Hyp or Pro residues in a nonribosomal linear peptide The compounds exhibited anti-bacterial and anti-fungal activity A novel immunosuppressant was isolated from the culture broth of Isaria sinclairii, the anamorph of C sinclairii, and characterized as (2S,3S,4R) -(E)-2-amino-3,4- dihydroxy-2-hydroxymethyl-14-oxoeicos-6-enoic acid, which was identical to anti-fungal substances, myriocin and ther- mozymocidin (Fujita et at, 1990) The suppressive activity was found to be equal to, or higher than cyclosporin, a which is used clinically The activities of the 10 derivatives were also examined, indicating the following relationships between structure and activity the: (a) lactone formation between the carboxy group at C-1 and the hydroxy group at C-4, and the reduction of the carbonyl group at C-14 to the hydroxy group do not affect the suppressive activity; (b) hydrogenation of the double bond at C-6 resulted in decreased activity; and (c) acetylation of the amino group and the thioketalization on the carbonyl group at C-14 ‘‘drastically” reduced the suppressive activity

Fig 9 (1 and 2) Cicapeptins I and II; (3) myriocin from Cordyceps heteropoda (Krasnoff et al., 2005) Also, the compound suppressed the production of anti-bodies to sheep red blood cells and induction of cytotoxic lymphocyte T cells more strongly than cyclosporin A Obviously, this is an important lead compound and hence one of the more satisfactory papers (Chen et al., 1999) isolated a pure compound (H1-A) from CS and investigated whether autoimmune disease progression in mice was affected by administration of the metabolite The authors are vague as to what the compound is and state that, ‘‘it is a kind of ergosterol and looks like testosterone”

The authors also provide a chemical structure, which confirms that it is a common sterol and a systematic name could have been provided Their results demonstrated that mice treated daily exhibited a progressive reduction in anti- ds-DNA production In clinical presentation, the treated group had a reduction in lymphadenopathy, a delayed progression of proteinuria, and an improvement in kidney function Histological analysis of kidney tissue indicated that H1-A inhibited mesangial proliferation that was evident in lupus nephritis However, there was no change in immune complex deposition H1-A ‘‘may be” useful for treating systemic lupus erythematosus in human patients However, more work is required H1-A was claimed to be effective in the treatment of autoimmune disorders (Yang et al., 2003) Results demonstrated inhibition of cell proliferation and promotion of apoptosis of activated human mesangial cells in vitro: the activities were not a result of cytotoxicity

Moving on to more interesting compounds, cordypyri-dones A and B were detected from the uncommon species, C nipponica This may indicate a particularly close taxonomic relationship between the two taxa The structure of ophiocordin was falsely assigned and balanol was the compound of interest

Balanol was under development as an anti-cancer agent as it established to be a selective inhibitor of protein kinase C see (Paterson et al., 2008) It is more common for pure compounds to be tested in the fields of anti-bacterial, anti- fungal, anti-malarial and insecticidal activity that is to be recommended more generally Ophiocordin is an anti-fungal antibiotic isolated from submerged cultures of C ophioglossoides

However, bioxanthracenes were isolated from C pseudomilitaris and appear to be anti-malarial (see also previously) (Jaturapat et al., 2001) Ten- membered mac- rolides, cepharosporolides C, E and F, cordycepin, pyri dine- 2,6-dicarboxylic acid and 2-carboxymethyl-4-(30- hydroxybutyl) furan were reported from C militaris by (Rukachaisirikul et al., 2004) An inhibitor of the prophenoloxidase activation was isolated from a culture filtrate of C militaris and identified as dipicolinic acid (DPA) The production of DPA in a range of Clavicipitaceae fungi was examined Entomogenous fungi that produce DPA were integrated into one group by a phylogenetic analysis based on 18S rDNA Interestingly, it was suggested that the group acquired an ability to produce DPA during its evolution from plant pathogenic fungi to entomogenous fungi (Watanabe et al., 2006) In a useful comparison of crude extracts and pure compound, the anti-diabetic effect of various fractions of C militaris, CCCA (crude cordycepin containing adenosine), CMESS (ethanol soluble supernatant), and cordycepin were evaluated in diabetic mice (Yun et al., 2003) CMESS showed a potent inhibitory activity of 34.7% in starch- loaded mice: CMESS reduced blood glucose level by 35.5%

