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-SPharmanex Pharmacology Center Beijing China; Institute of Materia Medica;

AbstractNatural 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 MDPA (Ref.)

PMEPi

γ-ATPα-ATP

β-ATP

31P NMR Spectroscopy Changes in Hepatic β-ATP

3 8

4.0

4.2+18.4%

P :

Ref

Pharmanex Pharmacology Center Beijing , China; Institute of Materia Medica; Peking Union Medical University, Beijing, China; Pharmanex Provo, USA

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

PCPDE

Note:MDPA methylene diphosphonic acid PME phosphomonoestersPi inorganic phosphate

3.0

3.2

3.4

3.6

3.8

Treatment

Placebo200 mg/kg400 mg/kg

0 7 14 (Days)

+12.3%

Rat

io o

f β-A

TP

D i H tiwere made at baseline, after 7 days of 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 receiving CordyMax: Group A 4.81±0.05 (+47.7% on

Pi inorganic phosphatePDE phosphodiestersPC phosphocreatine

31P NMR Spectroscopy on mouse liver: α-, β-, γ-ATP, other triphosphorate compounds, and inorganic phosphate; calculate cellular pH.

0.8

0.9

1.0

1.1

1.2

Placebo400 mg/kg

200 mg/kg

Rat

io o

f Pi :

Ref

-17.6%

-24.5%

Decreases in Hepatic Inorganic Phosphate

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 CordyMaxincreased 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.

3.4

3.6

3.8

io o

f β-A

TP :

Ref CordyMax

400 mg/kg/d

p>0.20p>0.20

+13.7%p<0.001

Increases in Hepatic β-ATPIn Response to Oral CordyMax

Treatment0.7

0 7 14 (Days)

4.0

5.0

P :

Pi

+47.7%

+41 4%

Increases in the Ratio of β-ATP : Pi

3.2

Rat Placebo

0 1 2 3 4 5 6 7 (Days)

Changes in Hepatic Tissue pH

Treatment2.0

3.0

4.0

Rat

io o

f β-A

T

Placebo400 mg/kg

200 mg/kg

0 7 14 (Days)

+41.4%

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

Experimental DesignPlacebo group No treatment (n=6mice)

CordyMax 200 mg/kg No treatment (n=5mice)

0

2

4

6

8

Pre-Rx

7-day Rx

7-day post-Rx

pH

Changes in Hepatic Tissue pH

CordyMax

Industrial Fermentation

Isolation & Purification

Cs-4 (A Paecilomyces hepialiChen strain)

At the time indicated, •Anaesthetized mice with Pentobarbital

•Preformed 31P NMR

0 (days) 7 14

31P NMR spectroscopy

CordyMax 400 mg/kg No treatment (n=5mice)

31P NMR spectroscopy

0Placebo 200 400 (mg/kg/d)

CordyMax

Summary7-day CordyMax Treatment:

↑ Hepatic ATP↓ Hepatic inorganic phosphate

CordyMax •Preformed 31P NMR spectroscopy on mouse liver

Introduction♦ CordyMax improves aerobic capability

and enhances endurance in older humans (Chin J Integrat Med 2004; 10:187-192; SH J Prevent Med2008, 20:367-369).↑ VO2max, ↑ Anaerobic Threshold,

Conclusion• CordyMax improved steady-state hepatic bio-energy status

↑ Hepatic ratio of ATP:PiNo ∆ in hepatic tissue pH

↑ 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).

