The traditional Chinese medicine Cordyceps sinensis and its effect in apoptotic homeostasis

Abstract

Cordyceps sinensis is a medicinal fungus of Traditional Chinese Medicine. While there are a wide range of reported uses of Cordyceps sinensis in the literature, the reports that extracts of this fungus may alter apoptotic homeostasis are most intriguing. However, there are significant challenges regarding research surrounding Cordyceps sinensis, such as the difficulty identifying the various species of Cordyceps and the many conflicting reports of pharmacological function in the literature. In this review we outline what is known about the ability of Cordyceps sinensis to alter apoptotic homeostasis, attempt to reconcile the differences in reported function, identify the challenges surrounding future Cordyceps sinensis research, and delineate options for overcoming these critical hurdles.

Full text

Journal of Ethnopharmacology 96 (2005) 19–29
Review
The traditional Chinese medicine Cordyceps sinensis and its effects on
apoptotic homeostasis
E.J. Buenza,b, B.A. Bauera,∗, T.W. Osmundsonc,d, T.J. Motleyd
aComplementary and Integrative Medicine Program, Mayo Clinic and Foundation, Rochester, Minnesota, USA
bMolecular Neuroscience Program, Mayo Clinic and Foundation, Rochester, Minnesota, USA
cDepartment of Ecology, Evolution and Environmental Biology, Columbia University, New York, New York, USA
dLewis B. and Dorothy Cullman Program for Molecular Systematics Studies, The New York Botanical Garden, Bronx, New York, USA
Received 23 May 2004; received in revised form 20 September 2004; accepted 20 September 2004
Available online 5 November 2004
Abstract
Cordyceps sinensis is a medicinal fungus of Traditional Chinese Medicine. While there are a wide range of reported uses of Cordyceps
sinensis in the literature, the reports that extracts of this fungus may alter apoptotic homeostasis are most intriguing. However, there are
significant challenges regarding research surrounding Cordyceps sinensis, such as the difficulty identifying the various species of Cordyceps
and the many conflicting reports of pharmacological function in the literature. In this review we outline what is known about the ability of
Cordyceps sinensis to alter apoptotic homeostasis, attempt to reconcile the differences in reported function, identify the challenges surrounding
future Cordyceps sinensis research, and delineate options for overcoming these critical hurdles.
© 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Cordyceps sinensis; Apoptosis; Traditional medicine; Plant; Fungus
Contents
1. Introduction .......................................................................................................... 20
2. Cordyceps sinensis background and ethnomedical use..................................................................... 20
2.1. Phylogenetic analysis of the genus Cordyceps ...................................................................... 21
2.2. Ethnomedical use of Cordyceps sinensis ........................................................................... 21
3. Apoptotic homeostasis and disease states ................................................................................ 21
3.1. Review of apoptosis............................................................................................. 21
3.2. Traditional medicines and apoptosis regulation..................................................................... 21
4. Cordyceps sinensis inhibits apoptosis.................................................................................... 22
5. Cordyceps sinensis induces apoptosis.................................................................................... 23
∗Corresponding author. Present address: Complementary and Integra-
tive Medicine Program, Mayo Clinic and Foundation, 200 First Street NW,
Rochester, Minnesota 55905, USA. Tel.: +1 507 284 8913;
fax: +1 507 284 5370.
E-mail address: bauer.brent@mayo.edu (B.A. Bauer).
0378-8741/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2004.09.029

20 E.J. Buenz et al. / Journal of Ethnopharmacology 96 (2005) 19–29
6. The challenge of identifying Cordyceps sinensis .......................................................................... 24
7. Strain typing and relations to product quality ............................................................................. 24
8. The future of apoptosis regulation through Cordyceps sinensis ............................................................. 25
Acknowledgements ........................................................................................................ 25
References ................................................................................................................ 26
1. Introduction
The past 20 years has seen a phenomenal growth in the
interest in and use of complementary or alternative medicine
(CAM), and in the United States 42% of people utilize some
form of CAM (Eisenberg et al., 1998). Of the total CAM
market, herbal and specialty dietary supplements command
a substantial portion with 29% of men and 36% of women re-
porting current use (Gunther et al., 2004). In 1994 the herbal
supplement market boomed, partly as a result of the passage
of the Dietary Supplement Health and Education Act (Center
for Food Safety and Applied Nutrition, 2004), driving sup-
plement sales up 70% from 1994 to 1997 (Radimer et al.,
2000) and leading to peak sales of US$ 3.3 billion in 1999
(Harnack et al., 2001). This interest in natural compounds is
certainly valid as these natural products undoubtedly contain
biologically active components: plant-based pharmaceuti-
cals have resulted in approximately one-half of the anti-
cancer drugs developed since 1960 (Kim and Park, 2002)
and have led to over 100 other successful pharmaceuticals
(Farnsworth, 1994). While there is a wide range of available
herbal supplements, one of the most interesting supplements
is the not yet well-characterized Cordyceps sinensis (Berk.)
Sacc.
The Cordyceps sinensis fungus first gained worldwide at-
tentionwhen itwas revealedthatseveral Chineserunners who
broke world records in 1993 had included this fungus as part
of their training program (Hollobaugh, 1993,Steinkraus and
Whitfield, 1994; Starr et al., 1993). In the ten years since the
initial reports, Cordyceps sinensis has received tremendous
attention from the public. Purported effects of the fungus
suggested a wide range of biological functions such as use as
an aphrodisiac (Bhattarai, 1993), analgesic (Koyama et al.,
1997)immune modulator (Gonget al.,2000), and freeradical
scavenger (Yamaguchi et al., 2000a). However, these effects
have not been well analyzed.
Recently, aseptic mycelial cultivation has resulted in es-
tablishing a number of Cordyceps sinensis derivative cul-
tures. The two most well studied of the cultures, referred
to by the anamorph names Paecilomyces hepiali (strain CS-
4) and Cephalosporium sinensis, may present the opportu-
nity to produce a Cordyceps sinensis derivative product in a
sustainable fashion. While these strains undoubtedly support
ecologically sustainable use of Cordyceps sinensis, the actual
similarities between wild Cordyceps sinensis and the cultures
are not clear.
2. Cordyceps sinensis background and ethnomedical
use
Cordyceps sinensis is endemic to alpine habitats
(3600–5000m in elevation) on the Tibetan plateau in south-
western China (Fig. 1), where it is a parasite on larvae of
moths (Lepidoptera) in the genera Hepialus and Thitarodes
(Kinjo and Zang, 2001). After a host larval infection with
either meiotic or mitotic spores, the fungus multiplies in the
host by yeast-like budding, eventually killing the host; the
fungus then grows in the form of threadlike hyphae. Follow-
ing overwintering, the fungus ruptures the host body, form-
ing a sexual sporulating structure (a perithecial stroma) that
is connected to the dead larva below ground and grows up-
ward to emerge above the soil surface. It is this stroma, either
with or without the host larva, which is traditionally used for
medicinal purposes (Fig. 2).
Fig. 1. Home range of Cordyceps sinensis.

E.J. Buenz et al. / Journal of Ethnopharmacology 96 (2005) 19–29 21
Fig. 2. Fruiting bodies of Cordyceps sinensis and dried caterpillars.
2.1. Phylogenetic analysis of the genus Cordyceps
Cordyceps is a genus of perithecial ascomycetes (Phylum
Ascomycota) classified in the Clavicipitaceae, a family sup-
portedby molecular phylogeneticanalyses asa monophyletic
group derived from the order Hypocreales (Artjariyasripong
et al., 2001; Rehner and Samuels, 1995; Spatafora and Black-
well, 1993; Suh et al., 1998). Cordyceps species are parasites
of insects or fungi, often exhibiting a high degree of host
specificity; as a result of this host specificity, the anamorphic
forms of some species (e.g., Beauvaria bassiana) are widely
used as insect biocontrol agents (Huang et al., 2002). Accord-
ing to molecular phylogenetic analyses, Cordyceps does not
represent a single evolutionary lineage; instead, Cordyceps
appears to represent several lineages within the Clavicipi-
taceae (Ito and Hirano, 1997; Artjariyasripong et al., 2001;
Sung et al., 2001; Zare et al., 2000). Similarly, the Cordy-
ceps species associated with Lepidopteran hosts do not rep-
resent a monophyletic group (Nikoh and Fukatsu, 2000; Park
et al., 2001; Sung et al., 2001). Furthermore, there appears
to be a high degree of genetic variation within the sinensis
species (Chen and Hseu, 1999). These levels of variation cre-
ate a significant challenge in verifying samples for analysis
of Cordyceps sinensis.
