Research Article Cordycepin Increases Nonrapid Eye ...downloads.hindawi.com/journals/ecam/2013/840134.pdfCordycepin ( 3 -deoxyadenosine) is a naturally occurring adenosine analogue

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2013 Article ID 840134 8 pageshttpdxdoiorg1011552013840134

Research ArticleCordycepin Increases Nonrapid Eye Movement Sleep viaAdenosine Receptors in Rats

Zhenzhen Hu12 Chung-Il Lee1 Vikash Kumar Shah1 Eun-Hye Oh1 Jin-Yi Han3

Jae-Ryong Bae4 Kinam Lee4 Myong-Soo Chong4 Jin Tae Hong1 and Ki-Wan Oh1

1 College of Pharmacy Chungbuk National University Cheongju 361-763 Republic of Korea2Department of Pathophysiology College of Medicine Nanchang University Nanchang Jiangxi 33006 China3 Research Institute of Veterinary Medicine Chungbuk National University Cheongju 361-763 Republic of Korea4College of Oriental Medicine Wonkwang University Iksan 570-749 Republic of Korea

Correspondence should be addressed to Ki-Wan Oh kiwanchungbukackr

Received 6 December 2012 Revised 22 March 2013 Accepted 29 March 2013

Academic Editor Shao Li

Copyright copy 2013 Zhenzhen Hu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Cordycepin (31015840-deoxyadenosine) is a naturally occurring adenosine analogue and one of the bioactive constituents isolated fromCordyceps militarisCordyceps sinensis species of the fungal genus Cordyceps It has traditionally been a prized Chinese folkmedicine for the human well-being Because of similarity of chemical structure of adenosine cordycepin has been focused onthe diverse effects of the central nervous systems (CNSs) like sleep regulation Therefore this study was undertaken to knowwhether cordycepin increases the natural sleep in rats and its effect is mediated by adenosine receptors (ARs) Sleep was recordedusing electroencephalogram (EEG) for 4 hours after oral administration of cordycepin in rats Sleep architecture and EEG powerspectra were analyzed Cordycepin reduced sleep-wake cycles and increased nonrapid eye movement (NREM) sleep Interestinglycordycepin increased 120579 (theta) waves power density during NREM sleep In addition the protein levels of AR subtypes (A1 A2Aand A2B) were increased after the administration of cordycepin especially in the rat hypothalamus which plays an important rolein sleep regulation Therefore we suggest that cordycepin increases theta waves power density during NREM sleep via nonspecificAR in rats In addition this experiment can provide basic evidence that cordycepin may be helpful for sleep-disturbed subjects

1 Introduction

Purines are ubiquitous molecules with important roles inthe regulation of metabolic networks and signal transductionevents In the central nervous systems (CNSs) adenosine andATP modulate the sleep-wake cycles acting as ligands ofspecific transmembrane receptors and as allosteric effectorsof key intracellular enzymes for brain energy expenditure[1] Adenosine levels are influenced by neuronal activityAdenosine is a secondary by-product of the breakdownof ATP and cAMP When ATP is coreleased with neuro-transmitters ectonucleotidases in the extracellular space canrapidly dephosphorylateATPADP andAMP into adenosineATP release from astrocytes also contributes to extracellularlevels of adenosine that have a powerful modulatory effect onsynaptic transmission [2] The role of this astrocyte-derived

adenosine in sleep-waking homeostasis was recently investi-gated Further research showed that systemic administrationof adenosine its analogs or inhibitors of its metabolismincrease nonrapid eye movement (NREM) sleep in rodentsespecially [3]

The local administration of adenosine andor adenosinereceptors (ARs) agonists into the medial preoptic area ofhypothalamus magnocellular cholinergic basal forebrainbrainstem cholinergic areas the laterodorsal and pedunculo-pontine tegmental nuclei (LDTPPT) and pontine reticularformation leads to sleep or reduction of wakefulness [4ndash6] Inthe basal forebrain both cholinergic and noncholinergic neu-ronal activities are associated with promoting wakefulness[7] The somnogenic effects of adenosine may be due to theinhibition of neuronal activity in both cholinergic and non-cholinergic neurons of the basal forebrain In addition the

2 Evidence-Based Complementary and Alternative Medicine

modulatory effects of sleep deprivation on the A1R mRNA

in the cholinergic basal forebrain suggest the significanceof an adrenergic pathway in the long-term effects of sleepdeprivation on the quality of ensuring sleep [8] Cholinergicneurons of LDT and PPT also comprise the cholinergicldquoarousal systemrdquo During the transition from waking to sleepthe firing rate of LDTPPT neurons markedly is decreasedreducing the cholinergic tone of their target sites and thusfacilitating the transition to sleep [9] In particular theventrolateral preoptic area of the hypothalamus contains apopulation of sleep-active neurons and is hypothesized to bean important part of the somnogenic process [10]

The four adenosine forms make up the family of G-protein-coupled AR A

1 A2AA2B and A3 [11]The difference

between the four ARs is found in their affinity for adenosinein the type of G proteins that they associate with and thesignaling pathways that are activated in the target cellsFor sleep-waking homeostasis A

1R and A

2AR have receivedthe most attention due to their expression pattern in thenervous system the availability of selective agonists andantagonists and selectivemolecular lesions of genes encodingthe receptor subtypes A

1and A

3Rs have high and low affinity

for adenosine [12] Possible changes in adenosine functioningdue to the aging process have been observed in animalmodels and abnormalities in the adenosine system couldalso explain primary insomnia or the reduced amount ofdelta waves sleep and increased sensitivity to caffeine in somesubjects with sleep deficits Caffeine is a methylated derivateof xanthine with profound effects on the onset and qualityof sleep episodes [13] This purine acts principally as anantagonist of the A

