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Molecular characterization, microscopic characteristics, and phylogenetic analysis of Ophiocordyceps sinensis from Sikkim, India

Authors:
  • Independent Researcher

Abstract

The zombie fungi are a group of entomo-parasitic fungi comprising of 400 diverse species having tremendous pharmaceutical virtues. They have their use in traditional practice among Himalayan highlanders and inhabitants across the globe living in high altitude areas. Ophiocordyceps sinensis is one such representative of the entomo-parasitic group. The current study aimed to identify Ophiocordyceps sinensis from Sikkim, India, following classical, molecular taxonomic approaches, and culture. The classical approach involved a microscopic study of asci, stroma, and mycelia and the macroscopic characters of the stroma and larva. The molecular approach involved the amplification of internal transcribed spacer (ITS) region from the stroma, cytochrome oxidase subunit-I (COI), and cytochrome b (Cytb) from host larva for phylogenetic studies. The pure culture was established on potato dextrose agar (PDA). The sequences were edited with Bioedit version 7.2.5 and subjected to multiple alignments using fast fourier transform (MAFFT) database. Model testing was performed using MegaX version 10.2.5, and the best model was utilized to construct the maximum likelihood tree. To confirm the results of the maximum likelihood tree, a Bayesian tree was also constructed using MrBayes 3.2.7. Subsequently, the study confirmed that the collected specimen is O. sinensis. The main bioactive compounds of O. sinensis are cordycepin and adenosine which has been explored for different therapeutic applications including treatment of cancer, diabetes, anemia, inflammation etc. Thus, such study provides the platform for their exploration for extensive pharmaceutical and nutraceutical future studies.
268
Content list available at http://epubs.icar.org.in, www.kiran.nic.in; ISSN: 0970-6429
Indian Journal of Hill Farming
Special Issue 2021, Volume-34, Page 268-275
Molecular characterization, microscopic characteristics, and phylogenetic analysis of
Ophiocordyceps sinensis from Sikkim, India
Vashkar Biswa1 Raju Ali1 Durga Kr. Pradhan2 Nitisha Boro3 Tikendrajit Baro1 Debajani Das1
Robin Subba4 Puja Das4 Sandeep Das1
1Department of Biotechnology, Bodoland University, Assam
2Sikkim State Forest Herbarium, Deorali, Gangtok, Sikkim
3Department of Molecular Biology & Biotechnology, Tezpur University, Assam
4College of Agricultural Engineering and Post-Harvest Technology, Ranipool, Sikkim
ARTICLE INFO ABSTRACT
Article history:
Received: 26th October 2021
Revision Received: 03rd Decembe 2021
Accepted: 06th December 2021
-----------------------------------------------
Key words: Medicinal Mushroom, Fungi,
Taxonomy, Phylogeny, Ophiocordyceps
sinensis.
----------------------------------------------
The zombie fungi are a group of entomo
-
parasitic fungi comprising of 400
diverse species having tremendous pharmaceutical virtues. They have their use in
traditional practice among Himalayan highlanders and inhabitants across the globe living
in high altitude areas. Ophiocordyceps sinensis is one such representative of the entomo-
parasitic group. The current study aimed to identify Ophiocordyceps sinensis from Sikkim,
India, following classical, molecular taxonomic approaches, and culture. The classical
approach involved a microscopic study of asci, stroma, and mycelia and the macroscopic
characters of the stroma and larva. The molecular approach involved the amplification of
internal transcribed spacer (ITS) region from the stroma, cytochrome oxidase subunit-I
(COI), and cytochrome b (Cytb) from host larva for phylogenetic studies. The pure culture
was established on potato dextrose agar (PDA). The sequences were edited with Bioedit
version 7.2.5 and subjected to multiple alignments using fast fourier transform (MAFFT)
database. Model testing was performed using MegaX version 10.2.5, and the best model
was utilized to construct the maximum likelihood tree. To confirm the results of the
maximum likelihood tree, a Bayesian tree was also constructed using MrBayes 3.2.7.