2.2. Ethnomedical use of Cordyceps sinensis
Cordyceps sinensis has a long history of medicinal use
in China. This fungus is thought to have been discovered
2000 years ago (Liu et al., 2001) and its use was docu-
mented formally in the Qing dynasty Bencao Congxin (New
Compilation of Materia Medica) in 1757. However, Cordy-
ceps sinensis is a unique traditional medicine in that there
exists little primary ethnomedical data describing medical
use in the literature. Current ethnomedical reports on the
uses of Cordyceps sinensis are limited to the application
as a general tonic in China (Huang et al., 1981; Jiang,
1991; Hanssen and Schadler, 1982) and as an aphrodisiac
in Nepal (Bhattarai, 1992a, 1993, 1992b, 1994, 1989). In
contrast to the ethnomedical data, the literature surround-
ing the biological effects of Cordyceps sinensis is diverse
(reviewed in Zhu et al., 1998a, 1998b). However, some of
the most intriguing reports regarding the biological functions
of Cordyceps sinensis center on its ability to alter apoptotic
homeostasis.
3. Apoptotic homeostasis and disease states
3.1. Review of apoptosis
Apoptosis, or programmed cell death, is an essential
event in organism development (Hidalgo and Ffrench-
Constant, 2003; Vaux and Korsmeyer, 1999) and homeosta-
sis (Kucharczak et al., 2003,Cory and Adams, 2002); how-
ever, it is becoming clear that numerous disorders such as
stroke (Zheng et al., 2003), myocardial infarction (Krijnen
et al., 2002), and HIV (Buenz and Badley, in press) incorpo-
rate apoptosis in their etiology and pathogenesis. There are
numerous events that can induce a cell to undergo apopto-
sis (Nagata, 1997) and since the implication of apoptosis in
various disease states as an effector mechanism, the ability
to inhibit apoptosis has emerged as an important potential
therapy. Interestingly, while inducing apoptosis has already
proven an efficient method to treat cancer (Hu and Kavanagh,
2003), the ability to inhibit apoptosis in a clinical setting is
just starting to be explored (Liston et al., 2003).
The potential for a cell to undergo apoptosis exists in a
balance between endogenous factors characteristic of apop-
tosis induction, such as Bax (Degli Esposti and Dive, 2003;
Scorrano and Korsmeyer, 2003), and factors characteristic of
apoptosis inhibition, such as Bcl-2 (Cory et al., 2003; Gross
et al., 1999). Once a cell receives sufficient pro-apoptotic
stimuli, or lack of anti-apoptotic stimuli, the effector cas-
pases, a family of cysteine aspartate proteases, are activated
(Fischer et al., 2003). Cells undergoing apoptosis experience
a cascade of events outlined in Fig. 3 that ultimately result in
phenotypic changes such as phosphotydylserine expression
on the outer cell leaflet (Maderna and Godson, 2003), nuclear
condensation, and DNA fragmentation (Nagata et al., 2003)
shown in Fig. 4.
3.2. Traditional medicines and apoptosis regulation
Many traditional medicines (e.g., Nelumbo nucifera (Jung
et al., 2003) and Bacopa monnieri (Russo et al., 2003)), are
reported to scavenge reactive oxygen species and the role of
reactive oxygen species in the apoptotic pathway is of partic-
ular interest regarding herbal supplements. Reactive oxygen
species and their regulatory molecules are important com-
ponents of the immune system and cell function, such as
superoxide radicals generated by activated neutrophils as a
pathogen defense mechanism (Babior, 1978), and cell signal-
ing (Ichiki et al., 2003). However, there are also reports of al-
tered oxygen-based free radical levels in disease states. These

22 E.J. Buenz et al. / Journal of Ethnopharmacology 96 (2005) 19–29
Fig. 3. Fas/CD95 apoptotic pathway. Death receptor trimerization induces
a signaling cascade that ultimately results in chromosomal condensation,
DNA fragmentation, and cellular apoptosis.
alteredlevels ofreactive oxygenspecies indisease states such
as cancer (Behrend et al., 2003) and stroke (Sugawara and
Chan,2003) haveresulted inhypotheses thatmitigation ofex-
cessive reactive oxygen species could be therapeutically im-
portant. While both the exact role of reactive oxygen species
in disease states and whether they are a primary insult or a
downstream result of the disease states have yet to be de-
termined. It appears that the regulation of reactive oxygen
species as therapy will be of significant interest in the future.
Undoubtedly there are herbal supplements that influence
apoptotichomeostasis. Forexample,an isolatedcompound of
thetraditional medicine European feverfew(Chrysanthemum
parthenium) has been shown to decrease susceptibility to
apoptotic stimuli through down regulation of the Fas receptor
and the Fas ligand agonist through inhibition of NF-␬B(Li-
Fig. 4. HeLa cells stained with Annexin V-FITC (green) and propidium iodine (red). (A) Control cells show little phosphatidylserine expression (green) on the
outer leaflet, no nuclear propidium iodine intercalation (red), and typical cellular morphology. (B) Cells treated with 30uM starurosporine for 4h are induced
to undergo apoptosis. (1) Early stage apoptotic cells show increased phosphatidylserine expression on the outer leaflet and slight morphological condensation;
however, the nuclear membrane is still intact and thus the cells do not stain propidium iodine positive. (2) In later stages of apoptosis, the integrity of the nuclear
membrane is compromised and the DNA stains propidium iodine positive. Nuclear condensation and blebbing are also evident (arrows). 3) In the final stages
of apoptosis, the cellular and nuclear membranes are totally compromised and the cell appears necrotic.
Weber et al., 2002). However, the ability of Cordyceps sinen-
sis to alter the apoptotic pathway is not so straightforward.
Currently, there are reports of Cordyceps sinensis extracts
both inhibiting (Table 1) and inducing apoptosis (Table 2).
Most of these reports reflect a phenomenon level of obser-
vation, such as treatment with Cordyceps sinensis resulting
in decreased caspase-3 activity (Shahed et al., 2001). While
these reports do not examine the mechanism of action, they
do facilitate a necessary foundation to allow examination of
the molecular mechanism.
4. Cordyceps sinensis inhibits apoptosis
The reports of clinical trials suggest that Cordyceps sinen-
sis potentially contains agents that may inhibit apoptosis (re-
viewedin Zhuetal., 1998a).These clinicalresults havedriven
work to assess the ability of Cordyceps sinensis to inhibit
apoptosis in vitro; however, the results of these studies are
conflicting. It has been reported that Cordyceps sinensis can
scavenge reactive oxygen species (Zhang et al., 1995)byin-
hibitingmalondialdehyde formationby theperoxynitrite gen-
erator SIN-1 (Yamaguchi et al., 2000a). These results have
been confirmed through in vitro xanthine oxidase, hemoly-
sis, and lipid peroxidation assays (Li et al., 2001). Further-
more, an isolated extract of Cordyceps sinensis H1-A has
been shown to inhibit apoptosis induced by dimethyl sulfox-
ide (DMSO), which is known to induce apoptosis through
permeabilizing the cell membrane and upregulating nitric
oxide synthase (Trubiani et al., 2003). However, extracts of
Cordyceps sinensis were unsuccessful in inhibiting hydrogen
peroxide-induced apoptosis (Buenz et al., 2004), a reactive
oxygen species model (Fauconneau et al., 2002).