However, CCCA, and cordycepin showed no difference After 7 days’ administrations of these drugs, CMESS, and cordycepin dramatically reduced blood glucose level CCCA with a high concentration of cordycepin did not reduce blood glucose level Proliferation of T-lymphocyte was significantly decreased; while NO production was increased more than two-fold in the cordycepin-administered group The proliferation of macrophages and NO production were significantly decreased in the CMESS administered group CMESS and cordycepin may be (a) useful tools in the control of blood glucose level in diabetes and (b) promising new drugs as an anti-hyperglycemic agent without the defects of lowered immune responses and other side effects, the authors suggest Furthermore, cordycepin, 30-amino-30-deoxyadenosine, homocitrullyl aminoadenosine, adenine, cordycepic acid and D-mannitol have been reported from Cordyceps spp (Kredich et al., 1961), (Liu et al., 1989) Ergosterol peroxide isolated from C cicadae inhibited phytohaemagglutinin- induced T cell proliferation, and arrested the progression of activated T cells from G1 to S phase of the cell cycle Early gene transcripts, in particular those of cyclin E, interferon, and inter- leukins were (Kuo et al., 2003) The glycosylated form of ergosterol peroxide from CS was more potent than the aglycone in inhibiting proliferation of tumour cells (Bok et al 1999) However, ergosterol peroxide is widespread in fungi and Cordyceps does not offer any particular advantage in its preparation It is worth noting that eight different

Commercial preparations

It is revealing to read the web sites of some of the commercial products: The best of them at least contain warnings that, ‘‘these statements have not been evaluated by the (US) Food and Drug Administration This product is not intended to diagnose, treat, cure or prevent any disease” (Colson et al., 2005) undertook a revealing experiment to determine the effect on male cyclists’ performance using a combination of CS and Rhodiola rosea ‘‘herbs” – a traditional herbal medicine Importantly, and unusually, the study followed a double blind, randomized, placebo-treatment, prepost test design Essentially no effect was observed This is an example of why scientists need to circumspect about making unsubstantiated claims about the powers of these preparations even if spin does help project funding ‘‘In this regard, the maintenance of a balance of Yin and Yang – two opposing components involved in life activities as exemplified by the antagonistic action of the sympathetic and parasympathetic nervous systemsis essential in achieving a healthy condition” (Leung et al., 2005) Statements such as these leaves the current reviewer bewildered Previous studies have shown that long-term treatment with a ‘‘Yang-invigorating” Chinese herbal formula (VI-28) could increase red cell CuZn- superoxide dismutase (SOD) activity in male human subjects Yet the authors conclude the beneficial effect of VI-28 treatment on mitochondrial functional ability and antioxidant capacity may have clinical implications in the prevention of age related diseases (Ka Wai Lee et al., 2006) investigated a commercial preparation of a cultivated strain of CS They state that the immunomodulatory activities have been renowned for centuries The report describes positively the immunomodulatory features: In vitro results demonstrated that the fungus induced the production of interleukin (IL)-1, IL-

6, IL- 10 and tumour necrosis factor alpha, augmented surface expression of CD25 on lymphocytes, and elevated macrophage phagocytosis and monocyte production of H2O2 The authors state that, ‘‘Our results possibly provide the biochemical basis for future clinical trials” The current author emphasises that such over extrapolation of data needs to be discouraged

Dai et al., 2001 founded that CordyMax Cs-4 (a mycelial fermentation product of CS) ‘‘improved the bioenergy sta- tus” in the mouse liver These findings may explain why CordyMax Cs-4 is claimed to alleviate fatigue and improve physical endurance especially in aged subjects Reports of athletes’ performance being improved may also be supported from these types of reports

CordyMax Cs-4 lowered fasting plasma levels of glucose and insulin, improved oral glucose tolerance and increased the glucose–insulin index, which measures insulin sensitivity, in rats (Zhao et al., 2002) Remarkably, a dried powder preparation of myceliacalled Bailing capsule (a) prevented rejection of renal transplants, (b) protected renal and hepatic function, (c) stimulated haematopoietic function, (d) improved hypoproteinaemia and hyperlipidaemia and (e) reduced the incidence of infections (Sun et al., 2004) However, such claims must surely carry considerable elements of doubt In another case of a combined treatment with as many as six other components in addition to CS, the treatment was more effective at preventing acute renal failure than chronic in rats Obviously, it is difficult or impossible to assess the efficacy of the individual components (Ngai et al., 2005).

