Chapter: 2 Review of Litrature - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/54938/9/09_chapter2.pdf · from insect larvae to adult. Insect’s epidermis is covered with

Chapter: 2

Review of Litrature

Review of literature

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2. Review

Winter worm and summer grass i.e Cordyceps militaris is one of the most

valued traditional Chinese medicines for fighting cancer, increasing longevity, and improving

immunity. In recent years number of studies had been carried out to cure dreadful

non -communicable diseases like cancer, HIV, diabetes etc. with the use of herbal or natural

medicine. Cordyceps militaris is such an entomopathogenic fungus, has been known to

have numerous therapeutic implications in terms of human health.

Cordycepin, the major bioactive constituent of Cordyceps militaris is known as first

nucleic acid antibiotic. Cordycepin is predominantly produced commercially via solid-state

fermentation and submerged cultivation of Cordyceps. Considerable effort is currently

focused on three aspects of cordycepin production: strain screening and improvement,

additives, and optimizing fermentation. However, high-efficiency batch fermentation of C.

Militaris is carried out in static culture for more than 30 days, which is too long to achieve

high production efficiency and low operational cost and energy consumption. Therefore, how

to reduce fermentation period is extremely vital. Various methods have been proposed to

extract and analyze bioactive metabolite like cordycepin from liquid culture as well as

fruiting body of C. militaris. This review reveals about the various production and extraction

strategy opted by the researchers for maximum gain of cordycepin. Furthermore review will

update us about the potential applications of Cordyceps sp. with special reference to

cordycepin as anti proliferatory and anti-angiogenic candidate.

2.1 Cordyceps Diversity and its interaction with host

There are more than 1200 entomopathogenic fungi reported (Humber, 2000) in

literature out of which Cordyceps constitutes one of the largest genus containing

approximately 500 species and varieties (Hodge et al., 1998; Hywel, 2002; Muslim and


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Rahman, 2010). Many different species of Cordyceps have been cultivated for their medicinal

and pharmaceutical properties including C. sinensis, C. militaris, C. ophioglossoides, C.

sobolifera, C. liangshanesis, and C. cicadicola. Simlarly, many other species of Cordyceps

have been documented like C. tuberculata, C. subsessilus, C. minuta, C. myrmecophila, C.

Canadensis, C. agriota, C. gracilis, C. ishikariensis, C. konnoana, C. nigrella, C. nutans, C.

pruinosa, C. scarabaeicola, C. sphecocephala, C. tricentri etc., although the molecular

evidence for their proper phylogenetic placement is still lacking (Shrestha and Sung, 2005;

Wang et al., 2008; Zhou et al., 2009).

Cordyceps usually infects insects at different stages of their development ranging

from insect larvae to adult. Insect’s epidermis is covered with a thick layer of cuticle

(procuticle and epicuticle) which is also known as integument. Infection begins with the

dispersion of fungus conidia on insect’s surface. Once conidia get settled, they start

germinating within few hours under suitable conditions. To get protection from

environmental ultraviolet radiations, protective enzymes like Cu-Zn superoxide dismutase

(SOD) and peroxidases are secreted by the fungal conidia. These enzymes were shown to

provide protection to the conidia from reactive oxygen species (ROS) generated due to UV

rays and heat in the environment (Wanga et al., 2005). Besides this, conidia secrete certain

hydrolytic enzymes like proteases, chitinases and lipases which lead to the dissolution of the

integument and play a very important role in infection to the host. These enzymes not only

provide a penetration path to the conidia but also provide nutrition to the germinating conidia

(Ali et al., 2010).

Further as infection proceeds, a short germ-tube protrudes out of the conidia which

starts thickening at the distal end and known as appressorium. This appressorium has been

known to maintain a kind of mechanical pressure on to the germinating germ tube which in

turn intensify the penetration effect of germ tube (Webster, 1980; Hajek and Leger, 1994). As


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the germ tube penetrates epicuticle layer of insect’s integument, it starts forming a plate like

structure called penetration plate. The penetration plate further produces secondary hyphae,

which cross the epidermal layer and reach into the haemocoel of insect’s body. From these

hyphae, protoplast bodies bud off and start circulating into the insect’s haemocoel. Fungus

now starts growing into a filamentous mode invading internal organs and tissues of the host.

During growth inside the host, fungus produces various kinds of toxic secondary metabolites,

which are insecticidal. These secondary metabolites are responsible to take insect into its

final stage of life and ultimately insect dies out. Finally fungal mycelium emerges out

through the cuticle and lead to the formation of fruiting body under suitable environmental

conditions. Morphological features of fruiting body has been described as stipitate,

yellowish-orange to orange to reddish-orange fruiting stroma which is cylindrical to slightly

clavate in shape. As shown in figure 2.1, stipes of 1.5 - 3 mm thickness with fertile clava

terminal (2.0 - 6.0 mm wide) are also commonly seen in the fruiting body with overall stroma

of about 1.5 - 7.0 cm tall which can vary in length depending on the size of the host (Das et

al., 2010a). The taxonomical data of C. militaris have been summarized in table 1.

Figure 2.1: The photograph showed morphological features of C. militaris (Das et al., 2010a).


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Table 2.1: Taxonomical data of Cordyceps militaris

Kingdom Fungi

Phylum Ascomycota

Sub-phylum Ascomycotina

Class Ascomycetes

Order Hypocreales

Family Clavicipataceae

Genus Cordyceps

Species Cordyceps militaris

2.2 Nutritional value of Cordyceps

Genus Cordyceps has always been carried with it an aura of class, prestige and worth

because of its health benefits. In Cordyceps, there occurs a wide range of nutritionally

important components including various types of essential amino acids, vitamins like B1, B2,

B12 and K, different kinds of carbohydrates such as monosaccharide, oligosaccharides and

various medicinally important polysaccharides, proteins, sterols, nucleosides, and other trace

elements (Hyun, 2008; Yang et al., 2009; Yang et al., 2010; Li et al., 2011). In the fruiting

body and in the corpus of C. militaris, the reported total free amino acid content is 69.32

mg/g and 14.03 mg/g, respectively. The fruiting body harbours many abundant amino acids

such as lysine, glutamic acid, proline and threonine as well. The fruiting body is also rich in

unsaturated fatty acids (e.g. linoleic acid), which comprises of about 70% of the total fatty

acids. There are differences in adenosine (0.18% and 0.06%) and Cordycepin (0.97% and

0.36%) contents between the fruiting body and the corpus respectively (Shiao et al., 1994;


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Hyun, 2008). More attention may need to be paid by scientific communities to analyse

bioactive peptide from Cordyceps.