CordyMax Improves Glucose Metabolism in Animals and in Humans

Wu ZM, Nicodemus K, Zhao CS, Zhu J-SPharmanex Pharmacology Center, Beijing, China; Fit Stop Human Performance Lab, Encinitas, CA, USA, Pharmanex Provo, USA

AbstractPreliminary reports demonstrated that supplementation witha mycelia fermentation product of Cordyceps sinensis(CordyMax) increased maximal O2 uptake and anaerobic Placebo (n=12 rats)

Experimental Design (1): Experimental Design (2):Randomized, Double-Blind, Placebo Controlled ( y ) 2 p

threshold in mid-age to elderly humans, and enhanced invivo bio-energy metabolisms in animals (J AlternatComplement Med 7:231, 2001; Chin J Integrat Med 10:187,2004; Shanghai J Prevent Med 20:367, 2008). We furtherstudied in a randomized, double-blind clinical trial the effectof CordyMax (4.5 g/day, 6 weeks) in highly-fit athletes onglucose metabolism. Male adventure racers and multi-sportendurance athletes (age 32±4 yrs; VO2peak 63±8ml/kg/min) were assigned to either a CordyMax or a controlgroup (n=15 each). We found a 7% decrease in fast bloodglucose within normal ranges after the CordyMax therapy

Placebo (n=12 rats)

0 (days) 18

CordyMax 500 mg/kg (n=12 rats)

CordyMax 250 mg/kg (n=12 rats)

6

7

8 Reduction of FBG

mol

/L)

Inclusion Criteria:

0 (weeks) 6

Placebo (n=15) (4.5 g/day)

CordyMax (n=15) (4.5 g/day)

Sub-maximal exercise-metabolism testing

glucose within normal ranges after the CordyMax therapy(92±1 to 87±2 mg/dL; p<0.01), but no change in placebocontrols. During prolonged sub-maximal exercise (70%VO2peak, 60 min), reductions of respiratory exchange ratiowere found in the CordyMax vs. control group (p=0.02). Inmice given CordyMax for 4 weeks, responses of seruminsulin and C-peptide to an oral glucose load werediminished and recovered to the pre-load levels quickly vs.control group (p<0.01 or 0.05) with no change in the glucosetolerant curve. The glucose-insulin index was lower in theCordyMax (7±1 x105 units) vs. control group (10±1 x105)(p<0 01) Our data suggest that CordyMax (1) safely lowers Improvement of oral Glu tolerance

0

1

2

3

4

5

6

Day

0

Day

8

Day

18

Vehicle Control

250 500 (mg/kg)CordyMax

p=0.

768

p<0.

001

p=0.

262

p<0.

001

Blo

od G

luco

se (m

m Inclusion Criteria:Healthy, highly fit, male endurance athletes under the age of 40 years Training Training vol. Number ofBackground Hrs./wk. Subjects

Adventure Racers 11 ± 7 9

Pro/Competitive Triathletes 15 ± 7 10

Active Military (Reco/SeALs) 11 ± 1 3(p 0.01). Our data suggest that CordyMax (1) safely lowersbasal glucose in normal humans, (2) improves glucosemetabolism by enhancing insulin receptor sensitivity, and (3)enhances fat mobilization and beta-oxidation therebysparing glycogen expenditure during prolonged enduranceexercise.

5

6

7

8

9

10

11

0 0.5 1 1.5 2Time (hours)

Blo

od G

luco

se (m

mol

/L) Vehicle

ControlCordyMax250 mg/kg

AUC:p=0.022 AUC:

p=0.005

pOlympic Rower/Kayak 10 1

Ultra-distance runner 14 1

High Fit Multi-sport 9± 4 6

Constant work-rate, sub-maximal exercise

Decrease in FBG

90

100

50

60

70

80

90

Placebo CordyMax

(mg/

dL)

p=0.870+0.89%

p=0.006 -7.22%

Pre-

Rx

Post

-Rx

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


Reduction of fasting serum insulin

0

10

20

30

40

Insu

lin (m

U/m

l)

p=0.

225

p=0.

012

0 18

0.20

0.22

0.24

n Se

nsiti

vity

008 01

Increase in insulin sensitivity

Reduction of RER during sub-maximal exercise

Pre-Rx0.90

0.95

1.00

1.05

RER

Placebo CordyMax

Post-RxCordyMax


Cs-4 (A Paecilomyces hepiali Chen strain)

0250 500

CordyMaxVehicle Control

0.14

0.16

0.18

Vehicle Control

250 500 (mg/kg)

Insu

lin

CordyMax

p=0.

0

p<0.

