ENHANCED BIO-ENERGY STATE IN MICE LIVER AFTER ADMINISTRATION OF CORDYMAX

Wu ZM, Dai DW, Bao TT, Xu CF, Zhu J-S
Pharmanex Pharmacology Center Beijing , China; Institute of Materia Medica; Peking Union Medical University, Beijing, China; Pharmanex Provo, USA
Abstract
Natural Cordyceps sinensis and its mycelial fermentation product CordyMax have been advocated for centuries to enhance human vitality. This study was to evaluate the effect of CordyMax on tissue energetics of male C57-BL/6 mice using non-invasive 31P NMR spectroscopy. Mice were divided into 3 groups. Groups A and B (n=5 each) received an extract of CordyMax , 200 or 400 mg/kg/day, and Group C (n=6) received placebo. All treatments were given by gavage for 7 days and then discontinued. Hepatic β-ATP and inorganic phosphate were measured using a 31P NMR spectroscope [Bruker], after mice been anesthetized with pentobarbitol (55 mg/kg, i.p.) and immobilized on shielding belt. An MDPA reference was placed on the back of the coil. Measurements were made at baseline, after 7 days of treatment, and 7 days after baseline treatment discontinuing treatment (washout phase). Tissue pH was calculated from chemical shift differences betweenα-ATP and Pi. At the end of the treatment phase, b-ATP was increased in relation to the MDPA reference in mice receiving CordyMax: Group A 3.81±0.03 (+12.3% on average); Group B 4.00±0.04 (+18.4%); compared with Group C 3.36±0.04 (p<0.001). Inorganic phosphate was decreased in Groups A and B, but not in Group C (p<0.001). Consequently, the ratio β-ATP/Pi was also significantly increased in mice receiving CordyMax: Group A 4.81±0.05 (+47.7% on g y p ( average); Group B 4.50±0.09 (+41.4%); compared with Group C 3.10±0.04 (p<0.001). At the end of the washout phase, β-ATP had returned to baseline in Groups A and B. Hepatic tissue pH was unchanged throughout the study. We conclude that CordyMax increased steady state levels of hepatic bio-energy when administered to mice for 7 days. Our findings may explain the reported energizing effect of CordyMax in human subjects.
31P

NMR Spectroscopy
α-ATP

Changes in Hepatic β-ATP
4.2

Ratio of β-ATP : Ref P

γ-ATP β-ATP
PC

4.0 3.8 38 3.6 3.4 3.2 3.0

+18.4%

MDPA (Ref.)

Pi PME PDE

+12.3%

400 mg/kg 200 mg/kg Placebo

Treatment 0 7 14 (Days)

Note: MDPA methylene diphosphonic acid PME phosphomonoesters Pi inorganic phosphate PDE phosphodiesters PC phosphocreatine

1.2

Decreases i H D in Hepatic ti Inorganic Phosphate
Placebo 400 mg/kg -17.6% 200 mg/kg

NMR Spectroscopy on mouse liver: α-, β-, γ-ATP, other triphosphorate compounds, and inorganic phosphate; calculate cellular pH.

31P

Ratio of Pi : Ref

1.1 1.0 0.9 0.8 0.7

-24.5%

Treatment 0 7 14 (Days)

Increases in Hepatic β-ATP In Response to Oral CordyMax
Ratio of β-ATP : Ref
3.8 CordyMax 400 mg/kg/d

3.6 p>0.20 3.4 p>0.20

5.0

Increases in the Ratio of β-ATP : Pi
+47.7%

Ratio of β-ATP : Pi

+13.7% p<0.001

4.0

Placebo
3.2
0 1 2 3 4 5 6 7 (Days)

+41.4% +41 4%

200 mg/kg 400 mg/kg

3.0

Placebo

Experimental Design
Cordyceps sinensis (Berk.) Sacc.
Placebo group CordyMax 200 mg/kg CordyMax 400 mg/kg No treatment (n=6mice) No treatment (n=5mice) No treatment

2.0

Treatment 0 7 14 (Days)

(Collected from Qinghai-Tibetan plateau of China)

Isolation & Purification
0

Changes in Hepatic Tissue pH
8 14 6

(n=5mice)

(days)

7

Cs-4

pH

(A Paecilomyces hepiali Chen strain)

31P

NMR spectroscopy

31P

NMR spectroscopy
4

Pre-Rx 7-day Rx 7-day post-Rx 2

Industrial Fermentation
CordyMax

At the time indicated, •Anaesthetized mice with Pentobarbital •Preformed 31P NMR spectroscopy on mouse liver

0 Placebo

200

400

(mg/kg/d)

CordyMax

Introduction
♦ CordyMax improves aerobic capability and enhances endurance in older humans (Chin J Integrat Med 2004; 10:187-192; SH J Prevent Med 2008, 20:367-369). ↑ VO2max, ↑ Anaerobic Threshold, ↑ Maximal Ventilation, ↓ RER, ↓ Lactic acid ♦ CordyMax improves glucose and lipid metabolisms (J Alternat Compl Med 2002; 8:309-314;
Chin J Clin Pharmacy 2007; 16:274-277; Proceedings 2008 Symposium Chin Asso Med Mycol. 2008, pp157-164).

Summary
7-day CordyMax Treatment: ↑ Hepatic ATP ↓ Hepatic inorganic phosphate ↑ Hepatic ratio of ATP:Pi No ∆ in hepatic tissue pH