Subsequently, the study confirmed that the collected specimen is O. sinensis. The main
bioactive compounds of O. sinensis are cordycepin and adenosine which has been explored
for different therapeutic applications including treatment of cancer, diabetes, anemia,
inflammation etc. Thus, such study provides the platform for their exploration for extensive
pharmaceutical and nutraceutical future studies.
1.
Introduction
Ophiordyceps sinensis (Berk.) is an entomo-
pathogenic fungus belonging to the genus Ascomycota [Zhou
et al. (2014)]. It consists of >400 different species worldwide,
which are parasitic, mainly on insects and larvae. Typically,
it exists in two stages: an asexual stage (mitosporic fungi) and
a sexual stage. The mitosporic fungi are parasitic on dead
caterpillars of the moth Hepialus spp. The spores of O.
sinensis germinate inside the caterpillars, colonizing with
hyphae and producing a stalked ascomata (sexual stage)
[Zeng, W., Yi, D.H. and Huang, T.F. (1998), Pu, Z.L. and Li,
Z.Z. (1996)]. Cordyceps are mainly found in China, Nepal,
and India at 3500 m above sea level. The fungus is a coveted
medicinal utility in traditional Tibetan and Chinese
medicine, which is commonly known in the West as
“Himalayan Viagra.” In India, the collection and trade of
Cordyceps by the Bhotiya community have been reported
from Garhwal, Uttarakhand [Caplins, L. B. (2016)]. It has
also been reported
______________
269
*Corresponding author: sandeep_dna2003@yahoo.co.in
from
hilly
region
of
district
Pithoragarh
(Uttarakhand)
at
an altitude of 3200 m from the snow meadows of Brahamkot,
Ultapara, Ghawardhappa, Chhipalakot, Najari in Dharchula,
Chetri Bugyal, and ChiplaKedar (4000 m), as well as from
Nagin Dhura, Ralam Bugyal at the base of Panchachuli
Hills, Laspa, Tolatop, Darti, Mapa top, Burfu top, and Milam
top in Johar Hills of Kumaun [Arora, R. K., (2014)]. Sikkim
is the organic state of India, comprising a vast diversity of
flora and fauna. The distribution of O. sinensis has been
reported from Lachen (ca.2750 masl;
27.7167°N;88.5577°E), Lachung (ca.2700 masl;
276891°N;88.7430°E) in North Sikkim, and Gnathang
valley (27.2986° N, 88.8173° E) in East Sikkim [Risley, H.
H. (1894)]. Lachenpas and Lachungpas are primary dwellers
of North Sikkim with a population of 3200 (Lachen) and
2495 (Lachung), according to the panchayat register of 2017
[Pradhan, B. K. et al. (2020)]. The Himalayan rural
population relies on herbal medicinal plants and fungi for 3–
58 percent of total yearly household income and 78 percent
of cash revenue [Pradhan, B. K. et al. (2020)]. Cordyceps
spp. has long been used to promote longevity, relieve
exhaustion, and treat numerous diseases in Chinese
traditional medicines [Russell, R.; Paterson, M. (2008)].
Recent studies have demonstrated that various species in this
genus possess multiple pharmacological properties,
including anti-tumor, anti-microbial, anti-inflammatory, and
immunomodulatory effects [Holliday, J.; Cleaver, M.
(2008), Agrawal, D. G., & Sandhu, S. S. (2020)].
The present study defines the taxonomic virtues of
the collected sample, identified as O. sinensis by both
classical and molecular taxonomy and characterized by
mycelial culture. Also, the host larva of the Cordyceps spp.
was characterized. Thus, this study could be significant for
distinguishing O. Sinensis from-related Cordyceps spp.
These features could be a measure for their systematic use as
nutraceuticals for treatments and beneficial effects. The
study can also be a tool to identify counterfeits to minimize
or regulate the trade of O. sinensis. Therefore, the current
study claims to be the first elaborate taxonomic study on O.
sinensis from Sikkim.
2. Materials and Methods
Samples were collected from Yumesamdong
(27°51'05.4"N 88°41'04.8"E), North Sikkim, in July 2021
and transported on an icebox to Bodoland University.