2AR Adenosine and ATP in the nervoussystems are the bridge between metabolic activity recoveryfunction and purinergic transmission that underlies the dailywake-sleep cycle in mammals Modulators of purine actionshave the potential to alleviate insomnia and other sleepdisorders based on their physiopathological role during thesleep process [14]

Cordycepin (31015840-deoxyadenosine Figure 1) is a naturallyoccurring adenosine analogue and one of the bioactiveconstituents of Cordyceps sinensismilitaris [15 16] C sinen-sismilitaris has been used for hundreds of years as a tradi-tional medicine in treating disorders of the lung and kidneythrough mechanisms of immunomodulation [17] Becauseof similarity of chemical structure of adenosine cordycepinhas been interested in the diverse effects of CNS like sleepregulation It also has traditionally been focused on thetreatment of insomnia In particular cordycepin is known tobe a bioactive constituent to regulate homeostatic function[18] Recently some herbs have been the charmingmedicinesfor considerable sufferers with sleep disabilities or insomnia[19] Most remedies of sleep aid originated from herbs havebeen targeted on GABAA systems However recent mor-phological and functional studies have identified AR in theventrolateral preoptic areas of the hypothalamus that plays animportant role in sleep regulation [20] Therefore this studywas designed to know whether cordycepin increases sleepvia ARThis experiment also can provide basic evidence thatcordycepin may be helpful for the treatment of insomnia

2 Materials and Methods

21 Experimental Animals MaleWistar rats (Samtako OsanKorea) weighing 250ndash300 g were used for sleep recordingand western blot Each rat was housed in each acrylic cage(45 times 60 times 25 cm) with water and food available ad libitumunder an artificial 12 h light-dark cycle (lights on at 700am) and at constant temperature (22 plusmn 2∘C) To ensureadaptation to the new environment mice and rats werekept in the departmental holding room for 1 week beforetesting This study was performed in accordance with theChungbuk National University Laboratory Animal ResearchCenter guidelines for the care and use of laboratory animals

22 Experimental Procedure After 7 days postsurgical recov-ery cordycepin (2 and 4mgkg in distilled saline) wasadministered to animals for 5 days once per day On the 5thday 1 h after final cordycepin treatment animals were cagedin recording system and spontaneous sleeping was recorded

23 Brain Surgery and EEG Recording Each rat was implant-ed with a transmitter (Data Sciences International TA11CTA-F40 MN USA) for recording EEG and activity via telemetryThe body of the transmitter was implanted subcutaneouslyoff the midline and posterior to the scapula and was attachedto the skin with 3 sutures for stabilization Leads from thetransmitter led subcutaneously to the skull and the bare endswere placed in contact with the dura through holes madein the skull (A 20 (Bregma) L 15 P 70 (Bregma) L 15contralateral) [21] The electrodes were anchored to the skullwith screws and dental cement All surgical procedures wereperformed stereotaxically under aseptic conditions Surgicalanesthesia was achieved with pentobarbital (50mgkg ip)and all efforts were made to minimize the suffering of theanimals Telemetric recording of cortical EEG and activitywas conducted using procedures similar to previous reportsFor the EEG signal the gain of transmitters was set atminus05+05 volts perunits times 2 and the raw signals generatedfrom the transmitter were in the range of 05ndash200Hz Thesignals were processed by a Data Sciences Internationalanalog converter and routed to anAD converter (Eagle PC30USA) housed in a PC computer The AD converter digitizedthe EEG and activity signals at 128Hz The digitized datawere transferred to the computer and displayed graphicallyAn online fast Fourier transformation (FFT) was performedon the EEG data at 10 sec intervals during data acquisition(1024 samples) after a Hanning window treatment The FFTanalysis generated power density values from00 to 200Hz ata resolution of 05HzThe FFT data were further averaged inthe range of 0 to 20Hz for every 10 secThe sleep data and FFTresults were saved to the hard disk every 10 sec for additionaloffline analysis Movement of the animal in relation to thetelemetry receiver generated transistor-transistor logic (TTL)pulses that were collected and counted as a measure ofactivity Cordycepin was administered 1 hour before the EEGrecording Recording began at 900 am for 4 h Seven or eightrats were used in each group

Evidence-Based Complementary and Alternative Medicine 3

HO HO

O O

O

O

OHOH OH

N N

N

N N

N N

N

NN N

N

NH2 NH2 H3C

CH3

CH3

Cordycepin Adenosine

Caffeine

Figure 1 Chemical structures of cordycepin adenosine and caffeine

24 Analysis of Sleep Architecture The amounts of time inwakefulness NREM and REM sleep were determined fromthe digitized data at 10 sec intervals using sleep analysissoftware SleepSign 21 (KISSEI Comtec Co Ltd MatsumotoJapan) Briefly the software discriminates wakefulness ashigh-frequency low-amplitude EEG NREM was scoredbased on the presence of spindles interspersed with slowwaves in the EEG EEG power during REM is signifi-cantly reduced in lower frequency 120575-wave (075ndash40Hz) andincreased in the range of 120579-wave activity (50ndash90Hz peakat 75Hz) The time spent (min) in NREM REM total sleeptime (NREM+REM) and numbers of sleep-wake cycle wereprocessed to obtain 4 h period totals for each rat We furthercalculated the time of each recording spent in the sleep-wakestate (wake NREM and REM) The absolute EEG powerduring wakefulness NREM and REM were calculated in05Hz bins from 05 to 20Hz for the entire 4 h reading of eachrecording process [22] Data from seven or eight rats in eachgroup were analyzed