Alternately, Cordyceps sinensis has also been reported to
down-regulate apoptotic genes and modulate apoptosis in a

E.J. Buenz et al. / Journal of Ethnopharmacology 96 (2005) 19–29 23
Table 1
Reported antiapoptotic effects of Cordyceps sinensis
Reported function Model Concentration Reference
Anticytotoxic activity Mouse 150 mg/kg Yu et al. (1993)
Antioxidant activity Mouse 50 mg/kg Yamaguchi et al. (2000b)
Antiproliferation activity Cell culture 100 ␮g/mL Zhao Long and Xiao Xia (2000)
Cell proliferation inhibition Cell culture 10 ␮g/mL Chen et al. (1997)
Cell proliferation inhibition Human adult 71 ␮g/mL Kuo et al. (1996)
Cell proliferation inhibition Human adult 40 ␮g/mL Lin et al. (1999)
Cell proliferation inhibition Mouse 1.0 % of diet Lin et al. (1999)
Gene expression inhibition Rat 0.5 mL per animal Shahed et al. (2001)
Hemolysis inhibitory activity Cell culture 1.5 mg/mL Li et al. (2001)
Lipid peroxide formation inhibition Cell culture 5.0 mg/mL Li et al. (2001)
Natural killer cell inhibition Human adult 12.9 ␮g/mL Kuo et al. (1996)
Radical scavenging effect Cell culture 5.0 mg/mL Shahed et al. (2001)
Radical scavenging effect Cell culture 0.08 mg/mL Li et al. (2001)
Tumor necrosis factor inhibition Human adult 2.7 ␮g/mL Kuo et al. (1996)
rat kidney ischemia reperfusion model. Shahed et al. (2001)
showed a significant decrease in Fas, Fas ligand, and Tu-
mor Necrosis Factor-␣(TNF-␣) expression along with de-
creased caspase-3 activity. Similarly, Cordyceps sinensis has
been reported to inhibit TNF-␣expression (Kuo et al., 1996).
However, when apoptosis was initiated through CH-11, a Fas
agonist antibody (Alderson et al., 1994), aqueous and alco-
hol extracts of Cordyceps sinensis were unable to rescue cells
induced through Fas receptor ligation (Buenz et al., 2004).
Furthermore, it has been shown that in certain cell types,
inhibition of proliferation or cell cycle arrest results in
cells becoming resistant to apoptosis (Chaturvedi et al.,
1999). In turn, the reports that Cordyceps sinensis inhibits
proliferation of leukemic U937 cells (Chen et al., 1997)
and glomerular mesangial cells (Zhao Long and Xiao Xia,
2000; Lin et al., 1999) may result by conferring a rela-
tively apoptotic resistant state to cells. While the mecha-
nisms of this inhibition have yet to be characterized, it is
reasonable to propose that the alteration may potentially in-
volve p53 (Fridman and Lowe, 2003)orNF-␬B(Karin et al.,
2004).
5. Cordyceps sinensis induces apoptosis
The ability to induce apoptosis has been identified and
utilized in successful cancer chemotherapeutics (Hu and Ka-
vanagh, 2003); Table 2 outlines the literature suggesting or
stating the ability of Cordyceps sinensis to induce apoptosis.
Recently, Yang et al. (Yang et al., 2003) described the abil-
ity of the previously isolated 410kDa polysaccharide frac-
tion of Cordyceps sinensis termed H1-A (Yang et al., 1999)
to induce apoptosis through inhibiting phosphorylation of
Bcl-2 and Bcl-xL. These anti-apoptotic Bcl-2 family mem-
bers are known to sequester cytosolic pro-apoptotic proteins
such as Bax. As addressed above, this fraction also inhibited
apoptosis induced by dimethyl sulfoxide (DMSO) (Yang et
al., 2003). However the report consisting of the inhibition of
Bcl-2 and Bcl-xL and subsequent inhibition of apoptosis by
DMSO (Yang et al., 2003) seems counterintuitive. Thus, fur-
ther work is necessary to clarify the physiologic role of the
H1-A extract.
Finally, there are reports of direct cytotoxic activity
(Nakamura et al., 1999; Kuo et al., 1994; Sato, 1989).
However, these studies report inhibition at the phenomena
level and do not address specific mechanisms. Yet it is in-
teresting that cordycepin, a compound originally isolated
from the Cordyceps sinensis relative Cordyceps militaris
(Cunningham et al., 1950), is known to exert cytotoxic effects
through nucleic acid methylation (Kredich, 1980). While
isolation of this single active compound may set the stage
for work to identify molecular mechanisms of action, the
actual presence of cordcycepin in Cordyceps sinensis has
been difficult to confirm. Cordycepin has been shown to
be present in Cordyceps sinensis through nuclear magnetic
resonance (Chen and Chu, 1996); however, other groups
have not been able to detect this compound (Shiao et al.,
1994).
Table 2
Reported apoptotic effects of Cordyceps sinensis
Reported function Model Dose Reference
Antitumor activity Mouse Not stated Zang et al. (1985)
Antitumor activity Mouse 5.0 g/kg Xu et al. (1992)
Cell proliferation stimulation Cell culture 10␮g/mL Chen et al. (1997)
Cytotoxic activity Cell culture 2.0 ␮g/mL Kuo et al. (1994)
Cytotoxic activity Cell culture 500 ␮g/mL Sato (1989)
Cytotoxic activity Cell culture 10 ␮g/mL Nakamura et al. (1999)
Metastasis inhibition Mouse 100 mg/kg Nakamura et al. (1999)

24 E.J. Buenz et al. / Journal of Ethnopharmacology 96 (2005) 19–29
6. The challenge of identifying Cordyceps sinensis
The most significant challenge working with Cordyceps
sinensis is the lack of a well-defined mechanism to iden-
tify sample material. Although Cordyceps species have been
included in a number of recent molecular phylogenetic anal-
yses (Artjariyasripong et al., 2001; Ito and Hirano, 1997;
Lumbsch et al., 2000; Nikoh and Fukatsu, 2000; Nikoh
and Fukatsu, 2001; Obornik et al., 2001; Park et al., 2001;
Spatafora and Blackwell, 1993; Suh et al., 2001; Suh et
al., 1998; Sung et al., 2001; Zare et al., 2000), most of
these studies have either not included Cordyceps sinen-
sis, or have included Cordyceps sinensis without enough
other taxa to allow phylogenetic resolution to be achieved
among Cordyceps sinensis and other closely related Cordy-
ceps species. Most DNA-based studies that include Cordy-
ceps sinensis have examined genetic differentiation at the
population level rather than at the species level (Chen et
al., 2001; Kinjo and Zang, 2001; Liu et al., 2001). Us-
ing a neighbor-joining analysis of ribosomal DNA internal
transcribed spacer (rDNA-ITS) sequences, Park et al. (Park
et al., 2001) determined Cordyceps sinensis to be closely
related to Cordyceps ophioglossoides, a parasite of “false
truffle” fungi in the genus Elaphomyces. Based on this ev-
idence, as well as the placement of a Hirsutella anamorph
(though with low bootstrap support) within a larger phyloge-
netic clade that includes Cordyceps ophioglossoides (Sung
et al., 2001), a possible phylogenetic placement for Cordy-
ceps sinensis is within a basal clade in the Clavicipitaceae
that contains both entomopathogenic and fungicolous fungi.
However, direct molecular evidence for the proper phyloge-
netic placement of Cordyceps sinensis in relation to a poly-
phyletic Cordyceps is currently lacking, and Cordyceps and
the family Clavicipitaceae are greatly in need of systematic
revision.
Many ascomycetes, including species of Cordyceps,have
both an asexual (anamorphic) and sexual (teleomorphic)
form. Cordyceps species have been shown in mycological
culture studies to be associated with a number of anamor-
phic genera including Paecilomyces,Beauvaria,Metarhiz-
ium,Verticillium, and Tolypocladium (Hodge et al., 1996;
Huang et al., 2002; Nikoh and Fukatsu, 2000; Nikoh and
Fukatsu, 2001; Obornik et al., 2001; Sung et al., 2001; Zare
et al., 2000), and phylogenetically related to several species
of yeast-like endosymbionts of insects (Suh et al., 2001). De-
termining the anamorphic state of Cordyceps sinensis has
posed difficulty for researchers; as a result, 22 names in
13 anamorph genera have previously been associated with
Cordyceps sinensis (Jiang and Yao, 2002). However, recent
molecular evidence (Chen et al., 2001; Chen et al., 2002;
Liu et al., 2001) and generation of the anamorphic state from
germinated Cordyceps sinensis ascospores (Liu et al., 2001)
supports Hirsutella sinensis Liu et al. as being the correct
anamorph of Cordyceps sinensis. Many anamorphic fungi,
including Hirsutella sinensis, can be cultured under labora-
tory conditions. Because Cordyceps sinensis is considered
to be declining in the wild due to overharvest (Liu et al.,
2003), culture of the anamorphic state may therefore be
a means of producing material for research and medicinal
preparations in the face of a shortage of teleomorph mate-
rial.
