Chapter: 2 Review of Litrature -...
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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’deoxy- adenosine 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
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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).
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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
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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
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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,
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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),
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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.
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Figure 2.9: Schematic representation of cordycepin as an anti-platelet aggregation agent.
Figure 2.10: Schematic representation of structure of embryonated chicken egg.
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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.