ANTIOXIDANT AND LIFESPAN-EXTENSION ACTIVITIES OF CORDYCEPS SINENSIS CS-4 IN OXIDATIVE STRESS AND AGING MODELS

Jia-Shi Zhu,1,2,3 Yan Zhang,2 Jieying Yang,2 Ningzhi Tan,2 Angela Mastaloudis,1 and Chunsheng Zhao2

1Pharmanex Research Institute, Provo, UT 84601; 2Pharmanex Beijing Clinical Pharmacology Center: 2Xinkang Road, Beijing 100088, China; 3School of Pharmacy, Shihezi University, Shihezi City, Xinjiang 832000, China

ABSTRACT

Cordyceps sinensis has traditionally been used in China as a strategy to combat aging. We have reported the e� ects of Cordyceps sinensis Cs-4 (Cs-4) a mycelia fermentation product of C. sinensis, in glucose-lipid-energy metabolisms, anti-fatigue, and endurance enhancement. In this study, we examined the e� ect of Cs-4 on lifespan extension and antioxidant status in mice. For the lifespan-extension study, 250 mice 12 months of age (both sexes) received either vehicle or Cs-4 (0.5, 1.0 or 1.5g/kg diet). Caloric intake was adjusted to match the levels for controls twice per week. Compared to controls, the Cs-4 dosage group’s 75% survival time was extended 94-108 days, 50% survival time extended 10-66 days, 25% survival time extended 29–44 days, and 12.5% survival time extended 7–50 days (86 weeks so far; treatment continues). The Kaplan-Meier Survivor analysis revealed the extended lifespan and the reduced risks of death by Cs-4. The antioxidant activity was tested in mice (6 months old) that received 60 days of vehicle or Cs-4 (0.5, 1.0 or 1.5 g/kg diet) and a single dose of 11Gy 60Co gamma-radiation on day 60. Compared to controls, Cs-4 prevented the depletion of plasma total thiol-groups, GSH and GSH-peroxidase, liver CAT, SOD, and GSH-reductase (p<0.05) caused by radiation exposure. Cs-4 also prevented the radiation-induced increases in liver protein carbonyls and 8-OHdG (p<0.05) observed in the control group. In conclusion, Cs-4 supplementation signifi cantly improves the body’s antioxidant capacity and extends the lifespan in mice, supporting the traditional anti-aging uses of Cs-4 in humans.

INTRODUCTION

1. Cordyceps sinensis is traditionally believed to have anti-aging activities and to promote longevity.

2. C. sinensis and its mycelia fermentation product C. sinensis Cs-4 have a broad spectrum of health benefi ts, including the therapeutic activities of improving cardiovascular, liver, lung, and kidney functions. (Zhu et al. J Altern Compl Med. 4:289–303, 429–457,1998)

3. We reported the endurance enhancement and anti-fatigue properties of Cs-4 and the improvement of energy, glucose, and lipid metabolisms by Cs-4 in animals and humans. (Dai et al. J Altern Compl Med. 7:231, 2001; Zhao et al. J Altern Compl Med. 8:309, 2002; Xiao et al. Chin J Integrat Med. 10:187, 2004; Li et al. Chin J Clin Pharmacy 16:274, 2007; Li et al. Shanghai J Prevent Med. 20:367, 2008; Wang et al. 2008 Symposium Chin Assoc Med Mycol. pp157–164)

4. The aim of this study was to test the lifespan-extension e� ect and antioxidant activity of Cs-4 in mice.

LIFESPAN EXTENSION (STUDY 1)

METHODS8-month-old mice fed normal chow until 12 months of age. At 12 months, mice were randomized into one of four groups matched for gender and weight.

GROUPS:• Control: Normal mouse chow• Treatment 1: Chow containing Cs-4: 500 mg/kg BW• Treatment 2: Chow containing Cs-4: 1000 mg/kg BW • Treatment 3: Chow containing Cs-4: 1500 mg/kg BW

DIET AND ENDPOINTS:• Caloric intake of treatment groups was adjusted bi-weekly to match

the intake of the control group• Deaths recorded daily • Lifespan study is ongoing

LIFESPAN STUDY IN MICE

DEATHS AND SURVIVORS AFTER 95 WEEKS OF CS-4

Vehicle control 46 Dead 2 Live Cs-4 500 mg/kg 40 Dead 8 Live Cs-4 1000 mg/kg 44 Dead 4 Live Cs-4 1500 mg/kg 44 Dead 4 Live

CS-4 DELAYS NORMAL DEATH OF MICE• Kaplan-Meier Cumulative Survival Plot for Time 1• Censor Variable: Censor 1• Grouping Variable: Group 1

METHODSMice were randomized into fi ve groups and treated for 64 consecutive days by gavage. Mice received 11Gy 60Co gamma-radiation on day 60 to induce oxidative stress and damage (except non-radiation control group). The study was completed on the fourth day and the liver and blood samples of all mice were collected and analyzed for antioxidant enzymes and biochemical markers of oxidative stress.

