Pharmanex's Cordymax Clinical Study

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 31P

Abstract

Changes in Hepatic β-ATP

NMR Spectroscopy α-ATP

MDPA (Ref.)

β-ATP

Pi PME PC

PDE

Ratio of β-ATP P : Ref

4.2

γ-ATP

+18.4%

4.0 38 3.8

+12.3%

3.6

200 mg/kg Placebo

3.2

Treatment 0

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

Ratio of Pi : Ref

1.2

31P

3.8

Placebo 3.2 1

2

3

4

5

6

7

(Days)

Placebo group

(Collected from Qinghai-Tibetan plateau of China)

Isolation & Purification

0.8

200 mg/kg

-24.5%

Treatment 7

14 (Days)

Increases in the Ratio of β-ATP : Pi

4.0

+41 4% +41.4%

200 mg/kg 400 mg/kg

3.0

Placebo

Treatment

2.0

Experimental Design Cordyceps sinensis (Berk.) Sacc.

-17.6%

0.9

+47.7%

p>0.20

0

Placebo 400 mg/kg

0

Ratio of β-ATP : Pi

3.4

Decreases iin H D Hepatic ti Inorganic Phosphate

1.0

5.0 +13.7% p<0.001

p>0.20

14 (Days)

1.1

CordyMax 400 mg/kg/d

3.6

7

0.7

Increases in Hepatic β-ATP In Response to Oral CordyMax

400 mg/kg

3.4

3.0

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

Ratio of β-ATP : Ref

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, baseline after 7 days of treatment, treatment and 7 days after 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 receivingg CordyMax: Groupp A 4.81±0.05 (+47.7% on y ( 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.

0

7

14 (Days)

No treatment (n=6mice)

CordyMax 200 mg/kg

No treatment (n=5mice)

CordyMax 400 mg/kg

No treatment

Changes in Hepatic Tissue pH

(n=5mice) 8

0

(A Paecilomyces hepiali Chen strain)

Industrial Fermentation CordyMax

7

14 6

31P

NMR spectroscopy

31P

NMR spectroscopy

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

pH

Cs-4

(days)

Pre-Rx 4

7-day Rx 7-day post-Rx

2

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

Experimental Design (1):

Experimental Design (2): Randomized, Double-Blind, Placebo Controlled

Placebo

(n=12 rats)

CordyMax

250 mg/kg

(n=12 rats)

CordyMax

500 mg/kg

(n=12 rats)

(n=15)

Placebo CordyMax 0

0 (days)

(4.5 g/day)

(n=15)

(4.5 g/day)

(weeks)

6

18

Sub-maximal exercise-metabolism testing

Reduction of FBG

8 7

Inclusion Criteria:

6

1

p<0.001

p=0.262

2

Day 8

3

Day 18

4

p<0.001

p=0.768

5

Day 0

mol/L) Blood Glucose (mm

0

Vehicle Control

250 500 CordyMax

(mg/kg)

Improvement p of oral Glu tolerance Blood Glucose (mmol/L)

Preliminary reports demonstrated that supplementation with a mycelia fermentation product of Cordyceps sinensis ((CordyMax) y ) increased maximal O2 uptake p and anaerobic 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<00.01). (p 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.

Vehicle Control

11 10

CordyMax 250 mg/kg

9 8

AUC: p=0.022

7

5 0.5

Training Background

Training vol. Hrs./wk.

Number of Subjects

Adventure Racers

11 ± 7

9

Pro/Competitive Triathletes

15 ± 7

10

Active Military (Reco/SeALs)

11 ± 1

3

Olympic Rower/Kayak

10

1

Ultra-distance runner

14

1

9± 4

6

High Fit Multi-sport

AUC: p=0.005

6 0

Healthy, highly fit, male endurance athletes under the age of 40 years

1 1.5 Time (hours)

2

Constant work-rate, sub-maximal exercise

Decrease in FBG 100

Isolation & Purification

20 10

Post-Rx

80 70

p=0.006 -7.22%

p=0.870 +0.89%

60

30

50

p=0.012

(Collected from Qinghai-Tibetan plateau)

40

p=0.225

Cordyceps sinensis (Berk.) Sacc.

