GANODERMA LUCIDUM (LING ZHI REISHI ) CAN PROLONG LIFE SAY RESEARCHERS IN CHINA AND JAPAN...PART 1

THE COOPERATIVE RESEARCH ON GANODERMA LUCIDUM
(Ling Zhi / Reishi )
The problem clarified by the cooperative research work of Shanghai Medical University,
Shanghai, China and Wakan Shoyaku Botany Institute, Tokyo, Japan is that Ling Zhi
(Reishi) has the effect of improving microcirculation and enhancing hemodynamics.
However, it was saying “Eating Reishi for a long period might make person comfortable
and prolong the life without senility” in “Sheng Nong Ben Cao Jing” and “Ben Cao Gang
Mu”.

* Comfort means no symptom.
* Prolonging life without senility means having a long long life.
Certainly, it would be impossible if there were no such basis of improving
microcirculation. The data collected in this book seems in expectation. It has proved that
Reishi (LZ) increased hemodynamics in microcirculation by decreasing the blood
viscosity. There are not only the data in medical research but also that of clinical
investigation. In addition, with the efforts of the teachers of Shanghai Medical
University, it has also proved that Reishi (LZ) has the effect of clearing radicals and the
effect of two-way immune modulation. I have paid cordially attention for the past 17
years. The fruitful result form the “Cooperative Research” makes me very excited.
Reishi could only be a food not a botany because of no scientific certificate in
Japan. However, as a few kind of drug Reishi might be used as a botany according to the
past experience in Eastern Medicine without any side effect.
“The Research on Ganoderma Lucidum, Part One” is not only a symbol to which
it had made a great effort, but also a basis of more understanding the traditional and
modern usage of Reishi (LZ) in the continuing research in future.
The great significance and rich contents of experimental medicine and pharmacy
were listed by this book. The efforts and instruction of the researchers in Shanghai
Medical University would be appreciated very much.
I would like to thank Professor and President Zhaoyou TANG, Professor and Vice
President Shineng ZHU, Shanghai Medical University again for their enhancing and
strengthening of the search work. I also say many thanks again to Mr. Masaru
KOBAYASHI, President, Wakan Shoyaku Botany Institute for his kindness of providing
the big financial support for research work.
Masao MORI
Academic Consultant,
Wakan Shoyaku Botany
Tokyo, Japan
CONTENTS
________________________________________________________________________
Effects of Ling Zhi on Lymphocyte Proliferation
— Immunopharmacological Study (1)…………..…………………XU Weimin et al
Effects of Ling Zhi on Antibody Productive Cells and Allergic Reaction
— Immunopharmacological Study (2)…………..…..…..….…ZHANG Luoxiu et al
Immune Suppressive Effects of Ling Zhi in Mice
— Immunopharmacological Study (3)…………..…….UN Bing & ZHANG Luoxiu
Effects of Ling Zhi on Macrophage Phagocytosis and Carbon Particles Clearance Test
— Immunopharmacological Study (4)………………………...ZHANG Luoxiu et al
Influence of Ling Zhi on Natural Killer Cells
— Immunopharmacological Study (5)……..……...ZHANG Luoxiu & YU Mingyan
Effects of Ling Zhi on the Production of Interleukin-1 (IL-1)
— Immunopharmacological Study (6)…………………………...JIA Yongfeng et al
Effects of Ling Zhi on the Production of Interleukin-2 (IL-2)
— Immunopharmacological Study (7)………………………...ZHANG Luoxiu et al
Influence of Ling Zhi on the Production of Tumor Necrosis Factor (TNF)
— Immunopharmacological Study (8)………….……ZHANG Luoxiu & XIE Xuhei
Effects of Ling Zhi on Cardiac Heterotopic Transplanation
— Immunopharmacological Study (9)……….…ZHANG Luoxiu & ZHAO Lianhua
Hepatoprotective Activity of Ling Zhi
— Immunopharmacological Study (10)……….…..…….….…ZHANG Luoxiu et al
Effects of Ling Zhi on Hemopoietic System in Mice
— Immunopharmacological Study (11)….………………………JIA Yongfeng et al
Effects of Ling Zhi on Sex Vitality and Longevity in DROSOPHILA
MELANOGASTER…………….……………………………………….….LI Huaiyi et al
Analgesic, Sedative Effects and Promoting Tolerance Activity of Ling Zhi in Mouse
…………………….…………………………………………………JIANG Minghua et al