Several Cordyceps species have been described that
appear morphologically similar to Cordyceps sinensis, in-
cluding Cordyceps nepalensis M. Zang & Kinjo, Cordy-
ceps multiaxialis M. Zang & Kinjo, Cordyceps gansuensis
Zhang et al., and Cordyceps crassispora Zang et al. How-
ever, these species may simply represent morphological vari-
ants of Cordyceps sinensis (Kinjo and Zang, 2001; Liu et
al., 2001). Whether these morphotypes correspond to differ-
ences in pharmacological activity has not been determined.
Other Cordyceps species (e.g., Cordyceps militaris)have
been shown to have some of the same medicinal properties
as Cordyceps sinensis (Wu et al., 2000), but appear to be less
highly regarded by consumers and practitioners.
7. Strain typing and relations to product quality
The lack of molecular evidence for proper phyloge-
netic placement of Cordyceps sinensis precludes establish-
ing a consensus strain of Cordyceps sinensis. Similarly,
conducting reliable clinical trials and ascertaining the qual-
ity of commercial herbal and other natural products depends
upon accurately identifying source materials. A number of
factors could contribute to poor quality of Cordyceps sinensis
products, including intentional substitution of other Cordy-
ceps species, substitution of counterfeit material, or unin-
tentional misidentification of field-collected Cordyceps.In
addition, as demand for Cordyceps sinensis products grows
and the supply of wild material declines, mycelium of
the asexual (anamorphic) stage grown under artificial cul-
ture conditions is increasingly used in medicinal products.
Among the source materials found to have been sold un-
der the name Chongcao in commercial markets are other
Cordyceps species (e.g., Cordyceps militaris,Cordyceps
liangshanensis,Cordyceps gunnii,Cordyceps hawkesii, and
Cordyceps ramosa), anamorphic fungi including Hirsutella
sinensis, the anamorph of Cordyceps sinensis, as well as
Paecilomyces sinensis and Tolypocladium sp., and rhizomes
from the plant species Stachys geobombycis and Stachys
sieboldii (Chen et al., 2002; Cheng et al., 1998). Even
within Cordyceps sinensis, differences in pharmacological
activity have been noted between strains (Kinjo and Zang,
2001).
Because commercial products generally contain dried,
powdered material, identifying source material using
morphological characters is normally impossible. DNA
fingerprinting methods offer a dependable means of identify-
ing source materials from fresh specimens and commercial
preparations (Zerega et al., 2002). Such methods are able
to distinguish isolates at the species, or even strain, level
depending upon the specific method used (e.g., (Terashima

E.J. Buenz et al. / Journal of Ethnopharmacology 96 (2005) 19–29 25
et al., 2002)). The effectiveness of DNA fingerprinting for
authenticating source material has been demonstrated for a
number of pharmacologically useful plants, including skull-
cap (Scutellaria spp.; (Hosokawa et al., 2000)), Echinacea
(Nieri et al., 2003), black cohosh (Actaea racemosa;(Zerega
et al., 2002)), Lycium (Zhang et al., 2001), ginseng (Panax
ginseng;(Hon et al., 2003,Ngan et al., 1999)), Withania
(Negi et al., 2000), St. John’s wort (Hypericum perforatum;
(Mayo and Langridge, 2003)), opium poppy (Papaver som-
niferum;(Saunders et al., 2001)), coca (Erythroxylum spp.;
(Johnson et al., 2003)), and yarrow (Achillea millefolium;
(Wallner et al., 1996)). DNA fingerprinting has been success-
fully used in fungi to distinguish strains of human pathogenic
fungi (Buffington et al., 1994; Mcewen et al., 2000), plant
pathogens (Barnes et al., 2001; Chen et al., 2000; Morris
et al., 2000; Schmidt et al., 2003; Zeller et al., 2003), food
spoilage agents (Kure et al., 2003), biocontrol agents (Avis
et al., 2001; Hermosa et al., 2001), and edible mushrooms
(Terashima et al., 2002).
Previous genetic analyses of multiple Cordyceps sinen-
sis populations have examined ribosomal DNA (rDNA) se-
quence diversity (Chen and Hseu, 1999; Chen et al., 2001;
Chen et al., 2002; Kinjo and Zang, 2001; Liu et al., 2001) and
patterns of genetic variability exhibited by randomly ampli-
fied polymorphic DNA (RAPD) markers (Chen et al., 1999;
Cheng et al., 1998). Variability in ribosomal ITS sequences
and 18S ribosomal DNA restriction fragment length poly-
morphism (RFLP) patterns has been shown to be informative
for differentiating Cordyceps sinensis from other Cordyceps
speciesand from marketcounterfeits (Chenet al.,1999; Chen
et al., 2001; Chen et al., 2002; Kinjo and Zang, 2001; Liu et
al., 2001), and for generating Cordyceps sinensis—specific
DNA probes (Chen et al., 2002); however, rDNA sequence
variation is too low to allow accurate genotyping at the strain
level (Chen et al., 2001; Kinjo and Zang, 2001; Liu et al.,
2001). Randomly amplified polymorphic DNA markers are
able not only to distinguish Cordyceps sinensis from other
Cordyceps species, but to distinguish individual Cordyceps
sinensis populations (Chen and Hseu, 1999; Cheng et al.,
1998).More recentlydeveloped DNAfingerprintingmethods
such as amplified fragment length polymorphisms (AFLP;
Vos et al., 1995) uncover more genetic polymorphism over a
larger part of the genome than do ribosomal DNA sequences.
Additionally, the results of AFLP fingerprinting have been
shown to have a higher degree of repeatability than those of
RAPDfingerprinting (Ranamukhaarachchiet al.,2000; Jones
et al., 1997; Barker et al., 1999). AFLP fingerprints have
thus far not been obtained for Cordyceps sinensis, and rep-
resent a potentially useful tool for characterizing Cordyceps
sinensis samples. Further population-level genetic character-
ization is extremely important for Cordyceps sinensis mate-
rial in order to determine the geographic origins of source
material, select standardized strains for clinical experiments,
distinguish anamorph cultures from fungal contaminants,
and facilitate conservation of genetic diversity in natural
populations.
8. The future of apoptosis regulation through
Cordyceps sinensis
There are three prominent factors that may contribute to
thediscrepancies inreports regardingtheability ofCordyceps
sinensis to inhibit apoptosis. First, as there is no consensus
strain, it is possible that certain populations contain different
biologically active compounds. Second, there is the potential
that Cordyceps sinensis extracts contain a pro-drug and there
is a necessary metabolism step in order to generate the bio-
logically active form of the drug. Third, as there are multiple
methods of extraction utilized in the literature such as ethanol
(Xu et al., 1992), methanol (Kuo et al., 1994), alkaline extract
(Kiho et al., 1996), hot water (Manabe et al., 1996), and cold
water (Chen et al., 1997), different constituents may be as-
sayed depending on extraction method. However, these chal-
lenges are certainly not intractable. The Cordyceps sinensis
consensus strain could be established through AFLP-based
DNA fingerprinting as has been done for other medicinal
plants. Similarly, the issue regarding an active metabolite
could be addressed through either establishing a liposome
system containing enzymes known to be important in drug
metabolism such as members of the P450 family (De Graaf
et al., 2002) or, alternately, it may be possible to treat the
Cordyceps sinensis extract with a liver homogenate to gen-
erate the active metabolites of the extracts (De Graaf et al.,
2002).