Conclusion

• CordyMax improved steady-state hepatic bio-energy status

CordyMax Improves Glucose Metabolism in Animals and in Humans
Wu ZM, Nicodemus K, Zhao CS, Zhu J-S
Pharmanex Pharmacology Center, Beijing, China; Fit Stop Human Performance Lab, Encinitas, CA, USA, Pharmanex Provo, USA
Abstract
Preliminary reports demonstrated that supplementation with a mycelia fermentation product of Cordyceps sinensis ( (CordyMax) increased maximal O2 uptake and anaerobic y ) p threshold in mid-age to elderly humans, and enhanced in vivo bio-energy metabolisms in animals (J Alternat Complement Med 7:231, 2001; Chin J Integrat Med 10:187, 2004; Shanghai J Prevent Med 20:367, 2008). We further studied in a randomized, double-blind clinical trial the effect of CordyMax (4.5 g/day, 6 weeks) in highly-fit athletes on glucose metabolism. Male adventure racers and multi-sport endurance athletes (age 32±4 yrs; VO2peak 63±8 ml/kg/min) were assigned to either a CordyMax or a control group (n=15 each). We found a 7% decrease in fast blood glucose within normal ranges after the CordyMax therapy (92±1 to 87±2 mg/dL; p<0.01), but no change in placebo controls. During prolonged sub-maximal exercise (70% VO2peak, 60 min), reductions of respiratory exchange ratio were found in the CordyMax vs. control group (p=0.02). In mice given CordyMax for 4 weeks, responses of serum insulin and C-peptide to an oral glucose load were diminished and recovered to the pre-load levels quickly vs. control group (p<0.01 or 0.05) with no change in the glucose tolerant curve. The glucose-insulin index was lower in the CordyMax (7±1 x105 units) vs. control group (10±1 x105) (p 0.01). (p<0 01) Our data suggest that CordyMax (1) safely lowers basal glucose in normal humans, (2) improves glucose metabolism by enhancing insulin receptor sensitivity, and (3) enhances fat mobilization and beta-oxidation thereby sparing glycogen expenditure during prolonged endurance exercise.

Experimental Design (1):
Placebo CordyMax CordyMax 250 mg/kg 500 mg/kg (n=12 rats) (n=12 rats)

Experimental Design (2):
Randomized, Double-Blind, Placebo Controlled Placebo CordyMax
(n=15) (4.5 g/day) (4.5 g/day)

(n=15)

(n=12 rats)

0
0 (days) 18

(weeks)

6

Sub-maximal exercise-metabolism testing
8

Reduction of FBG

mol/L) Blood Glucose (mm

7 6 5

Inclusion Criteria:
p=0.768 p=0.262

4

2 1 0

Day 18

3

Day 0

Day 8

p<0.001

p<0.001

Healthy, highly fit, male endurance athletes under the age of 40 years
Training Background Adventure Racers Pro/Competitive Triathletes Active Military (Reco/SeALs) Olympic Rower/Kayak Training vol. Hrs./wk. 11 ± 7 15 ± 7 11 ± 1 10 14 9± 4 Number of Subjects 9 10 3 1 1 6

Vehicle Control

250 500 CordyMax

(mg/kg)

Improvement of oral Glu tolerance p
Blood Glucose (mmol/L) 11 10 9 8 7 6 5 0 0.5 1 1.5 Time (hours) 2 AUC: p=0.022 AUC: p=0.005

Vehicle Control CordyMax 250 mg/kg

Ultra-distance runner High Fit Multi-sport

Constant work-rate, sub-maximal exercise

Decrease in FBG
100 90 Post-Rx

Cordyceps sinensis (Berk.) Sacc.
(Collected from Qinghai-Tibetan plateau)

Insulin (mU/ml)

40 30

70 60 50

p=0.870 +0.89%

p=0.225

Isolation & Purification
(A Paecilomyces hepiali Chen strain)

20 10 0

p=0.012

Placebo

Pre-Rx

Reduction of fasting serum insulin

(mg/dL)

80

p=0.006 -7.22%

CordyMax

Cs-4

Vehicle Control

250 500 CordyMax

Reduction of RER during sub-maximal exercise
1.05

Industrial Fermentation
Insulin Sensitivity n

Increase in insulin sensitivity
0.24

Placebo
1.00

CordyMax

RER

0.22 0.20

0.95

CordyMax

0.90

Post-Rx

Pre-Rx

p=0.0 008

0.18 0 18 0.16 0.14

p<0.001

Introduction
♦ CordyMax improves aerobic capability and enhances endurance in older humans (Chin J Integrat Med 2004; 10:187-192; SH J Prevent Med 2008, 20:367-369). ↑ VO2max, ↑ Anaerobic Threshold, ↑ Maximal Ventilation, ↓ RER ↓ Lactic acid RER, ♦ CordyMax improves lipid metabolism (Proceedings 2008 Symposium Chin Asso Med Mycol. 2008, pp157-164). ♦ CordyMax improves steady-state hepatic bio-energy status (J Alternat Compl Med 2001; 7:231-240).

0.85

Pre-Rx
0 10 20 30 40 50 60 0 10 20 30

Post-Rx

0.80 40 50 60

Vehicle Control

250

500 (mg/kg)

CordyMax

Exercise Time (min)

Summary and Conclusion
CordyMax Treatment: y ♦ Improves oral glucose tolerance, and reduces fasting blood glucose within normal ranges; ♦ Increases insulin sensitivity, and reduces fasting serum insulin; ♦ Enhances fat mobilization and beta-oxidation thereby sparing glycogen expenditure during prolonged submaximal exercise in highly-fit humans.

CORDYMAX INCREASES SERUM HDL-CHOLESTEROL AND REDUCES OXIDIZED LDL-CHOLESTROL IN HUMANS WITH REDUCED SERUM HDL-CHOLESTEROL
Wu ZM, Wang BE, Pei Y, Liu CZ, Li SQ, Zhang W, Xu ZB, Rippe J, Zhu JS
Pharmanex Clinical Center, Beijing; Beijing Friendship Hospital and Beijing Association for Medico-Pharmaceutical Research & Development; Rippe Lifestyle Institute, Shrewsbury, MA, USA;
Pharmanex Provo, UT, USA Ph P UT
Abstract
Literature reported CordyMax, a mycelial fermentation product of Cordyceps sinensis, regulated the blood lipids in hyperlipidemic patients and prevented the formation of atherosclerosis in animals and humans. (Administ Tradit Chin Med 1995;5:14-18). We tested the effect of CordyMax (3.0 g/day) in 133 dyslipidemic subjects on increasing HDL cholesterol y p j g (HDL-c) and reducing oxidized LDL cholesterol (ox-LDL). Subjects with reduced serum HDL-c (<40 mg/dL for males or <45 mg/dL for females) were randomized to a CordyMax or placebo group (double-blind). Eight weeks of CordyMax did not change total cholesterol and triglycerides significantly. At Week 8, LDL-c was reduced with CordyMax significantly by 5.4% (p=0.002). Most dramatically, ox-LDL was reduced by 22.9% in CordyMax group (p=0.001). Atherosclerosis Index [= (TC – HDL-c)/HDL-c] reduced with CordyMax by 29% (p<0.001). HDL-c was increased with CordyMax by 31.1% (p<0.001) in females and by 12.3% (p (p=0.003) in males. Apolipoprotein A1 was increased with ) p p p CordyMax by 34.7% (p=0.005). The ratio of ox-LDL/HDL-c was reduced by 10.5% (p=0.003). In conclusion, CordyMax  significantly lowers LDL-c, ox-LDL and Atherosclerosis Index, and increases HDL-c and ApoA1, reducing the risk of atherosclerosis and cardio- and cerebro-vascular diseases.