Insulin (mU/ml)

Reduction of fasting serum insulin

Pre-Rx

(mg/dL)

90

Placebo

CordyMax

0

Cs-4

(A Paecilomyces hepiali Chen strain)

Vehicle Control

250 500 CordyMax

Reduction of RER during sub-maximal exercise 1.05

Increase in insulin sensitivity

♦ 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, RER ↓ Lactic acid ♦ 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).

RER

0.22 0.20 0 18 0.18 0.16

p<0.001

Introduction

Placebo

CordyMax

1.00

0.24

p=0.0 008

CordyMax

Insulin n Sensitivity

Industrial Fermentation

250

500 (mg/kg)

0.14

0.95

0.85

Post-Rx

Pre-Rx

0.80 0

Vehicle Control

Pre-Rx

Post-Rx

0.90

10

20

30

CordyMax

40

50

60

0

10

20

Exercise Time (min)

Summary and Conclusion CordyMax y Treatment: ♦ 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.

30

40

50

60

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; Ph Pharmanex Provo, P UT, UT USA Abstract

Study Aims

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 y p subjects j on increasing g HDL cholesterol (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=0.003)) in males. Apolipoprotein (p p p p A1 was increased with 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.

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

Experimental Design Randomized; Double-blind; Placebo Controlled

-2

CordyMax 3 g/day

n=77

0 (weeks)

39

Week 0

41

Week 8

Week 0

Week 8

43

45 37 40

35

35

33

30 25

31

+14.7% p=0.015

+31.1% p<0.001

29

p=0.261

+12.3% p=0.003

27 25

Placebo CordyMax

Increases in Serum apoA1 1.8

8

Serum HDL-cholesterol Males ≤ 40 mg/dl

1.6 1.4 1.2 1.0 0.8

p=0.934

0.6

+34.7% p=0.005

0.4 0.2

females ≤ 45 mg/dl

0.0

Placebo

Reduction of Serum LDL-Cholesterol LDL Cholesterol

CordyMax

Reduction of Atherosclerosis Index 8

-5.4% p=0.002

130

1

-29.0% p<0.001

CordyMax

Reduction of Ratio of OxLDL:HDL

30

20 p=0.549 Week 8

-22.9% p=0.001

Ratio o of oxLDL : HDL

0.8

25

Week 0

Serum oxLDL (mg/dl)

2

Placebo

Reduction of Oxidized LDL-Cholesterol

5

p=0.309

3

CordyMax

Placebo

10

4

0

100

15

5

0.7 0.6 0.5

p=0.852

0.4 0.3 0.2 0.1

Week 8

110

Week 8

Week 0

120

6

Week 8

p=0.446

Week 0

140

7

Week 0

150

Atherosclerosis Index

160

♦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)

n=56

p=0.023

Placebo CordyMax

Inclusion Criteria:

Serum LDL (mg/dl)

♦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)

3 g/day

50

Males

20

Serum apoA1 (mg g/dl)

Lead-in Phase

Placebo

Females 55

Serum HDL (mg/dl)

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

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)

Increases in Serum HDL-Cholesterol

-10.5% p=0.003

0.0

0 Placebo

CordyMax

Placebo

CordyMax

Cordyceps sinensis (Berk.) Sacc.

(Collected from Qinghai-Tibetan plateau of China)

Isolation & Purification Cs-4

(A Paecilomyces hepiali Chen strain)

Industrial Fermentation CordyMax

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.

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 4.6

CM

((n=48 mice)) (n=48 mice)

CM 1000 mg/kg

(n=48 mice)

CM 1500 mg/kg

(n=48 mice) ~ 3 years

8m-o

Food intake (g)

16 Live 21 Live

10

20

30

40

50

60

70

Time ((weeks))

4.9

CM 1000 CM 500 Control

4.1 3.7 33 3.3 2.9

CM 1500 2.5

10

20

30

40

50

27 m-old

Kaplan-Meier Cumulative Survival Plot

Food intake of male mice 4.5

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

60

70

Cumulative Survival Rate

2.2

After 64 weeks of CM Treatment

1.0

CM 1500

0.8

CM 1000

0.6

Control

0.4

CM 500

0.2

p=0.027 0 0

Time (weeks)

100

200

300

400

500

Time (days)

CM 500

Body Weight (g)

