Reagents

LZ was prepared as before.

Heart grafts in the ears of anesthetized mice were monitored with physiograph.Electrodes for recording the electrocardiogram of heart in situ were made from pin needles and were inserted through the ear tissue on opposite sides of the heart graft on aline parallel to the long axis of the body. The end point of graft survival was taken as the time of cessation of electrical activity. The results were expressed as mean ± SD. The statistical analysis were performed by Student’s t test.

Recipients were treated with Ling Zhi 125 and 500 mg/kg po. qd. x 9 starting on the   day of transplantation. The duration of survival of viable grafts in Ling Zhi treatment groups were 11.6 ± 3.0 and 12.5 ± 1.7 days respectively. No significant prolongation of grafts-survival was observed when compared with untreated control group which survival time was 12.5 ± 2.6 days (Fig. 9-1).

LZ 125 and 500 mg/kg po. qd. x 9 in combination with CYA 10 mg/kg ip. qd. x 9 had no significant prologation in duration of survival time as compared with mice which received CYA 10 mg/kg alone group (Fig. 9-2). Cardiac allografted mice which received combination therapy of CYA 6 mg/kg and LZ 125 or 500 mg/kg po. starting on the day of transplantation had somewhat increase in graft survival as compared with mice which received CYA 6 mg/kg ip. alone (Fig. 9-3).

Treatment of cardiac allografted mice with CYA 15 mg/kg ip. q2d alone. Starting on the day of transplantation no significant effect on graft survival time could be detected. It was 12.1 ± 2.0 days when compared with 11.4 ± 2.4 days of controluntreated group. However, when LZ 125, 250 mg/kg po. qd. x 9 in combination with CYA 15 mg/kg ip. q2d resulted in a slight increase in duration of survival time as compared with untreated control group. It was 14.2 ± 3.7 and 14.9 ± 4.5 days in LZ combination group. But had no significant effect when compared with the CYA 15 mg/kg ip. q2d alone group. Even increasing the dosage of LZ to 600 mg/kg no better result could be obtained (Fig. 9-4).

Transplantation of cardiac tissue into the mouse ear has been demonstrated to be a

relatively simple model to investigate the interaction between host and grafts. It is a

more sensitive indicator of transplant rejection than the in vitro assays for cellular

immunity. The case with which numerous new born heart allograft can be performed and

the precision with which graft survival and function can be monitored by

electrocardiographic evaluation suggests that the mouse heart transplant model is greatly

it is a useful model to discover the effect of some immunosuppressive therapy on the

In this study electrical activity of grafted heart tissue can be discovered in almost all

of the recipience mice at 6 days after operation. The mean survival time of 20 mice of

untreated control group was 12.5 ± 2.6 days. LZ alone did not show any superior effect

on this model however when LZ in combination with suboptimal dosage of

Cyclophosphamide (6 mg/kg qd. x 9 or 15 mg/kg qod x 5) would express slight increase

in the duration of grafts survival time as compared mice which received suboptimal

dosage of Cyclophosphamide alone. This effect of LZ was probably attributable to its

regulative capacity on immune system, suppress the antibody response, DTH etc.

Besides the anticoagulative and vascular dilating activity of LZ may also contribute to a

more adequate blood supply to the graft. This is a primary study. Farther investigation is

necessary.

— Immunopharmacological Study (10)

ZHANG Luoxiu WU Wengrong CAO Zhifang

Lucidum, Fr. Karst) on experimental liver injury induced by D-galactosamine

combined with Endotoxin in mice were studied. Daily oral administration of

250 or 500 mg/kg LZ significantly decreased SGPT activity in Dgalactosamine

and Endotoxin intoxicated mice. It was also observed that LZ

obviously protected tetrachloride produced cytotoxicity in primary cultured

hepatocytes and reduced the release of lactate dehydrogenase (LDH) and

glutamic pyrovic transaminase (GPT) from rat liver cells. There was a

noticeable improvement in mice with liver injury induced by single oral

administration of tetrachloride. These results suggest that LZ has a

therapeutic effect on liver injury by improving liver functions.

Tetrachloride; D-galactosamine; Endotoxin.

LZ is the dried fruit body of a fungus Ganoderma Lucidum, Fr. Karst and has been

used in Chinese traditional medicine as a tonic and sedative drug for a long time. From

the Chinese ancient pharmacopea "Ben Cao" it was recorded that LZ possessed the

activity to reinforce and nourish the deficiencies of five parenchymatous viscera. In this

study we specially observed the activity of the hot water extract of LZ on experimental

pathological mode of liver injury.

It was reported in 1967 that D-galactosamine induced rat liver injury and the

pathological change was similar to those of human hepatitis. However, there was s large

inter and intra species variations with respect to the susceptibility of the animals. Mice

were less susceptible to D-galactosamine than rats. It was also known that Dgalactosamine

induced hepatitis in mice was potentiated by administration of

Dept. of Pharmacology, School of Pharmacy, Shanghai Medical University, Shanghai, China

Kunming (KM) Mice, 21 ~ 26 g, Sprague Danley (SD) rats, 180 ~ 220 g, were

supplied by the Animal Center, Shanghai Medical University.

The hot water extract from LZ planted in Japan was provided by Wakan Shoyaku

Botany Institute. LZ extract a brown powder was suspended in saline containing 0.2%

carboxymethylcellulose sodium (CMC) for in vivo study and for in vitro study 10 mg of

LZ was prepared in 10 ml NS then the tube put in 80°C water bath shaken for 2 h. After

centrifugation the soluble part was named Part A. If it was prepared by three times rapid

frozen and thawed then it was named as frozen thawed part (Part B) after centrifugation.

D-galactosamine was prepared by Chong Qing Medical University. Endotoxin

055B5 was provided by Second Millitary Medical University. Collagenase I type was

purchased from Sigma Chemical Co. RPMI – 1640 medium was purchased from GIBCO

Co. it contained 15 mmol/L Hepes, 100 U/ml Penicilline, 100 µg/ml Streptomycin and

10% NCS.

Mice were randomly divided into 4 groups. The first group was normal untreated

animals, the second group received D-galactosamine 500 mg/kg ip. 30 min later ip.

endotoxin 100 µg/mouse. The third group received Endotoxin only and the fourth group

received D-galactosamine only. 10 h after injection of toxic agent the serum was

separated by centrifugation (3 000 rpm x 10 min) from blood. The activity of glutamicoxaloacetic

transaminase (GOT) and glutamic pyruvic transaminase (GPT) was assayed.

Analysis of variance test followed by individual comparison by Student’s t test was

utilized to determine the significance of mean (± SD) values of various groups of

animals.