Study Aims
1. To examine the effect of CordyMax on serum HDL-cholesterol

Increases in Serum HDL-Cholesterol
Females
Week 0 Week 0
55

Males
Week 8 +12.3% p=0.003
43 41 39

Week 8

p=0.023

Serum HDL (mg/dl)

2. To examine the effect of CordyMax on serum oxidized LDL-cholesterol

50 45

37 40 35 30 25 20 35 33 31

Experimental Design
Randomized; Double-blind; Placebo Controlled
Lead-in Phase -2 Placebo 3 g/day n=56 n=77

+14.7% p=0.015

+31.1% p<0.001

29 27 25

p=0.261

Placebo CordyMax

Placebo CordyMax

CordyMax 3 g/day 0 (weeks)

Increases in Serum apoA1
1.8

Serum apoA1 (mg g/dl)

8

1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

Inclusion Criteria:
Serum HDL-cholesterol Males ≤ 40 mg/dl females ≤ 45 mg/dl

p=0.934

+34.7% p=0.005

Introduction
♦CordyMax increases activities of scavenging oxygen free radicals in elderly senescent patients (Administ Tradit Chin. Med. 1995;5:14-18) ↑ Superoxide Dismutase (SOD) activity ↓ Plasma malondialdehyde (MDA) ♦CordyMax interrupts the formation of atheromatous plaque in the aorta and inhibits thrombosis on the surface of experimentally injured endothelia of arteries in rabbits (Administ Tradit Chin. Med. 1995;5:6; Chapter 4, in: Advanced Study for TCM Herbs, Vol. 1. IMM, ed., 1995:91-113) ♦CordyMax decreases total and LDL-cholesterol in hyperlipidemic patients (Intl J Orient Med 1990; 15:77-80) ♦CordyMax improves lipid-glucose metabolisms in elite athletes (Chin J Clin Pharmacy 2007; 16:274-277)

Placebo

CordyMax

Reduction of Serum LDL-Cholesterol LDL Cholesterol
160 150

Reduction of Atherosclerosis Index
8

Atherosclerosis Index

7 6 5 4 3

Serum LDL (mg/dl)

140

p=0.446

-5.4% p=0.002

p=0.309

130 120

-29.0% p<0.001

Week 0

Week 0

110

Week 8

2 1 0

100

Week 8

Placebo

CordyMax

Placebo

CordyMax

Reduction of Oxidized LDL-Cholesterol
30

Reduction of Ratio of OxLDL:HDL
0.8

Serum oxLDL (mg/dl)

Ratio of oxLDL : HDL o

25 20 15 10 5 0 Placebo CordyMax p=0.549 -22.9% p=0.001 Week 0 Week 8

0.7 0.6 0.5 0.4

p=0.852

Week 0

0.2 0.1 0.0

Placebo

Week 8

0.3

-10.5% p=0.003

CordyMax

Cordyceps sinensis (Berk.) Sacc.

(Collected from Qinghai-Tibetan plateau of China)

Summary
CordyMax supplementation for 8 weeks
In all patients ↑ Serum HDL-cholesterol HDL cholesterol ↓ Serum oxLDL ↓ Ratio of oxLDL:HDL In patients with baseline serum LDL-c >130 mg/dL ↓ Serum LDL-cholesterol ↑ Serum apoA1 ↓ Atherosclerosis Index No ∆ serum triglycerides

Conclusion
CordyMax treatment increases serum HDL-c and decreases oxidized LDL-c in patients with reduced baseline serum HDL-c, indicating reduced risks of atherosclerosis, and ischemic cardio- and cerebrovascular diseases.

Isolation & Purification
Cs-4
(A Paecilomyces hepiali Chen strain)

Industrial Fermentation
CordyMax

CordyMax Extends the Lifespan of Mice — A preliminary report
Wu ZM, Zhang Y, Tan NZ, Zhao CS, Yang JY, Zhu J-S Pharmanex Beijing Pharmacology Center, Beijing China; Pharmanex Research Institute, Provo, USA
Abstract
Cordyceps sinensis and its mycelia fermentation product CordyMax have been used for Centuries for anti-fatigue and endurance enhancement. We reported anti-fatigue and endurance enhancement properties, and improvement of glucose, lipid and energy metabolisms by CordyMax in animals and humans in previous studies. In this study, we explored possible anti-aging effects of CordyMax in mice. A total of 192 healthy ICR mice (12 months of age, half males and half females) were randomized into 4 groups, receiving either vehicle or CordyMax at a dose of 500, 1000, or 1500 mg/kg.bw. All mice were fed with regular forage or forages contained CordyMax in different concentrations. Body weight was monitored once a week. Calorie intake was monitored twice per week and adjusted carefully to match the average calorie intake levels for vehicle controls, males and y y females respectively. CordyMax administration continues until all mice die. Mice have been treated for 64 weeks thus far. The preliminary results show: (1) no significant differences in body weight and calorie intake were observed amongst 4 groups. (2) Compared to controls, the survival time of 75% animals in the CordyMax groups (500, 1000, and 1500 mg/kg.bw) extends over 14, 14, and 16 weeks respectively, and the survival time of 50% animals in the CordyMax groups extends over 9, 1, and 5 weeks respectively. Analysis with use of Kaplan-Meier Cumuli Survivor Plot showed significantly extended lifespan of the mice and reduced death risks by CordyMax: p=0.049 (Week 36); p=0.036 (Week 40); p=0.059 (Week 48); p=0.027 (Week 64). The y (equivalent to the human dose) appeared to ) pp low dose CordyMax treatment ( q show the best survivor curve. This study demonstrates the lifespan-extending effects of CordyMax in mice, while the experiment is still ongoing. At the meanwhile, additional studies demonstrated that CordyMax has anti-oxidation activity, and improves glucose, lipid and energy metabolisms and aerobic exercise capacity in animals and in humans (reported separately), all of which support the general anti-aging function of CordyMax.