Effects of Ling Zhi on Stress Ulcer in Mice and Its Antagonism to Acetylcholine
……………………………………………………….………….…..HENG Zhanghua et al
Effects of Ling Zhi on Isolated Guinea-Pig Trachea………….…..MIAO Yongsheng et al
Effects of Ling Zhi on Superoxide Anion Radicals…………….…...JIANG Minghua et al
Anti-Lipid Peroxidative Effects of Ling Zhi in Mice……………….….PENG Hongli et al
Effects of Ling Zhi on Membranes Fluidity and Ghosts Reseal Ability of Rat Erythrocyte
…….…...…………………………………….…………………………….…LI Duan et al
Effects of Ling Zhi on Superoxide Dismutase Activity and Protein Components of Rate
Erythrocyte Membranes……..…………….…………………………………LI Duan et al
Effects of Ling Zhi on Experimental Thrombosis and Metamorphosis of Human
Erythrocyte…………….……………………………………………..WANG Jueying et al
Effects of Ling Zhi on Hemorrheology Parameters and Symptoms of Hypertension
Patients with Hyperlipidemia and Sequelae of Cerebral Thrombosis
………………………………………..……..…………………….CHENG Zhanghua et al

REGARDING TO THE MATERIAL LING ZHI ( REISHI ) USED IN
EXPERIMENTS
“The Cooperative Scientific Research on Botany Ling Zhi (Reishi)” accomplished
by Shanghai Medical University, Shanghai, China and Wakan Shoyaku Botany Institute,
Tokyo, Japan started in 1989. The total material Ling Zhi (Reishi) including seeds,
powder and its extract was produced in the special Reishi cultivated farm and was
provided by Wakan Shoyaku Botany Institute. It should be declared that the material
Reishi used in the experiments were taken randomly from the original material produced
in the Institute without and special ways.
The brief introduction of Reishi cultivated farm:
Tumagoi-Mura Farm, Wakan Shoyaku Botany Institute
The first farm 9 900m² The second farm 13 000m² Total 22 900m²
(Located in Imai-Sennoiri, Tumagoi-Mura, Agatuma-Gun, Gunma, Tapan)
Wakan Shoyaku Botany Institute is the biggest Reishi (LZ) produced and
researched organization in Japan. It includes 60 cultivated warm rooms, original wood
cultivated building, seeds dried building. Besides, there are general cultivated building
and office building with water cleared apparatus, big sterilized equipment and other
instruments.
The cultivated farm is located at the altitude of 1 030 meters above sea level. It is
a natural environment with little changed whether, good quality of water, fresh air and
getting the original wood at that place. It is benefit to the cultivation and treatment of
Reishi (LZ). The condition of original Reishi (LZ) produced with stable quality is
ensured in the farm. In addition, with the cultivated technique researched and created in
many years Wakan Shoyaku Botany Institute had the patent of management and
cultivation of sustaining the activity of original bacterial strain. All these might
guarantee the stable quality.

Effects of Ling Zhi on Lymphocyte Proliferation
— Immunopharmacological Study (1)
XU Weimin ZHANG Luoxiu MIAO Honghua
Abstract The study was to evaluate the process of lymphocyte
proliferation induced by the hot water extract of Ling Zhi (LZ, Ganoderma
Lucidum, Fr. Karst) cultivated in Tokyo, Japan. It was examined by assessing
the effect of LZ on ³H-TdR uptaken by cultured spleen cells alone or in the
presence of Con-A or Lps. LZ 300 mg/kg po. qd. x 10 in vivo or 10 ~ 500
μg/ml alone in vitro stimulated lymphocytes proliferation directly, but 1 000
μg/ml of LZ inhibited. The activity of frozen thawed part was stronger than
that of soluble part. The effect of LZ on lymphocyte proliferation induced by
Con A resulted in diverse regulatory activity. It was mainly related to both
concentrations of Con A and LZ. In general, LZ stimulated lymphocyte
proliferation in the presence of suboptimal concentration of Con A (0.625
μg/ml) and depressed when the cells were highly activated by optimal
concentration of Con A (2.5 μg/ml) in a concentration dependent fashion.
LZ antagonized the activity of cyclophosphamide and promoted the
lymphocyte proliferation in the immunosuppressed mice induced by
cyclophosphamide. However, LZ had no stimulative activity on lymphocyte
proliferation induced by Lps in vitro or in vivo. It implied that B cells may
have different mechanism from T cells to interact with LZ.