Mice were randomly divided into 3 groups. The first group received Dgalactosamine

500 mg/kg ip. and Endotoxin 100 µg/mouse only as a control group. The

second and third groups received 250 or 500 mg/kg LZ po. qd. x 2 respectively. 1h after

last administration of LZ ip. D-galactosamine and Endotoxin. 10h later blood was

removed from the mice to assay SGPT and SGOT activity.

The activity of IL-1 like substance of serum from the above experiment were

assayed by means of thymus cells proliferation test. The thymus cells of BALB/c mice

were measured.

SD rats (180 ~ 220 g) were used as liver donors. They were allowed food and

with minor modifications. Under ether anesthesia, the portal vein was cannulated and the

liver was perfused under a constant pressure. The flow rate was 9 ml/min with calcium –

(pH 7.4) and warmed at 37°C. Physiological solution contained 0.02% collagenase was

continued to perfuse for a further 8 min. Then the third physiological solution contained

liver was then placed in a beaker containing buffer solution plus bovine serum albumin

cycles/min) the cell suspension was then filtered through nylon gauze. The cells

suspension was centrifugated (500 rpm for 1 min) and washed twice. Finally cells

resuspended in RPMI – 1640 medium with 10% NCS. Viability of the cells was

seeded in 24 well microplate. Each group had triplicate cultures.

LZ Part A or Part B was added to make a final concentration 0.01 ~ 10 mg/ml. 20

After centrifugation (3 000 rpm x 10 min) the supernatants were determined the release

of lactate dehydrogenase and GPT from liver cells. The enzyme activity represented the

total value of 100% lysis was obtained by lysing hepatocytes with 1% Triton X-100 for 1

h.

Mice were randomly divided into 4 groups. Group A control, group B ip. 0.01%

intraperitoneal injection. 24 h later blood was removed and the serum were assayed for

SGPT activity.

The results showed the D-galactosamine alone resulted in a slight increase in GPT

activity (P < 0.05). However, when D-galactosamine combined with Endotoxin 100

µg/mouse produced an apparent increase in SGPT level (P < 0.01). While this amount of

Endotoxin alone did not produce significant liver injury (Tab. 10-1).

Group Galn

(mg/kg)

ET

(µg/mouse) n SGPT

(µg/ml ± SD)

SGOT

(µg/ml ± SD)

A B C D

„Ÿ

500

500

„Ÿ

„Ÿ

100

„Ÿ

100

5 5 5 5

92 ± 53

454 ± 209**

157 ± 88*

59 ± 17**

67 ± 25

106 ± 16*

74 ± 26*

48 ± 13*

B compared with A; C and compared with B. *P<0.05; **P<0.01

Mice were randomly divided into 3 groups. The first group received Dgalactosamine

500mg/kg ip. and Endotoxin 100 µg/mouse only as a control group. The

second and third groups received 250 or 500 mg/kg of LZ po. qd. x 2 respectively. 1 h

after last administration of LZ ip. D-galactosamine and Endotoxin, 10 h later blood was

removed from mice. The activities of SGPT demonstrated that LZ decreased the level of

SGPT in a dose dependent fashion (P < 0.05 ~ 0.01) (Tab. 10-2).

Group mg/kg Galn/Endotoxin n SGPT

(U/ml ± SD)

SGOT

(U/ml ± SD)

Control

LZ

LZ

„Ÿ

250

500

+ + +

6 6 6

1 474 ± 548

583 ± 733**

310 ± 182**

238 ± 101

127 ± 44*

162 ± 161

Galn: 500 mg/kg; Endotoxin: 100 µg/mouse. *P<0.05; **P<0.01

Dilution of serum Group N

1 : 40 1 : 20 1 : 10

Control

Galn / ET

LZ + G/E

5 4 5

16 319 ± 6 261

7 240 ± 4 843*

11 351 ± 7 384

5 828 ± 1 241

2 876 ± 2 439*

3 085 ± 1 746*

627 ± 158

480 ± 393

308 ± 88**

compare with control, *P<0.05; **P<0.01

Galn 500 mg/kg; Endotoxin 100 µg/mouse; LZ 500 mg/kg

The serum from each group of mice in above experiment was taken to study. It was

found that the level of IL-1 like substance in intoxicated mice was lower than that of

control group. LZ appeared to elevate the IL-1 level in serum. It suggested that LZ was

able to regulate the immune function of liver injured mice (Tab. 10-3).

From the results indicated in Tab. 10-4 it was shown that both part A and part B of

LZ induced an apparent decrease in GPT and LDH level released from liver cells (P<

0.01).

Group

µl / well

Mg/ml

Control

Part A

Part B

„Ÿ

20

20

20

20

20

20

20

20

20

„Ÿ „Ÿ

0.01

0.1

1

10

0.01

0.1

1

10

6 800 ± 200

14 483 ± 525

10 225 ± 225**

9 667 ± 289**

9 417 ± 144**

9 983 ± 475**

7 717 ± 375**

9 667 ± 289**

8 700 ± 260**

10 900 ± 450**

39.4

84.0

59.3

56.0

54.6

57.9

44.7

56.0

50.4

63.2

43 ± 4

145 ± 5

85 ± 6**

86 ± 6**

85 ± 2**

51 ± 5**

62 ± 3**

88 ± 4**

73 ± 6**

48 ± 3**

23.6

79.7

46.7

47.5

46.9

28.0

34.1

48.1

40.3

26.4

Hepatocytes: 1 : 40; culture time: 1 h

Part A: soluble part; Part B: frozen-thawed part

0.18 ml/10 g ip. groups C and D received LZ 250 or 500 mg/kg po. qd. x 2. 1 h after the

intraperitoneal injection. 24 h later blood was removed and the serum were assayed after

convenient dilution.

It was found that LZ decreased the SGPT level significantly (P < 0.05) (Tab. 10-5).

Group Dosage

(mg/kg)

(ml / 10g) n GPT

(U/ml ± SD)

GOT

(U/ml ± SD)

Control

LZ

LZ

„Ÿ „Ÿ

250

250

„Ÿ

0.18

0.18

0.18

9 9

10

10

52 ± 7

1 015 ± 0

883 ± 235

712 ± 396*

164 ± 96

1 426 ± 756

2 192 ± 1 366

1 660 ± 930

Although more than thousands of years ago the ancient Chinese medicinal work had

already noticed the activity of LZ on liver. But the liver described in Ben Cao implicated

a wider meaning. Our interest was to determine the protective activity of LZ on

experimental pathological model of liver injury. From the results stated above LZ

mice as well as the isolated primary cultured liver cells. These results suggest that LZ

possesses hepatoprotective activity.