48 46

Control

44 42 40

CM 1500

38 36

CM 1000

34 32 30

10

20

30

40

50

60

Cumulative Survival Rate

Kaplan-Meier Cumulative Survival Plot

50

1.0

0.8

CM 500 0.6 0.4

0.2 0

70

0

Time (weeks)

Bod dy Weight (g)

160

45 43 41

CM 1000 39

CM 1500

37

10

20

30

40

50

60

70

100

200

300

400

500

Time (days)

BW of male mice CM 500 Control

Control

p=0.013

Lifespan Extended (Days)  at the % Survival 

140 120 100 80 60 40 20 0 ‐20

500mg/kg

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

1000mg/kg ‐6 49 44 46 35 43 25 87 92 10 84 94 63 59 56 35 35 35 25 ‐2

‐6 13

1500mg/kg 75 87 88 95 78 13 13 12 12 13 12 10 74 63 60 35 35 32 28 ‐9

5

3

10 14 25 15

19 39 39 38 43 28

Summary

500 mg/kg

Treatment starts at the age of 12 months

32 dead 27 dead

CM 1500

Time (weeks)

Experimental Design: Lifespan studies in mice Vehicle control

CM 500

2.6

35

CordyMax

CM 1000 mg/kg CM 1500 mg/kg

Lifespaan (days) extended

Industrial Fermentation

26 Live

3.0

47

(A Paecilomyces hepiali Chen strain)

20 Live

22 dead

3.4

Cordyceps sinensis (Berk.) Sacc.

Isolation & Purification

28 dead

CM 500 mg/kg Control

3.8

BW of female mice

(Collected from Qinghai-Tibetan plateau of China)

Vehicle control CM 1000

4.2

Introduction 1. Cordyceps sinensis is traditionally believed as a medicinal herb with anti anti-aging aging activities and promoting longevity. 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.

Cs-4

Deaths in Each Group

Food intake of female mice

Food intake (g)

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 CordyMax y administration continues until all mice die. females respectively. 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 treatment ((equivalent q to the human dose)) appeared pp to low dose CordyMax 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.

all mice dead

1 Analysis with use of Kaplan 1. Kaplan-Meier Meier Cumuli Survivor Plot showed significant 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.

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

(A Paecilomyces hepiali Chen strain)

Vehicle

Vehicle control

(n=15)

CordyMax 500 mg/kg

(n=15)

CordyMax 1000 mg/kg

(n=15)

CordyMax 1500 mg/kg

(n=15)

Normal control

60Co

(11Gy)

+16% p<0.001

+15%; p=0.002

500

1000

1500(mg/kg)

CordyMax 60Co

-11Gy

-11Gy

Increases in liver GSH-Rd

4

2

0 Normal control

500

Vehicle

1000

35 30

+ 7.6% p=0.072 20

10

0

1500 (mg/kg)

C CordyMax 60Co

+11%; p=0.039

+29% p<0.001

GSH-Rd (nmol/min/mg pro o.)

+26.3%; p=0.023

6

+26.5%; p=0.026

8

Normal Vehicle control

500

1000

Increases in liver SOD 8.0

1500 (mg/kg)

CordyMax 60Co

-11Gy

-11Gy

Decreases in liver protein  carbonyl groups 

500 400 300

+6.0% p=0.018

200 100

500

1000

1500 (mg/kg)

CordyMax

-11Gy

Summary

Collect blood & liver

Vehicle

40

Normal Vehicle control

63

+16%; p=0.001

CAT (U/mg pro.)

0

1500 (mg/kg)

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

60

10

Increases in plasma GSH

Experimental Design: (n=15)

20

CordyMax

60Co

Non-radiation control

+ 25.0%; p<0.001

1000

60Co

CordyMax

(days)

500

0

Industrial Fermentation

0

+ 20.8%; p=0.001

Normal control

+ 22.9%; p=0.001

Thiol groups (mmol/L)

0.3

+ 24.7% p<0.001

↑ ↑ ↑ ↑ ↑ ↓

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

60 6.0

4.0

- 37% p<0.001

2.0

- 14%; p=0.001

Cs-4

0.4

30

- 8.7%; p=0.035

Isolation & Purification

0.5

Carbonyl group ps (nmol/mg pro o.)