ANTIOXIDANT ENZYMES:1. Superoxide dismutase (SOD)*2. Catalase (CAT)3. Glutathione peroxidase (GSH-Px)4. Glutathione reductase (GSH-Rd)*5. Glucose-6-phosphate dehydrogenase

(G6PD)**Data not shown

MARKERS OF OXIDATIVE STRESS:1. Total thiol groups and glutathione 2. Total protein carbonyl groups3. 8-OHdG in liver

SUMMARY & CONCLUSION

1. Supplementation with Cordyceps sinensisCs-4 prolongs lifespan in an aging mouse model.

2. Cordyceps sinensis Cs-4 supplementation increased antioxidant protection and reduced oxidative stress in mice exposed to radiation.

3. Cordyceps sinensis Cs-4 supplementation signifi cantly improves the body’s antioxidant capacity, protects against oxidative damage, and extends the lifespan in mice, supporting the traditional use of Cs-4 as an anti-aging strategy in humans.

ANTIOXIDANT & BIOCHEMICAL STUDIES (STUDY 2)

8-month-oldTreatment startsat age of 12 months

all mice deadTreatment starts all mice dead~3+ years

Cs-4 Prevented Depletion of Plasma Thiols

Mice randomized into fi ve groups:

Non-radiation control (n=15)

Radiation control (n=15) Cs-4 500 mg/kg (n=15) Cs-4 1000 mg/kg (n=15) Cs-4 1500 mg/kg (n=15)

0 (days) 60 63

60Co Collect (11Gy) blood & liver

Cs-4 Prevented DepletionLiver Catalase

Cs-4 Prevented Depletionof Plasma GSH

Cs-4 Attenuated Increases in Liver Protein Carbonyls and 8-OHdG

Cs-4 Prevented Depletion of Liver GSH Peroxidase

Cordyceps sinensis (Berk.) Sacc. 冬虫夏草(Collected from Qinghai-Tibetan plateau of China)

Isolation & Purifi cation

(A fungal strain of Paecilomyces hepiali Chen et Dai)

Industrial Fermentation

Cordyceps sinensis Cs-4

Cs-4

冬虫夏草 Cordyceps sinensis

POSTER WAS PRESENTED AT THE OXYGEN CLUB OF CALIFORNIA 2010 WORLD CONGRESS IN SANTA BARBARA, CALIFORNIA; MARCH 2010 WHERE IT RECEIVED THE DSM NUTRACEUTICAL RESEARCH AWARD.

Time

(control)

(Cs-4 500 mg/kg)

0 100 200 300 400 500 600 700

1.0

0.8

0.6

0.4

0.2

0

Cum

. sur

viva

l

Thio

l gro

ups (

mm

ol/L)

-21%

; p<0

.05

0.7

0.6

0.5

0.4

0.3Non

radiation control

radiation control

500 1000 1500Cs-4 (mg/kg)

60Co-11Gy

GSH

-Px (

U/m

g pr

o.)

-22%

; p<0

.05

700

600

500

400

300

200

100

0Non

radiation control

radiation control

500 1000 1500Cs-4 (mg/kg)

60Co-11Gy

GSH

-Px (

U/m

g pr

o.)

8-O

HdG

(ng/

g liv

er ti

ssue

)

+26%

; p<0

.05

+22%

; p<0

.05

-12.5

%; p

<0.0

5

+22%

; p<0

.05

-33%

; p<0

.05

-15.5

%; p

=0.0

5

-21%

; p=0

.05

8 –

7 –

6 –

5 –

4 –

3 –

2 –

1 –

0 –

30 –

25 –

20 –

15 –

10 –

5 –

0 –Non

radiation control

radiation control

500 1000 1500Cs-4 (mg/kg)

60Co-11Gy

Non radiation control

radiation control

500 1000 1500Cs-4 (mg/kg)

60Co-11Gy

CAT

(U/m

g pr

o.)

-23.

5%; p

<0.0

5

40

30

20

10

0Non

radiation control

radiation control

500 1000 1500Cs-4 (mg/kg)

60Co-11Gy

Glu

tath

ione

s (m

g/L)

-25.

3%; p

<0.0

5

8

6

4

2

0Non

radiation control

radiation control

500 1000 1500Cs-4 (mg/kg)

60Co-11Gy