Photographs were taken before and after cleaning the
samples. DNeasy plant genomic, PCR purification, and gel
extraction kits were purchased from Qiagen, Germany.
Wizard SV Genomic purification system (Promega,USA)
was obtained from the USA. Taq Polymerase was procured
from Thermo Fischer, USA, and 100-bp ladder was
purchased from TaKaRa, Japan.
Isolation and Culture
The samples were cleaned with a sterile art brush and detached
from the larva, followed by washing in sterile distilled water.
0.1% mercuric chloride was used for surface sterilization.
Subsequently, the samples were cut vertically using a sterilized
scalpel. The tissue from the center of the fruiting body was
inoculated on potato dextrose agar (PDA) with 0.05% MgCl2
[Barseghyan, G. S. et al. (2011)]. The culture plates were
incubated at 18 °C in a Bio-oxygen Demand (BOD) incubator.
Mycelial growth was observed after 4-5 days. In order to obtain
pure culture, small chunks of mycelia were sub-cultured on PDA
plates containing MgCl2 and 500 mg/L ampicillin. The mycelia
were then transferred to liquid media for DNA extraction to
confirm the culture.
Microscopic Characteristics
The microscopic characteristics, such as the total diameter of the
transverse section of stroma and the length and breadth of the
asci and mycelia, were studied [Liu, H et al. (2011)].
DNA Barcoding Analysis
The genomic DNA of the sample was extracted from stroma
(fruiting body), mycelia, and host (larva), separately. The DNA
isolation from stroma and mycelia was carried out using the
DNeasy plant genomic kit (Qiagen), and the isolation from the
host was carried out with the Wizard SV Genomic purification
kit (Promega) with some minor modifications. The DNA
samples were quantified on Qubit4 fluorometer (Thermo Fisher
Scientific). The genomic DNA was visualized on 0.8% agarose
gel electrophoresis. The primers used for rDNA internal
transcribed region (ITS) amplification were as follows: forward
primer ITS-5 (5’-GGAAGTAAAGTCGTAACAAGG-3’) and
reverse primer ITS-4 (5’-TCCTCCGCTTATTGATATGC-3’)
[Wu, D. et al. (2016)]. The amplification of the cytochrome
region from the genomic DNA of host larva was carried using
forward primer Cytb-1 (5’-
TATGTACTACCATGAGGACAAATATC-3’) and reverse
primer Cytb-2 (5’-ATTACACCTCCTAATTTATTAGGAAT-
3’) [Quan, X., & Zhou, S. L. (2011) and Wu, D. et al. (2016)].
The amplification of COI gene used forward primer COI-F (5’-
GGTCAACAAATCATAAAGATATTG-3’) and reverse primer
COI-R (5’-TAAACTTCAGGGTGACCAAAAAAT3’). The
polymerase chain reaction (PCR) for the ITS region of the
genomic DNA from stroma (fruiting body) was as follows: an
initial denaturation step of 95 °C for 5 min, followed by 35
cycles of 95 °C for 30 s, 51 °C for 2 min, and 72°C for 1 min,
and a final extension step of 72 °C for 10 min. The PCR
conditions for the amplification of Cytb gene of host larva
consisted of an initial denaturation step of 95 °C for 5 min,
followed by 35 cycles of 95 °C for 30 s, 50 °C for 2 min, and 72
°C for 1 min, and a final extension step of 72 °C for 10 min.
270
The PCR amplification of
COI
gene of host larva consisted of an
initial denaturation step of 95 °C for 5 min, followed by 35 cycles
of 95 °C for 30 s, 48 °C for 2 min, and 72°C for 1 min, and a
final extension step of 72 °C for 10 min [Wu, D et al. (2016)].