Key words Ling Zhi (LZ, Ganoderma Lucidum, Fr. Karst); Lymphocyte
proliferation

The fungus Ling Zhi (LZ, Ganoderma Lucidum, Fr. Karst) exhibits wide
pharmacological effects and has been used as a health protective drug for a long time.
However, the investigation of LZ on immune system was rare and the mechanism was
poorly understood. The aim of this study was to evaluate the effect of LZ on lymphocyte
proliferation.
Dept. of Pharmacology, School of Pharmacy, Shanghai Medical University, Shanghai, China
Materials and Methods
Animals
Kunming mice, male, 18 ~ 22 g, were supplied by the Animal Center, Shanghai
Medical University.
Reagents
The hot water extract of LZ was provided by Wakan Shoyaku Botany Institute,
Tokyo, Japan. A certain amount of LZ extract was grinded and dissolved in normal
saline containing 0.2% carboxymethyl cellulose sodium (CMC) shaken in 80ºC water
bath for 4h. This suspension was used for in vivo study. For in vitro study, a little
amount (about 10mg) of LZ extract was dissolved in normal saline and rotated slowly in 80ºC
water bath for 2h then centrifugated 1 600 rpm 10 min. The supernatant was kept at 4ºC until
study. Before experiment this solution was diluted to desire concentration. It was named as
soluble part (Part A). When LZ was dissolved and shaken in 80ºC water bath then rapidly frozen
(-30ºC) and thawed (37ºC) 3 times, it was named as frozen thawed part (Part B).
PRMI-1640 was purchased from GIBCO Co. It contained Hepes 15 mM, Penicillin 100
U/ml, Streptomycin 100 μg/ml, NCS 10%, 2-Mercaptoethanol (2-ME) 5 x 10¯5 mmol, pH 7.3.
Con A, S were purchased from Sigma Co.
³H-TdR was provided by Shanghai Institute of Nuclear Research, Chinese Academy of
Sciences and the specific activity was 1 100 GBq. Mmol.
Lymphocyte proliferation in vitro (1)
Mice were sacrificed by cervical dislocation and the spleens were removed with sterile
technique. The spleen cells were collected after passing through a nylon screen and red blood
cells in the cell suspension were hemolyzed with distilled water for 30 s, then osmoregulated.
After washing with RPMI-1640 for 3 times the cells were seeded into each well of 90-well
microplate and various concentrations of LZ and Con A or LPS were added in alone or
combination. The plates were cultured at 30ºC with 5% CO2 in a humidified atmosphere for 48 h.
For the last 6 h each well as pulsed with 9.25 kBq ³H-TdR. The cells were harvested using a
multiple automatic sample harvester onto a fibroglass filter paper which was then dried and the
radioactivity incorporated were counted by a liquid scintillation counter. All counts/min values
shown were the mean of triplicate sample ± SD. Statistical analysis was carried out by student’s
test.
Mitogenic activity of LZ in vivo(2)
Mice were randomly divided into 2 groups. The mice in control group were given 0.2%
CMC 0.5 ml po. qd. x 10 and treated group with LZ 300 mg/kg po. qd. x 10. The spleen cells
were prepared as mentioned above.
The effects of LZ on immunosuppressed mice
40 mice were divided into 4 groups randomly. The mice in control group were given 0.2%
CMC 0.5 ml po. qd. x 10. In cyclophosphamide (CYA) group, mouse were taken CYA 10 mg/kg
ip. qod. x 2, q3d x 2. The mice of LZ groups were given LZ 150, 300 mg./kg, po. Combined
with CYA 10 mg/kg, ip. qd. x 2, qod. x 2 respectively. The spleen cells were prepared as the
same technique mentioned above.
Results
Effect of LZ on lymphocytes proliferation in vitro
In vitro, LZ alone stimulated the ³H-TdR uptake at 1 ~ 500 μg/ml (Part A) and at 1 ~ 10 μg/ml
(Part B) without mitogen. And turned to inhibition at right concentration. The activity of Part B
was stronger than that of Part A. Part B inhibited lymphocyte proliferation at less concentration
(Tab. 1-1).
Tab. 1-1 Effect of LZ alone on proliferation of lymphocytes in vitro
LZ cpm / 1 x 106 cells
(μg/ml) Part A Part B
0
1
10
100
500
1 000
2 991 ± 445
3 744 ± 529
5 997 ± 699**
8 054 ± 1 013**
7 668 ± 1 181**
1 126 ± 156**
2 991 ± 445
11 208 ± 554**
10 532 ± 1 301**
1 595 ± 18*
1 552 ± 264*
499 ± 48**
Part A: soluble part; Part B: frozen thawed part; X ± SD; n = 3;
*P<0.05,>599 μg/ml) resulted in inhibitive effect (Tab. 1-2, Fig. 1-1).