Food intake of female mice
4.6

Deaths in Each Group Vehicle control 28 dead 22 dead 32 dead 27 dead 20 Live 26 Live 16 Live 21 Live

Food intake (g)

4.2 3.8 3.4 3.0 2.6 2.2

CM 1000 Control

CM 500 mg/kg CM 1000 mg/kg CM 1500 mg/kg

CM 500
10 20 30

After 64 weeks of CM Treatment
CM 1500
40 50 60 70

Time (weeks) ( )

12 m-old (n=48) 8 m-old

27 m-old

Food intake of male mice
4.9 4.5 4.1 3.7 3.3 33 2.9

Kaplan-Meier Cumulative Survival Plot
Cumulative Survival Rate
1.0 0.8

CM 1500 CM 1000 Control CM 500

Food intake (g)

CM 1000 CM 500 Control

0.6 0.4

0.2

CM 1500
2.5 10 20 30 40 50 60 70

p=0.027
0 0 100 200 300 400 500

Time (weeks)

Time (days)

Introduction
1. Cordyceps sinensis is traditionally believed as a medicinal herb with anti-aging activities and promoting longevity. anti aging 2. Literature indicates the therapeutic functions of Cordyceps sinensis and its mycelia fermentation product CordyMax in improving energy, glucose, and lipid metabolisms and benefiting to cardiovascular, the liver, the lungs and kidneys health. (Zhu et al. J. Altern Compl Med. 1998; 4: 289-303, 429-457) 3. We have reported anti-fatigue and vitality-endurance enhancement properties of CordyMax, and improvement of energy, glucose, and lipid metabolisms by CordyMax in animals and humans in previous studies. 4. The aim of this study is to test the lifespan extension effect of CordyMax in mice.
50 48

BW of female mice
CM 500

Kaplan-Meier Cumulative Survival Plot
Cumulative Survival Rate
1.0

Body Weight (g)

46 44 42 40 38 36 34 32 30 10 20 30 40 50 60 70

0.8

Control CM 1500 CM 1000

CM 500
0.6 0.4

0.2 0 0

p=0.013
100 200

Control

Time (weeks)

300

400

500

Time (days)

BW of male mice
Lifespa an (days) extended
47

Cordyceps sinensis (Berk.) Sacc.

CM 500 Control

160 140 120 100 80 60 40 20 0 ‐20
500mg/kg

Lifespan Extended (Days)  at the % Survival 

Isolation & Purification
Cs-4
(A Paecilomyces hepiali Chen strain)

Bod Weight (g) dy

(Collected from Qinghai-Tibetan plateau of China)

45 43 41

CM 1000
39 37 35

CM 1500

Industrial Fermentation
CordyMax

10

20

30

40

50

60

70

Time (weeks)

97. 95. 93. 91. 89. 87. 85. 83. 81. 79. 77. 75. 72. 70. 68. 66. 64. 62. 60. 58. 56. 54. 52. 50. 47. 45. 43. 9% 8% 8% 7% 6% 5% 4% 3% 3% 2% 1% 0% 9% 8% 8% 7% 6% 5% 4% 3% 3% 2% 1% 0% 9% 8% 8% 23 35 85 76 50 85 98 94 85 12 10 98 60 80 80 78 82 82 74 45 42 75 65 66 69 72 57 ‐6 13 5 3 10 14 25 15 19 39 39 38 43 28

1000mg/kg ‐6 49 44 46 35 43 25 87 92 10 84 94 63 59 56 35 35 35 25 ‐2 1500mg/kg 75 87 88 95 78 13 13 12 12 13 12 10 74 63 60 35 35 32 28 ‐9

Experimental Design: Lifespan studies in mice
Vehicle control CM 500 mg/kg CM 1000 mg/kg CM 1500 mg/kg ( (n=48 mice) ) (n=48 mice) (n=48 mice) (n=48 mice) ~ 3 years
8m-o Treatment starts at the age of 12 months

Summary 1. 1 Analysis with use of Kaplan-Meier Cumuli Survivor Plot showed significant Kaplan Meier extension of mouse lifespan and reduced death risks by CordyMax: p=0.049 (Week 36); p=0.036 (Week 40); p=0.059 (Week 48); p=0.027 (Week 64). 2. The low dose CordyMax treatment (equivalent to the human dose) appeared to show the best survivor curve. 3. This study demonstrates the lifespan-extending effects of CordyMax in mice, while the experiment is still ongoing.

all mice dead

Anti-oxidation activities of CordyMax: a mechanism of its anti-aging property
Wu ZM, Yang JY, Tan NZ, Zhang Y, Zhao C, Zhu J-S Pharmanex Beijing, China; Pharmanex Research Institute, Provo Utah, USA
Abstract
Cordyceps sinensis and its mycelia fermentation product CordyMax have g been used for Centuries for anti-fatigue and endurance enhancement. We reported anti-fatigue and endurance enhancement properties, and improvement of glucose, lipid and energy metabolisms by CordyMax in animals and humans in previous studies. We also demonstrated the antiaging effect of CordyMax in mice. To explore the mechanism of the antiaging actions, we tested anti-oxidation activity of CordyMax in mouse models with oxidative damage. Mice were randomized into 5 groups, receiving vehicle or CordyMax at a dose of 500, 1000, or 1500 mg/kg.bw for 60 days. Mice in vehicle and 3 CordyMax groups were given a single dose of 11 Gry 60Co γ-rays radiation, and sacrificed in Day 4 after radiation. We found that plasma glutathione (GSH) and the thiol groups, and liver superoxide dismutase (SOD) and catalase (CAT) were p ( ) ( ) significantly reduced by 29.0%, 22.6%, 6.0% and 24.7%, and liver protein carbonyl groups was significantly increased by 37.3% in vehicle controls, compared to normal mice. As compared to vehicle controls, CordyMax therapy at a dose of 500, 1000, or 1500 mg/kg.bw increased plasma thiol groups by 22.9%, 20.8%, and 25% respectively (p=0.001, 0.001, and <0.001), and liver CAT by 16%, 14.8%, and 16.4% respectively (p=0.001, 0.002, and<0.001). CordyMax at a dose of 1000 or 1500 mg/kg.bw reduced liver protein carbonyl groups by 8.7% and 13.5% respectively (p=0.035 and 0.001), and increased plasma GSH by 26.3% and 26.5% (p=0.023 and 0.026). Liver SOD was increased with CordyMax (1000 and 1500 mg/kg.bw) by 9.4% and 5.7% ( y (both p<0.05). Liver GSH-reductase was increased with p ) CordyMax (500mg/kg) by 10.5% (p=0.039). The results indicate that CordyMax improves antioxidant capacity in mice with radiation-induced oxidative injure, representing one of the mechanisms of anti-aging functions of CordyMax.