GPT and GOT are enzymes usually released from liver cells which injured by

intoxication or infection. These enzymes represent the degenertion and necrosis of liver

cells. Because a great amount of GOT enzyme exists within the cells of heart, kidney and

Lactic acid dehydrotenase (LDH) reflected the liver injury. LZ could decrease the

IL-1 is an important monocytrkine with wide biological activities relating its

important role in immune and certain pathological events. The relationship between IL-1

and liver injury is not available. In this study it was found that the IL-1 like substance in

serum of D-galactosamine and Endotoxin intoxicated mice was lower than that of normal

mice. LZ which protects mice against liver injury would be able to elevate IL-1 like

substance in serum. This further indicates that LZ regulates immune function.

— Immunopharmacological Study (11)

JIA Yongfeng ZHOU Xianbiao MENG Hong ZHANG Luoxiu

normal mice and in mice with suppressed function induced by

cyclophosphamide (CYA) were studied. LZ 250, 500 or 1 000 mg/kg po. qd.

x 6 could enhance the bone marrow nucleated cells (BMNC) proliferation,

increase the number of white blood cell (WBC) and the amount of

hematoglobin (Hb) of peripheral blood. LZ also improved BMNC, WBC, Hb

of mice treated with CYA (40 mg/kg). 250 mg/kg of LZ had the best

therapeutic effectiveness. Our results indicated that LZ could stimulate the

hemopoietic function of mice.

Hematoglobin; Bone marrow nucleated cell.

Ling Zhi (LZ, Ganoderma Lucidum, Fr. Karst), a Chinese medicinal herb, has been

used to protect or cure many kinds of diseases for two thousand years. The clinical data

showed: LZ had affects on leukopenia and could improve blood depression caused by

other disorders. However, there was no experimental data of LZ effecting on

hemopoietic system.

Our purpose was to investigate the effect of LZ from Japan on hemopoietic system,

filling the blank of basic pharmacological data on this aspect.

Kunming (KM) mice, male, 20 ± 2g, supplied by the Animal Center, Shanghai

Medical University.

Dept. of Pharmacology, School of Pharmacy, Shanghai Medical University, Shanghai, China

Blood from tail vein of mice was taken twice and its WBC was counted before

experiment. The animals were chosen and randomly divided into 4 groups, including

control group, CYA group and LZ groups with different dosages. Control group was oral

administration of 0.2% CMC; CYA group was intrapertioneal injection of CYA 40

LZ hot water extract form Ganoderma Lucidum was provided by Wakan Shoyaku

Bnotany Institute, Tokyo, Japan. LZ extract was grinded and dissolved in normal saline

containing 0.2% CMC, shaken at 80°C water bath for 4 h, stored at 4°C.

Cyclophosphamide (No. 12 Pharmaceutical Factory of Shanghai). CYA was

dissolved in sterile saline. Final concentration was 0.2%, stored at 4°C.

Reference heamatin hydrochloride (R.H.Hc) solution. 5.055mg R.H.Hc (Shanghai

Research Institute of Biochemistry) were dissolved with 0.1 mol/L NaOH, total volume

was 0.5L. The concentration of Hb was 250 g/L.

Kunming mice Cyclophosphamide 40 mg/kg ip. d0 ~ d3 and d6.

The reference heamatin hydrochloride solution was diluted with 0.1 mol/L NaOH to

a series of difference concentrations.

The absorption of these solutions was done with spectrophotometer Model 721, ë

395nm. The data showed in Tab. 11-1. Linear regression line was drawn according to

the absorption and the amount of Hb. This regression line was called Hb standard curve.

concentration of H.HC 64 458 x 1 000 Hb(g/L) = 4 x 1 000

No. of tube

1 2 3 4 5 6 7 8 9 10

Volume of R. H.

Hc (ml)

Volume of

NzOH (m)

Hb (g/L)

Absorption

(x 1 000)

Y1

Y2

¯

1.0

9.0

25

62

32

47

2.0

8.0

50

122

89

105

3.0

7.0

75

164

156

160

4.0

6.0

100

264

303

283

5.0

5.0

125

370

341

355

6.0

4.0

150

426

448

437

7.0

3.0

175

468

513

491

8.0

2.0

200

586

580

583

9.0

1.0

225

614

671

642

10.0

0.0

250

718

700

709

Regression Line: Y = 327.51X ± 12.59

20 µl blood from mice tail vein was mixed with 380 µl 0.1 mol/L HCI and took out

40 µl for cell count, another 100 µl mixture was added into 4.9 ml 0.1 mol/L NaOH, the

absorption of those solutions was done using spectrophotometer Model 721 at ë 395nm

and its concentration of Hb was read from the standard curve of Hb.

Mice were killed and a femur was taken. After getting rid of femural surface tissue,

the bone marrow cavity was opened and washed with saline, then centrifuged 1 500 rpm

10 min. The pallet was collected and mixed with 2ml 3% HAc, this solution was further

10 times diluted with 3% Hac and take 50 µl for cells count.

Cell counts were expressed in number as the mean ± SD, these data were compared

for statistical significance by Student’s t test.

Leukocyte count level was within the normal range during the first week. On d10,

WBC count of groups treated with LZ exceeded. The mice in group of 250 mg/kg LZ

had significant difference with that in control group (Tab. 11-2).

(mg/kg) d0 d2 d4 d6 d10

Control

250

500

10 744 ± 1 628

10 763 ± 994

10 780 ± 1 086

11 155 ± 2 068

12 223 ± 746

12 000 ± 1 828

10 510 ± 1 557

8 364 ± 1 242

9 433 ± 3 027

11 328 ± 1 923

12 490 ± 1 401

13 000 ± 2 211

11 962 ± 311

13 783 ± 1 637*

13 300 ± 1 517

The number of BMNC of LZ groups was significantly higher than that of control

group on d8, and the action of LZ 250 mg/kg group was still existed on d10 (Tab. 11-3).

(mg/kg) d5 d8 d10

Control

250

500

1 188 ± 198

1 172 ± 62

1 343 ± 344

987 ± 43

1 557 ± 163**

1 377 ± 168**

1 044 ± 99

1 566 ± 112*

1 014 ± 106

The result was given on Tab. 11-4. The amount of Hb of LZ groups had increasing

tendency compared with that of control group on d10, without statistic significance. The

data indicated the change of amount of Hb in mice was less distinct than that of the white

cells.

Hb (g/100ml) LZ

(mg/kg) D2 d4 d10

Control

250

500

152.8 ± 31.8

140.0 ± 12.1

133.9 ± 11.4

151.2 ± 13.4

145.7 ± 17.2

138.6 ± 9.04

158.2 ± 12.9

177.9 ± 21.8

170.3 ± 11.1

When CYA was injectived the general finding consisted of an initial fall in the

number of circulating leukocytes followed by a period of sustained depression and a

subsequent return to normal level. When CYA was injectived after CYA was stopped,

blood count of LZ groups dirapiy rise on d6 and significantly higher than that of CYA

group (Tab. 11-5).