2.3 Bio-metabolites isolated from Cordyceps

Cordyceps, especially its extract has been known to contain many biologically active

compounds like Cordycepin, cordycepic acid, adenosine, exo-polysaccharides, vitamins,

enzymes etc. (Table 2.2). Out of these, Cordycepin i.e. 3’-deoxyadenosine (Figure 2.2)

isolated from ascomycetes fungus C. militaris, is the main active constituent which is most

widely studied for its medicinal value having a broad spectrum biological activity

(Cunningham, 1950).

2.4 Cordycepin: Mechanism of Action

The structure of Cordycepin is very much similar with cellular nucleoside, adenosine

(Figure 2.3) and acts like a nucleoside analogue. Cordycepin comprises a purine (adenine)

nucleoside molecule attached to a ribose sugar (ribofuranose) moietyvia aβ-N9-glycosidic

bond. The chemical synthesis of cordycepin is achieved mainly through the replacement of

the OH group at the 3′ position in the ribofuranosyl moiety with H, generating a deoxy

analogue of adenosine.

Figure 2.2: The figure elucidates the difference in the chemical structures of bioactive compounds, Cordycepin and adenosine, produced by C. militaris.


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Table 2.2: Bioactive compounds isolated from Cordyceps sp.

S.No Bioactive Compounds

References

1. Cordycepin

Cunningham (1950)

2. Cordycepic acid Chatterjee et al., (1957)

3. N-acetylgalactosamine Kawaguchi et al., (1986)

4. Adenosine Guo et al., (1998)

5. Ergosterol & ergosteryl esters Yuan et al., 2007

6. Bioxanthracense Isaka et al., (2001)

7. Hypoxanthine Huang et al., (2003)

8. Acid Deoxyribonuclease

Ye et al., (2004)

9. Polysaccharide &

Exopolysaccharide

Yu et al., (2007); Yu et al., 2009; Xiao et al.,

2010; Yan et al., 2010

10. Chitinase Lee and Min (2003)

11. Macrolides (C10H14O4) Rukachaisirikul et al., (2004)

12. Cicadapeptins and myriocin Krasnoff et al., (2005)

13. Superoxide dismutase Wanga et al., (2005)

14. Protease Hattori et al., (2005)

15. Naphthaquinone Unagul et al., (2005)

16. Cordyheptapeptide Rukachaisirikul et al., (2006)

17. Dipicolinic acid Watanabe et al., (2006)

18. Fibrynolytical enzyme Kim et al., (2006a)

19. Lectin Jung et al., (2007)

20. Cordymin Wonga et al., (2011)


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2.4.1 Inhibition of Purine Biosynthesis Pathway

Once inside the cell, Cordycepin gets converted into 5’ mono, di- and tri-phosp hates

that inhibits the activity of enzymes like ribose-phosphate pyrophosphokinase and 5-

phosphoribosyl-1-pyrophosphate amidotransferase which are used in de-novo biosynthesis of

purines (Figure 2.4) (Klenow, 1963; Overgaard, 1964; Rottman and Guarino, 1964).

Figure 2.3: The figure shows the inhibitory effect of Cordycepin in mono and tri- phosp hate states on the enzymes, Phosphoribosyl pyrophosphate synthase and phosphoribosyl pyrophosphate amidotransferase, involved in purine biosynthesis pathway.

2.4.2 Cordycepin Provokes RNA Chain Termination

Cordycepin lacks 3’ hydroxyl group in its structure (Figure 2.2), which is the only

difference from adenosine. Adenosine is a nitrogenous base and act as cellular nucleoside,

which is needed for the various molecular processes in cells like synthesis of DNA and/or

RNA. During the process of transcription (RNA synthesis), some enzymes are not able to

distinguish between an adenosine and Cordycepin which leads to incorporation of 3’-

deoxyadenosine or Cordycepin, in place of normal nucleoside preventing further


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incorporation of nitrogenous bases (A, U, G, and C), leading to premature termination of

transcription (Figure 2.4) (Chen et al., 2008; Holbein et al., 2009). Recently, genotoxic

effect of cordycepin reported in cancer cells mediated through induction of DNA double

strand breaks (Lee et al., 2012)

Figure 2.4: The addition of Cordycepin as a Co-TP (Cordycepin tri-phosphate) leads to transcriptional termination.


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2.4.3 Cordycepin Interferes in mTOR Signal Transduction

Cordycepin has been reported to shorten the poly A tail of m-RNA which further

affects its stability inside the cytoplasm. It was observed that inhibition of polyadenylation

with Cordycepin of some m-RNAs made them more sensitive than the other mRNAs. At

higher doses, Cordycepin inhibits cell attachment and reduces focal adhesion. Further

increase in the dosage of Cordycepin may shut down mTOR (mammalian target of

rapamycin) signaling pathway (Fig. 2.5) (Wong et al., 2010). The name mTOR has been

derived from the drug rapamycin because this drug inhibits mTOR activity. The mTOR

inhibitors such as rapamycin and CCI-779 have been tested as anticancer drugs because they

inhibit or block mTOR signaling pathway. mTOR is a 298 kDa serine/threonine proteine

kinase from the family PIKK (Phosp hatidylinositol 3-kinase-related kinase). The mTOR

plays a very important role to regulate proteins synthesis. However mTOR itself is regulated

by various kinds of cellular signals like growth factors, hormones, nutritional environment,

and cellular energy level of cells. As growth factors bind with cell receptor, Phosphatidyl

inositol 3 kinase (PI3K) gets activated converting Phosphatidyl inositol bisphosphate (PIP2)

to Phosphatidylinositol trisphosphate (PIP3). PIP3 further activates PDK1 (Phosphoinositide