0

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

Exercise Time (min)

Post-Rx

0.80

0.85

0 10 20 30 40 50 60 0 10 20 30 40 50 60

Pre-Rx

Summary and ConclusionCordyMax Treatment:↓ 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 Med2001; 7:231-240).

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-CHOLESTEROLWu 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 P UT USA

AbstractLiterature 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

Study Aims1. To examine the effect of CordyMax

on serum HDL-cholesterol2. To examine the effect of CordyMax

on serum oxidized LDL-cholesterol

Increases in Serum HDL-Cholesterol

40

45

50

55

DL

(mg/

dl)

35

37

39

41

43

Wee

k 0

Wee

k 8

Females Males

p=0.023

Wee

k 0

Wee

k 8

Pharmanex Provo, UT, USA

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=0.003) in males. Apolipoprotein A1 was increased with

Experimental DesignRandomized; Double-blind;

Placebo Controlled

-2 0 (weeks) 8

CordyMax 3 g/day n=77

Placebo 3 g/day n=56Lead-inPhase

20

25

30

35

Placebo CordyMax

Seru

m H

25

27

29

31

33

Placebo CordyMax

+14.7%p=0.015

+31.1%p<0.001

p=0.261 +12.3%p=0.003

Increases in Serum apoA1

1.4

1.6

1.8

g/dl

)

(p ) p p pCordyMax 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.

Introduction♦CordyMax increases activities of scavenging oxygen

free radicals in elderly senescent patients

Inclusion Criteria:Serum HDL-cholesterol

Males ≤ 40 mg/dl

females ≤ 45 mg/dl

Reduction of Serum LDL-Cholesterol

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Placebo CordyMaxSe

rum

apo

A1 (m

g

p=0.934+34.7%p=0.005

Reduction of Atherosclerosis Index(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

Reduction of Serum LDL Cholesterol

100

110

120

130

140

150

160

Seru

m L

DL

(mg/

dl)

Wee

k 0

Wee

k 8

p=0.446 -5.4%p=0.002

0

1

2

3

4

5

6

7

8

Athe

rosc

lero

sis

Inde

x

p=0.309-29.0%

p<0.001

Wee

k 0

Wee

k 8

♦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 Placebo CordyMax

Reduction of Oxidized LDL-Cholesterol

10

15

20

25

30

m o

xLD

L (m

g/dl

)

p=0.549-22.9%

p=0.001

0 8

Reduction of Ratio of OxLDL:HDL

0.3

0.4

0.5

0.6

0.7

0.8

o of

oxL

DL

: HD

L

p=0.852-10.5%

p=0.0030 8

0

5

Placebo CordyMax

Seru

Wee

k 0

Wee

k 8

0.0

0.1

0.2

Placebo CordyMax

Rat

io

Wee

k

Wee

k

SummaryCordyMax supplementation for 8 weeks

In all patients ↑ Serum HDL cholesterol

ConclusionCordyMax treatment increases

serum HDL-c and decreases oxidized



↑ Serum HDL-cholesterol↓ Serum oxLDL↓ Ratio of oxLDL:HDL

In patients with baseline serum LDL-c >130 mg/dL↓ Serum LDL-cholesterol↑ Serum apoA1↓ Atherosclerosis IndexNo ∆ serum triglycerides

LDL-c in patients with reduced baseline serum HDL-c, indicating reduced risks of atherosclerosis, and ischemic cardio- and cerebro-vascular diseases.

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

AbstractCordyceps 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 4.6

Food intake of female mice Deaths in Each Group

Vehicle control 28 dead 20 Liveimprovement 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 females respectively. CordyMax administration continues until all mice die.