(mg/kg)

CYA

(mg/kg) D0 d2 d4 d6 d0

„Ÿ „Ÿ

250

500

1 000

„Ÿ

40

40

40

40

10744 ± 1628

10670 ± 1313

10728 ± 1457

10737 ± 1384

10700 ± 1444

11155 ± 2 068

6900 ± 682

8130 ± 1210

6353 ± 1320

8325 ± 1587

10510 ± 1557

3715 ± 992

3433 ± 574

2621 ± 592

2903 ± 567

11327 ± 1923

7562 ± 453

11160 ± 1783*

10688 ± 3480

11271 ± 1827*

11962 ± 311

10550 ±1257

11044 ± 867

9588 ± 1594

11641 ± 2222

The range of number of BMNA was seen in Tab. 11-6. In CYA group from d1 to

d8 the BMNC counts dropped rapidly and continued slowly to rise. The number of

BMNC in LZ groups was significantly higher than that of CYA group on d10.

(mg.kg)

CYA

(mg/kg) d5 d8 d10

„Ÿ „Ÿ

250

500

1 000

„Ÿ

40

40

40

40

1 188 ± 198

245 ± 94

245 ± 40

310 ± 77

192 ± 20

987 ± 43

668 ± 80

762 ± 161

762 ± 303

377 ± 131

1 044 ± 99

678 ± 199

1 234 ± 155**

1 115 ± 212*

1 137 ± 220*

On d10, all animals of LZ groups (250, 1 000 mg/kg) showed the amount of Hb

similar to normal, but that of CYA group was still in lower degree and significantly lower

than that of LZ groups (Tab. 11-7).

Hb (g/100mml) LZ

(mg.kg)

CYA

(mg/kg) d2 d4 d10

„Ÿ „Ÿ

250

500

1 000

„Ÿ

40

40

40

40

152.8 ± 31.8

135.1 ± 11.4

134.2 ± 19.0

127.8 ± 12.5

132.0 ± 8.8

151.2 ± 13.4

143.0 ± 8.0

138.7 ± 6.1

131.1 ± 10.6

131.8 ± 13.9

158.2 ± 12.9

126.2 ± 13.5

151.2 ± 12.0*

141.3 ± 7.2

152.0 ± 19.0*

Blood picture depression was a common clinical phenomenon. It was caused by

various diseases such as virus infection, malignant tumor or autoimmune diseases. At

present, the ordinary methods of killing tumor cells were chemotherapy or radiotherapy.

But these methods could cause the decrease of WBC. In our work therapeutic

effectiveness of LZ was investigated on the animal model of leukopinia. The data

showed that LZ not only enhanced the number of leukocyte and the amount of Hb, but

also promoted the proliferation of BMBC. These results were consistent with clinical

information.

Our experimental data had also shown, either in normal mice or in

immunosuppressed mice by CYA, the therapeutic effectiveness of LZ in low dosage (250

mg/kg) was better than that of higher dosages.

LI Huaiyi* WANG Xinmin* JIANG Minhua** ZHANG Peng**

PENG Hongli** Masao MORI***

showed that 0.5% and 1.0% of LZ could significantly increase the number of

feeding in female flies and 1.0% of LZ increase the number of mating with

dose-effectiveness. In addition, the favorable effects of 0.5% ~ 3.0% of LZ in

prolonging the mean life span, the maximum life span and the median life

span were observed in tested flies.

Longevity.

Ling Zhi (LZ) was used in treatment for chronic diseases and improvement of

The extract in hot water of LZ was provided by Wakan Shoyaku Botany Institute,

Tokyo, Japan. The drug is the powder in yellow-brown color. In the treated groups, the

drug was incorporated into medium and the flies fed on the medium at whole adult stage.

* Dept. of Special Toxicology, Second Military Medical University, Shanghai, China

** School of Pharmacy, Shanghai Medical University, Shanghai, China

*** Wakan Shoyaku Botany Institute, Tokyo, Japan

LZ proved to be nontoxic as measured by the mortality of adult flies when they

were reared on medium containing of 0.5% ~ 5.0% of LZ. Thus, LZ, at doses of 0.5%,

1.0%, 2.0% and 3.0%, were selected for the study.

The mean life span, maximum life span and median life span were tested in survival

experiment. In addition, both tests for the number of feeding and for the number of

mating were also conducted to evaluate the effects of LZ on aging process. All tests

mentioned above were performed in 4 groups with concentrations of 0.5%, 1.0%, 2.0%

survival experiment at same concentration of LZ was conducted by means of dividing

into female flies group and male flies group. The number of feeding in female flies was

scored in this study only. Mean body weight range 1.10 mg ~ 1.15 mg in female flies

and 0.78 mg ~ 0.82 mg in male ones.

Test for the number of feeding: To score the number of feeding at interval of 5 min

was performed over a period of 4 h.

Test for sex vitality: To score the number of mating at intervals of 1 min was

performed over a period of 11 min.

Survival test: To score the number of natural dead flies at intervals of 6 h was

performed over a period of the whole adult stage.

LZ significantly increased the number of feeding in female flies compared with that

of control group. There was a 5% ~ 24% increase in the number of feeding with doseeffectiveness

(Tab. 12-1).

LZ (%) Number of feeding

0

0.5

1.0

2.0

3.0

343

463***

554***

374*

350*

÷² test; n = 60; Š *P >0.05, ***P<0.01 vs control

LZ significantly increased the number of mating of the flies compared with that of

control group. There was a 2% 6 ~ 2% increase in the number of mating with doseeffectiveness

(Tab. 12-2).

LZ (%) Number of mating

0

0.5

1.0

2.0

3.0

265

304*

329***

283*

278*

÷² test Š : ‰ = 60 : 40; *P >0.05, ***P<0.01 vs control

LZ significantly prolonged the mean life span, the maximum life span and the

median life span compared with that of the control group with dose-effectiveness. The

mean life span was increased 7% ~ 13% in the female flies and 8% ~ 15% in the male

flies, the maximum life span increased 3% ~ 23% in the female flies and 13% ~ 21% in

the male flies, the median life span increased 3% ~ 10% in the female flies and 6% ~

13% in the male flies (Tab. 12-3).

life span (d)

Median

life span (d) LZ

% Š ‰

Š ‰

Š ‰

0

0.5

1.0

2.0

3.0

55.12 ± 14.78

58.82 ± 14.83*

62.19 ± 13.20***

59.38 ± 16.43**

61.07 ± 12.86***

50.37 + 14.36

54.33 + 15.16**

55.70 ± 12.54***

57.45 ± 14.42***

57.96 ± 13.69***

80

90

98

82

83

72

81

87

84

84

60

62

66

64

62

53

56

57

59

60

u test; n = 105; *P >0.05, **P<0.05, ***P<0.01 vs control

As shown in Figure 12-1 and 2, the survival curves of the treated groups were

located at the right side of that of the control group. Furthermore, the effects were

observed in both female and male flies, and continued for 60 days (i.e. from 20 ~ 30 days

to 80 ~ 100 days at adult stage).