25 Membrane Protein Preparation After deep anesthesia(induced by diethyl ether) animals were decapitated andthe brain was quickly removed and chilled in ice cold salineCoronal sections were made using a Rodent Brain Matrix(ASI Instruments) This was immediately followed by a1200120583m section containing the medial basal hypothalamusand samples were immediately frozen on dry ice and storedat minus80∘C [23] Frozen tissue samples were homogenizedin PRO-PREPTM protein extraction solution (iNTRONBiotechnology Inc) The homogenate was centrifuged at15000 g at 4∘C for 20min and the supernatant was recoveredA portion of the supernatant was collected to determinethe protein concentration and for western blot analysis Theconcentration of total protein was determined by a modifiedLowry method using bovine serum albumin as a standardThe samples were stored at minus20∘C

26 Western Blot The total proteins of hypothalamus wereloaded in each lane and sodium dodecyl sulphate poly-acrylamide gel electrophoresis (SDSPAGE) was performedusing 12 polyacrylamide gels Proteins were transferred toPVDF membranes (Amersham Hybond-P GE Healthcare)using a semidry transfer system Receptors were detectedwith the following primary antibodies rabbit antiadenosinereceptor A

1(diluted 1 1000 in PBS containing 05 Tween

20 Abcam) mouse antiadenosine receptor A2A (diluted

1 1000 in PBS containing 05 Tween 20) and goat anti-adenosine receptor A

2B (diluted 1 1000 in PBS containing05 Tween 20) The following horseradish peroxidase sec-ondary antibodies were used goat antrabbit IgG (diluted1 5000) rabbit antimouse IgG and antigoat IgG (1 5000)After stripping membranes were developed with rabbitanti-GAPDH (1 1000 Santa Cruz Biotechnology Inc USA)followed by goat antirabbit IgG to confirm equal proteinloading Immunoreactive bands were developed with aBM Chemiluminescence Detection Kit (Roche Diagnostics)Quantitative analysis of detected bands was performed withdensitometric scanning and all values were normalized usingGAPDH as a standard [24]

27 Statistical Analysis All data were analyzed using SPSS170 software (SPSS) Significant differences after one-wayANOVAs were measured by post hoc Holm-Sidak test 119875 lt005was considered to be significantThe values are expressedasmean plusmn SEMAll statistical analyses were conducted usingSigmaStat software

3 Results

31 Effects of Cordycepin on the Number of Sleep-Wake CyclesCordycepin (2mgkg 119875 lt 005) and (4mgkg 119875 lt001) significantly reduced the number of sleep-wake cyclesrespectively compared with that of the control (Figure 2)

32 Effects of Cordycepin on Sleep Architecture Cordycepin(2 and 4mgkg) significantly increased NREM (119875 lt 0005and 119875 lt 005) and decreased REM sleep (119875 lt 001 and 119875 lt001) However both wakefulness and total sleep time werenot changed significantly compared with that of the control(Figure 3)

33 Effects of Cordycepin on EEG Power Density duringTotal Sleep Time No significant changes in delta wave thetawave and alpha waves power density during total sleep timewere observed cordycepin- (2 and 4mgkg) treated groupscompared with that of the control (Figure 4)

34 Effects of Cordycepin on EEG Power Density duringNREMSleep Interestingly cordycepin decreased delta waves

4 Evidence-Based Complementary and Alternative MedicineN

umbe

r of t

rans

ition

s

600

400

200

0

Control 2 4

lowast

lowastlowast

Cordycepin (mgkg)

Figure 2 Effects of cordycepin (2 and 4mgkg) on sleep-wakecycles Values are expressed as the mean plusmn SEM lowast119875 lt 005 andlowastlowast

119875 lt 001 compared with that of the control Five to six animalswere used in each group

Wake Sleep NERM REM

ControlCordycepin 2 mgkgCordycepin 4 mgkg

200

150

100

50

0

Tim

e spe

nt (m

in)

lowast

lowastlowastlowastlowast

lowastlowastlowast

Figure 3 Effects of cordycepin (2 and 4mgkg) on rat sleeparchitecture Values are expressed as the mean plusmn SEM NREMnonrapid eye movement REM rapid eye movement lowast119875 lt 005lowastlowast

119875 lt 001 and lowastlowastlowast119875 lt 0005 compared with that of the controlFor more details refer Figure 1

(119875 lt 0005) during NREM sleep However theta waves (119875 lt0005) during NREM sleep significantly were increased bycordycepin (4mgkg) No changes in alpha waves powerdensity during NREM sleep were observed compared withthat of control (Figure 5)

35 Effects of Cordycepin on EEG Power Density during REMSleep No significant changes were observed in delta wavetheta wave or alpha wave power density during REM sleep incordycepin- (2 and 4mgkg) treated groups compared withthat of the control (Figure 6)

120575 waves 120579 waves 120572 waves

80

100

60

40

20

0

Pow

er d

ensit

y (

)


Figure 4 Effects of cordycepin (2 and 4mgkg) treatment onEEG power density during total sleep time EEG power densitiesin delta wave theta wave and alpha wave spectral bandwidthswere evaluated during total sleep time No significant differencescompared with that of the control For more details refer Figure 1


80

100

60

40

20

0

Pow

er d

ensit

y (

)

lowastlowastlowast

lowastlowastlowast


Figure 5 Effects of cordycepin (2 and 4mgkg) on EEG powerdensity during NREM sleep EEG power densities in delta wavetheta wave and alpha wave spectral bandwidths were evaluatedValues are expressed as the mean plusmn SEM of EEG power densities inthree selected frequency bands during NREM sleep lowastlowastlowast119875 lt 0005compared with that of the control For more details refer Figure 1

36 Effects of Cordycepin on the Protein Levels of the A1R

A2119860R and A

2119861R Subtypes in the Rat Hypothalamus The

protein levels of the A1R A2AR and A

2BR subtypes in thehypothalamusweremeasured bywestern blot after codycepinadministration Cordyceps (4mgkg) increased the proteinlevels of A