2.2

2.6

3.0

3.4

3.8

4.2

10 20 30 40 50 60 70

Time (weeks)

Food

inta

ke (g

)

Control

CM 500

CM 1000

CM 1500After 64 weeks of CM Treatment

27 m-old12 m-old (n=48)8 m-old

Vehicle control

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

28 dead22 dead32 dead27 dead

20 Live26 Live16 Live21 Live

y yMice 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 low dose CordyMax treatment (equivalent to the human dose) appeared to

( )

3 3

3.7

4.1

4.5

4.9

Food intake of male mice

d in

take

(g)

ControlCM 500

CM 1000

8 m-old

Kaplan-Meier Cumulative Survival Plot

0.4

0.6

0.8

1.0

CM 500

CM 1000

CM 1500

e Su

rviv

al R

ate

Controly ( q ) ppshow 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.

Introduction1. Cordyceps sinensis is traditionally believed as a medicinal

herb with anti-aging activities and promoting longevity. 48

50

BW of female mice

CM 500

2.5

2.9

3.3

10 20 30 40 50 60 70

Time (weeks)

Foo

CM 15000

0.2

0 100 200 300 400 500Time (days)

CM 500

Cum

ulat

iv

p=0.027

Kaplan-Meier Cumulative Survival Plot1.0

ate

herb with anti 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

30

32

34

36

38

40

42

44

46

10 20 30 40 50 60 70Time (weeks)

Bod

y W

eigh

t (g)

Control

CM 1000

CM 1500

0.2

0.4

0.6

0.8

0

0 100 200 300 400 500

Cum

ulat

ive

Surv

ival

R

Time (days)

p=0.013

CM 500

Control

animals and humans in previous studies. 4. The aim of this study is to test the lifespan extension effect

of CordyMax in mice. BW of male mice

39

41

43

45

47 CM 500

dy W

eigh

t (g)

Control

CM 1000CM 1500 40

60

80

100

120

140

160

an (d

ays) exten

ded

Lifespan Extended (Days) at the % Survival



Cs-4 (A Paecilomyces hepiali

Experimental Design:Lifespan studies in miceVehicle control (n=48 mice)

Time (weeks)

35

37

10 20 30 40 50 60 70

Bod

CM 1500

97.9%

95.8%

93.8%

91.7%

89.6%

87.5%

85.4%

83.3%

81.3%

79.2%

77.1%

75.0%

72.9%

70.8%

68.8%

66.7%

64.6%

62.5%

60.4%

58.3%

56.3%

54.2%

52.1%

50.0%

47.9%

45.8%

43.8%

500mg/kg 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 3 10 14 25 15

1500mg/kg 75 87 88 95 78 13 13 12 12 13 12 10 74 63 60 35 35 32 28 ‐9 5 19 39 39 38 43 28

‐20

0

20

40

Lifespa

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

CordyMax

Industrial FermentationChen strain)

( )

CM 1500 mg/kg (n=48 mice)CM 1000 mg/kg (n=48 mice)CM 500 mg/kg (n=48 mice)

~ 3 years

all mice dead

Treatment startsat the age of 12 months8m-o

1. Analysis with use of Kaplan 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

AbstractCordyceps sinensis and its mycelia fermentation product CordyMax have been used for Centuries for anti-fatigue and endurance enhancement. We

Increases in plasma thiol groups0.7

Increases in liver CAT

Pharmanex Beijing, China; Pharmanex Research Institute, Provo Utah, USA

greported 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 anti-aging effect of CordyMax in mice. To explore the mechanism of the anti-aging 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 Th

iol g

roup

s (m

mol

/L)

0.5

0.6

0.4

+ 22

.9%

; p=0

.001

+ 20

.8%

; p=0

.001

+ 25

.0%

; p<0

.001

+22.6% p<0.001

CAT

(U/m

g pr

o.)

10

20

30

0

40

+ 24.7% p<0.001

+16%

; p=0

.001

+15%

; p=0

.002

+16%

p<0

.001

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% (both p<0.05). Liver GSH-reductase was increased with

Increases in plasma GSH

500 1000 1500 (mg/kg)CordyMax

60Co -11Gy

Normal control

Vehicle0.3

Increases in liver GSH-Rd

o.)

35

40

500 1000 1500(mg/kg)CordyMax

0Normalcontrol

Vehicle

60Co -11Gy

y ( 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.