Compounds of fungi isolated from Cordyceps

A factor which is not often considered is whether the activities of extracts and pure compounds are from the Cordyceps of interest or from other contaminating fungi The issues of anamorph/teleomorph associations are relevant here For example, epicoccins A–D were isolated from cultures of a

Cordyceps -colonizing isolate of Epicoccum nigrum (Zhang et al., 2007)

Gliocladinins A and B are of an isolate of Gliocladium sp that colonized CS (Guo et al., 2007) It is essential that such activity is differentiated from that obtained from the traditional medicine or Cordyceps perse In a series of papers,

(a) Paecilomyces militaris is shown to possess militarinones A, B, C, D and (b) farinosomes and a deoxymilitarinone substance were detected from

Paecilomyces farinosus (Cheng et al., 2004) The authors do not make statements as to the holomophic connections to Cordyceps (Hamburger 2007) does make this link to C militaris (teleomorph) from P militaris (anamorph) apparently on the basis of the same pigment production However, it is having been suggested that Paecilomyces does not contain anamophic species of Cordyceps (Chen et al., 2001) Hence, comment on these reports is avoided by the current author because the relationships between Paecilomyces and Cordyceps remain illdefined.

What about the insect

Another complicating facet is that the medicine may contain a proportion of the insect host and so what does this bring to the activity This is an area of investigation which simply is not reported These aspects are almost totally ignored in the case of the medicinal Cordyceps It appears as if entomologists have simply not been involved in the field Hence, it is worth reporting what is known about the differences between the insect and fungus parts: Interestingly, galactomannans isolated from the insect portion of C cicadae demonstrate potent hypoglycaemic activity in mice (Kiho et al., 1990.) The fruiting body portion, but not the carcass portion, of Cordyceps reduced weight loss, polydipsia and hyperglycaemia in diabetic rats (Lo et al., 2004) Aqueous methanol extracts of C cicadae ascocarps enhanced human mononuclear cells (HMNC) proliferation (Weng et al., 2002) In contrast, the methanol (100%) extracts of the C cicadae insect-body portion suppressed HMNC proliferation Interestingly, the ergosterol esters concentrations were much higher in the (Marwah et al.) caterpillar than the fruiting bodies although ergosterol was similar Finally, the fruiting body and the caterpillar parts of CS are claimed to be similar in chemical composition and hence antioxidant activity because the fungus had presumable replaced the insect constituents with fungal (Li et al.,

The water extracts from the fruiting body and ‘‘worm” of natural

Cordyceps were analyzed for their content of nucleosides and polysaccharides; the results showed that the worm had a chemical composition similar to the fruiting body (Li et al., 2002) In addition, both the fruiting body and worm of

Cordyceps showed similar potency in their antioxidation activities in the xanthine oxidase assay, the induction of hemolysis assay and the lipid peroxidation assay These results suggest that the function of the worm is to provide a growth medium for the fruiting body, and that eventually, the worm is totally invaded by mycelia Aqueous methanol extracts of C cicadae ascocarps enhanced human mononuclear cells HMNC proliferation (Weng et al., 2002) In contrast, the methanol (100%) extracts of the C cicadae insect- body portion suppressed HMNC proliferation This is a most interesting result in the current author’s opinion.

Profiling chemical constituents

The following are references concerning the chemical constituents as determined by analytical profiles (Li and Li 1991) (Guo et al., 1998) described an HPLC method for quantitative determination of adenosine and deoxya- denosine The two active compounds were identified as guanosine and adenosine (Li et al., 2001) found that cultured CS mycelia have a much higher content of nucleosides than natural CS (Yu et al., 2007) developed a method involving ‘‘biospecific” extraction and HPLC for potential immunological components in CS (Li et al., 1999) determined adenosine in fermented products of Cordyceps by reversed phase HPLC Ergosterol in CS can be determined by HPLC (Li et al., 2004), and, of course, ergosterol is present in all fungi.