When the lymphocytes were highly activated by optimal concentration of Con A (2.5 μg/ml)
LZ mainly expressed an inhibitive activity.
The activity of LZ was in the middle between two conditions mentioned above when
lymphocytes were stimulated by Con A (1.25 μg/ml).
Tab. 1-2 Effect of LZ on ConA-induced lymphocyte proliferation in vitro
LZ cpm / 1 x 106 cells
(μg/ml) ConA 0.625 μg/ml ConA 1.25 μg/ml ConA 2.5 μg/ml
Part A 0
1
10
100
500
1 000
Part B 1
10
100
500
1 000
3 589 ± 44
5 886 ± 3 020
13 366 ± 5 955*
19 788 ± 4 285**
9 375 ± 1 677*
984 ± 117**
16 546 ± 3 217**
15 944 ± 4 801*
7 407 ± 585**
4 196 ± 460
685 ± 28**
19 559 ± 3 087
35 293 ± 9 642
19 954 ± 8 656
16 944 ± 6 768
4 948 ± 2 044**
764 ± 166**
20 509 ± 4 701
61 059 ± 5 666**
56 432 ± 7 227**
4 512 ± 470**
1 918 ± 98**
69 991 ± 1 894
58 956 ± 12 175
38 487 ± 2 523**
28 640 ± 10 150**
15 496 ± 1 254**
4 732 ± 832**
26 034 ± 7 193**
35 337 ± 13 165*
17 476 ± 2 351**
16 996 ± 4 123**
7 347 ± 825**
Part A: soluble part; Part B: frozen thawed part; X ± SD; n = 3; *P<0.05, n=" 3;" n =" 3;" n="3;" n =" 9" acetone =" 3" v="VL-VR" n="6~7;"> 0.05 when compared with control.
Effect of LZ on DTH test
It was found that LZ 500 mg/kg significantly inhibited the SRBC induced DTH
reaction at 24 h after antigen challenge. CYA 10 mg/kg ip. qd. x 8 also inhibited DTH
reaction (P < 4w =" WL" n =" 10;"> 0.05 Compared with control. n = 5
Lymphocyte proliferation
T lymphocyte proliferation induced by 5 μg/ml Con A was suppressed by LZ in vivo
(Tab. 3-3).
Tab. 3-3 Lymphocyte proliferation in vivo (cpm)
Group Dose (mg/kg) Con A 5 μg/ml Without mitogen
Control
LZ I
LZ II
CYA
0.2% CMC
125
250
200
9 166 ± 4 578ΔΔΔ
3 052 ± 1 585**
4 115 ± 716**
1 626 ± 533***
1 116 ± 200
**P<0.05, n =" 10" ns =" 1:2." a =" 3√k" n =" 4;" n =" 4;" n =" 5;" cytotoxicity =" cpmcom" release =" cpmtotal" release =" cpmtotal" n="3;" n =" 4" n =" 4;" n =" 4;" n =" 3;" t =" 100" n="3;" t =" 100" n="3;" n =" 3;" n =" 3;" n =" 3;" n =" 4;" n="4;" n =" 4;" n =" 4;" n =" 4;" n="4" n =" 4;" n="4;􀁕􀁕􀁕P<0.01" n="4;**P<0.05"> 95%) were seeded in 96 well plates and cultured at 37°C 5%
CO2 for 20 h (final 200 μl/well). Triplicate wells were used for each experimental
condition. At intervals, plates were removed from incubator and medium was removed
by rapid decantation. 100 μl of RPMI – 1640 medium containing actinomycin D yielding
a final concentration of 1 μg/ml. Plates were similarly reincubated for 20 h followed by
removal of culture supernatants and staining with 0.5% crystal violet for 20 min. Rinsed
and dried plates were enumerated by measuring absorbance at 590 nm on Elisa
autoreador.
% cytotoxicity was calculated using the formula:
% cytotoxicity = ODcon ─ ODtest ODcon x 100%
ODcon = absorbance in control well
ODtest = absorbance at a particular dilution of test sample.
TNF (U/ml): a unit is defined on that amount of cytotoxin necessary to cause 50%
destruction of the cell-culture.
Results
The effect of LZ on the growth of L929 cells
As shown in Tab. 8-1 LZ 1 ~ 100 μg/ml had no significant influence on the growth
of L929 cells.