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:0

2

4

6

8

Normal control

Vehicle 500 1000 1500 (mg/kg)C

+26.

3%;

p=0.

023

+26.

5%;

p=0.

026

Glu

tath

ione

(mg/

L)

+29% p<0.001

10

0Normal control Vehicle 500 1000 1500 (mg/kg)

+ 7.6% p=0.072

+11%

; p=

0.03

9

GSH

-Rd

(nm

ol/m

in/m

g pr

o20

30

35

sinensis, has the following functions:o Anti-fatigue and endurance enhancemento 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 SOD

ro.)

500

control CordyMax

60Co -11Gy

Decreases in liver protein carbonyl groups

ps o.)

6 0

8.0

control CordyMax60Co -11Gy

Cordyceps sinensis (Berk.) Sacc.

SOD

(U/m

g pr

Normal control

300

200

100

0

400

500 1000 1500 (mg/kg)CordyMax

Vehicle

+ 9.

4%;

p<0.

05

+ 5.

7%;

p<0.

05

+6.0% p=0.018

Carb

onyl

grou

p(n

mol

/mg

pro

Normal control

2.0

0

4.0

6.0

Vehicle 500 1000 1500 (mg/kg)CordyMax

- 37% p<0.001

-8.7

%;

p=0.

035

-14%

; p=

0.00

1y p ( )

(Collected from Qinghai-Tibetan plateau of China)




Experimental Design:Non-radiation control (n=15)

Vehicle control (n=15)CordyMax 500 mg/kg (n=15)

60Co -11Gy 60Co -11GyCordyMax

SummaryCordyMax Treatment for 60 days in

radiation-induced oxidative injured mice: ↑ Plasma thiol groups↑

Conclusion♦ CordyMax improves

antioxidant capacity in mice with radiation-induced oxidative injure.

CordyMax 1000 mg/kg (n=15)CordyMax 1500 mg/kg (n=15)

0 (days) 60 63

Collect blood & liver

60Co(11Gy)

↑ Plasma glutathione↑ Liver SOD↑ Liver CAT↑ Liver GSH-Rd↓ Liver protein carbonyl groups

j♦ The results from this study

supports the concept of CordyMax as an anti-aging product that has been demonstrated by the lifespan-extending study.

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

AbstractCordyceps sinensis and a standardized mycelial fermentation product of C. sinensis, CordyMax, are known as medicinal herbal products for invigoration, h lth ti ti i d ti f ti Th i ti f ti d

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

To Examine the Effects of CordyMax on Aerobic Capacity, Endurance Enhancement, and Exercise metabolism +2%

+3%

art R

ate

p=0.05

Heart Rate during CWR

health preservation, anti-aging, and anti-fatigue. Their anti-fatigue and 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

i d th ti f ti d d h t ti f Exercise tests Exercise tests

H lth ld bj t & thl t

0 (weeks) 6 or 12

Placebo

CordyMax

in Humans Randomized, Double-blind, Placebo-controlled

-4%

-3%

-2%

-1%

0%

+1%

+2%

Aver

age %

Cha

nge i

n He

a

CordyMax Placebo

p 0.05

O P l d i CWRexamined the anti-fatigue and endurance enhancement properties of 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

Healthy older subjects & athletes

1.8

2.0

2.2

(L/m

in)

0 05%

+6.7%, p=0.05

k 0

k 6

Peak Oxygen Uptake

-4%

-2%

0%

+2%

+4%

+6%

+8%

+10%

+12%

Aver

age %

Cha

nge o

f O2 P

ulse

p=0.05

O2 Pulse during CWR

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.