1R A2AR and A

2BR subtypes ( lowastlowastlowast119875 lt 0005lowastlowast

119875 lt 001 and lowast119875 lt 005) compared with that of thecontrol (Figure 7)



80

100

60

40

20

0

Pow

er d

ensit

y (

)


Figure 6 Effects of cordycepin (2 and 4mgkg) on EEG powerdensity during REM sleep EEG power densities in delta wavetheta wave and alpha wave spectral bandwidths were evaluatedValues are expressed as the mean plusmn SEM of EEG power densitiesin three selected frequency bands for the REM sleep No significantdifferences compared with that of the control Five to six animalswere used in each group For more details refer Figure 1

4 Discussion

C sinensismilitaris which contain cordycepin (31015840-deoxyade-nosine) a naturally occurring adenosine analogue and oneof the bioactive constituents have been used for hundreds ofyears as traditional medicines in treating insomnia [15] Inthis study cordycepin reduced sleep-wake cycles increasedNREM sleep and decreased REM sleep It also increasedtotal sleep time in rodents and decreased wakefulnessInterestingly power spectral analysis showed that cordycepinsignificantly increased theta waves power density duringNREM sleep Therefore it will be suggested that cordycepinan adenosine analogue plays a role in the modulation oftheta oscillations in NREM sleep Sleep in most mammalsis divided into two major types of sleep REM sleep andNREM sleep REM sleep is characterized by fast waves sleepwith muscle atonia activation of brain and eye movementNREM sleep characterized by slow waves sleep is emergedin delta waves [25] During NREM sleep neuronal activitymetabolic rate and brain temperature are low From the EEGexperiment cordycepin increased NREM sleep and reducedREM sleep However it tended to increase slow waves intotal sleep although they were not significantly increased Onthe contrary theta waves in NREM sleep were significantlyincreased delta waves in NREM sleep slightly decreased andno change in alpha waves was observedTheta waves betweenalpha and delta waves in wavelength specially were increasedin NREM sleep [26]

Multiple interacting neurotransmitter systems in thebrain stem hypothalamus and basal forebrain converge ontocommon effector systems in the thalamus and cortex Sleepresults from the inhibition of wake-promoting systems by

Rela

tive O

D

00

25

50

75

100

ControlCordycepin 4 mgkg

lowastlowastlowastlowastlowastlowast

A1 A2A A2B

Figure 7 Expression of AR A1 A2A and A

2B in the rat hypothala-mus after cordycepin treatment AR subtypes in the rat hypothala-mus after cordycepin treatment were analyzed by western blottingThe intensity of the immunoreactive bands of 3 to 4 independentexperiments was measured by densitometry scanning and normal-ized using glyceraldehyde 3-phosphate dehydrogenase (GAPDH)as a standard (bar graph) Results are presented as the percentageimmunoreactivity detected in the hypothalamus with respect tothe GAPDH protein loading control Values are expressed as themean plusmn SEM lowast119875 lt 005 lowastlowast119875 lt 001 and lowastlowastlowast119875 lt 0005 comparedwith that of the control

homeostatic sleep factors such as adenosine nitric oxide andGABAergic neurons in the preoptic area of the hypothalamusresulting in large-amplitude slow EEG oscillations [27] Oneubiquitous neuromodulatory system in the CNS is operatedby adenosine Adenosine is an important homeostatic sleepfactor acting in basal forebrain and preoptic areas throughA1R and A

2AR Recent morphological and functional studieshave identified AR in the ventrolateral preoptic areas of thehypothalamus that plays an important role in sleep regulation[28] Adenosine neuromodulation is mostly conceived asan inhibitory system that restrains excitatory transmissionthrough activation of inhibitory A

1R Furthermore adeno-

sine modulates the neuronal activity of basal forebrain andpreopticanterior hypothalamic in the control of behavioralstate [7] The sleep-inducing effect of adenosine is most effi-cient when adenosine or its agonists are applied locally intothe basal forebrain and preopticanterior hypothalamus [2930] The compensatory sleep response to sleep deprivationis significantly reduced when the increase in adenosine orA2R activation is blocked [31] A

2AR is expressed in the brainareas such as striatum nucleus accumbens and olfactorytubercle [32] Selective A

2AR agonist administration into thesubarachnoid space adjacent to the basal forebrain and thelateral preoptic area indduces NREM sleep [33] In contrastto other brain areas increases in extracellular adenosinelevels in the basal forebrain are very sensitive to even shortperiods of prolonged waking adenosine levels continue torise throughout the waking period and do not decline untilsleep is initiated As the basal forebrain is essential for


wake promotion the sleep-inducing effect is thought to bemediated via inhibition of the wake-active basal forebraincells [34 35] Prolonged waking activates inducible nitricoxide synthase in the basal forebrain which causes adenosinerelease and recovery sleep through energy depletion Thereare numerous reports indicating that endogenous adenosineis a candidate for the homeostatic sleep factor theory induct-ing sleep after prolonged wakefulness [35]

Adenosinersquos pivotal role in sleep modulation is stronglysupported by the subjective and EEG-defined arousal pro-duced by its antagonists caffeine and theophylline as wellas by the fact that extracellular adenosine concentration islinked to neuronal metabolic activity [1 36] Researchesshowed that systemic or local administration of adenosineits analogs or inhibitors of its metabolism increase especiallyNREM sleep in rodents [37] The mechanism by whichcordycepin increased NREM sleep and decreased wakeful-ness was probably achieved through activation of adenosinereceptors Thereby we suggest that cordycepin mediatedeffects on sleep-wake states are site and receptor dependentFurthermore cordycepin may pharmacologically act in thesame manner as that of adenosine as cordycepin has affinityat the A

1 A2 and A

3receptors [38] For sleep-waking

homeostasis A1R and A

2AR have received the most attentiondue to their expression pattern in the nervous system A