Furthermore, the research surrounding Cordyceps sinen-
sis is lacking molecular studies. Rather, there are numerous
reports of treating model systems with Cordyceps sinensis
and measuring outcome. However, establishing a consensus
strain is an essential foundation to defining molecular mech-
anisms. Currently the use of voucher specimens and marker
compounds does allow identification of species phenotypi-
cally and biochemically, respectively. However, a principle
marker compound for Cordyceps sinensis is cordycepic acid
(mannitol-D) and while the presence of cordycepic acid is
indicative of Cordyceps sinensis, the inclusion of a single
marker compound does not necessarily guarantee the pres-
ence of other potentially active compounds. Thus, develop-
ing an AFLP based DNA fingerprinting program would allow
positive identification of not just Cordyceps sinensis, but also
identification of sub-populations of the species.
Cordyceps sinensis hasbeen usedas atraditional medicine
throughout history and, undoubtedly, as investigation into
this fungus continues, more active components with potential
therapeutic value will be isolated.
Acknowledgements
We would like to thank Jacklynn Conway and Jane Meyer,
Mayo Clinic, Rochester, Minnesota, USA, for their admin-
istrative assistance; Holly Johnson, University of Illinois at
Chicago / National Institutes of Heath Center for Botani-
cal Dietary Supplements Research, Chicago, Illinois, USA,

26 E.J. Buenz et al. / Journal of Ethnopharmacology 96 (2005) 19–29
for her assistance with the Natural Products Alert Database
queries; and Sy Chalpin, Hi-Health, Scottsdale, Arizona,
USA, for providing Cordyceps sinensis for our work.
References
Alderson, M.R., Tough, T.W., Braddy, S., Davis-Smith, T., Roux, E.,
Schooley, K., Miller, R.E., Lynch, D.H., 1994. Regulation of apoptosis
and T cell activation by Fas-specific mAb. International Immunology
6, 1799–1806.
Artjariyasripong, S., Mitchell, J.I., Hywel-Jones, N.L., Jones, E.B.G.,
2001. Relationship of the genus Cordyceps and related genera, based
on parsimony and spectral analysis of partial 18S and 28S ribosomal
gene sequences. Mycoscience 42, 503–517.
Avis, T.J., Caron, S.J., Boekhout, T., Hamelin, R.C., Belanger, R.R., 2001.
Molecular and physiological analysis of the powdery mildew antag-
onist Pseudozyma flocculosa and related fungi. Phytopathology 91,
249–254.
Babior, B.M., 1978. Oxygen-dependent microbial killing by phagocytes
(first of two parts). New England Journal of Medicine 298, 659–
668.
Barker, J.H.A., Matthes, M., Arnold, G.M., Edwards, K.J., Ahman, I.,
Larsson, S., Karp, A., 1999. Characterisation of genetic diversity in
potential biomass willows (Salix spp.) by RAPD and AFLP analyses.
Genome/National Research Council Canada 42, 173–183.
Barnes, I., Gaur, A., Burgess, T., Roux, J., Wingfield, B.D., Wingfield,
M.J., 2001. Microsatellite markers reflect intra-specific relationships
between isolates of the vascular wilt pathogen Ceratocystis fimbriata.
Molecular Plant Pathology 2, 319–325.
Behrend, L., Henderson, G., Zwacka, R.M., 2003. Reactive oxygen
species in oncogenic transformation. Biochemical Society Transac-
tions 31, 1441–1444.
Bhattarai, N.K., 1989. Traditional phytotherapy among the Sherpas of
Helambu, central Nepal. Journal of Ethnopharmacology 27, 45–54.
Bhattarai, N.K., 1992a. Folk use of plants in veterinary medicine in central
Nepal. Fitoterapia 63, 497–506.
Bhattarai, N.K., 1992b. Medical ethnobotany in the Karnali Zone, Nepal.
Economic Botany 46, 257–261.
Bhattarai, N.K., 1993. Folk herbal medicines of Dolakha District, Nepal.
Fitoterapia 66, 387–395.
Bhattarai, N.K., 1994. Folk herbal remedies for gynaecological complaints
in central Nepal. International Journal of Pharmacognosy 32, 13–26.
Buenz, E., Badley, A., in press. Impact of mitochondrial regulation of
apoptosis on the pathogenesis of HIV-1 induced immunodeficiency.
Mitochondrion. In press.
Buenz, E.J., Weaver, J.G., Bauer, B.A., Chalpin, S.D., Badley, A.D., 2004.
Cordyceps sinensis extracts do not prevent Fas-receptor and hydrogen
peroxide-induced T-cell apoptosis. Journal of Ethnopharmacology 90,
57–62.
Buffington, J., Reporter, R., Lasker, B.A., Mcneil, M.M., Lanson, J.M.,
Ross, L.A., Mascola, L., Jarvis, W.R., 1994. Investigation of an epi-
demic of invasive aspergillosis: utility of molecular typing with the
use of random amplified polymorphic DNA probes. The Pediatric
Infectious Disease Journal 13, 386–393.
Center for Food Safety and Applied Nutrition, Dietary Supplement Health
and Education Act of 1994. 2004.
Chaturvedi, V., Qin, J.Z., Denning, M.F., Choubey, D., Diaz, M.O.,
Nickoloff, B.J., 1999. Apoptosis in proliferating, senescent, and im-
mortalized keratinocytes. The Journal of Biological Chemistry 274,
23358–23367.
Chen, C.-S., Hseu, R.-S., 1999. Differentiation of Cordyceps sinensis
(Berk.) Sacc. specimens using restriction fragment length polymor-
phism of 18S rRNA gene. Journal of the Chinese Agricultural Chem-
ical Society 37, 533–545.
Chen, S.Z., Chu, J.Z., 1996. NMR and IR studies on the characterization
of cordycepin and 2-deoxyadenosine. Zhongguo kang sheng su za
shi (Chinese Journal of Antibiotics) 21, 9–12.
Chen, W.-D., Grau, C.R., Adee, E.A., Meng, X.-Q., 2000. A molecu-
lar marker identifying subspecific populations of the soybean brown
stem rot pathogen, Phialophora gregata. Phytopathology 90, 875–
883.
Chen, Y.J., Shiao, M.S., Lee, S.S., Wang, S.Y., 1997. Effect of Cordyceps
sinensis on the proliferation and differentiation of human leukemic
U937 cells. Life Sciences 60, 2349–2359.
Chen, Y.-J., Zhang, Y.-P., Yang, Y.-X., Yang, D.-R., 1999. Genetic diver-
sity and taxonomic implication of Cordyceps sinensis as revealed by
RAPD markers. Biochemical Genetics 37, 201–213.
Chen, Y.-Q., Wang, N., Qu, L.-H., Li, T.-H., Zhang, W.-M., 2001. De-
termination of the anamorph of Cordyceps sinensis inferred from the
analysis of the ribosomal DNA internal transcribed spacers and 5.8 S
rDNA. Biochemical Systematics and Ecology 29, 597–607.
Chen, Y.-Q., Wang, N., Zhou, H., Qu, L.-H., 2002. Differentiation of
medicinal Cordyceps species by rDNA ITS sequence analysis. Planta
Medica 68, 635–639.
Cheng, K.-T., Su, C.-H., Chang, H.-C., Huang, J.-Y., 1998. Differentia-
tion of genuines and counterfeits of Cordyceps species using random
amplified polymorphic DNA. Planta Medica 64, 451–453.
Cory, S., Adams, J.M., 2002. The Bcl2 family: regulators of the cellular
life-or-death switch. Nature Reviews Cancer 2, 647–656.
Cory, S., Huang, D., Adams, J., 2003. The Bcl-2 family: roles in cell
survival and oncogenesis. Oncogene 22, 8590–8607.
Cunningham, K.G., Manson, W., Spring, F.S., Hutchinson, S.A., 1950.
Cordycepin, a metabolic product isolated from cultures of Cordyceps
militaris (Linn.) Link. Nature 166, 949.
De Graaf, I.A., Van Meijeren, C.E., Pektas, F., Koster, H.J., 2002.
Comparison of in vitro preparations for semi-quantitative prediction
of in vivo drug metabolism. Drug Metabolism and Disposition 30,
1129–1136.
Degli Esposti, M., Dive, C., 2003. Mitochondrial membrane permeabili-
sation by Bax/Bak. Biochemical and Biophysical Research Commu-
nications 304, 455–461.