The amplification products were purified using the Qiaquick
PCR purification kit (Qiagen, Germany) and confirmed on 1.5%
agarose gel electrophoresis with a 100-bp ladder. The fragments
were excised and purified using QIAEX gel extraction kit
(Qiagen), and the concentration was measured on a Qubit 4
Fluorometer (Invitrogen, USA). Subsequently, the DNA was
used for sequencing in a AB13730XL, Applied Biosystem
Sequencer following sanger sequencing method. The ITS, Cytb,
and COI sequences were subjected to homology search by
BLAST tool in the NCBI nucleotide blast portal
(https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE
=BlastSearch). Finally, the sequences were submitted, and the
accession number was obtained from the NCBI database
Phylogenetic analysis
Sequences were edited using BioEdit version 7.2.5. Phylogenetic
analysis was performed using MegaXversion 10.2.5, and
reference sequences were downloaded from NCBI. The
evolutionary history was obtained using the maximum
likelihood method and Kimura 2-parameter model [Kimura, M.
(1980)]. The tree with the highest log likelihood (-3549.73) is
shown in figure-2. The percentage of trees in which the
associated taxa are clustered together is shown next to the
branches. The initial tree(s) for the heuristic search were
obtained by applying the neighbor-joining (NJ) method to a
matrix of pairwise distances estimated using the maximum
composite likelihood (MCL) approach. A discrete gamma
distribution was used to model the evolutionary rate differences
among the sites (5 categories (+G, parameter = 1.1526)). This
analysis involved 23 nucleotide sequences and a total of 880
positions in the final dataset. The evolutionary analysis was
conducted in MEGA X10.2.5 [Kumar S et al, 2018].
3. Results and Discussion
The collected samples were submitted to Sikkim State
Forest Herbarium (SSFH), Deorali, Sikkim vide (SSFH
SK005007) and cultured in Petri plates containing PDA media
supplemented with 0.5 g/L and 50 mg thiamine hydrochloride.
The mycelia started to grow after 5–7 days, followed by a
subculture to obtain a pure culture of the sample. Then, the
collected samples were cleaned, and DNA was extracted
(DNeasy Plant Genomic kit, Qiagen). The DNA from stroma,
pure-cultured mycelia, and host larva was isolated independently
and amplified on a Thermal Cycler (2720, Applied Biosystems).
The PCR product of ITS, COI,
and
Cyt
b
amplification
was
541
bp
,
650
bp,
and
419
bp,
respectively. These were then sequenced (AB13730XL, Applied
Biosystem Sequencer) following sanger sequencing method.
The sequencing data of stroma, mycelia, and larva were blasted
in the NCBI database
(https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGETYPE=BlastSea
rch). The ITS amplicon of sample Cordyceps Bodoland
University Sikkim-3 (CBUS3 fruiting body and culture) covered
a query of 97% and 99%, which is identical with O. sinensis. The
sequencing data of COI gene had query cover of 100% and 93%
identity to Thitarodes spp, while Cytb gene had a 92% query
coverage and 96% identity to Thitarodes spp. The presence of
grouped, cylindrical, and embedded asci confirmed O. sinensis.
The phylogenetic analysis of the ITS region of stroma and COI
gene of the host was performed separately. A total of 22
sequences of Cordyceps spp. were retrieved from NCBI for the
phylogenetic analysis of the ITS region of the stroma, and 19
sequences were retrieved for the phylogenetic analysis of the
COI region of the host. The sequences were subject to multiple
alignment using fast fourier transform (MAFFT) online tool.
The alignment was optimized visually, and ambiguous regions
were excluded from subsequent phylogenetic analyses. The best
model was calculated by the model testing in Mega (version
10.2.5), and the K2+G model was chosen [Kimura, M. (1980)].
Pairwise distance matrices were generated using Kimura models
of nucleotide substitutions [Kimura, M. (1980); Kumar S. et al.