Dependent Protein Kinase 1). The activated PDK1 then phosphorylates AKT 1 kinase and

makes it partially activated which is further made fully activated by mTORC2 complex. The

activated AKT 1 kinase now activates mTORC1 complex that leads to the phosphorylation of

4EBP1 (translational repressor) and makes it inactive, switching on the protein synthesis

(Wong et al., 2010). The study confirmed that under low nutritional stress, Cordycepin

activates AMPK which blocks the activity of mTORC1 and mTORC2 complex by some

unknown mechanism. The inactivated mTORC2 complex can not activate AKT 1 kinase

fully, which in turn blocks mTOR signal transduction inhibiting translation and further cell

proliferation and growth (Figure 2.5).


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Figure 2.5: Cordycepin presumably activates the AMPK by some unknown mechanism which further negatively regulates the translation of mTOR signaling transduction pathway by the formation of a translational repressor,4-E-Binding Protein-1 (4EBP1).

2.5 Fermentation strategy for cordycepin production

The medicinal mushrooms are abundant sources of useful natural products with

various biological activities. Therefore, many researchers in the field of physiology,

pharmacology and medical science, etc., are interested in the contents of mushrooms (Das et

al., 2010b). Nearly 80 to 85% of all medicinal mushroom products are extracted from their

fruiting bodies while only 15% are derived from mycelium culture (Lindequist et al., 2005).

Fruiting body of Cordyceps is a very small blade-like structure, making its collection difficult

and expensive. Since there is a huge requirement of medicinal mushroom bio-metabolites, it

is necessary to cultivate mycelium biomass artificially for which variety of methods for its

cultivation have been proposed by many research groups (Masuda et al., 2007; Das et al.,

2008; Das et al., 2010d). Cordyceps mycelium can grow on different nutrients containing

media, but for commercial fermentation and cultivation, insect larvae (silkworm residue) and

various cereal grains have been used in the past. It has been seen consistently that from both


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insect larvae and cereal grains, fruiting body of fungus can be obtained with almost

comparable medicinal properties (Holliday et al., 2004).

There are basically two fermentation techniques by which the cultivation of mycelium

biomass of Cordyceps can be achieved including surface and submerged fermentation. In

surface fermentation, the cultivation of microbial biomass occurs on the surface of liquid or

solid substrate. This technique, however, is very cumbersome, expensive, labor intensive and

rarely used at the industrial scale. While in submerged fermentation, micro-organisms are

cultivated in liquid medium aerobically with proper agitation to get homogenous growth of

cells and media components. However, there is a loss of extra-cellular compounds (after

harvesting mycelium) from the broth which makes it necessary to improve the culture

medium composition and downstream processing technology to get large-scale production of

the secondary bio-metabolites (Ni et al., 2009). Some reports are mentioned below describing

the cordycepin production using submerged and surface fermentations.

Mao et al., (2005) studied the effects of various carbon sources and carbon/nitrogen

ratios on production of a useful bioactive metabolite, cordycepin by submerged cultivation.

The highest cordycepin production, i.e. 245.7 ± 4.4 mg/L on day 18, was obtained in medium

containing 40 g glucose/L. Further using central composite design and response surface

analysis, cordycepin production & productivity was increased up to 345.4 ± 8.5 mg/L and

19.2 ± 0.5 mg/L per day respectively.

Masuda et al., (2006) gave the production conditions of cordycepin using surface

culture technique. They reported under the optimal conditions, the maximum cordycepin

concentration in the culture medium reached 640 mg/L, and the maximum cordycepin

productivity was 32 mg/L day-1. Further Masuda et al., (2011) studied the effects of

adenosine on cordycepin production in a surface liquid culture of the mutant and the wild


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type strains. For the mutant strain, the maximum levels of production with adenosine and

without adenosine were 8.6 g/L and 6.7 g/L, respectively.

Effects of nitrogen sources (NH4+) on cell growth and cordycepin production by

submerged cultivation of Cordyceps militaris were studied by Mao et al., (2006). Researcher

found that by optimizing the feeding time and feeding amount of NH4+, a maximal

cordycepin concentration of 420.5 ± 15.1 mg/L was obtained.

Shin et al., (2007) investigated the influence of initial pH value, various nitrogen

sources, plant oils, and modes of propagation (shake-flask and static culture) on the

production of biomass, exopolysaccharide (EPS), adenosine and cordycepin using Cordyceps

militaris CCRC 32219. A Box–Behnken experimental design was employed to optimize the

production of cordycepin up to 2214.5 mg/L.

Effect of ammonium feeding on cordycepin production was investigated by Leung

and Wu (2007). Authors reported that cordycepin production increase nearly fourfold (from

28.5 to 117l µg/g) by the supplementation of 10 mM NH4Cl. However at higher

concentration, they found the negative its effect on mycelium growth.

Das et al., (2009) studied a mutant of the medicinal mushroom Cordyceps

militaris for higher cordycepin production. Among all the mutants, G81-3 had the

highest cordycepin production of 6.84 g/L using optimized conditions compared to that

of the control of 2.45 g/L (2.79 times higher). In addition, influences of different

additives on the cordycepin production such as glycine and adenosine were studied and

found cordycepin production increase up to 8.57 g/L.

Effects of culture conditions on the growth of mycelium and the production of

intracellular cordycepin using Cordyceps militaris was investigated by Xie et al., (2009).


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Researchers optimized fermentation temperature, pH and medium capacity, by Box-Behnken

design. Results showed that highest dry mycelium weight (19.1 g/L) and Cordycepin (1.8

mg/g) can be obtained at temperature 28°C, pH 6.2 and medium capacity 57ml.