LZ at concentration less than 5% was proved to be nontoxic and might increased

the number of feeding and mating, and might prolong the mean life span, the maximum

data analysis showed that LZ had a dose-effectiveness in the antiaging action. Wang et al

reported that LZ significantly increased the mean life span but did not significantly

and American wild type). This data were not consistent with that of Wang’s. LZ was not

only significantly prolonged the mean life span and the median life span but also

used in their study in efficient ingredient or in preparation techniques. The real cause

remains to be determined.

There were the different opinions on the criteria of the antiaging drugs. Walford

postulated that the drug so long as prolonged the maximum life span might be considered

suggested that the drug which only prolonged the mean life span and did not prolong the

demonstrate that LZ was able to prolong the longevity including the mean life span,

that LZ belongs to the antiaging drug according to two criteria mentioned above.

JIANG Minhua* LIN Chung* FANG Xiang* MIAO Yongsheng*

Masao MORI**

k/kg measured by chemical stimulus method. LZ also cooperates with using

activities of LZ is 2.65 g/kg. LZ (3.5 g/kg) is obviously to prolong the

swimming time of mice. The ability to stand hypoxia is increased by using

5.0 g/kg and 7.5 g/kg of LZ. These results further confirm that LZ possesses

analgesic, sedative effects and promoting tolerance activity.

LZ has been used in Chinese traditional medicine for centuries. It was reported that

LZ had been used clinically to alleviate pain sensation such as headache, backache,

neuralgic pain, cancerous pain, and to be a hypnotic as well as a mild energetic remedy.

Following the clinical observation, it is suggested that LZ have many pharmacological

Recently, we are interested in the evaluation of the analgesic and sedative effects of

LZ. The study was designed to investigate the activities of LZ on analgesia and sedation.

Meanwhile, the work was carried out the promoting tolerance activity of LZ either.

Kunming (KM) mice, 20 ± 2.0g, supplied by the Animal Center, Shanghai Medical

University.

* Dept. of Pharmacology, School of Pharmacy, Shanghai Medical University, Shanghai, China

** Wakan Shoyaku Botany Institute, Tokyo, Japan

LZ extracta were provided by Wakan Botany Institute, Tokyo, Japan. It was diluted

by 0.5% CMC and prepared in a shaking bath at 80°C, 4 h.

Aminopyrine was purchased from Shanghai Pharmaceutical Company. Acetic acid

(analytical grade) was made by Suzhou Jincheng Chemical Factory. Diazepam was the

product of Shanghai Haipu Pharmaceutical Factory. Sodium pentobarbitol was obtained

from Sigma Chemical Company. Propranolol was the product of Beijing Pharmaceutical

Factory.

Writhing inhibition test

Seventy male mice were randomly divided into 6 groups. The mice were

pretreatment with LZ 0, 3.4, 4.9, 7, 10 g/kg (0.5% CMC as Vehicle) and aminopyrine 0.3

g/kg po. respectively. 10 min later, the mice were treated with 0.7% acetic acid (prepared

counted in 15 min after 5 min injection of acetic acid. The writhing inhibition rate (1%)

was calculated as following:

Writhing inhibition rate (%)

The analgesic activity was accessed by a type Y SD-4 hot plate apparatus with

water bath of 55 ± 0.2°C. The first signs of discomfort shown by a mouse on the hot

plate are that it stands up on its hind legs and licks or blow its front paw to cool them. In

a few seconds the pain is too heavy to be borne by the back paws, and the mouse either

kicks its leg and dances around the restraining cylinder, or attempts to jump out of the

cylinder. As normal mice with often standing up and grooming their front paws, the

standard time of exposure to the pain stimulus has been 30s. It were tested at regular

intervals (30 min) after injection, then at intervals of 30 min for at least 1.5 h. The results

were expressed as pain threshold increment rate:

Pain threshold increment rate (%)

For this experiment, forty five female mice were arranged to 3 groups with 0, 5, 7.5

g/kg of LZ po. respectively. The pain threshold were measured before and after

treatment of LZ 30, 60, 90 min respectively. Each pain threshold was measured twice,

the mean value was taken and the increment rate was calculated.

Seventy six mice of either sex were divided into 6 groups and placed in a

having drugs 15 min, the spontaneous motor activities in 5 min were measured in groups

of mice which have received the following doses of drugs: LZ 0, 2.06, 2.94, 4.2, 6 g/kg

Twenty two mice of either sex were arranged to 2 groups. The mice of the first

group were given Pentobarbitol 25 mg/kg ip. The mice of the second group were given

LZ 4.2 g/kg po., 20 min later, each mouse was additionally received Pentobarbitol 25

mg/kg ip. Compared the difference of the righting reflex between 2 groups for 20 min

Eighty male mice were divided into 6 groups with LZ in the doses of 0, 0.15, 1.5,

3.5, 5, 7.5 g/kg/day x 7 po., respectively. 1 h after the last drug given, the mice with a tail

load (10% body weight) were placed in bath (deep 30 cm, 27°C) for swimming until the

mice subsided. Recorded the swimming time, i.e., from swimming to subsiding

(maintain 10 s) of mice. If the swimming time of mouse was more than 60 min, the test

Forty male mice were randomly divided into 4 groups. The mice were pretreatment

with LZ 0, 3.5, 5, 7.5 g/kg, po. bid x 7. 30 min after the last administration of drug, the

mice were placed into a tighting cylinder (250 ml) with 10 g of calx natrica. The results

Tab. 13-1 showed that the writhing response was inhibited by LZ. LZ (10 g/kg) is

The results by hot plate test was indicated that LZ could significantly increase the

pain threshold. It was found that the analgesic effect appeared after LZ given 30 min,

and the effect was still maintained until 90 min (Tab. 13-2, Fig. 13-1, 2).

N Writhing response

Inhibition (%)

CMC

Aminopyrine 0.3 g/kg

LZ 10 g/kg

LZ 7 g/kg

LZ 4.9 g/kg

LZ 3.4 g/kg

20

10

10

10

10

10

54.5 ± 19.5

0.1 ± 0.3

4.0 ± 3.6

12.3 + 10.9

30.8 ± 16.7

41.9 ± 14.4

99.8

92.7

77.4

43.5

23.1

Increment of pain

threshold (%)

n

0 min 30 min 60 min 90 min 30min 60min 90min

CMC

LZ 5g/kg

LZ 7.5g/kg

15

15

15

25.9±2.5

23.8±2.7

22.5±3.3

26.8±5.4*

28.3±7.9**

36.0±11.9**

26.5±4.6*

33.1±8.2***

38.4±10.5***

28.7±7.3*

34.0±10.5***

40.4±10.3***

3.5

18.9

60.0

2.3

39.1

70.7

10.8

42.9

79.6

0 min: before administration; 30, 60 and 90 min: after administration.