Increases in plasma thiol groups
0.7

Increases in liver CAT
40

Thiol groups (mmol/L)

+ 20.8%; p=0.001

+ 22.9%; p=0.001

+ 25.0%; p<0.001

+16%; p=0.001

+15%; p=0.002

0.5

20

0.4

10

0.3

0

Normal control

Vehicle

500
60Co

1000 CordyMax

1500 (mg/kg)

Normal control

Vehicle

500
60Co

1000 CordyMax

1500(mg/kg)

-11Gy

-11Gy

Increases in plasma GSH
GSH-Rd (nmol/min/mg pro o.) Glutathione(mg/L)
8

Increases in liver GSH-Rd
40 35 30

Introduction
♦ Cordyceps sinensis is believed as an “Yin-Yang” double invigorating and anti-aging TCM herb ♦ CordyMax, the mycelia fermentation product of Cordyceps sinensis, has the following functions: o Anti-fatigue and endurance enhancement o Improvement of glucose, lipid & energy metabolisms ♦ Oxidative stress and free radical damages are generally believed as the most important cause of aging and induce aging-related biochemical/molecular changes ♦ Isotope radiation induces production of large amount of free radicals and oxidative injury, causing acute aging and death

4

20

2

10

0 Normal control Vehicle

500

1000 CordyMax C
60Co

1500 (mg/kg)

0

Normal Vehicle control

500
60Co

+11%; p=0.039

+29% p<0.001

+26.3%; p=0.023

+26.5%; p=0.026

6

+ 7.6% p=0.072

1000 CordyMax

1500 (mg/kg)

-11Gy

-11Gy

Increases in liver SOD
8.0

Decreases in liver protein  carbonyl groups 

Carbonyl group ps (nmol/mg pro o.)

Cordyceps sinensis (Berk.) Sacc. y p ( )

SOD (U/mg pr ro.)

500 400
6.0 60

(Collected from Qinghai-Tibetan plateau of China)

+ 9.4%; p<0.05

- 8.7%; p=0.035

4.0

200 100 0

- 37% p<0.001

Cs-4

(A Paecilomyces hepiali Chen strain)

2.0

Industrial Fermentation
CordyMax

Normal Vehicle control

500

1000 CordyMax
60Co

1500 (mg/kg)

0 Normal control Vehicle 500
60Co

1000

1500 (mg/kg)

CordyMax

-11Gy

-11Gy

Experimental Design:
Non-radiation control Vehicle control CordyMax 500 mg/kg CordyMax 1000 mg/kg CordyMax 1500 mg/kg 0 (days) (n=15) (n=15) (n=15) (n=15) (n=15) 60
60Co

Summary
CordyMax Treatment for 60 days in radiation-induced oxidative injured mice:

Conclusion
♦ CordyMax improves antioxidant capacity in mice with radiation-induced oxidative injure. j ♦ The results from this study supports the concept of CordyMax as an anti-aging product that has been demonstrated by the lifespanextending study.

63 Collect blood & liver

(11Gy)

↑ ↑ ↑ ↑ ↑ ↓

Plasma thiol groups Plasma glutathione Liver SOD Liver CAT Liver GSH-Rd Liver protein carbonyl groups

- 14%; p=0.001

Isolation & Purification

300

+ 5.7%; p<0.05

+6.0% p=0.018

+16% p<0.001

+22.6% p<0.001

CAT (U/mg pro.)

0.6

30

+ 24.7% p<0.001

ANTI-FATIGUE AND ENDURANCE ENHANCEMENT PROPERTIES OF CORDYMAX IN HUMANS
Zhu JS, Xiao Y, Li CL, Huang XZ, Hagan RD
Pharmanex Research Institute Provo UT USA; Shihezi University Xinjiang; Pharmanex Institute, UT, University, Clinical Center, Beijing; Peking Union University, Beijing; Peking University, Beijing; The Fit Stop Human Performance & Health Enhancement Laboratory, Encinitas, CA, USA
Abstract
Cordyceps sinensis and a standardized mycelial fermentation product of C. sinensis, CordyMax, are known as medicinal herbal products for invigoration, health h lth preservation, anti-aging, and anti-fatigue. Th i anti-fatigue and ti ti i d ti f ti Their ti f ti d endurance enhancement functions have been reported. By use of a traditional symptom-analysis method in a double-blind clinical trial, elderly patients with senescence-related symptoms reported improvement of fatigue, dizziness, intolerance to cold temperature, sexual dysfunction, etc. by CordyMax supplementation in majority of patients (J Appl Tradit Chin Med 1993;1:32). Animal studies demonstrated that CordyMax improved steady state bio-energy ATP levels in mouse liver by use of in vivo serial 31P NMR spectroscopy (J Alternat Compl Med 2001, 7:231), and promoted efficient use of limited oxygen supply to support body’s essential physiological activities and greater tolerance to hypoxia-induced acidosis (Chin Tradit Herbal Drugs 1986;17: 209). We examined th anti-fatigue and endurance enhancement properties of i d the ti f ti d d h t ti f CordyMax with use of sports physiology methods. In double-blind clinical trials with use of an incremental work rate protocol on a cycle ergometer and/or treadmill, we found that CordyMax increased VO2max by 7.0%, anaerobic threshold by 12.6%, maximal ventilation by 10.4% and maximal work rate by 5.9%, indicating improvement of aerobic exercise capacity in healthy sedentary adults of advance ages. In healthy young athletes and by use of a constant work rate protocol, CordyMax therapy increased O2 pulse by 7.6% and reduced heart rate (HR) by 2.2%, RER by 2.5%, and lactic acid by 10.5% during endurance exercise, indicating improvement of cardiovascular and metabolism functions during endurance exercise. CordyMax also accelerated HR recovery 3 min post maximal exercise by 6.3%. In summary, CordyMax supplementation influences favorably aerobic capacity and cardiovascular, pulmonary, and metabolic functions during maximal and endurance exercise, improves fatigue and endurance performance, and facilitates recovery from exercise. Average % Change in Hea Rate art