Das et al., (2010c) explored the effect of inoculation on cordycepin production in

surface fermentation using Cordyceps militaris. Results showed that cordycepin production

in control, double, triple inoculants were 5.05, 6.11 and 5.61 g/L respectively.

The effect of ferrous sulphate addition on production of cordycepin (3′-

deoxyadenosine) was investigated in submerged cultures of Cordyceps militaris in shake

flasks (Fan et al., 2012). They showed that the optimal addition condition (1 g/L) of ferrous

sulfate, cordycepin production reached 596.59 ± 85.5 mg/L which was about 70% higher than

the control.

The effect of liquid culture conditions on extracellular secretion of cordycepin from C

militaris was investigated by Zhong et al., (2011). They reported the optimal cultural

conditions as follows: initial pH- 7, cultivation temperature- 24ºC, shaking speed- 180 rpm

and cultivation period 9 d. By use of these culture conditions, the content of cordycepin in the

culture fluid attained to 0.537g/L.

Zhang et al., (2013), applied response surface methodology (RSM) to optimize the

medium components for the cordycepin production by submerged liquid culture. They found

64.15 mg/L of cordycepin which was very close to the model prediction value. Further they

suggested repeated batch operation an efficient method to increase the cordycepin yield.

Kang et al., (2014) studied single factor design, using Plackett-Burman and central

composite design to establish the key factors responsible for cordycepin production. They


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reported that maximum cordycepin up to 2 g/L could be achieved with working volume of

700 ml in the 1000 ml glass jar.

Similarly, Jiapeng et al., (2014) carried out fermentation to optimize maximum

production of cordycepin in static culture using single-factor experiments with Placket–

Burman and a central composite design. They demonstrated a maximum cordycepin yield of

7.35 g/L can be achieved in a 5-L fermenter under the optimized conditions.

2.6 Analysis tool for cordycepin detection

Nowadays different products of Cordyceps are available in the market, as health

supplement or neutraceuticals. Hence, it is very important to analyse the presence of

cordycepin for its quantitative as well as qualitative analysis (Zheng et al., 1999). Several

techniques such as thin layer chromatography, high performance liquid chromatography

(HPLC) and Capillary electrophoresis have been reported for the analysis of the cordycepin

present in medicinal herb Cordyceps. Herein, the development on bio-chemical analysis of

cordycepin is reviewed and discussed.

2.6.1 Thin layer chromatographic analysis

Kim et al., (2006b) developed thin layer chromatography (TLC) plates in chloroform/

methanol/water (64:14:1). The spots of separated molecules were stained with 10% sulphuric

acid solution (in ethanol) for visualization. Ma and Wangh (2008) established a dual

wavelength TLC-scanning method for the determination of nucleosides in the preparation of

Cordyceps sinensis and analyzed the samples on silica GF254 thin layer plate using 1%

CMC-Na (carboxyl-methyl-cellulose) as adhesive and chloroform-ethylacetate-isopropanol-

water-ammonia (8∶2∶6∶0.5∶0.12) as developing agent. Hu and Fang (2008) compared the

similarity between chemical components of Cordyceps sinensis and solid fermentation of


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Cordyceps militaris by TLC. They showed that solid fermentation of Cordyceps militaris and

Cordyceps sinensis were basically similar in terms of their TLC spots occurred at the

corresponding place except a slight difference in size.

2.6.2 Spectrometric analysis of cordycepin

Heating of cordycepin with a slightly modified anthrone reagents resulted in the

production of a cherry-red color and this was investigated further as a means of quantitatively

estimating of cordycepin by Kredich and Guarino (1960). The reaction was negative with

adenine. The reagent was prepared fresh daily by dissolving 0.2 g anthrone (Eastman Kodak

Co.) in 100 ml 90 % H2SO4 (150 ml water plus 900 ml conc. H2SO4).

2.6.3 HPLC analysis of cordycepin

A simple HPLC with UV detection (HPLC–UV) method was also proposed for the

detection of cordycepin (Zhang et al., 2005; Song et al., 2008; An et al., 2008; Huang et al.,

2009). Chang et al., (2005) determined the concentrations of adenosine and cordycepin,

3’deoxyadenosine in the hot water extract of a cultivated Antrodia camphorate by high

performance liquid chromatography (HPLC) method. The procedure was carried out on a

reversed-phase C-18 column. Meena et al., (2010) compared the cordycepin content in

natural and artificial cultured mycelium of Cordyceps using reverse phase HPLC. In addition,

though UV detection is widely used for chromatographic analysis, MS detection allows more

definite identification and quantitative determination of compounds which may not be fully

separated. ESI-MS in positive mode is most commonly used in analysis of nucleosides in

Cordyceps (Huang et al., 2003, Xie et al., 2010).


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2.6.4 Capillary electrophoresis

Ling et al., (2002) determined the content of cordycepin by capillary zone

electrophoresis in ultrasonic extracted Cordyceps for the first time. Similarly, Rao et al.,

(2006) investigated a modified capillary electrophoresis (CE) procedure with UV detection at

254 nm for determination of cordycepin. They found 20 mM sodium borate buffer with

28.6% methanol, pH 9.5, separation voltage 20 kV, hydrodynamic injection time 10 sec and

temperature 25ºC were the optimal condition for cordycepin detection. Furthermore, Yang et

al., (2009) developed capillary electrophoresis-mass spectrometry (CE-MS) method for the

simultaneous analysis of 12 nucleosides and nucleobases including cytosine, adenine,

guanine, cytidine, cordycepin, adenosine, hypoxanthine, guanosine, inosine, 2'-deoxyuridine,

uridine and thymidine in natural and cultured Cordyceps using 5-chlorocytosine arabinoside

as an internal standard (IS). They optimized systematically for achieving good CE resolution

and MS response tested compounds and found optimum parameters as follows: 75 % (v/v)

methanol containing 0.3 % formic acid with a flow rate of 3 µl/min as the sheath liquid; the

flow rate and temperature of drying gas were 6 L/min and 350ºC, respectively.