(Collected from Qinghai-Tibetan plateau of China)

+22.6% p<0.001

+ 5.7%; p<0.05

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

40 0.6

+ 9.4%; p<0.05

♦ 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

Increases in liver CAT

0.7

Glutathione(mg/L)

Introduction

Increases in plasma thiol groups

SOD (U/mg prro.)

Abstract Cordyceps sinensis and its mycelia fermentation product CordyMax have g and endurance enhancement. We been used for Centuries for anti-fatigue 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 p dismutase (SOD) ( ) and catalase (CAT) ( ) were 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) byy 9.4% and 5.7% ((both p<0.05). p ) Liver GSH-reductase was increased with 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.

0 Normal control

Vehicle

500

1000

1500 (mg/kg)

CordyMax 60Co

-11Gy

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.

ANTI-FATIGUE AND ENDURANCE ENHANCEMENT PROPERTIES OF CORDYMAX IN HUMANS Zhu JS, Xiao Y, Li CL, Huang XZ, Hagan RD

Placebo CordyMax 0

(weeks)

6 or 12

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

Peak Oxygen Uptake 2.2

+6.7%, p=0.05

1.8 1.6

Week k6

2.0

-0.05% 0 05% p=0.80

work rate: +25 watt every 2 min

+12.6%, p=0.012

13 1.3

Week 0

1.1 1.0 0.9 0.8

Time (min)

Week 6

1.2

CordyMax

Placebo

CordyMax

Placebo

+3.1%, p=0.033

1.4 1.3 1.2 1.1 1.0

-4.9% p=0.626

+2% 0% -2% -4%

CordyMax

Placebo

1-Mile Walk Time 5%

p=0.05

3% 2% 1% 0% -1% -2% -3% -4% -5%

CordyMax

Placebo

CordyMax

RER during CWR +4% +3%

p=0.025

+2% +1% 0% -1% -2% -3% -4%

CordyMax

Placebo

Lactic Acid during CWR

+30% +20%

p=0.031

+10% 0% -10% -20%

CordyMax

Placebo

Summary y & Conclusion

0.9

4%

p=0.05

+4%

-2.4% p=0.41

W k Output Work O t t 1.5

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

VO2 (L/min)

Work Rate at 70% VO2max

-4%

+6%

Average % Change in Lactic Acid

1.4

Time (min) Constant Work Rate (CWR) Sub-Maximal Exercise

-3%

+8%

Averagee % Change in RER

Anaerobic Threshold

Week 12

VO2 (L/min)

Incremental work-rate (IWR) exercise on cycle ergometer

-2%

Placebo

CordyMax

Week 0

♦ 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)

0% -1%

1.4

Anaerobic Threshold ((L/min)

♦ CordyMax extends lifespan and enhances body’s antioxidant activities

p=0.05 p 0.05

+1%

+10%

Work Output (KJ/kg)

♦ CordyMax, the mycelia fermentation product of C. sinensis, p senescence-related symptoms, y p , including g fatigue, g , improves and increases activities of scavenging oxygen free radicals in elderly senescent patients (Administ Tradit Chin Med 1995; 5:14-18)

+2%

+12%

Introduction ♦ Cordyceps sinensis is traditionally believed as an antiaging tonic herb

Heart Rate during CWR +3%

O2 Pulse P l during d i CWR

Average % Change of O2 Pulse

To Examine the Effects of CordyMax on Aerobic Capacity, Endurance Enhancement, and Exercise metabolism in Humans Randomized, Double-blind, Placebo-controlled

Week k0

Cordyceps sinensis and a standardized mycelial fermentation product of C. sinensis, CordyMax, are known as medicinal herbal products for invigoration, h lth preservation, health ti anti-aging, ti i andd anti-fatigue. ti f ti Th Theiri anti-fatigue ti f ti andd 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 i d th the anti-fatigue ti f ti andd endurance d enhancement h t properties ti off 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.

(L/min)

Abstract

Average % Change in Heaart Rate

Pharmanex Research Institute, Institute Provo UT UT, USA; Shihezi University, University Xinjiang; Pharmanex Clinical Center, Beijing; Peking Union University, Beijing; Peking University, Beijing; The Fit Stop Human Performance & Health Enhancement Laboratory, Encinitas, CA, USA

Placebo

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 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

Template: 1

2

1

2

3

Fresh C. sinensis

4

5

6

7

8

9

10

11

AccI

(bp)

AccI

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

500 400 300 200 100

AccI

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 y p sinensis,, 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.