Eisenberg, D.M., Miller, F.H., Curto, D.A., Kaptchuk, T.J., Brennan,
T.A., 1998. Trends in alternative medicine use in the United States,
1990–1997: results of a follow-up national survey [comment]. JAMA:
The Journal of the American Medical Association 280, 1610–1615.
Farnsworth, N., 1994. Ethnopharmacology and drug development. Eth-
nobotany and the search for new drugs. Ciba Foundation Symposium
185, 42–59.
Fauconneau, B., Petegnief, V., Sanfeliu, C., Piriou, A., Planas, A.M.,
2002. Induction of heat shock proteins (HSPs) by sodium arsenite in
cultured astrocytes and reduction of hydrogen peroxide-induced cell
death. Journal of Neurochemistry 83, 1338–1348.
Fischer, U., Janicke, R.U., Schulze-Osthoff, K., 2003. Many cuts to ruin:
a comprehensive update of caspase substrates. Cell Death and Differ-
entiation 10, 76–100.
Fridman, J.S., Lowe, S.W., 2003. Control of apoptosis by p53. Oncogene
22, 9030–9040.
Gong, X.J., Ji, H., Lu, S.G., Li, S.P., Li, P., 2000. Effects of polysac-
charides from cultured Cordyceps sinensis on murine immunologic
function. Zhongguo Yaoke Daxue Xuebao 31, 53–55.
Gross, A., Mcdonnell, J.M., Korsmeyer, S.J., 1999. BCL-2 family mem-
bers and the mitochondria in apoptosis. Genes and Development 13,
1899–1911.
Gunther, S., Patterson, R.E., Kristal, A.R., Stratton, K.L., White, E., 2004.
Demographic and health-related correlates of herbal and specialty sup-
plement use. Journal of the American Dietetic Association 104, 27–34.
Hanssen, H.P., Schadler, M., 1982. Mushrooms as folk remedy from Chi-
nese medicine. Deutsche Apotheker-Zeitung 122, 1844–1848.
Harnack, L.J., Rydell, S.A., Stang, J., 2001. Prevalence of use of herbal
products by adults in the Minneapolis/St Paul, Minn, metropolitan
area. Mayo Clinic Proceedings 76, 688–694.

E.J. Buenz et al. / Journal of Ethnopharmacology 96 (2005) 19–29 27
Hermosa, M.R., Grondona, I., Diaz-Minguez, J.M., Iturriaga, E.A., Monte,
E., 2001. Development of a strain-specific SCAR marker for the detec-
tion of Trichoderma atroviride 11, a biological control agent against
soilborne fungal plant pathogens. Current Genetics 38, 343–350.
Hidalgo, A., Ffrench-Constant, C., 2003. The control of cell number dur-
ing central nervous system development in flies and mice. Mechanisms
of Development 120, 1311–1325.
Hodge, K.T., Krasnoff, S.B., Humber, R.A., 1996. Tolypocladium inflatum
is the anamorph of Cordyceps subsessilis. Mycologia 88, 715–719.
Hollobaugh, J., 1993. A giant awakens. Track & Field News 46.
Hon, C.C., Chow, Y.C., Zeng, F.Y., Leung, F.C.C., 2003. Genetic au-
thentication of ginseng and other traditional Chinese medicine. Acta
Pharmacologica Sinica 24, 841–846.
Hosokawa, K., Minami, M., Kawahara, K., Nakamura, I., Shibata, T.,
2000. Discrimination among three species of medicinal Scutellaria
plants using RAPD markers. Planta Medica 66, 270–272.
Hu, W., Kavanagh, J.J., 2003. Anticancer therapy targeting the apoptotic
pathway. The Lancet Oncology 4, 721–729.
Huang, B., Li, C.-R., Li, Z.-G., Fan, M.-Z., Li, Z.-Z., 2002. Molecular
identification of the teleomorph of Beauveria bassiana. Mycotaxon
81, 229–236.
Huang, H.T., Chou, S.H., Ho, H.L., 1981. Comparison of chemical con-
stituents between Cordyceps hawkesii and Corcyceps sinensis. Chinese
Pharmaceutical Bulletin 16, 53.
Ichiki, T., Tokunou, T., Fukuyama, K., Iino, N., Masuda, S., Takeshita, A.,
2003. Cyclic AMP response element-binding protein mediates reactive
oxygen species-induced c-fos expression. Hypertension 42, 177–183.
Ito, Y., Hirano, T., 1997. The determination of the partial 18S ribosomal
DNA sequences of Cordyceps species. Letters in Applied Microbiol-
ogy 25, 239–242.
Jiang, S.J., 1991. Immunomodulating effects of cordyceps sinensis. Inter-
national Journal of Oriental Medicine 16, 128–130.
Jiang, Y., Yao, Y.-J., 2002. Names related to Cordyceps sinensis
anamorph. Mycotaxon 84, 245–254.
Johnson, E.L., Saunders, J.A., Mischke, S., Helling, C.S., Emche, S.D.,
2003. Identification of Erythroxylum taxa by AFLP DNA analysis.
Phytochemistry 64, 187–197.
Jones, C.J., Edwards, K.J., Castaglione, S., Winfield, M.O., Sala, F., Van
De Wiel, C., Bredemeijer, G., Vosman, B., Matthes, M., Daly, A.,
Brettschneider, R., Bettini, P., Buiatti, M., Maestri, E., Malcevschi,
A., Marmiroli, N., Aert, R., Volckaert, G., Rueda, J., Linacero, R.,
Vazquez, A., Karp, A., 1997. Reproducibility testing of RAPD, AFLP
and SSR markers in plants by a network of European laboratories.
Molecular Breeding 3, 381–390.
Jung, H.A., Kim, J.E., Chung, H.Y., Choi, J.S., 2003. Antioxidant princi-
ples of Nelumbo nucifera stamens. Archives of Pharmacal Research
26, 279–285.
Karin, M., Yamamoto, Y., Wang, Q.M., 2004. The IKK NF-kappa B
system: a treasure trove for drug development. Nature Reviews. Drug
Discovery 3, 17–26.
Kiho, T., Yamane, A., Hui, J., Usui, S., Ukai, S., 1996. Polysaccharides
in fungi. XXXVI. Hypoglycemic activity of polysaccharide (CS-F30)
from the cultural mycelium of Cordyceps sinensis and its effect on
glucose metabolism in mouse liver. Biological & Pharmaceutical Bul-
letin 19, 294–296.
Kim, J., Park, E.J., 2002. Cytotoxic anticancer candidates from natu-
ral resources. Current Medicinal Chemistry. Anti-Cancer Agents 2,
485–537.
Kinjo, N., Zang, M., 2001. Morphological and phylogenetic studies on
Cordyceps sinensis distributed in southwestern China. Mycoscience
42, 567–574.
Koyama, K., Imaizumi, T., Akiba, M., Kinostita, K., Takahashi, K.,
Suzuki, A., Yano, S., Horie, S., Watanabe, K., Naoi, Y., 1997.
Antinociceptive components of ganoderma lucidum. Planta Medica
63, 224–227.
Kredich, N.M., 1980. Inhibition of nucleic acid methylation by cordy-
cepin. In vivo synthesis of S-3-deoxyadenosylmethionine by WI-
L2 human lymphoblasts. The Journal of Biological Chemistry 255,
7380–7385.
Krijnen, P.A., Nijmeijer, R., Meijer, C.J., Visser, C.A., Hack, C.E.,
Niessen, H.W., 2002. Apoptosis in myocardial ischaemia and infarc-
tion. Journal of Clinical Pathology 55, 801–811.
Kucharczak, J., Simmons, M., Fan, Y., Gelinas, C., 2003. To be, or not
to be: NF-kappaB is the answer—role of Rel/NF-kappaB in the reg-
ulation of apoptosis. Oncogene 22, 8961–8982.
Kuo, Y.C., Lin, C.Y., Tsai, W.J., Wu, C.L., Chen, C.F., Shiao, M.S., 1994.
Growth inhibitors against tumor cells in Cordyceps sinensis other
than corydcepin and polysaccharides. Cancer Investigation 12, 611–
615.