2.7 Extraction strategy for cordycepin

The studies have shown that cordycepin is one of the most important effective

naturally existing constituents (Patel and Ingalhalli, 2013; Fan and Zhu, 2012; Yue et al.,

2013). Because of the importance of its biological properties, a large quantity of pure

materials is urgently needed for further studies. Several extraction methods have been

developed to extract cordycepin from a fermentative solution and from fruiting bodies of C.

militaris, including ultrasound- or microwave-assisted extraction, pressurized extraction,

soxhlet extraction and reflux extraction. Some of them are discussed as follows.


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Kredich & Guarino (1960) gave first report on cordycepin extraction from liquid

culture of Cordyceps militaris. They concentrated the fermented broth in an evaporator at

50ºC followed by cold precipitation and removal of impurities. Further the obtained sample

was passed through a column of 10 cm high × 71 cm dimensions packed with Dowex-I-

chloride of 200-400 mesh size.

Wang et al., (2004) compared supersonic water extraction, supersonic ethanol

extraction, hydrothermal refluxing extraction and ethanol thermal refluxing extraction for

cordycepin and polysaccharide extraction using an orthogonal design experiment. They

showed that hydrothermal refluxing extraction was the best extraction method of cordycepin

and polysaccharide and its optimal technological conditions were optimized.

Rukachaisirikul et al., (2004) isolated and analysed nine compounds from fungal

mycelium as well as from liquid culture of Cordyceps militaris. Out of these, three were 10-

membered macrolides, two were cepharosporolides, 2-carboxymethyl-4-(3′-hydroxybutyl)

one was, furan, one was cordycepin, and one was pyridine-2,6-dicarboxylic acid. Kim et al.,

(2006b) extracted cordycepin from dried fruiting bodies of Cordyceps militaris using solvent-

solvent extraction. They extracted aqueous layer with hexane, butanol and ethyl acetate.

Similarly, Rao et al., (2010) extracted and purified 10 pure compounds from fruiting body of

Cordyceps militaris including cordycepin. Still all these methods, needs to optimization and

was unsuitable for industrial applicability.

Jiansheng (2008) extracted and purified cordycepin from Cordyceps militaris using

ion exchange resin and silica gel column chromatography. They detected cordycepin on

HPLC, LC/MS and CE.

Wei et al., (2009) presented an efficient method of extracting and purifying

cordycepin from the waste of fruiting body production medium. This method included


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continuous counter current extraction followed by column chromatography using 732 cation

exchange resins. They found under optimized conditions cordycepin extraction yield reached

up to 66.0%.

Ni et al., 2009, developed column chromatographic extraction (CCE) method for the

extraction of cordycepin from solid waste medium of Cordyceps militaris. The dried waste

material was imbibed in water for 6 h, and transferred to columns and eluted with water.

Eluates were directly separated with macroporous resin DM130 columns followed by

purification steps with more than 95 % extraction yield.

Song et al., (2007), investigated Optimization of cordycepin extraction from cultured

Cordyceps militaris by HPLC-DAD coupled method. They reported that, cordycepin

extraction yield reached a peak with ethanol concentration 20.21 %, extraction time 101.88

min, and volume ratio of solvent to sample 33.13 ml/g.

Ling et al., (2009) used supercritical fluid extraction (SFE) to extract cordycepin and

adenosine from Cordyceps kyushuensis. They applied orthogonal array design (OAD) test,

L9(3)4 followed by preparative SFE extracts using high-speed counter-current

chromatography (HSCCC). Their results yielded 8.92 mg of cordycepin and 5.94 mg of

adenosine with purities of 98.5 and 99.2 % from 400 mg SFE crude extraction respectively.

Yong et al., (2010) compared 6 kinds of cordycepin extraction method from

Cordyceps militaris medium. Authors got higher extraction rate of cordycepin using

microwave extraction method.

Zhang et al., (2012) did optimization of cordycepin extraction from fruiting body of

Cordyceps militaris YCC-01 using water, ethanol, ultrasonic, and synergistic approaches.


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They found synergetic approach was more efficient with cordycepin content of 9.559 mg/g.

Results suggested, the yield was 66.2 % higher than the control group.

The microwave assisted extraction of cordycepin from cultured mycelium of

Cordyceps militaris was investigated by Chen et al., (2012a). The prepared extract was

purified using a cation exchange resin (CER) of LSD-001.They found optimal desorption

conditions as follows: 0.2M of NH3 combined with 80 % ethanol (v/v), desorption time- 2 h,

temperature- 25°C and pH- 14.

Yu et al., (2013) investigated the optimal conditions for cordycepin extraction from

waste medium of Cordyceps militaris using column chromatography. Initially, they did hot

water leaching of Cordyceps waste at 70℃ for 8 h with dried feed and water ratio of 1g∶ 20

ml followed by separation of cordycepin on macroporus resin XAD16 and polyamide column

chromatography.

2.8 Therapeutic functions of cordycepin related to anti-cancer potential

Cytotoxic nucleoside analogues were the first chemotherapeutic agents for cancer

treatment. Cordycepin is a type of nucleoside analogue that is structurally similar to

adenosine, except that it lacks a 3’ hydroxyl group, which increases its potency, and it is

known to interfere with a number of biochemical and molecular processes, such as purine

biosynthesis (Overgaard, 1964; Rottman and Guarino, 1964), DNA/RNA synthesis (Holbein

et al., 2009) and mTOR (mammalian target of rapamycin) signal transduction (Wong et al.,

2010). In addition to these classical activities, other molecular targets of cordycepin include

apoptosis, cell cycle arrest, anti-metastasis, inhibition of platelet aggregation and

inflammation mediator pathways. However, the cure of diseases such as cancer remains

elusive despite the availability of a variety of chemotherapeutics agents that exhibit

sophisticated mechanisms of action. Therefore, in-depth knowledge of nucleoside analogues,


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such as cordycepin, is essential not only to understand the biology behind cancer but also to

develop novel therapeutic strategies. The descriptions of cordycepin as anticancer agent in

various cell lines have been summarized below.