1R is

widely distributed in CNS and inhibits especially cholinergicneurons from forebrain and LDTPPT [39] Stimulation ofA1R results in adenylate cyclase inhibition and phospholipase

C activation Higher level of A1R agonist or higher expres-

sion of A1R potentiates the phospholipase C pathway [40]

The activation of presynaptic A1R inhibits neurotransmitter

release mostly of excitatory neurotransmitter such as gluta-mate and acetylcholine From this study cordycepin activatedAR subtypes So the present report strongly suggests thatcordycepin enhances NREM sleep in activating upon AR

Therefore administration of cordycepin an adenosineanalogue widely modulated the power spectral densities ofEEG architecture Increase of total sleep and NREM sleepby cordycepin might be related to the action upon ARMoreover themechanismswhich cordycepinmodulates EEGarchitecture and sleep behaviors may be attributed to thesimilar chemical structure of adenosine The use of severalherbs in the treatment of sleep disorder such as insomnia isregarded as a developing market for agents acting on GABAreceptors St Johnrsquos Wort valerian extracts and ginsengspecies are the best examples of this issue [41ndash43] In additionginseng extract enhanced the total sleep and NREM sleepand reduced the number of sleep-wake cycles [44 45]Nevertheless this is the first experiment that cordycepin anatural product for the treatment of sleep disorders such asinsomnia acts on AR

Cordycepin is quickly deaminated by adenosine deami-nase and rapidly metabolized to an inactive metabolite 31015840-deoxyhypoxanthinosine in vivo [46] Adenosine is otherwisetransported into cells by several nucleoside permeases andthen the adenosine will be phosphorylated to form ATP byadenosine kinase If the cell does not utilize the adenosine itwill be deaminated to hypoxanthinosine by adenosine deam-inase inside the cell [47] So adenosine and cordycepin had

short elimination half-lives and high rates of clearance [18]We also presume that cordycepin has short-acting compoundin the regulation of sleep Therefore EEG recording wasperformed for 4 hours

5 Conclusions

Cordycepin targeting on AR can be helpful for sleep dis-turbance subjects Thus the results also indicated a role forcordycepin an adenosine analogue in the regulation of sleepand held promise for a new class of compounds as potentialagents in the treatment of sleep disorders such as insomniaCordycepin as an adenosine analogue increased NREM sleepand decreased REM sleep suppressing waking Cordycepinalso increases theta waves power density during NREM sleepvia nonspecific AR in the ventrolateral preoptic area of thehypothalamus of the rats Further research would be neces-sary to understand the cellular andmolecular mechanisms ofcordycepin-induced sleep modulation in the brain

Conflict of Interest

The authors declare that they have no conflict of interests

Acknowledgments

This work was supported by the research grant of ChungbukNational University in 2011 and was kindly gifted from Si-Kwan Kim a Professor of Konkuk University

References

[1] M Diaz-Munoz and R Salin-Pascual ldquoPurine molecules ashypnogenic factors role of adenosine ATP and caffeinerdquo Cen-tral Nervous System Agents in Medicinal Chemistry vol 10 no4 pp 259ndash268

[2] O Pascual K B Casper C Kubera et al ldquoNeurobiologyastrocytic purinergic signaling coordinates synaptic networksrdquoScience vol 310 no 5745 pp 113ndash116 2005

[3] M Radulovacki ldquoRole of adenosine in sleep in ratsrdquo Reviewsin Clinical and Basic Pharmacology vol 5 no 3-4 pp 327ndash3391985

[4] GAMarks andCG Birabil ldquoEnhancement of rapid eyemove-ment sleep in the rat by cholinergic and adenosinergic agonistsinfused into the pontine reticular formationrdquoNeuroscience vol86 no 1 pp 29ndash37 1998

[5] CM PortasMThakkarDG Rainnie RWGreene andRWMcCarley ldquoRole of adenosine in behavioral state modulation amicrodialysis study in the freely moving catrdquo Neuroscience vol79 no 1 pp 225ndash235 1997

[6] S R Ticho andM Radulovacki ldquoRole of adenosine in sleep andtemperature regulation in the preoptic area of ratsrdquo Pharmacol-ogy Biochemistry and Behavior vol 40 no 1 pp 33ndash40 1991

[7] R E Strecker S Morairty M MThakkar et al ldquoAdenosinergicmodulation of basal forebrain and preopticanterior hypotha-lamic neuronal activity in the control of behavioral staterdquoBehavioural Brain Research vol 115 no 2 pp 183ndash204 2000

[8] D Stenberg ldquoNeuroanatomy and neurochemistry of sleeprdquoCellular and Molecular Life Sciences vol 64 no 10 pp 1187ndash1204 2007


[9] M Steriade S Datta D Pare G Oakson and R Curro DossildquoNeuronal activities in brain-stem cholinergic nuclei related totonic activation processes in thalamocortical systemsrdquo Journalof Neuroscience vol 10 no 8 pp 2541ndash2559 1990

[10] S Morairty D Rainnie R McCarley and R Greene ldquoDis-inhibition of ventrolateral preoptic area sleep-active neuronsby adenosine a new mechanism for sleep promotionrdquo Neuro-science vol 123 no 2 pp 451ndash457 2004

[11] M G Collis and S M O Hourani ldquoAdenosine receptorsubtypesrdquo Trends in Pharmacological Sciences vol 14 no 10 pp360ndash366 1993

[12] P G Baraldi M A Tabrizi F Fruttarolo R Romagnoli andD Preti ldquoRecent improvements in the development of A2Badenosine receptor agonistsrdquo Purinergic Signalling vol 5 no 1pp 3ndash19 2009

[13] S H Synder and P Sklar ldquoBehavioral and molecular actions ofcaffeine focus and adenosinerdquo Journal of Psychiatric Researchvol 18 no 2 pp 91ndash106 1984