(2018); Swofford, D. L. (1998)], and the phylogenetic analysis
was performed in the Mega version 10.2.5. An NJ tree [Jukes,
T.H. and Cantor, C.R. (1969)] with bootstrapping was
constructed with distance measured by the Jukes Cantor distance
[Jukes, T.H. and Cantor, C.R. (1969)] model and Kimura’s two-
parameter distance. To assess the confidence of phylogenetic
relationships, the bootstrap test [Felsenstein, J. (1985)] was
conducted with 1000 resampling for NJ analysis. The
phylogenetic relationships of O. sinensis were analyzed using
the Bayesian method [Ronquist, F. et al. (2012)]. Volvariella
volvacea is used as an outgroup for phylogenetic analysis of the
ITS region and Anthera assama as an outgroup for COI region
of host larva. The comparative phylogenetics results using ITS
sequence indicated that the presence of the sample collected
from Sikkim clubbed with the sequences of O. Sinensis
sequences (China) retrieved from NCBI. The presence in the
same clade diverging from the other related species confirmed
that the sample belong to O. Sinensis, which is an entomo-
parasitic fungus infecting larva of various genera (Thitarodes
spp., Endoclita spp., Napialus spp.). The phylogenetic analysis
was performed to confirm the larva. The sequence of COI of
larva from the sample was compared to that of the common larva
of O. Sinensis. The sequences were retrieved from NCBI, and
phylogenetic analysis was carried out. Subsequently, the sample
clubbed and grouped
271
with the sequences from
Ahamus
spp.,
Hepialus
spp., and
Thitarodes spp., which are synonyms, confirmed that the larva
belongs to Thitarodes spp. The tree from Bayesian inference
showed an identical tree topology. The recent studies have
shown cordycepin from cordyceps spp. are capable of reducing
pain, inflammation and joint pathology in rodent model
[Ashraf, S, et al. (2019)] also cordycepin have been known to
be involve in activation of AMPK and induction of apoptosis
in
prostate
carcinoma
cells
[
Hawley,
S.
A
,
et
al.
(2020);
Lee,
H.
H. et al. (2013); Zhang, Y.et al. (2018); Li, S. Z. et al. (2019)].
Although O. sinensis has been reported, a detailed microscopic
and phylogenetics analysis of O. sinensis from Sikkim is reported
in this study. Thus, this study will be helpful in properly
characterizing and differentiating different species of cordyceps
which have diverse therapeutic potential.
Table 1. Details of macro-morphological characters of O. sinensis
Species
Microscopic
characteristics
of
stroma
Morphological
characteristics
of
larva
Morphological
and
microscopic
characteristics of
mycelia
O.
sinensis
(CBUS3
-
Stroma),
Thitarodes sp. (CBUS3-
larva),
Stroma
sparingly
cylindrical,
dark
pinkish
(fresh)
and brown when dried, 38–40 mm in length, and
about 2.5–3 mm in diameter. The total diameter of
the transverse section is 298 µm, length and
breadth of the asci were 84 µm and 33 µm,
Larva
body
resembling
a
silkworm, 29–32 mm in length
and 4–5 mm in breadth.
Yellowish in color with 8 pairs of
the leg.
Light
-
creamish.
Mycelia branched
with 3-µm diameter.
Fig 1.
(A)
O. sinensis
. (B)
O. sinensis
cleaned. (C)
O. Sinensis
larva with 8 pairs of the legs on the abdomen and 4 pairs at the center. (D)
Transverse section of the stroma. (E) Perithecia at 20x (F) Perithecia at 40x (G) Mycelial culture of O. sinensis
grown on PDA media. (H)
Mycelia of O. sinensis. (I) Central portion of the stroma.
272
O.
sinensis
(CBUS3
-
mycelia)
respectively.
Perithecia
oval
to
elliptical,
elongated, and grouped at the fertile portion of
stroma. Mycelia embedded with the asci. The
outer layer of the stroma stained dark with Congo
red.
Gene
Bank
Accession
Number
MW990119
MZ956161
OK041477
4.
Conclusions
The collection and trade of O. sinensis in Lachung and
Lachen valley of North Sikkim is a common practice for
livelihood management [Pradhan, B. K. et al. (2020)].
Traditional use of O. sinensis as aphrodisiac, treating fatigue,
anti-inflammation, immune booster and treatment of
respiratory ailments is a practice of choice by the traditional
healers in Himalayan region [Panda, A. K., & Swain, K. C.