2.8.1 Induction of apoptosis

Apoptosis, also referred to as programmed cell death, is characterized mainly by a

series of distinct changes in cell morphology, such as blebbing, loss of cell attachment,

cytoplasmic contraction, DNA fragmentation and other biochemical changes including the

activation of caspases through extrinsic and/ or intrinsic mitochondrial pathways. Past

research has demonstrated the role of cordycepin in the induction of apoptosis through

regulating the expression of various proteins. This process involves programmed cell death,

for example, the Bcl-2 family of proteins has both anti-apoptotic and pro-apoptotic members.

Cordycepin has been known to induce apoptosis in human colorectal cancer cell lines

(SW480 and SW620) by enhancing the protein expression levels of JNK, p38 kinase and Bcl-

2 pro-apoptotic molecules (He et al., 2010). Similarly, cordycepin-mediated apoptosis has

been observed in the breast cancer cell line MDA-MB-231 by increasing the mitochondrial

translocation of Bax and the release of cytochrome C, followed by the activation of caspases-

9 and -3 (Choi et al., 2011). The apoptotic effect of cordycepin has also been investigated in

human leukemia cells (U937 and THP-1) through the reactive oxygen species (ROS)-

mediated activation of caspase (-3,-8 and -9) and the cleavage of the poly (ADB-ribose)

polymerase protein (Jeong et al., 2011). Baik et al., (2012) suggested the induction of active

caspase-3 and cleavage of poly (ADB-ribose) polymerase protein (PARP) by cordycepin,

leading to apoptotic cell death in human the neuroblastoma SK-NBE(2)-C (CRL-2268) and

human melanoma SK-Mel-2 (HTB-68) cell lines. Furthermore, Lee et al., (2012)

demonstrated the anti-proliferative effect of cordycepin in human breast cancer cells through


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the induction of DNA damage, PARP cleavage and the phosphorylation of ATM, ATR and

histone γH2AX. A study on human colonic cancer cell HT-29 revealed apoptotic effect of

cordycepin in dose and time dependent through Death receptor (DR3) pathway (Lee et al.,

2013a). Treatment of human prostate carcinoma PC3 cells with cordycepin leads to activation

of apoptotic features such as caspase- 3, 9 and degradation of poly (ADP-ribose) polymerases

via ROS mediated pathway (Lee et al., 2013b). Chen et al., 2014, reported time and dose

dependent apoptotic effect of cordycepin on rat glioma C6 cell lines. They investigated that

cordycepin treatment in cells leads to increase in p53 expression which results apoptotic

effect by cleaving of Caspase 7 and PARP.

Hong et al., 2004, investigated the anti-proliferatory and apoptotic effect of aqueous

extract of Cordyceps militaris in A549 human lung cancer cells. They found that AECM has

dose dependent anti-proliferatory effect and associated with morphological changes such as

membrane shrinking and cell rounding up. Park et al., (2009) investigated the potential of the

water extract of Cordyceps militaris (WECM) to induce apoptosis in human lung carcinoma

A549 cells and its effects on telomerase activity. They indicated that WECM induces the

apoptosis of A549 cells through a signaling cascade of death receptor-mediated extrinsic and

mitochondria-mediated intrinsic caspase pathways. They also concluded that apoptotic events

of WECM were might be mediated with diminished telomerase activity through the inhibition

of hTERT transcriptional activity. Thakur et al., (2011) reported the apoptotic effect of

aqueous extract of Cordyceps sinensis on A549 human lung cancer. They found that 2mg/ml

of aqueous extract doses was able to induce apoptosis after 24h of treatment.

A synergetic approach was carried out to study the apoptotic effect of cisplatin and

cordycepin in OC3 human oral cancer cells (Chen et al., 2013). Data demonstrated that co-

administration of cisplatin (2.5 μg/mL) and cordycepin (10 or 100 μmol/L) resulted in the

enhancement of OC3 cell apoptosis compared to cisplatin or cordycepin alone treatment (24


Page 26

h). Similarly, Cui et al., (2013) proposed a novel therapeutic treatment using cordycepin and

cladribine synergy to decrease adverse treatment effects in various cancer cell lines. Results

revealed that Cordycepin/Cladribine combination experienced similar levels of cancer cell

inhibition as cladribine alone, but exhibited a significant increase in healthy cells left

unharmed.

2.8.2 Cell cycle regulation

In addition to its involvement in apoptotic pathways, cordycepin is also known to

arrest cell cycle at certain checkpoints. The cell cycle is regulated by cyclin-dependent

kinases (CDKs), and cyclins and cyclin-dependent kinase inhibitors (CDKIs) in turn,

positively and negatively control the CDKs. Studies of the cell cycle in a human oral

squamous cancer cell line (OEC-MI) demonstrated that cordycepin decreased the percentage

of G1 phase cells in a dose dependent manner, whereas the percentage of G2M and sub-G1

phase cells was increased (Wu et al., 2007). Another study reported the anti-cancer activity of

cordycepin in human bladder carcinoma cell lines (5637 and T-24) through the activation of

JNK and cell cycle arrest at G2/M transition via the inhibition of the cyclin B/ CDC-complex

and the up-regulation of P21WAF1 expression (Lee et al., 2009). Similar results were

observed in a study in which colon cancer HCT116 cells were treated with cordycepin (Lee et

al., 2010a). In 11z human immortalized epithelial endometriotic cells (Imesch et al., 2011),

cordycepin demonstrated an anti-proliferative effect through the up-regulation of p21 and the

downregulation of cyclin D1, followed by the reduced phosphorylation of p38 MAPK and

retinoblastoma protein (pRb). Jung et al., (2012) demonstrated that cordycepin arrests

vascular smooth muscle cell (VSMC) growth at G1 phase by decreasing cyclin/CDK

complexes through the up-regulation of p27KIPI via the Ras/ERK1 signaling pathway.