Kuo, Y.C., Tsai, W.J., Shiao, M.S., Chen, C.F., Lin, C.Y., 1996. Cordyceps
sinensis as an immunomodulatory agent. American Journal of Chinese
Medicine 24, 111–125.
Kure, C.F., Skaar, I., Holst-Jensen, A., Abeln, E.C.A., 2003. The use of
AFLP to relate cheese-contaminating Penicillium strains to specific
points in the production plants. International Journal of Food Micro-
biology 83, 195–204.
Li, S.P., Li, P., Dong, T.T.X., Tsim, K.W.K., 2001. Anti-oxidation activity
of different types of natural Cordyceps sinensis and cultured Cordy-
ceps mycelia. Phytomedicine: International Journal of Phytotherapy
and Phytopharmacology 8, 207–212.
Lin, C.Y., Ku, F.M., Kuo, Y.C., Chen, C.F., Chen, W.P., Chen, A., Shiao,
M.S., 1999. Inhibition of activated human mesangial cell proliferation
by the natural product of cordyceps sinensis (H1-1): an implication for
treatment of IGA mesangial nephropathy. The Journal of Laboratory
and Clinical Medicine 133, 55–63.
Liston, P., Fong, W., Korneluk, R., 2003. The inhibitors of apoptosis:
there is more to life than Bcl2. Oncogene 22, 8568–8580.
Liu, P.-G., Wang, X.-H., Yu, F.-Q., Zheng, H.-D., Chen, J., 2003. Key
taxa of larger members in higher fungi of biodiversity from China.
Acta Botanica Yunnanica 25, 285–296.
Liu, Z.-Y., Yao, Y.-J., Liang, Z.-Q., Liu, A.-Y., Pegler, D.N., Chase, M.W.,
2001. Molecular evidence for the anamorph-teleomorph connection in
Cordyceps sinensis. Mycological Research 105, 827–832.
Li-Weber, M., Giaisi, M., Baumann, S., Treiber, M.K., Krammer, P.H.,
2002. The anti-inflammatory sesquiterpene lactone parthenolide sup-
presses CD95-mediated activation-induced-cell-death in T-cells. Cell
Death and Differentiation 9, 1256–1265.
Lumbsch, H.T., Lindemuth, R., Schmitt, I., 2000. Evolution of filamentous
ascomycetes inferred from LSU rDNA sequence data. Plant Biology
2, 525–529.
Maderna, P., Godson, C., 2003. Phagocytosis of apoptotic cells and the
resolution of inflammation. Biochimica et Biophysica Acta 1639,
141–151.
Manabe, N., Sugimoto, M., Azuma, Y., Taketomo, N., Yamashita, A.,
Tsuboi, H., Tsunoo, A., Kinjo, N., Huang, N.L., Miyamoto, H., 1996.
Effects of the mycelial extract of cultured Cordyceps sinensis on in
vivo hepatic energy metabolism in the mouse. Japanese Journal of
Pharmacology 70, 85–88.
Mayo, G.M., Langridge, P., 2003. Modes of reproduction in Australian
populations of Hypericum perforatum L. (St. John’s wort) revealed
by DNA fingerprinting and cytological methods. Genome/National
Research Council Canada 46, 573–579.
Mcewen, J.G., Taylor, J.W., Carter, D., Xu, J., Felipe, M.S.S., Vilgalys,
R., Mitchell, T.G., Kasuga, T., White, T., Bui, T., Soares, C.M.A.,
2000. Molecular typing of pathogenic fungi. Medical Mycology: Of-
ficial publication of the International Society for Human and Animal
Mycology 38, 189–197.
Morris, P.F., Connolly, M.S., St. Clair, D.A., 2000. Genetic diversity of
Alternaria alternata isolated from tomato in California assessed using
RAPDs. Mycological Research 104, 286–292.
Nagata, S., 1997. Apoptosis by death factor. Cell 88, 355–365.
Nagata, S., Nagase, H., Kawane, K., Mukae, N., Fukuyama, H., 2003.
Degradation of chromosomal DNA during apoptosis. Cell Death and
Differentiation 10, 108–116.

28 E.J. Buenz et al. / Journal of Ethnopharmacology 96 (2005) 19–29
Nakamura, K., Yamaguchi, Y., Kagota, S., Kwon, Y.M., Shinzuka, K.,
Kunitomo, M., 1999. Inhibitory effect of Cordyceps sinensis on spon-
taneous liver metastasis of Lewis lung carcinoma and B16 melanoma
cells in syngeneic mice. Japanese Journal of Pharmacology 79,
335–341.
Negi, M.S., Singh, A., Lakshmikumaran, M., 2000. Genetic variation and
relationship among and within Withania species as revealed by AFLP
markers. Genome/National Research Council Canada 43, 975–980.
Ngan, F., Shaw, P., But, P., Wang, J., 1999. Molecular authentication of
Panax species. Phytochemistry 50, 787–791.
Nieri, P., Adinolfi, B., Morelli, I., Breschi, M.C., Simoni, G., Martinotti,
E., 2003. Genetic characterization of the three medicinal Echinacea
species using RAPD analysis. Planta Medica 69, 685–686.
Nikoh, N., Fukatsu, T., 2000. Interkingdom host jumping underground:
phylogenetic analysis of entomoparasitic fungi of the genus Cordy-
ceps. Molecular Biology and Evolution 17, 629–638.
Nikoh, N., Fukatsu, T., 2001. Evolutionary dynamics of multiple group I
introns in nuclear ribosomal RNA genes of endoparasitic fungi of the
genus Cordyceps. Molecular Biology and Evolution 18, 1631–1642.
Obornik, M., Jirku, M., Dolezel, D., 2001. Phylogeny of mitosporic ento-
mopathogenic fungi: is the genus Paecilomyces polyphyletic? Cana-
dian Journal of Microbiology 47, 813–819.
Park, J.-E., Kim, G.-Y., Park, H.-S., Nam, B.-H., An, W.-G., Cha, J.-
H., Lee, T.-H., Lee, J.-D., 2001. Phylogenetic analysis of caterpillar
fungi by comparing ITS 1-5.8S-ITS 2 ribosomal DNA sequences.
Mycobiology 29, 121–131.
Radimer, K.L., Subar, A.F., Thompson, F.E., 2000. Nonvitamin, non-
mineral dietary supplements: issues and findings from NHANES III.
Journal of the American Dietetic Association 100, 447–454.
Ranamukhaarachchi, D.G., Kane, M.E., Guy, C.L., Li, Q.B., 2000. Mod-
ified AFLP technique for rapid genetic characterization in plants.
BioTechniques 29, 858–866.
Rehner, S.A., Samuels, G.J., 1995. Molecular systematics of the Hypocre-
ales: a teleomorph gene phylogeny and the status of their anamorphs.
Canadian Journal of Botany 73, S816–S823.
Russo, A., Borrelli, F., Campisi, A., Acquaviva, R., Raciti, G., Vanella,
A., 2003. Nitric oxide-related toxicity in cultured astrocytes: effect of
Bacopa monniera. Life Sciences 73, 1517–1526.
Sato, A., 1989. Studies on anti-tumor activity of crude drugs. I. The
effects of aqueous extracts of some crude drugs in shortterm screening
test. Yakugaku Zasshi. Journal of the Pharmaceutical Society of Japan
109, 407–423.
Saunders, J.A., Pedroni, M.J., Penrose, L.D.J., Fist, A.J., 2001. AFLP
analysis of opium poppy. Crop Science 41, 1596–1601.
Schmidt, H., Ehrmann, M., Vogel, R.F., Taniwaki, M.H., Niessen, L.,
2003. Molecular typing of Aspergillus ochraceus and construction
of species specific SCAR-primers based on AFLP. Systematic and
Applied Microbiology 26, 138–146.
Scorrano, L., Korsmeyer, S.J., 2003. Mechanisms of cytochrome c release
by proapoptotic BCL-2 family members. Biochemical and Biophysical
Research Communications 304, 437–444.
Shahed, A.R., Kim, S.I., Shoskes, D.A., 2001. Down-regulation of apop-
totic and inflammatory genes by Cordyceps sinensis extract in rat kid-
ney following ischemia/reperfusion. Transplantation Proceedings 33,
2986–2987.