To Examine the Effects of CordyMax on Aerobic Capacity, Endurance Enhancement, and Exercise metabolism in Humans Randomized, Double-blind, Placebo-controlled Placebo CordyMax 0 (weeks) 6 or 12

Heart Rate during CWR
+3% +2% +1% 0% -1% -2% -3% -4%

p 0.05 p=0.05

CordyMax

Placebo

Average % Change of O2 Pulse

Exercise tests Exercise tests Healthy ld H lth older subjects & athletes bj t thl t

O2 P l during CWR Pulse d i
p=0.05
+8% +6% +4% +2% 0% -2% -4%

+12% +10%

Peak Oxygen Uptake
2.2 2.0 (L/min)

+6.7%, p=0.05

Week 0 k

Week 6 k

1.8 1.6 1.4

-0.05% 0 05% p=0.80 Placebo

CordyMax

Placebo

CordyMax

RER during CWR
Average % Change in RER e
+4% +3% +2% +1% 0% -1% -2% -3% -4%

Introduction
♦ CordyMax, the mycelia fermentation product of C. sinensis, p y p , g g , improves senescence-related symptoms, including fatigue, and increases activities of scavenging oxygen free radicals in elderly senescent patients (Administ Tradit Chin Med 1995; 5:14-18) ♦ CordyMax extends lifespan and enhances body’s antioxidant activities ♦ CordyMax improves energy, glucose and lipids metabolisms in humans and animals (J Alternat Compl Med 2001; 7:231-240; J Alternat Compl Med 2002; 8:309-314; Chin J Clin Pharmacy 2007; 16:274-277; Proceedings 2008 Symposium Chin Asso Med Mycol. 2008, pp157-164) Anaerobic Threshold (L/min) ( ♦ Cordyceps sinensis is traditionally believed as an antiaging tonic herb

p=0.025

Anaerobic Threshold
1.4 1.3 13 1.2 1.1 1.0 0.9 0.8

+12.6%, p=0.012

Week 0

Week 6

-2.4% p=0.41 Placebo
Average % Change in Lactic Acid

CordyMax

Placebo

CordyMax

Lactic Acid during CWR
p=0.031

+30% +20% +10% 0% -10% -20%

Work O t t W k Output
1.5

Work Output (KJ/kg)

Incremental work-rate (IWR) exercise on cycle ergometer
VO2 (L/min)

+3.1%, p=0.033

1.4 1.3

Week 12

Week 0

1.2 1.1 1.0 0.9

-4.9% p=0.626 Placebo

CordyMax

Placebo

work rate: +25 watt every 2 min
Time (min) Constant Work Rate (CWR) Sub-Maximal Exercise

Summary & Conclusion y
CordyMax Treatment:
↑ ↑ ↑ ↑ ↓ ↓ ↓ ↓
Peak O2 uptake Anaerobic threshold Work output O2 pulse Peak heart rate RER Blood lactate Time for 1-mile walk and for recovery

CordyMax

% Change in Time for 1-Mile Walk e From Week 0 to Week 12

1-Mile Walk Time
5% 4% 3% 2% 1% 0% -1% -2% -3% -4% -5%

p=0.05

VO2 (L/min)

Work Rate at 70% VO2max
Time (min)

CordyMax

Placebo

CordyMax Treatment: In healthy subjects, improved aerobic exercise capacity, endurance, glucose-lipid metabolisms during endurance exercises

Maturation of Cordyceps sinensis associates with co-existence of Hirsutella sinensis and Paecilomyces hepiali DNA
Zhu J-S, Guo Y, Yao YS, Zhou YJ, Zheng TY, Chen W
Pharmanex Research Institute, Provo UT, USA; Shihezi University, Xinjiang; Pharmanex Clinical Center, Beijing; Institute of Microbiology, Chinese Academy of Sciences, Beijing
Abstract
A decades-long debate has not given a consensus on the anamorph-telemorph connection for Cordyceps sinensis (Cs). Literature reported isolations of Paecilomyces hepiali and Hirsutella sinensis from natural Cordyceps sinensis, y p , and evidence of molecular existence of H. sinensis in C. sinensis. We tested a hypothesis in this study that P. hepiali and H. sinensis co-exist in natural C. sinensis, and their proliferation predominance changes during maturation of C. sinensis. Mycological and molecular approaches were employed to examine the growth of P. hepiali and H. sinensis and their genes in freshly collected C. sinensis after thorough prior cleaning and surface sterilization. A nested PCR method with use of a touch-down program was used to identify the genes of P. hepiali and H. sinensis in the caterpillar body and stroma of natural C. sinensis. We found that P. hepiali and H. sinensis were detected simultaneously in freshly collected C. sinensis by mycological and molecular examinations. Maturation of C. sinensis after it is visible above ground associates with a large decrease in the ability of competitive growth of H. sinensis (p<0.001). P. hepiali and H. sinensis genes were found in both caterpillar body and stroma of natural C. sinensis. We conclude that C. sinensis is a complex traditional Chinese herb with multiple fungi living in its caterpillar and stroma. Its maturation from early May to late June associates with dynamic changes in proliferation predominance of the fungi.

ITS PCR Products

Nested PCR Products Using Phsp3/5Primers
Template:
1 500 400 300 200 100 2 3 4

Fresh C. sinensis
5 6 7 8 9 10 11

ITS4/5 DNA segments by single-step PCR. rDNA was extracted from P. hepiali (Lane 1), H. sinensis (Lane 2), and C. sinensis (Lane 3) as the templates, and ITS4 & ITS5 primers were used.

AccI

AccI

AccI

(bp)

DNA Sequences
1 2 3 4 5 6 7 8 9

PCR products were amplified from rDNA templates extracted from caterpillars and stromata of C. sinensis with use of a nested PCR protocol (upper panel), and digested with AccI (lower panel).