Therefore, the induction of apoptosis (Figure 2.6) and cell cycle arrest (Figure 2.7) comprise

an important mechanism in many anti-cancer drugs, including cordycepin.


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Figure 2.6: Schematic representation of the various apoptotic pathways controlled by cordycepin in cancer cells.

Figure 2.7: Cordycepin has been shown to arrest cell cycle, which is regulated by cyclin dependent kinases (CDKs).


Page 28

2.8.3 In vivo anticancer studies

The first in vivo study to demonstrate the anticancer properties of cordycepin was

performed in mice and demonstrated that cordycepin leads to increased survival time in mice

bearing the Ehrlich mouse ascites tumor (Jeggar et al., 1961). Furthermore, a phase 1 clinical

trial was completed (Dec 1997- Mar 2001) using cordycepin and pentostatin to treat patients

with refractory acute lymphocytic or chronic myelogenous leukemia (NCT00003005). Phase

2 clinical trials are ongoing, and the last update was in Jan 2009 (NCT00709215). Despite

these promising results, there a number of limitations are associated with the application of

cordycepin in clinical settings because it requires the co-administration of ADA inhibitors,

such as Cladribine / deoxycoformycin (dcf)/ coformycin (cf). However, research is ongoing

to identify a less toxic, synergistic co-drug for cordycepin that works via mechanisms other

than adenosine deaminase resistance (Janek et al., 2007; Cui et al., 2013).

2.8.4 Anti metastatic activity

The major cause of cancer mortality is the metastasis of the cancer from the native

site to other parts of the body. Metastasis typically includes the detachment of tumor cells

from the primary site, invasion through the extracellular matrix (ECM), followed by

intravasation, survival and extravasation from the blood vessels and finally, invasion of the

ECM of distant target organs. The ECM is made up of type IV collagen, laminin, heparan

sulfate, proteoglycan, nidogen and fibronectin (Nakajima et al., 1987). Metalloproteinases

(MMPs) have been shown to play a critical role in the degradation of the ECM. MMPs are a

family of zinc- and calcium-dependent endopeptidases that can be divided into four

subclasses based on their ECM specificity, including collagenases, gelatinases, stromelysins

and matrilysins. Among the MMP family, MMP-2 and 9 are the two key enzymes known to

degrade type IV collagen, which is an essential component of the basement membrane. The


Page 29

elevated expression of these MMPs has been functionally correlated with elevated tumor

invasion capacity in various cancers, including brain, prostrate, bladder and breast cancer

(Egeblad and Werb, 2002; Hunter et al., 2008). The activity of MMPs can be regulated by

several mechanisms (Woessner, 1991), such as transcription inhibition, pro-enzyme

activation or MMP inhibitors, i.e., tissue inhibitors of metalloproteinases (TIMPs). Cytokines

and TPA have been known to induce MMP-9 synthesis and secretion via the activation of

transcription factors, such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1)

during tumor invasion (Chung et al., 2004; Hong et al., 2005; Woo et al., 2005). Cordycepin

has been known to inhibit MMP expressions levels (Figure 2.8) through the deactivation of

AP-1 via the MAPK signaling pathway (Noh et al., 2010). A study demonstrated that

cordycepin blocks TNF-α-induced MMP-9 expression by decreasing the DNA-binding

activity of transcription factors, such as NF-κB and AP-1 (Lee et al., 2010b). Yet another

study concluded that the combination of cisplatin and cordycepin might act as a potent

inhibitor of peritoneal tumor dissemination and VEGF expression (Cai et al., 2011).

Furthermore, it has been suggested that cordycepin inhibits the migration and invasion of

LNCaP (human prostate carcinoma) cells by down regulating the activity of TJs (tight

junctions) and MMPs, possibly in association with the suppression of Akt activation (Jeong et

al., 2012).

2.8.5 Anti-platelets aggregation

Platelets are known to regulate tumor angiogenesis, growth and metastasis. Earlier

evidence suggested that cancer cells have the ability to activate platelets, which facilitates

their survival in the blood circulation during hematogenous metastasis by preventing tumor

cell lysis by natural killer (NK) cells and cytotoxic lymphocytes (Tsuruo and Fujita, 2008;

Goubran and Bournouf, 2012). A variety of molecular mechanisms (Figure 2.9), such as

GPIIb/IIIa, GP Ib-IX-V, P2Y receptors, and PAR receptors, have been proposed to describe


Page 30

tumor cell-induced platelet aggregation (TCIPA) (Jackson, 2007; Bambace and Holmes,

2011). Cho et al., (2006; 2007a) reported that cordycepin inhibits collagen-induced platelet

aggregation by lowering [Ca2+] ions and thromboxane A2 [TXA2] and through the up-

regulation of intracellular levels of cAMP and cGMP. Furthermore, the study demonstrated

that 500-μM cordycepin blocked the up-regulation of [Ca2+] ions and TXA2 by 74% and

46%, respectively. Another study demonstrated that cordycepin exerted a potent inhibitory

effect on thapsigargin and U46619-elevated intracellular Ca2+, which is known to induce a

wide variety of ligands that further facilitate platelet aggregation (Cho et al., 2007b). In

addition to [Ca2+] ions and TXA2, cordycepin has been known to reduce the hematogenic

metastasis of cancer cells via the inhibition of adenosine diphosphate (ADP), which is a

potent inducer of platelet aggregation, during TCIPA (Yoshikawa et al., 2009).