Shiao, M.-S., Wang, Z.-N., Lin, L.-J., Lien, J.-Y., Wang, J.-J., 1994. Pro-
files of nucleosides and nitrogen bases in Chinese medicinal fun-
gus Cordyceps sinensis and related species. Botanical Bulletin of
Academia Sinica New Series 35, 261–267.
Spatafora, J.W., Blackwell, M., 1993. Molecular systematics of unituni-
cate perithecial ascomycetes: the Clavicipitales-Hypocreales connec-
tion. Mycologia 85, 912–922.
Starr, M., Post, T., Huus, K., 1993. The Games China Plays. Newsweek,
62–63.
Steinkraus, D.C., Whitfield, J.B., 1994. Chinese Caterpillar Fungus
and World Record Runners. American Entomologist. Winter, 235–
239.
Sugawara, T., Chan, P.H., 2003. Reactive oxygen radicals and pathogen-
esis of neuronal death after cerebral ischemia. Antioxidants & Redox
Signaling 5, 597–607.
Suh, S.-O., Noda, H., Blackwell, M., 2001. Insect symbiosis: derivation
of yeast-like endosymbionts within an entomopathogenic filamentous
lineage. Molecular Biology and Evolution 18, 995–1000.
Suh, S.-O., Spatafora, J.W., Ochiel, G.R.S., Evans, H.C., Blackwell, M.,
1998. Molecular phylogenetic study of a termite pathogen Cordyce-
pioideus bisporusogia. Mycologia 90, 611–617.
Sung, G.-H., Spatafora, J.W., Zare, R., Hodge, K.T., Gams, W., 2001.
A revision of Verticillium sect. Prostrata, II. Phylogenetic analy-
ses of SSU and LSU nuclear rDNA sequences from anamorphs
and teleomorphs of the Clavicipitaceae. Nova Hedwigia 72, 311–
328.
Terashima, K., Matsumoto, T., Hasebe, K., Fukumasa-Nakai, Y., 2002.
Genetic diversity and strain-typing in cultivated strains of Lentinula
edodes (the shii-take mushroom) in Japan by AFLP analysis. Myco-
logical Research 106, 34–39.
Trubiani, O., Salvolini, E., Staffolani, R., Di Primio, R., Mazzanti, L.,
2003. DMSO modifies structural and functional properties of RPMI-
8402 cells by promoting programmed cell death. International Journal
of Immunopathology and Pharmacology 16, 253–259.
Vaux, D.L., Korsmeyer, S.J., 1999. Cell death in development. Cell 96,
245–254.
Vos, P., Hogers, R., Bleeker, M., Reijans, M., Van De Lee, T., Hornes, M.,
Frijters, A., Pot, J., Peleman, J., Kulper, M., Zabeau, M., 1995. AFLP:
a new technique for DNA fingerprinting. Nucleic Acids Research 23,
4407–4414.
Wallner, E., Weising, K., Rompf, R., Kahl, G., Kopp, B., 1996. Oligonu-
cleotide fingerprinting and RAPD analysis of Achillea species: charac-
terization and long-term monitoring of micropropagated clones. Plant
Cell Reports 15, 647–652.
Wu, Z.-L., Wang, X.-X., Cheng, W.-Y., 2000. Inhibitory effect of Cordy-
ceps sinensis and Cordyceps militaris on human glomerular mesan-
gial cell proliferation induced by native LDL. Cell Biochemistry and
Function 18, 93–97.
Xu, R., Peng, X.E., Chen, G.Z., Chen, G.L., 1992. Effects of Cordy-
ceps sinensis on natural killer activity and colony formation of B16
melanoma. Chinese Medical Journal 105, 97–101.
Yamaguchi, Y., Kagota, S., Nakamura, K., Shinozuka, K., Kunitomo, M.,
2000a. Antioxidant activity of the extracts from fruiting bodies of
cultured Cordyceps sinensis. Phytotherapy Research: PTR 14, 647–
649.
Yamaguchi, Y., Kagota, S., Nakamura, K., Shinozuka, K., Kunitomo,
M., 2000b. Inhibitory effects of water extracts from fruiting bodies
of cultured Cordyceps sinensis on raised serum lipid peroxide levels
and aortic cholesterol deposition in atherosclerotic mice. Phytotherapy
Research: PTR 14, 650–652.
Yang, L.Y., Chen, A., Kuo, Y.C., Lin, C.Y., 1999. Efficacy of a pure
compound H1-A extracted from Cordyceps sinensis on autoimmune
disease of MRL LPR/LPR mice. The Journal of Laboratory and Clin-
ical Medicine 134, 492–500.
Yang, L.Y., Huang, W.J., Hsieh, H.G., Lin, C.Y., 2003. H1-A extracted
from Cordyceps sinensis suppresses the proliferation of human mesan-
gial cells and promotes apoptosis, probably by inhibiting the tyrosine
phosphorylation of Bcl-2 and Bcl-XL. The Journal of Laboratory and
Clinical Medicine 141, 74–83.
Yu, L., Chen, G.Z., Jiang, D.Z., 1993. Combined traditional Chinese and
western medicine. Effect of Cordyceps sinensis on erythropoiesis in
mouse bone marrow. Chinese Medical Journal 106, 313–316.
Zang, Q., He, G., Zheng, Z., Liu, J., Wang, S., Huang, J., Du, D., Zeng,
Q., Al, E., 1985. Pharmacological action of the polysaccharide from
cordyceps (Cordyceps sinensis). Zhong cao yao (Chinese Traditional
and Herbal Drugs) 16, 306–311.
Zare, R., Gams, W., Culham, A., 2000. A revision of Verticillium sect.
Prostrata, I. Phylogenetic studies using ITS sequences. Nova Hedwigia
71, 465–480.

E.J. Buenz et al. / Journal of Ethnopharmacology 96 (2005) 19–29 29
Zeller, K.A., Bowden, R.L., Leslie, J.F., 2003. Diversity of epidemic
populations of Gibberella zeae from small quadrats in Kansas and
North Dakota. Phytopathology 93, 874–880.
Zerega, N.J.C., Mori, S., Lindqvist, C., Zheng, Q., Motley, T.J., 2002.
Using amplified fragment length polymorphisms (AFLP) to identify
black cohosh (Actaea racemosa). Economic Botany 56, 154–164.
Zhang, K.Y.B., Leung, H.-W., Yeung, H.W., Wong, R.N.S., 2001. Differ-
entiation of Lycium barbarum from its related Lycium species using
random amplified polymorphic DNA. Planta Medica 67, 379–381.
Zhang, Z.H.W., Liao, S., Li, J., Lei, L., Lui, J., Leng, F., Gong, W.,
Zhang, H., Wan, L., Wu, R., Li, S., Luo, H., Zhu, F., 1995. Clinical
and laboratory studies of JinShuiBao in scavenging oxygen free radi-
cals in elderly senescent XuZheng patients. Journal of Administration
Traditional Chinese Medicine, 5.
Zhao Long, W., Xiao Xia, W., Wei Ying, C., 2000. Inhibitory effect
of Cordyceps sinensis and Cordyceps militaris on human glomerular
mesangial cell proliferation induced by native LDL. Cell Biochemistry
and Function 18, 93–97.
Zheng, Z., Lee, J.E., Yenari, M.A., 2003. Stroke: molecular mechanisms
and potential targets for treatment. Current Molecular Medicine 3,
361–372.
Zhu, J.S., Gm, H., K.J., 1998a. The scientific rediscovery of an ancient
Chinese herbal medicine: Cordyceps sinensis: part I. Journal of Alter-
native and Complementary Medicine (New York, NY). The Journal
of Alternative and Complementary Medicine: Research on Paradigm,
Practice, and Policy 4, 289–303.
Zhu, J.S., Halpern, G.M., Jones, K., 1998b. The scientific redis-
covery of a precious ancient Chinese herbal regimen: Cordy-
ceps sinensis: part II. Journal of Alternative and Complementary
Medicine (New York). The Journal of Alternative and Complemen-
tary Medicine: Research on Paradigm, Practice, and Policy 4, 429–
457.