Sequences of the Nested PCR Products

Scientific Background
♦ Cordyceps sinensis (Berk.) Sacc. is a precious tonic herb used in china for centuries, having broad nutritional and health benefits (Zhu et al., J Alternat Compli Med 4:289-303 & 429-457, 1998). Its life cycle is complex and its natural supply is scarce. ♦ Although on the basis of DNA sequence Hirsutella sinensis is hypothesized as the anamorph (asexual stage) of C. sinensis, >10 fungi were isolated from natural C. sinensis, possibly suggesting a multifungi symbiosis in natural C. sinensis. ♦ Through competitive proliferation, live Paecilomyces hepiali and Hirsutella sinensis were identified simultaneously (Guo and Zhu, FASEB J. 19: A1033, 2005).

Endonuclease Digestions of P. hepiali & H. sinensis ITS PCR Products
BamHI Acc I BamHI Acc I Ava I Ava I Alu I Alu I

Experimental Methods
♦ 100 pieces of fresh C. sinensis were collected from Qinghai-Tibetan Plateau in early May, following by immediate on-site clean, surface sterilized and freeze in liquid nitrogen for transportation and further process. ♦ Caterpillars and stromata of natural C. sinensis were separately grounded into powder i li id nitrogen. Rib d in liquid it Ribosomal DNA l was extracted. ♦ A nested, dual-step PCR technique was used (Guo and Zhu, FASEB J. 20: A431, 2006). 1. A pair of primers, ITS4/5, were used for the 1st PCR: annealing at 40 for 30 cycles. 2. A pair of Paecilomyces hepiali primers, Phsp3 and Phsp5, and a touch down PCR touch-down protocol were used for the 2nd PCR: initially annealing at 70 with 0.3 decrement for each cycle, for 35 cycles. 3. DNA sequencing and restrictive endonuclease digestions with BamHI and AccI specific for Paecilomyces hepiali and Aval and AluI for Hirsutella sinensis.

Lane: 1

2

3

4

5

6

7

8

9

10

11

DNA template:

P. hepiali ITS4/5

H. sinensis ITS4/5
P. hepi iali

PCR Products Using Phsp3/5 Primers
DNA Template: Fresh C. sinensis

Summary
♦ P. hepiali genes were amplified from caterpillar and stroma of natural C. sinensis (GenBank #EF555097), in addition to the detections of H. sinensis genes.

600 500 400 300 200 100

Lane:

1

2

3

4

5

6

7

8

9

10

Discussions
♦ We found simultaneous existence of live P hepiali and H sinensis P. H. and their genes in nature C. sinensis. ♦ The multi-fungi existence in natural C. sinensis may suggest complex anamorph-telemorph connections to C. sinensis.
AccI AccI AvaI AvaI

Lane:

1

2

3

4

5

PCR products were amplified from rDNA templates extracted from C. sinensis and P. hepiali with use of P. hepiali “specific” primers Phsp3-Phsp5 (upper panel), and digested with AccI and AvaI (lower panel).

AccI

Maturational alterations of differential expressions of GC:AT-biased Cordyceps sinensis mutants and Paecilomyces hepiali in natural Cordyceps sinensis
Zhu J-S, Gao L, Yao YS, Zhou YJ, Zheng TY Chen W J-S L YS YJ TY,
Pharmanex Research Institute, Provo, UT, USA; Shihezi University, Xinjiang; Pharmanex Clinical Center, Beijing
Abstract
A decades-long debate has not given a consensus on the anamorph-telemorph connection for Cordyceps sinensis (Cs). Literature reported simultaneous detections of Paecilomyces hepiali (Ph) and Hirsutella sinensis (Hs) and their DNA in freshly collected natural Cs and a >50% decline of colony-forming ability of caterpillar Hs along with Cs maturation under competitive proliferation conditions. In this study, we tested the expressions of Ph and multiple Hs-related Cs genes during Cs maturation. Southern blotting analysis revealed dramatic increases in expressions of Ph and Hs genes with Cs maturation, with use of Ph (ITS5-179) and Hs (ITS5-162) specific probes. Two Hs-related species on Hs blot were seen after the genomic Cs DNA was digested with EcoRI, probably representing the GC:ATbiased mutants. They expressed differentially in stroma and caterpillar body at different stages of Cs maturation. The AT bias does not express in premature Cs caterpillar, but highly predominately in premature stroma; while the GC bias expresses oppositely in the premature Cs compartments. The differential expressions altered non-proportionally in the caterpillar body and stroma when Cs matures. The expression pattern of the AT-bias species is highly similar to that for Ph. EcoRI digestions of PCR products in ITS1/5.8S rDNA g genomic DNA used as the template showed increased q p quantity of y regions when Cs stroma g digestible species in proportion to the increased length of stroma during Cs maturation. In conclusion, Cs maturation associates with augmented expressions of both Ph and Hs genes and altered differential expressions of GC:AT-biased Cs mutants. These maturationrelated changes in the expressions of Cs-associated fungi represent the important elements in Cs life cycle, opening an avenue to unmask the anamorph-telemorph connection of this precious Chinese herb.

rDNA preparation, Southern blotting and PCR analysis
♦ Caterpillar and stroma of the sterilized C. sinensis were grounded separately into powder in liquid nitrogen for genomic DNA extraction with DNeasy Plant Mini Kit (Qiagen). ♦ AvaI, DraI, & EcoRI were used for the genomic DNA and PCR product digestions ♦ Probes: ― P. hepiali probe: 175 bp (ITS5-179) ― H. sinensis probe: 158 bp (ITS5-162) ― Internal control probes: a 18S fragment ♦ Probes were labeled with DIG High Prime DNA Labeling and Detection Kit (Roche)

Restrictive digestions of Hsprp1/3 PCR products

Scientific Background
♦ Cordyceps sinensis (Berk.) Sacc. has been used as a precious medicinal herb in china for centuries ♦ It has broad nutritional and health benefits (Zhu et al., J Alternat Compli Med 4:289-303 & 429-457, 1998) ♦ Co-existence of fungi P. hepiali and H. sinensis was found in fresh C. sinensis (Zhu et al., J Mycol Res 5:214-224, 2007)

Southern blots: P. hepiali (upper) & H. sinensis (lower) rDNA in Type 1 or 2 C sinensis C. Caterpillar Stroma
Collected in: May Jun May Jun Type of C. sinensis: 1 2 1 2
P. hepiali 18S Internal Control