(2011)]. A detailed scientific characterization and
authentication of the collected sample of O.sinensis shall
facilitate differentiation between other counterfeit species
and establish the collected Cordyceps spp. from Sikkim to be
Ophiocordyceps sinensis.
5.
Acknowledgement
We are thankful to DBT Sponsored Advanced Level
Institutional Biotech Hub & Technology Incubation Centre,
Bodoland University for providing us the research facility.
We are grateful to Sikkim Biodiversity Board & Sikkim State
Forest Herbarium, Deorali, Sikkim for granting us the
research permission. We sincerely thank Bidur Biswa, Sajesh
Chhetri, Sonam Wangyal Bhutia, Aruna Rai, Dhanmaya
Poudyal and Sewantika Sharma for their assistance.
6. Conflict of Interest
The authors declare no conflict of interest.
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Ophiocordyceps sinensis, commonly known as the caterpillar fungus or yartsa gunbu, is indeed a fascinating organism with a complex life cycle and significant medicinal value. This fungus infects caterpillars of certain moth species, primarily Hepialus armoricanus, with the formation of the characteristic fungus-caterpillar complex, which is harvested for medicinal purposes. O. sinensis is primarily concentrated in regions of higher elevations in the Tibetan Plateau, along with parts of China, India, Nepal, and Bhutan. However, due to factors like habitat loss, overexploitation, and climate change, its wild population is under severe threat, leading to its classification as vulnerable on the IUCN Red List. The species’ habitat preferences, particularly its association with specific altitudes and climatic conditions, are crucial for understanding its distribution patterns. Its distribution and population dynamics are intricately linked to environmental factors such as temperature, precipitation, and topography, making it an interesting subject for ecological modeling studies. Ecological niche modeling (ENM), habitat suitability modeling (HSM), or species distribution modeling (SDM) techniques have played a pivotal role in predicting both the current and future distribution of O. sinensis under diverse scenarios of climate change. These models have helped to identify suitable habitats and potential areas for conservation efforts. Projections indicate that the distribution of O. sinensis may be influenced by both favorable and adverse effects of climate change, with potential expansions in some regions but contractions in others. Moreover, the presence of bioactive compounds like adenosine and cordycepin in O. sinensis underscores the significance of environmental covariates in shaping the quality and effectiveness of this medicinal fungus. The alterations in patterns of temperature and precipitation induced by climate change can profoundly affect the production of these bioactive compounds, consequently influencing the medicinal properties of O. sinensis. Despite significant progress in modeling O. sinensis distribution, there are still challenges related to data limitations and model selection. Incorporating factors like species interactions and dispersal capabilities could improve the accuracy of predictions and inform conservation strategies. The review underscores the importance of interdisciplinary research efforts involving ecology, climatology, and traditional knowledge to understand and conserve species like O. sinensis during ongoing environmental changes. It highlights the need for comprehensive conservation strategies that consider both ecological and socio-economic factors to ensure the sustainability of this valuable bioresource.
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Cordycepin (3'-deoxyadenosine) is a major bioactive agent in Cordyceps militaris, a fungus used in traditional Chinese medicine. It has been proposed to have many beneficial metabolic effects by activating AMP-activated protein kinase (AMPK), but the mechanism of activation remained uncertain. We report that cordycepin enters cells via adenosine transporters and is converted by cellular metabolism into mono-, di-, and triphosphates, which at high cordycepin concentrations can almost replace cellular adenine nucleotides. AMPK activation by cordycepin in intact cells correlates with the content of cordycepin monophosphate and not other cordycepin or adenine nucleotides. Genetic knockout of AMPK sensitizes cells to the cytotoxic effects of cordycepin. In cell-free assays, cordycepin monophosphate mimics all three effects of AMP on AMPK, while activation in cells is blocked by a γ-subunit mutation that prevents activation by AMP. Thus, cordycepin is a pro-drug that activates AMPK by being converted by cellular metabolism into the AMP analog cordycepin monophosphate.