*P > 0.05; ** P > 0.05; *** P > 0.01 compared with control (0 min)

Tab. 13-3 demonstrated that LZ significantly decreased the spontaneous motor

It was obvious that LZ could cooperate the effect of pentobarbital. The righting

reflex in the group of Pentobarbitol was normal, but it disappeared in the pentobarbitol

Group Animals

(n)

Spontaneous motor activities

Inhibition (%)

CMC

LZ 2.058 g/kg

LZ 2.94 g/kg

LZ 4.2 g/kg

LZ 6.0 g/kg

Diazepam 10 mg/kg

10

14

13

10

17

12

57.1 ± 16.5

39.0 ± 22.2

20.9 ± 12.8

15.2 ± 14.8

14.1 ± 10.2

2.2 ± 3.6

32

63

74

75

95

Tab. 13-4 showed that the swimming time of mice could be prolonged by LZ (3.5

g/kg), but decreased by LZ (5, 7.5 g/kg). It was found that the swimming time of mice on

hypoxia test could be prolonged in the group treated with LZ (5, 7.5 g/kg) (Tab. 13-5).

Group Animals

(n)

Swimming time (min)

P value

CMC

LZ 0.15 g/kg

LZ 1.5 g/kg

LZ 3.5 g/kg

LZ 5.0 g/kg

LZ 7.5 g/kg

15

12

12

15

14

12

26.0 ± 21.7

12.7 ± 15.0

24.7 ± 26.3

43.1 ± 22.2

5.2 ± 2.1

4.9 ± 1.9

> 0.05

> 0.05

< 0.05

< 0.01

< 0.01

Group Animals

(n)

Survival time (min)

P value

CMC

LZ 3.5 g/kg

LZ 5.0 g/kg

LZ 7.5 g/kg

10

10

14

12

19.65 ± 6.18

22.87 ± 5.50

27.02 ± 4.58

29.47 ± 6.38

> 0.05

> 0.05

< 0.01

< 0.01

Most tests for analgesia depend on the application of an external stimulus on an

animal and the observation of typical change in behavior by which the animal attempts to

acid) and physical stimulus (hot place) to induce pain. It was obvious that LZ could

However, it is not possible to say that the sensations in the mouse are similar to those

experienced by human and the mouse feels pain in exactly the same degree as human

does. Results obtained from mice, however, may give some guide to the drug using in

human.

The righting reflex in mice can be used to assess whether slept or not. If they are

placed on their backs, they normally turn over immediately. If they are given a suitable

dose of a drug they can be placed on their backs and the time after the administration of

the drug at which they are right themselves is taken as the sleeping time. In this study, it

was found that the mice with sedative dose of pentobarbitol (25 mg/kg) could not go to

sleep because of their righting reflex was normal. If they additionally received LZ (4.2

cooperative to combine use. In this way, we may assess the sedative activity of drugs

indirectly.

After administration of LZ, we have observed that the mice can stand hypoxia, and

can prolong the swimming time. It is suggested that LZ possesses the promoting

tolerance activity. The swimming time was shortened in the larger dose groups of LZ (5,

7.5 g/kg). However, the mechanism of that is unclear. It may be related to the toxicity of

LZ.

CHENG Zhanghua* WEN Jingyuan* CHEN Binling* Masao MORI**

g/kg, po. 3d respectively. 1 h after last dosage given, mice were kept in

restraint plus water immersion stress for 22 h. The results revealed that LZ

significantly lowered the mucosal lesion incidence and mucosal hemorrhage

induced by stress at doses of 1.0 g/kg and 2.0 g/kg. The contractile responses

LZ was markedly effective on suppressing the stress ulcer formation. It

suggested that LZ might block the peripheral parasympathetic nervous system.

Anticholinergics.

LZ has depressant effects on the central nervous system. It was reported that LZ

had been used clinically to improve the patients’ sleep. In this paper, we observed the

effects of LZ on the parasympathetic nervous system.

Kunming (KM) male mice, 22 ± 2g, supplied by the Animal Center, Shanghai

Medical University. Guinea-pigs of both sexes, weighing 350 ± 50g, purchased from

market.

LZ extract was provided from Wakan Shoyaku Botany Institute, Tokyo, Japan. It

was diluted by 0.5% CMC and 5% LZ suspension was obtained, oscillated 4 h in 80°C

water bath and cooled in 4°C refrigerator for using. Atropine sulphate was the product of

Shanghai No. 10 Pharmaceutic Factory. It was diluted by 0.5% CMC and 0.1% atropine

solution was prepared and kept at 4°C.

* Dept. of Pharmacology, School of Pharmacy, Shanghai Medical University, Shanghai, China

** Wakan Shoyaku Botany Institute, Tokyo, Japan

Fifty male mice were randomly divided into 5 groups, ten mice in each group. The

mice were pretreated with LZ 0.4 g, 1.0 g, 2.0 g/kg; atropine sulphate 0.04 g/kg; CMC

0.2 g/kg, po. respectively in the same volume of 0.4 ml / 10 g body weight

intragastrically for 3 days. Mice were deprived of food but allowed to take tap water

freely for 24 h before stress. 1 h after last dosage given, mice were put into individual

close-fitting tubular cages of wire mesh and immersed in a water bath (23°C) to the depth

of the xiphoid to be stressed. The mice were killed for autopsy after 22 h stress.

After cardia occluded, the stomach was excised. The gastric contents were

squeezed out through pylorus and diluted 10 times with the Hayem’s solution for

determination of red blood cells. Total number of RBC was counted on the

hemocytometer. Then the stomach was inflated with 2 ml of 1% formalin, occluded

pylorus and put in 1% formalin for 1 h to fix both inner and outer layers. Stomach was

opened along the greater curvature and rinsed with normal saline to remove adherent

substance on mucosa. The stomach walls were observed carefully under dissecting

microscope and examined for mucosal lesions.

Guinea-pigs were stunned and the abdomen opened. Isolated ileum 1.5 cm in

length was excised, suspended in 20 ml organ bath containing Tyrode’s solution bubbled

with air at 37°C. The resting tension was 1.0 g. Maximum contractile responses of

accetylcholine were recorded by force-displacement transducer. Antagonisms in

different dosages of LZ to contraction of acetylcholine were measured.

Student’s t test. P value of less than 0.05 was considered significant.