[14] R Sharma S Engemann P Sahota and M M ThakkarldquoRole of adenosine and wake-promoting basal forebrain ininsomnia and associated sleep disruptions caused by ethanoldependencerdquo Journal of Neurochemistry vol 115 no 3 pp 782ndash794 2010

[15] J Melling F C Belton D Kitching andW R Stones ldquoProduc-tion of pure cordycepin (31015840-deoxyadenosine) from Cordycepsmilitarisrdquo Journal of Pharmacy and Pharmacology vol 24 p125 1972

[16] B S Pan C Y Lin and B M Huang ldquoThe effect of cordycepinon steroidogenesis and apoptosis inMA-10mouse leydig tumorcellsrdquo Evidence-based Complementary andAlternativeMedicinevol 2011 Article ID 750468 2011

[17] N Yoshikawa M Kunitomo S Kagota K Shinozuka and KNakamura ldquoInhibitory effect of cordycepin on hematogenicmetastasis of B16-F1 mouse melanoma cells accelerated byadenosine-51015840-diphosphaterdquo Anticancer Research vol 29 no 10pp 3857ndash3860 2009

[18] Y J Tsai L C Lin and T H Tsai ldquoPharmacokinetics ofadenosine and cordycepin a bioactive constituent of cordycepssinensis in ratrdquo Journal of Agricultural and Food Chemistry vol58 no 8 pp 4638ndash4643 2010

[19] Y KWing ldquoHerbal treatment of insomniardquoHong KongMedicalJournal vol 7 no 4 pp 392ndash402 2001

[20] S Ferre I Diamond S R Goldberg et al ldquoAdenosine A2Areceptors in ventral striatum hypothalamus and nociceptivecircuitry implications for drug addiction sleep and painrdquoProgress in Neurobiology vol 83 no 5 pp 332ndash347 2007

[21] G Paxinos C Watson M Pennisi and A Topple ldquoBregmalambda and the interaural midpoint in stereotaxic surgery withrats of different sex strain and weightrdquo Journal of NeuroscienceMethods vol 13 no 2 pp 139ndash143 1985

[22] A J Lim and W D Winters ldquoA practical method for auto-matic real-time EEG sleep state analysisrdquo IEEE Transactions onBiomedical Engineering vol 27 no 4 pp 212ndash220 1980

[23] F M Semenenko and B M Lumb ldquoProjections of anteriorhypothalamic neurons to the dorsal and ventral periaqueductalgrey in the ratrdquo Brain Research vol 582 no 2 pp 237ndash245 1992

[24] L Gimenez-Llort S N Schiffmann T Shmidt et al ldquoWork-ing memory deficits in transgenic rats overexpressing humanadenosine A2A receptors in the brainrdquoNeurobiology of Learningand Memory vol 87 no 1 pp 42ndash56 2007

[25] S Uchida T Maloney J D March R Azari and I FeinbergldquoSigma (12ndash15Hz) and delta (03ndash3Hz) EEG oscillate recipro-cally within NREM sleeprdquo Brain Research Bulletin vol 27 no 1pp 93ndash96 1991

[26] D Menicucci A Piarulli U Debarnot P drsquoAscanio A Landiand A Gemignani ldquoFunctional structure of spontaneous SleepSlow Oscillation activity in humansrdquo PLoS ONE vol 4 no 10article e7601 2009

[27] R E Brown R Basheer J T McKenna R E Strecker and RW McCarley ldquoControl of sleep and wakefulnessrdquo PhysiologicalReviews vol 92 no 3 pp 1087ndash1187 2012

[28] T E Bjorness C L Kelly T Gao V Poffenberger and RW Greene ldquoControl and function of the homeostatic sleepresponse by adenosine A

1receptorsrdquo Journal of Neuroscience

vol 29 no 5 pp 1267ndash1276 2009[29] T Gallopin P H Luppi B Cauli et al ldquoThe endogenous

somnogen adenosine excites a subset of sleep-promoting neu-rons via A2A receptors in the ventrolateral preoptic nucleusrdquoNeuroscience vol 134 no 4 pp 1377ndash1390 2005

[30] R Basheer R E Strecker M M Thakkar and R W McCarleyldquoAdenosine and sleep-wake regulationrdquo Progress in Neurobiol-ogy vol 73 no 6 pp 379ndash396 2004

[31] S W Wurts and D M Edgar ldquoCaffeine during sleep depriva-tion sleep tendency and dynamics of recovery sleep in ratsrdquoPharmacology Biochemistry and Behavior vol 65 no 1 pp 155ndash162 2000

[32] D L Rosin A Robeva R L Woodard P G Guyenet andJ Linden ldquoImmunohistochemical localization of adenosineA2A receptors in the rat central nervous systemrdquo Journal ofComparative Neurology vol 401 no 2 pp 163ndash186

[33] D McGinty and R Szymusiak ldquoBrain structures and mecha-nisms involved in the generation of NREM sleep focus on thepreoptic hypothalamusrdquo Sleep Medicine Reviews vol 5 no 4pp 323ndash342 2001

[34] A J Morin and A Beaudet ldquoOrigin of the neurotensinergicinnervation of the rat basal forebrain studied by retrogradetransport of cholera toxinrdquo Journal of Comparative Neurologyvol 391 no 1 pp 30ndash41 1998

[35] J Marino and J Cudeiro ldquoNitric oxide-mediated corticalactivation a diffuse wake-up systemrdquo Journal of Neurosciencevol 23 no 10 pp 4299ndash4307 2003

[36] A Nehlig J L Daval and G Debry ldquoCaffeine and the centralnervous system mechanisms of action biochemical metabolicand psychostimulant effectsrdquoBrain Research Reviews vol 17 no2 pp 139ndash169 1992