Nested PCR Products Using Phsp3/5Primers

ITS PCR Products

Abstract

DNA Sequences 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.

Alu I

Ava I

Acc I

Alu I

Ava I

BamHI

Endonuclease Digestions of P. hepiali & H. sinensis ITS PCR Products BamHI

♦ Through competitive proliferation, live Paecilomyces hepiali and Hirsutella sinensis were identified simultaneously (Guo and Zhu, FASEB J. 19: A1033, 2005).

Acc I

♦ 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.

Experimental Methods

2. A pair of Paecilomyces hepiali primers, Phsp3 and Phsp5, and a touch touch-down down PCR protocol were used for the 2nd PCR: initially annealing at 70 Ԩ with 0.3 Ԩ 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.

4

5

6

7

8

9

10

11

H. sinensis ITS4/5

DNA Template:

P. hepiiali

PCR Products Using Phsp3/5 Primers Fresh C. sinensis

600 500 400 300 200 100

Lane:

1

2

3

4

5

6

7

8

9

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.

10

Discussions

Lane:

1

AvaI

1. A pair of primers, ITS4/5, were used for the 1st PCR: annealing at 40 Ԩ for 30 cycles.

3

P. hepiali ITS4/5

AccI

♦ A nested, dual-step PCR technique was used (Guo and Zhu, FASEB J. 20: A431, 2006).

2

AvaI

♦ Caterpillars and stromata of natural C. sinensis were separately grounded into powder d iin li liquid id nitrogen. it Rib Ribosomal l DNA was extracted.

Lane: 1

DNA template:

AccI

♦ 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.

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).

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

Maturational alterations of differential expressions of GC:AT-biased Cordyceps sinensis mutants and Paecilomyces hepiali in natural Cordyceps sinensis Zhu J-S, J-S Gao L, L Yao YS, YS Zhou YJ, YJ Zheng TY, TY Chen W 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 when Cs stroma ggenomic DNA used as the template p showed increased qquantityy of regions 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.

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)

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

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

Maturation of C. C sinensis and the Temperature on Qinghai-Tibet Plateau Less Matured Harvest Season

Early May

Temperature on the Plateau

0-10 Ԩ

Stroma

Short A: “Type 1”

A

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

C t ill Stroma Caterpillar St Collected in: May Jun May Jun Type of C. sinensis: 1 2 1 2

Reversely orientated rDNA sequences (GC-bias)

H. sinensis 18S Internal Control

GC and AT-biased GCAT biased mutants of C. sinensis

B

Summary & Conclusions ↑ Biomass of P. hepiali and C. sinensis mutants rDNA with C. sinensis maturation ♦ Differential expressions of C. sinensis mutants:

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

¾ Premature caterpillar: only AB067721 (GC-bias) ¾ Premature stroma: predominated with AT-biased mutants (AB067740 or AB067744) ¾ Mature caterpillar: still predominated by AT-biased mutants (AB067740 or AB067744), but more GC-bias expresses (AB067721) ¾ Mature stroma: equally express with AT-bias (AB067740 or AB067744) and GC-bias (AB067721) ¾ With C. sinensis Maturation: ƒ In stroma: expression of GC-bias increases ƒ In caterpillar: expression of AT-biases increases

♦ 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, S Zhang, LJ, Qi Q Y, Liu XJ, Zhang L, Wu ZM, Zheng TY, Chen W Pharmanex Research Institute, Provo UT, USA; Shihezi University, Xinjiang; Pharmanex Clinical Center, Beijing; Pharmanex R&D Center, Shanghai; Fudan University, Shanghai Collection of fresh C. sinensis

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 and Methods: SELDI-TOF MS and HPLC analyses y were used to Design 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 g in the component p organic g chemicals. The compounds p from all component fungi may contribute jointly to the overall pharmacological functions of natural 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 Ԩ. ♦ 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

Matured

Harvest Season

Early May

Temperature on the Plateau

0-10 Ԩ

10-20 Ԩ

Stroma

Short

Long

A: “Type 1” A

Late June

B: “Type 2”

B

Summary & Conclusions ♦ Distinct profiles of proteins and small molecule chemicals were noted for caterpillars and stromata during C. C sinensis maturation. maturation The 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.