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Cordycepin, or 3'-deoxyadenosine, is a derivative of the nucleoside adenosine. Initially extracted from the fungus Cordyceps militaris, cordycepin exhibits antitumor activity against certain cancer cell lines; however, the mechanism by which cordycepin counteracts colorectal cancer (CRC) remains poorly understood. The aim of the present study was to explore the underlying mechanisms of cordycepin against human CRC. To investigate the molecular mechanisms of cordycepin against colon cancer and in driving apoptosis, p53 and Bcl-2-like protein 4-null (Bax-/-) colon cancer HCT116 cell lines were used. Cell viability and growth were repressed in a dose-dependent manner in cells treated with cordycepin. Treatment with cordycepin resulted in increased apoptosis in HCT116 cells; however, flow cytometic analysis demonstrated that apoptosis was notably decreased in the Bax-/- HCT116 cell lines, but not in the p53-/- HCT116 cell lines. Furthermore, cordycepin exposure resulted in the translocation of Bax from the cytosol to the mitochondria and the subsequent release of cytochrome c from the mitochondria. Results from the present study demonstrated that cordycepin inhibited colon cancer cell growth in vitro and this appears to be through the endogenous Bax-dependent mitochondrial apoptosis pathway, which suggested a molecular mechanism for cordycepin against human colon cancer. These results indicated the possibility of cordycepin as a novel drug for the prevention of colon cancer.
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Background Cordycepin, the main active ingredient of a traditional Chinese herbal remedy – extracted from Cordyceps sinensis – has been demonstrated as a very effective anti-inflammatory and antitumor drug. The present study investigated its antitumor effect on pancreatic cancer, a highly aggressive cancer with extremely poor prognosis due to malignancy, and clarified its underlying mechanism both in vitro and in vivo. Methods The antitumor viability of cordycepin on human pancreatic cancer MIAPaCa-2 and Capan-1 cells was determined by colony formation assays. Annexin V/PI double staining and flow cytometry assay were used to investigate whether cordycepin induced apoptosis and cell cycle arrest. The mitochondrial membrane potential (ΔΨm) was analyzed by Rhodamine 123 staining, and expression of related proteins evaluated by Western blot and immunohistochemistry, both on pancreatic cancer cells and tumor xenografts to reveal the potential mechanism for the effect of cordycepin. Furthermore, the in vivo efficacy was examined on nude mice bearing MIAPaCa-2 cell tumors treated by intraperitoneal injection of cordycepin (0, 15, and 50 mg/kg/d) for 28 days. Results Cordycepin inhibited cell viability, proliferation and colony formation ability and induced cell cycle arrest and early apoptosis of human pancreatic cancer cells (MIAPaCa-2 and Capan-1) in a dose- and time-dependent manner. The same effect was also observed in vivo. Decrease of ΔΨm and upregulation of Bax, cleaved caspase-3, cleaved caspase-9, and cleaved PARP as well as downregulation of Bcl-2 both in vitro and in vivo indicated that the mitochondria-mediated intrinsic pathway was involved in cordycepin’s antitumor effect. Conclusion Our data showed that cordycepin inhibited the activity of pancreatic cancer both in vitro and in vivo by regulating apoptosis-related protein expression through the mitochondrial pathway and suggest that cordycepin may be a promising therapeutic option for pancreatic cancer.
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Natural Cordyceps collected in Bhutan has been widely used as natural Cordyceps sinensis, an official species of Cordyceps used as Chinese medicines, around the world in recent years. However, whether Cordyceps from Bhutan could be really used as natural C. sinensis remains unknown. Therefore, DNA sequence, bioactive components including nucleosides and polysaccharides in twelve batches of Cordyceps from Bhutan were firstly investigated, and compared with natural C. sinensis. Results showed that the fungus of Cordyceps from Bhutan was C. sinensis and the host insect belonged to Hepialidae sp. In addition, nucleosides and their bases such as guanine, guanosine, hypoxanthine, uridine, inosine, thymidine, adenine, and adenosine, as well as compositional monosaccharides, partial acid or enzymatic hydrolysates, molecular weights and contents of polysaccharides in Cordyceps from Bhutan were all similar to those of natural C. sinensis. All data suggest that Cordyceps from Bhutan is a rational alternative of natural C. sinensis, which is beneficial for the improvement of their performance in health and medicinal food areas.