[37] RWMcCarley ldquoNeurobiology of REMandNREMsleeprdquo SleepMedicine vol 8 no 4 pp 302ndash330 2007

[38] P J M Van Galen A H Van Bergen C Gallo-Rodriguez etal ldquoA binding sitemodel and structure-activity relationships forthe rat A

3adenosine receptorrdquoMolecular Pharmacology vol 45

no 6 pp 1101ndash1111 1994[39] Y Kim Y Bolortuya L Chen et al ldquoDecoupling of sleepiness

from sleep time and intensity during chronic sleep restrictionevidence for a role of the adenosine systemrdquo Sleep vol 35 no6 pp 861ndash869 2012

[40] F Okajima H Tomura K Sho M Akbar M A Majid and YKondo ldquoIntracellular cross-talk between thyrotropin receptorandA

1adenosine receptor in regulation of phospholipaseC and

adenylate cyclase in COS-7 cells transfected with their receptorgenesrdquo Biochemical Journal vol 306 no 3 pp 709ndash715 1995


[41] K Chung-Soo H Ji-Yi and K Seunghwan ldquoHerbs for thetratment of insomniardquo Biomoecules amp Therapeutics vol 19 no3 pp 274ndash281 2011

[42] M Cott ldquoIn vitro receptor binding and enzyme inhibition byhypericum perforation extractrdquo Pharmacopsychiatry vol 30supplement 2 pp 108ndash112 1997

[43] S L Yang S Y Nam J Y Han et al ldquoAlterations of spontaneoussleep architecture and cortical electroencephalogram powerspectra by red ginseng extract via GABAA ergic systemsrdquoJournal of Ginseng Research vol 34 no 4 pp 304ndash313 2010

[44] S M Cho M Shimizu C J Lee et al ldquoHypnotic effectsand binding studies for GABAA and 5-HT2C receptors oftraditional medicinal plants used in Asia for insomniardquo Journalof Ethnopharmacology vol 132 no 1 pp 225ndash232 2010

[45] L Chung-Il C S KimH Jin-Yi et al ldquoRepeated administrationofKorea red ginseng extract increases non-rapid-eyemovementsleep via GABAAergic systemsrdquo Journal Ginseng Research vol36 no 4 pp 403ndash410 2012

[46] E A Kaczka N R Trenner B Arison R W Walker andK Folkers ldquoIdentification of cordycepin a metabolite ofCordyceps militaris as 31015840-deoxyadenosinerdquo Biochemical andBiophysical Research Communications vol 14 no 5 pp 456ndash457 1964

[47] G W Zieve and E J Roemer ldquoCordycepin rapidly collapsesthe intermediate filament networks into juxtanuclear caps infibroblasts and epidermal cellsrdquo Experimental Cell Research vol177 no 1 pp 19ndash26 1988

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


modulatory effects of sleep deprivation on the A1R mRNA

in the cholinergic basal forebrain suggest the significanceof an adrenergic pathway in the long-term effects of sleepdeprivation on the quality of ensuring sleep [8] Cholinergicneurons of LDT and PPT also comprise the cholinergicldquoarousal systemrdquo During the transition from waking to sleepthe firing rate of LDTPPT neurons markedly is decreasedreducing the cholinergic tone of their target sites and thusfacilitating the transition to sleep [9] In particular theventrolateral preoptic area of the hypothalamus contains apopulation of sleep-active neurons and is hypothesized to bean important part of the somnogenic process [10]

The four adenosine forms make up the family of G-protein-coupled AR A

1 A2AA2B and A3 [11]The difference

between the four ARs is found in their affinity for adenosinein the type of G proteins that they associate with and thesignaling pathways that are activated in the target cellsFor sleep-waking homeostasis A

1R and A

2AR have receivedthe most attention due to their expression pattern in thenervous system the availability of selective agonists andantagonists and selectivemolecular lesions of genes encodingthe receptor subtypes A

1and A

3Rs have high and low affinity

for adenosine [12] Possible changes in adenosine functioningdue to the aging process have been observed in animalmodels and abnormalities in the adenosine system couldalso explain primary insomnia or the reduced amount ofdelta waves sleep and increased sensitivity to caffeine in somesubjects with sleep deficits Caffeine is a methylated derivateof xanthine with profound effects on the onset and qualityof sleep episodes [13] This purine acts principally as anantagonist of the A

2AR Adenosine and ATP in the nervoussystems are the bridge between metabolic activity recoveryfunction and purinergic transmission that underlies the dailywake-sleep cycle in mammals Modulators of purine actionshave the potential to alleviate insomnia and other sleepdisorders based on their physiopathological role during thesleep process [14]

Cordycepin (31015840-deoxyadenosine Figure 1) is a naturallyoccurring adenosine analogue and one of the bioactiveconstituents of Cordyceps sinensismilitaris [15 16] C sinen-sismilitaris has been used for hundreds of years as a tradi-tional medicine in treating disorders of the lung and kidneythrough mechanisms of immunomodulation [17] Becauseof similarity of chemical structure of adenosine cordycepinhas been interested in the diverse effects of CNS like sleepregulation It also has traditionally been focused on thetreatment of insomnia In particular cordycepin is known tobe a bioactive constituent to regulate homeostatic function[18] Recently some herbs have been the charmingmedicinesfor considerable sufferers with sleep disabilities or insomnia[19] Most remedies of sleep aid originated from herbs havebeen targeted on GABAA systems However recent mor-phological and functional studies have identified AR in theventrolateral preoptic areas of the hypothalamus that plays animportant role in sleep regulation [20] Therefore this studywas designed to know whether cordycepin increases sleepvia ARThis experiment also can provide basic evidence thatcordycepin may be helpful for the treatment of insomnia