Tab. 8-1 Effect of LZ on L929 cells activity
Drug Dose
(μg/ml) OD
Control
LZ
LZ
LZ
─
1
10
100
0.380 ± 0.02
0.357 ± 0.015
0.370 ± 0.036
0.363 ± 0.021
Note: Dilution of LZ was 1 : 20 L929 cells seeded density was 5 x 104 / well. X ± SD; n = 3
The effect of LZ on γTNF mediated cytotoxicity on tumor cells
Tab. 8-2 indicated that 5U of γTNF resulted in 76.3% of cytotoxicity. Addition of
LZ 1 ~ 100 μg/ml to the cultures with 5U of γTNF showed that LZ had no effect on the
cytotoxic activity of 5U γTNF.
Tab. 8-2 Effect of LZ on the reaction of L929 cells to γTNF
Drug Dose
(μg/ml)
γTNF
(U) γTNF cytotoxicity %
Control
LZ
LZ
LZ
─
1
10
100
5
5
5
5
76.3 ± 1.3
75.6 ± 0
74.8 ± 3.4
77.0 ± 5.6
Note: Dilution of drug was 1 : 20. L929 collected density was 5 x 104 / well.
Result expressed as cytotoxicity %, X ± SD; n = 3
LZ stimulated the production of TNF from murine peritoneal macrophages
Tab. 8-3, 8-4 and Fig. 8-1 showed that LZ 0.01 ~ 1 μg/ml stimulated murine
peritoneal macrophages to produce TNF in a concentration dependent fashion. Higher
concentration of LZ (100 μg/ml) resulted in an inhibitive activity. Unfiltered LZ solution
showed a stronger stimulative activity to produce TNF than filtered LZ solution in vitro.
Tab. 8-3 Effect of LZ (unfiltered preparation ) on TNF production
OD
Drug Dose Dilution of test supernatant
(μg/ml) 1 : 40
1 : 80 1 : 160
TNF production
(U/ml)
Control
LZ
LZ
LZ
LZ
LZ
─
0.01
0.1
1
10
100
0.137 ± 0.015
0.150 ± 0
0.133 ± 0.006
0.120 ± 0
0.143 ± 0.025
0.147 ± 0.006
0.210 ± 0.01
0.170 ± 0.01*
0.177 ± 0.006*
0.180 ± 0.02
0.173 ± 0.015
0.207 ± 0.015
0.260 ± 0.01
0.210 ± 0.017*
0.220 ± 0.01*
0.200 ± 0.026*
0.207 ± 0.012**
0.247 ± 0.015
63.4 ± 2.1
89.9 ± 6.01*
86.7 ± 4.0*
103.8 ± 5.3**
93.9 ± 6.0**
61.8 ± 2.5
Notes: OD of control well (only AntiD.): 0.363 ± 0.006
Control NS. X ± SD; n = 3; *P<0.05; **P<0.01, compared with control
Tab. 8-4 Effect of LZ (filtered preparation ) on TNF production
OD
Drug Does Dilution of test supernatant
(μg/ml) 1 : 40
1 : 80 1 : 160 1 : 320
TNF
production
(U/ml)
Control
LZ
LZ
LZ
LZ
LZ
─
0.01
0.1
1
10
100
0.347 ± 0.025
0.330 ± 0.017
0.323 ± 0.015
0.263 ± 0.012*
0.283 ± 0.024*
0.350 ± 0.01
0.420 ± 0.053
0.473 ± 0.047
0.463 ± 0.02
0.420 ± 0.035
0.390 ± 0.035
0.507 ± 0.04
0.473 ± 0.015
0.540 ± 0.062
0.503 ± 0.02
0.493 ± 0.025
0.517 ± 0.04
0.650 ± 0.017
0.493 ± 0.07
0.623 ± 0.03
0.590 ± 0.053
0.603 ± 0.05
0.610 ± 0.06
64.8 ± 6.8
55.2 ± 3.1
60.3 ± 3.8
81.7 ± 3.9*
79.4 ± 3.8*
48.4 ± 1.7*
Notes: OD of control well (only AntiD.): 0.787 ± 0.015
Control NS. X ± SD; n = 3; *P<0.05, compared with control
125
75
50
25
100
control
TNF production (U/ml)
Ling Zhi concentration (μg/ml)
0.01 0.1 1 10 100
Fig. 8-1 Effects of unfiltered ( 􀁊 􀁊 ) or filtered ( 􀁆 ) LZ on the Lps-indused release of TNF from
5% TG-primed macrophages
X
±SD; n=3;*P<0.05; **P<0.01 compared with control
**
*
**
*
**
*
**
The effect of pretreatment of macrophages with LZ
Then we examined the effect of preincubation of macrophages with LZ on the
production of TNF. Macrophages were incubated with LZ (0.01 ~ 100 μg/ml) or without
of LZ for 8 h, then the medium were removed, cells were washed and reincubated with
LPS 10 μg/ml at 37°C for 24 h. The TNF activity of tested supernatants were assayed.