Maturation of C sinensis and the C. Temperature on Qinghai-Tibet Plateau
Less Matured Harvest Season Temperature on the Plateau Stroma Early May 0-10 Short A: “Type 1” Matured Late June 10-20 Long B: “Type 2”

Caterpillar Stroma C t ill St
Collected in: May Jun May Jun Type of C. sinensis: 1 2 1 2
H. sinensis 18S Internal Control

Reversely orientated rDNA sequences (GC-bias)

GCGC and AT-biased mutants AT biased of C. sinensis

A

B

Summary & Conclusions
↑ Biomass of P. hepiali and C. sinensis mutants rDNA with C. sinensis maturation ♦ Differential expressions of C. sinensis mutants:
Premature caterpillar: only AB067721 (GC-bias) Premature stroma: predominated with AT-biased mutants (AB067740 or AB067744) Mature caterpillar: still predominated by AT-biased mutants (AB067740 or AB067744), but more GC-bias expresses (AB067721) Mature stroma: equally express with AT-bias (AB067740 or AB067744) and GC-bias (AB067721) With C. sinensis Maturation: In stroma: expression of GC-bias increases In caterpillar: expression of AT-biases increases

Collection of fresh C. sinensis
♦ Type 1 C. sinensis: 50 pieces ♦ Type 2 C. sinensis: 50 pieces ♦ C. sinensis were collected from QinghaiTibet Plateau, followed by on-site cleaning, surface sterilizing and freezing in liquid nitrogen.

Restrictive digestions of Hsprp1/3 PCR products

♦ C. sinensis exists reversely orientated GC-bias rDNA, indicating reverse chromosome exchanges ♦ Expressions of P. hepiali and AT-biased mutants are synchronized with C. sinensis maturation

MATURATION OF CORDYCEPS SINENSIS ASSOCIATES WITH CHANGES IN PROFILES OF PROTEINS AND SMALL MOLECULAR WEIGHT ORGANIC CHEMICALS
Zhu J-S, Zhang, LJ, Q Y, Liu XJ, Zhang L, Wu ZM, Zheng TY, S Qi Chen W
Pharmanex Research Institute, Provo UT, USA; Shihezi University, Xinjiang; Pharmanex Clinical Center, Beijing; Pharmanex R&D Center, Shanghai; Fudan University, Shanghai
Abstract
Background: Literature reported differential expressions of Paecilomyces hepiali and Hirsutella sinensis DNA in the caterpillar body and stroma of natural Cordyceps sinensis during C. sinensis maturation. Hypothesis: Differential expressions of P. hepiali and H. sinensis in natural C. sinensis may alter the profiles of proteins and other organic chemicals during maturation of C. sinensis. g y Design and Methods: SELDI-TOF MS and HPLC analyses were used to profile proteins and small molecular weight organic chemicals in caterpillar body and stroma of natural C. sinensis and mycelia of P. hepiali and H. sinensis. Results: Maturation of C. sinensis after it is visible above ground associates with altered profiles of proteins and small organic chemicals. The profiles for the mycelia of fungi do not completely match to those for the stroma or caterpillar body of natural C. sinensis during its maturation. The profiles for H. sinensis mycelia appear to be more distinct from those for natural C. sinensis, than the profiles for P. hepiali mycelia. Conclusions: The maturation of C. sinensis associates with significant changes in the component organic chemicals. The compounds from all g p g p component fungi may contribute jointly to the overall pharmacological functions of natural C. sinensis.

Collection of fresh C. sinensis
♦ Type 1 C. sinensis: 90 pieces ♦ Type 2 C. sinensis: 90 pieces ♦ Fresh C. sinensis were collected from Qinghai-Tibet Plateau in early May for Type 1 C. sinensis and in late June for Type 2, followed by on-site cleaning, surface sterilizing and freezing in liquid nitrogen for further process.

HPLC profiling of small molecule chemicals
♦Caterpillars and stromata of C. sinensis, and freeze-dried P. hepiali and H. sinensis mycelia were individually grinded into powder in liquid nitrogen. Small molecules were extracted in either methanol or water, and analyzed for total quality profiling on HPLC with an ODS-C18 (262 nm). ♦The mobile phase was a linear gradient of Acetonitrile (A) and 0.1% H3PO4 with 5mM Sodium salt of 1-Hexanesulflonic Acid (B).

Scientific Background
♦ Cordyceps sinensis (Berk.) Sacc. has been used in china as a precious tonic herb for centuries ♦ It has broad nutritional and health benefits (Zhu et al., J Alternat Compli Med 4:289-303 & 429457, 1998). , ) ♦ Co-existence of P. hepiali and H. sinensis was found in fresh C. sinensis (Zhu et al., J Mycol Res 5:214-224, 2007) ♦ Competitive proliferation abilities are altered associated with unparallel alterations of gene copies of the fungi (EB2008 Program #704.4, Apr. 6 Poster Board #C303)

SELDI-TOF MS profiling of proteins
♦ Caterpillars and stromata of C. sinensis and freeze-dried P. hepiali & H. sinensis mycelia were individually grinded into powder in liquid nitrogen. The powder (0.5 g each) was dissolved in 600 ml Tris-Glycine (pH 8.3) & centrifuged at 14,000 rpm for 5 min at 4 . ♦ Supernatants were diluted with PBS to about 200-300 nM, before being subjected to a normal-phase chip and analyzed on a PBS-II protein chip reader (Surface-enhanced laser desorption ionization – time of flight mass spectra, or SELDI-TOF MS; Ciphergen Biosystems Inc.).

Maturation of C. sinensis and the Temperature on Qinghai-Tibet Plateau
Less Matured Harvest Season Temperature on the Plateau Stroma Early May 0-10 Short A: “Type 1”
A B

Matured Late June 10-20 Long B: “Type 2”

Summary & Conclusions
♦ Distinct profiles of proteins and small molecule chemicals were noted for caterpillars and stromata during C sinensis maturation The maturation C. maturation. associates with dynamic changes in profiles of component chemicals, supporting its superior function profiles. ♦ The profiles for P. hepiali and H. sinensis mycelia do not completely match to those for C. sinensis. ♦ The profiles for P. hepiali mycelia appear to be closer to those for C. sinensis, than those for H. sinensis.

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