Introduction♦Cordyceps sinensis is traditionally believed as an anti-

aging tonic herb♦CordyMax, the mycelia fermentation product of C. sinensis,

improves senescence-related symptoms, including fatigue, (L/m

in) +12.6%, p=0.012

1 31.4

Anaerobic Threshold

1.4

1.6-0.05%p=0.80

CordyMax Placebo

Wee

k

Wee

k

-1%

0%

+1%

+2%

+3%

+4%

e % C

hang

e in

RER p=0.025

RER during CWR

CordyMax Placebo

p y p , g g ,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 anti-oxidant activities

♦CordyMax improves energy, glucose and lipids metabolisms in humans and animals (J Alternat Compl Med2001; 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)

CordyMax Placebo

Anae

robi

c Thr

esho

ld (

-2.4%p=0.41

0.80.91.01.11.21.3

Wee

k 0

Wee

k 6

W k O t t

-4%

-3%

-2%

Aver

age

CordyMax Placebo

+20%

+30%

Lac

tic A

cid

p=0.031

Lactic Acid during CWR

Incremental work-rate (IWR) exercise on cycle ergometer

VO2

(L/m

in)

work rate: +25 watt

Work Output

0.9

1.0

1.1

1.2

1.3

1.4

1.5

Wor

k Out

put (

KJ/kg

) +3.1%, p=0.033

-4.9%p=0.626

Wee

k 0

Wee

k 12

-20%

-10%

0%

+10%

Aver

age %

Cha

nge i

n

CordyMax Placebo

Summary & Conclusion

Time (min)

+25 watt every 2 min

Constant Work Rate (CWR) Sub-Maximal Exercise

CordyMax Placebo

1%2%3%4%5%

e for

1-Mi

le W

alk0 t

o W

eek 1

2 p=0.05

1-Mile Walk Time

yCordyMax Treatment:↑ Peak O2 uptake↑ Anaerobic threshold↑ Work output↑ O2 pulse↓ Peak heart rate↓ RER↓ Blood lactate↓ Time for 1-mile walk and for

Time (min)

VO2

(L/m

in)

Work Rate at 70% VO2max-5%-4%-3%-2%-1%0%

% C

hang

e in

Tim

eFr

om W

eek 0

CordyMax Placebo

recoveryCordyMax Treatment:

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

Zhu J-S, Guo Y, Yao YS, Zhou YJ, Zheng TY, Chen W

Maturation of Cordyceps sinensis associates with co-existence of Hirsutella sinensis and

Paecilomyces hepiali DNA

AbstractA decades-long debate has not given a consensus on the anamorph-telemorph connection for Cordyceps sinensis(Cs). Literature reported isolations of Paecilomyces hepialiand Hirsutella sinensis from natural Cordyceps sinensis,

Pharmanex Research Institute, Provo UT, USA; Shihezi University, Xinjiang; Pharmanex Clinical Center, Beijing; Institute of Microbiology, Chinese Academy of Sciences, Beijing

ITS PCR Products Nested PCR Products Using Phsp3/5Primers

1 2 3 4 5 6 7 8 9 10 11 500400

Template: Fresh C. sinensis

y p ,and evidence of molecular existence of H. sinensis in C. sinensis. We tested a hypothesis in this study that P. hepialiand 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. sinensisand 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

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.

DNA Sequences

300200100

(bp)

1 2 3 4 5 6 7 8 9

Acc

I

Acc

I

Acc

I

Acc

I

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.

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 proliferation predominance of the fungi.

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 AlternatCompli 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 Hirsutellasinensis is hypothesized as the anamorph (asexual sinensis is hypothesized as the anamorph (asexual stage) of C. sinensis, >10 fungi were isolated from natural C. sinensis, possibly suggesting a multi-fungi 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).

Experimental Methods

Acc

I

Bam

HI

Ava

I

Alu

I

Acc

I

Bam

HI

Ava

I

Alu

I

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

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

d i li id it Rib l DNA

Lane: 1 2 3 4 5 6 7 8 9 10 11DNA template: P. hepiali ITS4/5 H. sinensis ITS4/5

PCR Products Using Phsp3/5 PrimersDNA ia

li ♦ P. hepiali genes were amplified

Summary

powder in liquid nitrogen. Ribosomal DNA 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 Ac

cI

AvaI

AccI

AvaI

DNA Template: Fresh C. sinensis

P. h

epi

Lane: 1 2 3 4 5 6 7 8

600500400300200100

9 10

from caterpillar and stroma of natural C. sinensis (GenBank #EF555097), in addition to thedetections of H. sinensis genes.