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Macroscopic and microscopic studies were applied to distinguish Cordyceps sinensis (Berk.) Sacc. and its 5 common counterfeits. Transverse sections of stroma and larvae and surface sections of stroma of C. sinensis, Cordyceps gunnii, Cordyceps barnesii, Cordyceps gracilis, Cordyceps liangshanensis and Cordyceps militaris were examined and their morphological and microscopic features photographed. The main morphological and microscopic features of the 6 species of Cordyceps were basically similar except for certain diagnostic differences. These included macroscopic differences from C. sinensis as follows: the stroma of C. gunnii is stout and rough with sterile bulgy or branched apex; the larvae of C. barnesii has a pair of teeth on the head; the stroma of C. liangshanensi is thread-like; C. gracilis is without stroma; and C. militaris is without larvae. There were also microscopic differences: from C. sinensis as follows: the stroma of C. barnesii is without perithecia; C. gunnii, C. liangshanensis and C. gracilis are without bristles on the larva body. These differences allow C. sinensis and its counterfeits to be easily distinguished.
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Abstract Ophiocordyceps sinensis (syn. Cordyceps sinensis), a traditional Chinese medicine called DongChongXiaCao (DCXC) in Chinese, is well known and has been used in Asia countries since the fifteenth century, and it contains some valuable medicinal component defined by modern pharmacological science. DCXC only appears at high altitudes on the Qinghai-Tibetan Plateau. Consequently, it is difficult to find and harvest. Because of its rarity and medicinal value, DCXC has always been one of the most expensive medicines known. As the price of DCXC has risen in recent years, thousands of migrants have entered into the various grasslands to search for them in season, which makes ecological environments of the grassland more fragile. In order to relieve the environmental pressures and protect this valuable resource, the artificial cultivation of DCXC involving two aspects of the genus Hepialus and the fungi of the host larvae should be employed and applied at the first available time point. In this article, the reproduction of moth larvae of the genus Hepialus is first described, which includes their ecological characteristics and the methods of artificial feeding. Second, the generation and isolation method of the fungi from DCXC are subsequently summarized, and then the mechanism of fungal spores to attack the moth larvae are restated. Finally, the basic model of artificial cultivation of DCXC is introduced; meanwhile, the potential application of modern biotechnology to the artificial cultivation is analyzed in prospect. This review article will not only expand people's knowledge regarding the artificial cultivation of DCXC, but also hopefully provide an informative reference for the development of this valuable resource and the environmental protection of alpine meadows.
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This review looks in depth at the history and medicinal value of the Cordyceps species, especially C. sinensis. The C. sinensis medicinal species, with a long history of use, has only been found growing from the head of one type of subterranean caterpillar, at high altitudes, in the Qinghai-Tibetan plateau. Because of this highly specific growth environment and restricted geographical distribution, C. sinensis has a long reputation of being the single-most expensive raw material used in Oriental Medicine. Due to environmental and ecological factors, the annual harvest has been steadily declining, while at the same time the worldwide demand has been increasing. This situation has driven Cordyceps spp. prices into an ever-increasing spiral over the last few years, driving research to determine ways of cultivating it to make it a more affordable material for commercial trade. Part of the goal of this research has been to understand the complex biological niche such an organism fills. This is a mushroom that is only found in cohabitation with the larvae of an insect, and it is this unique growth parameter that has made it challenging to produce Cordyceps spp. in artificial cultivation. Further complicating this cultivation issue is the rarefied atmosphere, mineral-rich soil, and low temperature in which Cordyceps naturally grows, resulting in a unique profile of secondary metabolites possessing interesting biological potential for medical exploitation, but which are not readily reproduced in normal laboratory cultivation. In this article, we attempt to unravel many of the mysteries of Cordyceps spp., detailing the history, medicinal uses, chemical composition, and cultivation of Cordyceps spp., with special attention to C. sinensis, the world's most costly medicinal mushroom.
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