Tab. 8-5 showed that preincubation of macrophages with LZ slightly increased the
activity of TNF.
Tab. 8-5 Effect of preincubation with LZ on release of TNF
Drug Dose
(μg/ml) TNF cytotoxicity (%)
Control
LZ
LZ
LZ
LZ
LZ
─
0.01
0.1
1
10
100
45.6 ± 2.5
57.5 ± 5.9*
47.8 ± 2.5
57.2 ± 2.5**
53.9 ± 1.9*
51.1 ± 3.8
Note: 5% TG-primed macrophages (from 2.5 x 106 /ml PECS) incubation with drug for 8 h. Thereafter,
cells were washed to remove drug and fresh medium containing 10 μg/ml Lps was added. Then incubating
for 24 hours, tested supernatant diluted to 1 : 160.
X ± SD; n = 3; *P<0.05; **P<0.01, compared with control
Effect of LZ on TNF production in vivo
Tab. 8-6, Fig. 8-2 indicated that LZ 100, 200, 300 mg/kg po. qd. x 14 stimulated the
level of TNF in serum of BCG primed and Endotoxin treated mice.
Tab. 8-6 Effect of LZ on TNF production in vivo
Drug Dose
(μg/ml) TNF cytotoxicity (%)
Control
LZ
LZ
LZ
─
100
200
300
2.90 ± 1.2
50.0 ± 7.5**
40.0 ± 9.9**
26.5 ± 8.9*
Note: Serum were diluted to 1 : 1000 and tested
LZ: po. qd. x 14; 0.2% CMC: po. qd. x 14
X ± SD; n = 3; *P<0.05; **P<0.01, compared with control
0.5
0.3
0.2
0.1
0.4
1 : 1000
Dilution of serrum containing TNF
control 0.2%CMC P.O. qd X 14
LZ 200mg/kg -1 P.O .qd X 14 LZ 300mg/kg P.O. qd X 14
LZ 100mg/kg P.O. qd X 14
1 : 2000 1 : 4000
Fig. 8-2 Effects of LZ on TNF production in vivo
X
±SD; n=3;*P<0.05, **P<0.01 compared with control
OD
**
*
**
**
**
*
*
Discussion
Since TNF has been widely noticed, several investigators have developed in vitro
cell cytotoxicity assays for TNF. Flick et al compared four published in vitro assays
which measured cell cytotoxicity of TNF(4). These included determination of residual cell
number by crystal violet staining in the presence and absence of Actinomycin D, lack of
viability as determined by neutral red uptake and ³H-Thymine release in cytotoxin treated
L929 cells. Flick compared these methods and discovered that treatment of L929 cells with
Actinomycin D followed by crystal violet staining was the most sensitive method
measured. We set up this method of assay TNF activity. We observed various
conditions, L929 cell density, the amount of TG, the dosage cause of Actinomycin D and
the mice strain. We found that crystal violet culture was a sensitive and repeatable assay.
Addition of Actinomycin D to the cell cultures greatly enhanced killing and shortens the
necessary incubation time to 20 h. We used this method for all the experiments.
From this study it was shown that LZ enhanced the production of TNF in vitro. LZ
itself had no direct cytotoxic activity against L929 cells. There was less pronounce
synergistic activity between LZ and γTNF under this condition. However LZ increased
the TNF production both in vitro and in vivo. Even preincubation of macrophages with
LZ for 8 h promoted the production of TNF. We have reported that LZ stimulated
macrophage phagocytosis and enhanced the carbon particles clearance activity. Now we
further demonstrated that LZ elevated macrophage activity to increase the capacity of the
host defense mechanism against cancer and infection diseases through indirect
mechanism.
One must emphasize that from the in vitro study we can see LZ modulated TNF
production form 0.01 ~ 1 μg/ml LZ stimulated TNF release but higher concentration (100
μg/ml) resulted in an inhibitive activity. This further demonstrated LZ played a role as an
immunomodulator.