♦ We found simultaneous existence of live P hepiali and H sinensis

Discussions

Phsp3 and Phsp5, and a touch down PCR 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

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).

of live P. hepiali and H. sinensisand their genes in nature C. sinensis.

♦ The multi-fungi existence innatural C. sinensis may suggestcomplex anamorph-telemorph connections to C. sinensis.

Zhu J-S Gao L Yao YS Zhou YJ Zheng TY Chen W

Maturational alterations of differential expressions of GC:AT-biased Cordyceps sinensis mutants and

Paecilomyces hepiali in natural Cordyceps sinensis

AbstractA 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

Zhu J-S, Gao L, Yao YS, Zhou YJ, Zheng TY, Chen WPharmanex Research Institute, Provo, UT, USA; Shihezi University, Xinjiang; Pharmanex Clinical Center, Beijing

rDNA preparation, Southern blotting and PCR analysis

♦ Caterpillar and stroma of the sterilized C.

Restrictive digestions of Hsprp1/3 PCR products

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:AT-biased 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 regions when Cs stroma genomic DNA used as the template showed increased quantity of

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)g g p q y

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 maturation-related 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

― 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)

Southern blots: P. hepiali (upper) & H. sinensis (lower) rDNA in

Type 1 or 2 C sinensisfor 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)

Maturation of C sinensis and the

Type 1 or 2 C. sinensisCaterpillar Stroma

Collected in: May Jun May JunType of C. sinensis: 1 2 1 2

18S Internal Control

P. hepiali

C t ill StMaturation of C. sinensis and the Temperature on Qinghai-Tibet Plateau

Less Matured Matured

Harvest Season Early May Late JuneTemperature on 0-10 10-20 the PlateauStroma Short Long

Caterpillar StromaCollected in: May Jun May Jun

Type of C. sinensis: 1 2 1 2

18S Internal Control

H. sinensis

Reversely orientated rDNA sequences (GC-bias)

GC and AT biased mutants

A B

Stroma Short LongA: “Type 1” B: “Type 2”

GC- and AT-biased mutants of C. sinensis

Collection of fresh C. sinensis

↑ Biomass of P. hepiali and C. sinensis mutants rDNAwith 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)

Summary & Conclusions

Restrictive digestions of ♦ Type 1 C. sinensis: 50 pieces♦ Type 2 C. sinensis: 50 pieces♦ C. sinensis were collected from Qinghai-

Tibet Plateau, followed by on-site cleaning, surface sterilizing and freezing in liquid nitrogen.

Mature stroma: equally express with AT-bias (AB067740 or AB067744) and GC-bias (AB067721)With C. sinensis Maturation:

In stroma: expression of GC-bias increasesIn 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

Hsprp1/3 PCR products

MATURATION OF CORDYCEPS SINENSIS ASSOCIATES WITH CHANGES IN PROFILES OF PROTEINS AND SMALL

MOLECULAR WEIGHT ORGANIC CHEMICALSS QZhu J-S, Zhang, LJ, Qi 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

AbstractBackground: 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. Design and Methods: SELDI-TOF MS and HPLC analyses were used to

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 chemicalsg yprofile 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 forH. 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

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).

g p g pcomponent fungi may contribute jointly to the overall pharmacological functions of natural C. sinensis.

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 & 429-457, 1998). , )

♦ Co-existence of P. hepiali and H. sinensis was found in fresh C. sinensis (Zhu et al., J MycolRes 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 Matured

Harvest Season Early May Late JuneTemperature on 0-10 10-20 the Plateau

A B

the PlateauStroma Short Long

A: “Type 1” B: “Type 2”

Summary & Conclusions♦ Distinct profiles of proteins and small molecule chemicals were noted for

caterpillars and stromata during C sinensis maturation The maturationcaterpillars and stromata during C. sinensis 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.

pharmanex's cordymax clinical study

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