2.8.6 Anti-inflammatory activity

It is understood that inflammatory activities are related to cancer progression. Cancer

cells are known to express variety of cytokines, chemokines and their receptors, which play

an important role in mediating inflammatory responses (Arias et al., 2007; Nelson et al.,

2004; Farrow et al., 2004). Many anti-cancer compounds can also be used to treat

inflammatory diseases (Rayburn et al., 2009). Previous studies have reported that the

expression of cytokines, such as IL-6, IL-8, G-CSF, IFN-γ, and MIP-1β, is up-regulated in

carcinoma (Chavey et al., 2007; Wang et al., 2009). Therefore, to develop a potent strategy to

tackle and prevent cancer, it is essential to inhibit inflammatory mediators. Kim et al.,

(2006b) evaluated the anti-inflammatory effect of cordycepin in LPS-stimulated macrophages

by inhibiting nitric oxide (NO) production. The study suggested that cordycepin

downregulates iNOS, COX-2 and TNF-α gene expression via the suppression of NF-κB

activation and Akt and p38 phosphorylation. Similar studies have shown that cordycepin

significantly attenuated the release of inflammatory mediators, such as NO, PGE2, TNF-α,


Page 31

and IL-1β in LPS-induced microglia cells by inactivating NF-κB through the inhibition of

IκB-α degradation (Jeong et al., 2010). In lipo-polysaccharide-stimulated macrophages (Shin

et al., 2009), cordycepin suppressed the gene expression of M1 cytokines (IL-1β and TNF-α)

and chemokines (CX3CR1 and RANTES), whereas the expression of M2 cytokines (IL-10,

IL-1ra, TGF-β) was increased. Furthermore, cordycepin has been shown to increase the

expression of the interleukin-10 protein in human peripheral blood mononuclear cells, which

is known to inhibit the secretion of proinflammatory and inflammatory cytokines (Moore et

al., 1993; Armstrong et al., 1996; Cunha et al., 1992; Mertz et al., 1994; Dokka et al., 2001;

Zhou et al., 2002). Rao et al., (2010) demonstrated that cordycepin is a potent inhibitor of

free radical NO and cytokine (TNF-and IL-12) production. The study suggested that

cordycepin might be useful for the prevention of cancer by acting as an anti-inflammatory

agent. Kim and colleagues demonstrated in a mouse model that cordycepin blocks acute lung

inflammatory (ALI) responses and the expression of related genes, including adhesion

molecules (ICAM and VCAM), cytokines / chemokines and the chemokine receptor CXCR2.

The study suggested that cordycepin blocks LPS-induced VCAM expression in A549 cells

through a remarkable reduction in p65-NF-B, which is directly linked to PARP inhibition

(Kim et al., 2011).

2.8.7 Anti-angiogenic potential of Cordyceps

Angiogenesis is a complex and tightly controlled physiological process by which new

blood vessels are originated from pre-existing capillaries. Angiogenesis plays an important

role in physiological and pathological processes such as wound healing, placentation,

embryogenesis, diabetic retinopathy, inflammatory disorders and tumor growth (Folkman

1995; Risau 1997). Tumor cells can induce angiogenesis through the activation of endothelial

cells followed by pro-angiogenic factors such as vascular endothelial growth factor (VEGF),


Page 32

fibroblast growth factor (FGF) and epidermal growth factor (EGF) (Jain, 2002; Kaya et al.,

2000; Weidner et al., 1991). These factors are highly expressed and associated with growth of

various types of human tumors (Ferrara et al., 2003; Breier and Risau, 1996). Therefore,

compounds with anti-angiogenic properties have been identified as an attractive strategy for

the treatment and prevention of cancer. Researchers have been trying to screen novel herbal

preparations with anti angiogenic potential.

Among various animal model systems designed to study the mechanisms underlying

angiogenesis, chick embryo models have been useful tools in analyzing the angiogenic

potential of purified factors and intact cells. The chorioallantoic membrane (CAM), a

specialized, highly vascularized tissue of the avian embryo, serves as an ideal indicator of the

anti- or pro-angiogenic properties of test compounds. chorio-allantoic membrane is formed

by the chorion, the allantois and the inner shell membrane which takes part in the metabolic

processes such as respiration, digestion, excretion.

Figure 2.8: The inhibitory effect of cordycepin on tumor metastasis through the MAPK signaling pathway.


Page 33

Figure 2.9: Schematic representation of cordycepin as an anti-platelet aggregation agent.

Figure 2.10: Schematic representation of structure of embryonated chicken egg.


Page 34

Yoo et al., (2004) studied the antiangiogenic effect of Cordyceps militaris extract

(CME) on human umblical vein endothelial cells (HUVEC). They investigated CME

inhibited growth of HUVECs at 100 and 200 mg/L and also reduce the bFGF gene expression

up to 41.3%.

Won and park (2005) elucidated the pharmacological activities of Cordyceps

militaris. They prepared ethanolic extracts of cultured mycelia (CME) and fruiting bodies

(FBE) and studied its dose-dependent inhibitory effect by chorioallantoic membrane (CAM)

assay.

Chia et al., (2010) investigated the inhibitory effect of Chinese herbal cocktail Tien-

Hsien liquid (THL) against metastasis, angiogenesis, and tumor growth. THL was an aqueous

preparation of herbal mixture of extracts from 14 Chinese medicinal herbs including

Cordyceps. They found, THL inhibited the growth of human MDA-MB-231 breast cancer

xenografts in NOD-SCID mice and reported that suppression of tumor growth was associated

with decreased microvessel formation and increased apoptosis caused by THL.

Lu et al., (2014) studied the anti-proliferatory, anti metastatic and antiangiogenic

effect of cordycepin in human umbilical vein endothelial cell line (EA-hy926). They

investigated that cordycepin inhibited EA-hy926 cell migration (percentage of wound healing

area, 2,000 µg/ml: 3.45±0.29% vs. 0 µg/ml: 85.48±0.84%), as well as tube formation (total

length of tubular structure, 1,000 µg/ml: 107±39 µm vs. 0 µg/ml: 936±56 µm).

Ruma et al., (2014) demonstrated that Cordyceps militaris extract remarkably

suppressed tumor growth via induction of apoptotic cell death in culture which was linked

angiogenesis in melanoma cells. They treated melanoma cells xenografted mouse model with

Cordyceps extract which resulted in a dramatic antitumor effect due to down regulation of

VEGF expression.

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