2 Materials and Methods

21 Experimental Animals MaleWistar rats (Samtako OsanKorea) weighing 250ndash300 g were used for sleep recordingand western blot Each rat was housed in each acrylic cage(45 times 60 times 25 cm) with water and food available ad libitumunder an artificial 12 h light-dark cycle (lights on at 700am) and at constant temperature (22 plusmn 2∘C) To ensureadaptation to the new environment mice and rats werekept in the departmental holding room for 1 week beforetesting This study was performed in accordance with theChungbuk National University Laboratory Animal ResearchCenter guidelines for the care and use of laboratory animals

22 Experimental Procedure After 7 days postsurgical recov-ery cordycepin (2 and 4mgkg in distilled saline) wasadministered to animals for 5 days once per day On the 5thday 1 h after final cordycepin treatment animals were cagedin recording system and spontaneous sleeping was recorded

23 Brain Surgery and EEG Recording Each rat was implant-ed with a transmitter (Data Sciences International TA11CTA-F40 MN USA) for recording EEG and activity via telemetryThe body of the transmitter was implanted subcutaneouslyoff the midline and posterior to the scapula and was attachedto the skin with 3 sutures for stabilization Leads from thetransmitter led subcutaneously to the skull and the bare endswere placed in contact with the dura through holes madein the skull (A 20 (Bregma) L 15 P 70 (Bregma) L 15contralateral) [21] The electrodes were anchored to the skullwith screws and dental cement All surgical procedures wereperformed stereotaxically under aseptic conditions Surgicalanesthesia was achieved with pentobarbital (50mgkg ip)and all efforts were made to minimize the suffering of theanimals Telemetric recording of cortical EEG and activitywas conducted using procedures similar to previous reportsFor the EEG signal the gain of transmitters was set atminus05+05 volts perunits times 2 and the raw signals generatedfrom the transmitter were in the range of 05ndash200Hz Thesignals were processed by a Data Sciences Internationalanalog converter and routed to anAD converter (Eagle PC30USA) housed in a PC computer The AD converter digitizedthe EEG and activity signals at 128Hz The digitized datawere transferred to the computer and displayed graphicallyAn online fast Fourier transformation (FFT) was performedon the EEG data at 10 sec intervals during data acquisition(1024 samples) after a Hanning window treatment The FFTanalysis generated power density values from00 to 200Hz ata resolution of 05HzThe FFT data were further averaged inthe range of 0 to 20Hz for every 10 secThe sleep data and FFTresults were saved to the hard disk every 10 sec for additionaloffline analysis Movement of the animal in relation to thetelemetry receiver generated transistor-transistor logic (TTL)pulses that were collected and counted as a measure ofactivity Cordycepin was administered 1 hour before the EEGrecording Recording began at 900 am for 4 h Seven or eightrats were used in each group


HO HO

O O

O

O

OHOH OH

N N

N

N N

N N

N

NN N

N

NH2 NH2 H3C

CH3

CH3


Caffeine










3 Results






umbe

r of t

rans

ition

s

600

400

200

0

Control 2 4

lowast

lowastlowast

Cordycepin (mgkg)



Wake Sleep NERM REM


200

150

100

50

0

Tim

e spe

nt (m

in)

lowast


lowastlowastlowast






80

100

60

40

20

0

Pow

er d

ensit

y (

)




80

100

60

40

20

0

Pow

er d

ensit

y (

)

lowastlowastlowast

lowastlowastlowast




A2119860R and A




1R A2AR and A





80

100

60

40

20

0

Pow

er d

ensit

y (

)



4 Discussion



Rela

tive O

D

00

25

50

75

100



A1 A2A A2B












1 A2 and A




1R is









5 Conclusions




Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















HO HO

O O

O

O

OHOH OH

N N

N

N N

N N

N

NN N

N

NH2 NH2 H3C

CH3

CH3


Caffeine










3 Results






umbe

r of t

rans

ition

s

600

400

200

0

Control 2 4

lowast

lowastlowast

Cordycepin (mgkg)



Wake Sleep NERM REM


200

150

100

50

0

Tim

e spe

nt (m

in)

lowast


lowastlowastlowast






80

100

60

40

20

0

Pow

er d

ensit

y (

)




80

100

60

40

20

0

Pow

er d

ensit

y (

)

lowastlowastlowast

lowastlowastlowast




A2119860R and A




1R A2AR and A





80

100

60

40

20

0

Pow

er d

ensit

y (

)



4 Discussion



Rela

tive O

D

00

25

50

75

100



A1 A2A A2B












1 A2 and A




1R is









5 Conclusions




Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















umbe

r of t

rans

ition

s

600

400

200

0

Control 2 4

lowast

lowastlowast

Cordycepin (mgkg)



Wake Sleep NERM REM


200

150

100

50

0

Tim

e spe

nt (m

in)

lowast


lowastlowastlowast






80

100

60

40

20

0

Pow

er d

ensit

y (

)




80

100

60

40

20

0

Pow

er d

ensit

y (

)

lowastlowastlowast

lowastlowastlowast




A2119860R and A




1R A2AR and A





80

100

60

40

20

0

Pow

er d

ensit

y (

)



4 Discussion



Rela

tive O

D

00

25

50

75

100



A1 A2A A2B












1 A2 and A




1R is









5 Conclusions




Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















80

100

60

40

20

0

Pow

er d

ensit

y (

)



4 Discussion



Rela

tive O

D

00

25

50

75

100



A1 A2A A2B












1 A2 and A




1R is









5 Conclusions




Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















1 A2 and A




1R is









5 Conclusions




Acknowledgments


References





























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article Cordycepin Increases Nonrapid Eye ...downloads.hindawi.com/journals/ecam/2013/840134.pdfCordycepin ( 3 -deoxyadenosine) is a naturally occurring adenosine analogue