The incidences of ulcer and hemorrhage caused by water-immersing stress were

100%. Mucosal lesions and acute hemorrhagic erosions on the stomach walls were

observed. Mucosal lesion with size less than 1 mm in length and hemorrhagic erosion

were expressed as the petechial lesions. Mucosal lesion with size more than 1 mm in

length was expressed as an ulcer. Three petechial lesions were considered equivalent to

an ulcer. Experiments revealed that LZ 1.0 g, 2.0 g/kg, Atropine 0.04 g/kg groups

compared with control group markedly decreased ulcer formations and hemorrhage

incidences (Tab. 14-1, Tab. 14-2).

Group No. of

animals

No. of

petechial lesions

Decreasing

rate (%) No. of ulcer Decreasing

rate (%)

LZ

0.4g/kg

LZ

1.0g/kg

LZ

2.0g/kg

Atropine

0.4g/kg

Control

CMC

0.2g/kg

10

10

10

10

10

31.4 ± 8.7*

13.7 ± 7.7***

13.7 ± 7.1***

4.5 ± 3.9***

33.6 ± 13.1

6.5

59.2

59.2

86.7

„Ÿ

20.2 ± 5.4*

8.1 ± 5.8***

6.8 ± 4.1***

1.8 ± 2.0***

21.1 ± 8.8

4.3

61.6

67.8

91.5

„Ÿ

Group No. of

Animals

No. of

RBC from mucosal hemorrhage

Decreasing

rate (%)

LZ

0.4g/kg

LZ

1.0g/kg

LZ

2.0g/kg

Atropine

0.4g/kg

Control

CMC

0.2g/kg

10

10

10

10

10

357.7 ± 245.8*

66.6 ± 90.1***

32.9 ± 14.5***

88.1 ± 112.2**

451.1 ± 404.9

20.8

85.2

92.7

80.5

LZ in different dosages could inhibit ileum contractile responses produced by

Acetylcholine. Inhibition rate 9%) in each dosage group was the average of five

Atropine was 4.3 x 10-3 g/ml obtained from the same experimental procedure.

The highest incidence of ulcer and lowest mortality of animal were observed when

the stress to fix plus water-immersion in mice. Ulcer formation under stress conditions is

due to stimulation of the excessive central hypothalamus and parasympathetic nervous

The following pathogenetic mechanisms have been reported to account for the stressinduced

gastric mucosal lesions: abnormal gastric motility; alteration in gastric secretion;

ischaemic changes in the gastric mucosa, muscular contractions and extrinsic

compression of intramural vessels, reduction in mucosal resistance to acid-peptic

muscular contraction. The increase of acid secretion during water-immersion stress was

was markedly effective on suppressing the ulcer formation. LZ at dose of 1.0 g/kg

markedly antagonized stress ulcer formation as the same as Atropine. Lin et al reported

peripheral actions mainly with little effect on the central nervous system at dose of 1.0

g/kg. LZ also showed antagonism to Acetylcholine on isolated ileum preparation. The

LZ prevented the stress ulcer formation significantly and antagonized contraction of

the smooth muscle induced by Acetylcholine. These results demonstrated that LZ

possesses the blocking effects on peripheral parasympathetic nervous system

MIAO Yongsheng* ZHANG Luoxiu* JIANG Minghua* Masao MORI**

on isolated guinea-pig trachea was demonstrated in this study. LZ can also

antagonize the contraction of the above-mentioned trachea caused by

histamine in non-competitive manner. LZ relaxes the contraction of the

trachea induced by slow reacting substance anaphylaxis (SRS-A) when the

concentration of LZ is higher than 0.001 mg/ml.

It has been well known for more than 2 000 years in clinical medical practice that

Ling Zhi (LZ) is effective in the treatment of chronic bronchitis and asthma but its

mechanism is not clear so far. This study was designed for clarifying the mechanism,

using isolated guinea-pig trachea and taking another Chinese traditional medicine Han Ji

Song (Tetrandrini Dimethiodidum) as a positive control.

Guinea-pig, male 250 ~ 300 g, supplied by the Animal Center, Shanghai Medical

University.

LZ extract was provided by Wakan Shoyaku Botany Institute, Tokyo, Japan. The

powder 30 g was treated in 100 ml 80°C distilled water for 2 h in an constant vibrating

state. Then it was contrifugalized with 1 600 rpm for 10 min. The upper supernatant was

separated and kept at -30°C (Part A). For Part B, after LZ powder was treated in 80°C

distilled water for 2 h in constant vibrating state, it was put in a refrigerator at -30°C for 1

h, then thawed at 37°C. This course was conducted for three times. It was

centrifugalized with 1 600 rpm for 10 min. The supernatant was taken and kept at -37°C

for further use.

* Dept. of Pharmacology, School of Pharmacy, Shanghai Medical University, Shanghai, China

** Wakan Shoyaku Botany Institute, Tokyo, Japan

Han Ji Song (Tetrandrini Dimethiodidum) was provided by Department of Natural

Chemistry, School of Pharmacy, Shanghai Medical University.

Guinea-pig was sacrificed by head knocked. The trachea was taken and put into

the trachea was cut into sectors and connected as a chain. The preparation was then put

into a bath containing 20 ml Krebs’ solution. The solution remained at 37 ± 0.5°C and

bubbled with oxygen. One end of the preparation was fixed to a steel needle at the

bottom of the bath and the other end was binded to muscle force displacement equipment

which connected to recorder. The rest tension was adjusted to 1.5 g and remained

unchanging for 2 h. During this period, the Krebs'’solution in the bath was replaced

every 15 min. After equilibrium, the experiment commenced.

LZ was added to the bath accumulatively with 5 min interval. The concentration of

LZ was from 0.00001 ~ 0.1 mg/ml.

Histamine (0.001 ~ 0.1 mmol/ml) was added to the bath accumulatively with 5 min

interval. The concentration-response curve of histamine was drawn. The preparation

was washed by fresh Krebs’ solution for several times and equilibrated for 30 min. LZ in

different concentrations (0.001 ~ 0.01, 0.1 mg/ml) or chlorpheniramine (0.00001

mmol/ml) was added to the bath. After the preparation had contacted the drug for 10

min, the concentration-response curves of histamine were made again. The

After equilibrium, the Krebs’ solution in bath was replaced and 1 µg/ml Atropine

and 0.06 µg/ml Chlorpheniramine were added to the bath to antagonize the possible

effect of acetylcholine and histamine. 5 min later, SRS-A 72 unit/ml was added to the

bath. When the contraction of the preparation reached to the maximum, LZ (0.001 ~ 0.1

mg/ml) or Han Ji Song (Tetrandrini Dimethiodidum 0.00001 ~ 0.1 mg/ml) were added to

bath in an accumulative manner. The SRS-A-induced contraction of the preparation was

steady, lasting for 45 min without change.

Both LZ Parts A and B had slight relaxation effects on isolated guinea-pig trachea

when their concentrations were higher than 0.0001 mg/ml (Fig. 15-1). The relaxation

effect of Part B is stronger than that of Part A.

Chlorpheniramine competitively antagonized the histamine-induced contraction of

B (0.1 mg/ml) both antagonized the contraction of the trachea in a non-competitive

manner with the maximum relaxation 37.5% and 29.6% respectively. (Fig. 15-2).

LZ showed relaxation effect when its concentration was higher than 0.001 mg/ml.

LZ Part A (0.1 mg/ml), B(0.1 mg/ml), and Han Ji Song (Tetrandrini Dimethiodidum) (0.1

g/ml) all reduced the maximum contraction of the trachea by 20.4%, 24.2% and 40.5%,

respectively (Fig. 15-3).

It is known that bronchial smooth muscle tone is controlled by humoral factors as

well as the autonomic nervous system (ANS). The humoral factors, such as histamine,

SRS-A, serotonin, prostaglandin and bradykinin may cause bronchoconstriction. It may

influence the calibre of the airways in ill situation. In healthy condition the calibre is

mainly controlled by the ANS, both parasympathetic and sympathetic. The present study

was designed to investigate the model of LZ effect. Even it was carried out on trachea

preparation, it is proved to be an easy-doing, valid and reliable experiment used to

estimate the effect of a drug on muscle.

This study demonstrated that LZ has relaxation effects on the isolated guinea-pig

trachea in normal condition or contacted by histamine or SRS-A. Part A is more

effective than Part B in relaxing the trachea contracted by histamine, while preparation B

is more effective in relaxing SRS-A induced contraction. The results may be due to the

different treatment of LZ, related to the pharmaceutical substances.

LZ shows its antagonism against histamine and SRS-A which are the key factors in

LZ interfering with other humoral factors or/and ANS remains to be studied.

JIANG Minghua* MIAO Yongsheng* WANG Zhengyue* HU Tianxi**

CHEN Jiwu** ZHOU Chenming*** GUO Huiju*** Masao MORI****

superoxide anion radicals was investigated. It showed that LZ inhibited the

xanthine/xanthine oxidase reaction with its dose-dependency in Luminoldependent

B of LZ were 48.5 µg/ml and 30.4 µg/ml, respectively.

Electron spin resonance (ESR) detection was also used to

determine the effect of LZ on superoxide anion radicals. It has been indicated

that Parts A and B (0.25 g/ml) of LZ can decrease the intensities of the ESR

signals about 84% and 86%.

It was suggested that LZ might act as an antioxidant in scavenging

the superoxide anion radicals.

chemiluminoscence; ESR.

It has been reported that LZ were used in the prevention and restoration of senile

change, cardiovascular diseases and also possessed anticancer, anti-inflammatory

owing to free radicals from the metabolism. In this paper, we attempted to elucidate the

ESR detection and CL system were widely used in studying free radicals formed in

biological systems. This report describes the application of CL system and ESR

* School of Pharmacy, Shanghai Medical University, Shanghai, China

** East China Normal University, Shanghai, China

*** Shanghai Institute of Organic Chemistry, China Academy of Science, Shanghai, China

**** Wakan Shoyaku Botany Institute, Tokyo, Japan

Xanthine, luminol were purchased from Sigma Co. Xanthinoxidase was extracted

(DMSO) was purchased from Nan Xin Solvent Factory.

LZ extract was provided by Wakan Shoyaku Botany Institute, Tokyo, Japan. It was

diluted by distilled water and prepared 0.25 g/ml in the shaker bath at 80°C for 4 h. The

mixture was centrifugalized at 30 000 g for 15 min, the supernatant was taken as Part A.

Freezing Part A at -30°C for 1 h, then thawed at 37°C, 3 times, after centrifuge at 30 000

Parts A and B with distilled water diluted 20 times.

CL measurement were performed in a SHG-1 Luminometer. ESR spectra were

recorded at liquid nitrogen 77K on a Varian E 112 X-band spectrometer with a field set a

mw, time constant of 0.25s.

The control value was measured with a tube (1.2 x 5 cm) containing 50 µl of

For LZ determinations, the following conditions were used: prior to measurement, 400

detected at liquid nitrogen 77K after adding 100 µl of LZ Part A or B. The total volume

was 500 µl.

The result of kinetic light emission has been shown that the max of light emission

was occurred at 10s (Fig. 16-1). After adding 5 ~ 100 µg/ml of LZ, the light emission

B were 48.5 µg/ml, respectively.

LZ concentration

(µg/ml) mean value Light emission

Intensity (%) Inhibition (%)

0

100

80

50

40

20

49 803

11 340

12 067

24 669

28 403

38 528

100.0

22.8

24.2

49.5

57.0

77.4

0

77.2

75.8

50.5

43.0

22.6

LZ concentration

(µg/ml) mean value Light emission

Intensity (%) Inhibition (%)

0

80

40

30

20

10

5

53 114

9 799

22 093

27 230

29 431

40 092

44 078

100.0

18.4

41.6

51.3

55.4

75.5

83.0

0.0

81.6

58.4

48.7

44.6

24.5

17.0

(control = 24.1 cm) (Fig. 16-3 ~ 6).

It means that the addition of Parts A and B of LZ can decrease the ESR intensities

Free radicals are atoms, ions or molecules containing an unpaired electron. Most of

oxygen consumed by cells produced water with cytochrome oxidase. However, part of

radicals might damage cellular-constituents, such as to inact enzymes, break DNA, crosslink

proteins and peroxidize lipid. Furthermore, it has been postulated that free radicals

The potentially disastrous consequences of free radical reactions might be expected

to protect the cell of plants and animals (i.e. antioxidant defenses). LZ is a well known

Chinese traditional medicine herb and available for treatment of some disease related to

free radicals. In order to research the mechanism of LZ, we observe the effect of LZ on

superoxide anion radical.

The xanthine/xanthine oxidase (x/xo) reaction, widely used as a superoxide

generating system, was employed in the present study. It was demonstrated that LZ

A and B were 48.5 µg/ml and 30.4 µg/ml, respectively. However, x/xo reaction may be

inhibited by xanthine oxidase inhibitor, it should be carefully considered whether LZ

Since ESR technique was employed for direct detection and identification of free

radicals, and the sequence of the ESR spectra is essential for successful and correct

interpretation of the results. We observed that the effect of LZ exhibited on ESR

detection. The results of ESR detection have been indicated that the ESR signal

intensities are decreased with LZ. The experiments are related to the various

concentrations of LZ shown in Fig. 15-3 ~ 6.

In summary, it suggested that LZ might be used as an antioxidant in possessing the