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N u t r i t i v e Q u a l i t i e s o f F o o d

JFS S: Sensory and Nutritive Qualities of Food

Potential of a Novel Polysaccharide Preparation(GLPP) fromAnhui-GrownGanoderma lucidumin Tumor Treatment and ImmunostimulationX. PANG, Z . CHEN, X . GAO, W. LIU, M . SLAVIN, W. Y AO, AND L.L. YU

ABSTRACT: Growing evidence indicates the potential of developing novel polysaccharide-based adjuvant for tu-mor therapy from edible mushrooms, including Ganoderma lucidum. In the present study, a novel polysaccharidepreparation (GLPP) was isolated from the fruiting body of G. lucidum grown in Anhui, China, and characterizedfor its physicochemical properties. GLPP had an average molecular weight of 6600 and a specific optical rotation of+25.6, contained 10.6% protein, and had a molar ratio of 0.9:15:1 formannose, glucose, and galactose, respectively.GLPP was also investigated and compared with PSP (polysaccharopeptide preparation), a commercial antitumorand immunostimulating agent, for its antitumor and immunostimulation capacity, and potential in reducing thetoxic effects induced by cyclophosphamide (Cy) treatment and Cobalt-60 (60Co) radiation in mice. GLPP at levels of100 and 300 mg/kg body weight (BW)/d significantly inhibited the growth of inoculated S 180, Heps, and EAC tumorcells in mice. GLPP at a dose of 300 mg/BW kg/d showed stronger growth inhibition against all 3 tested tumor cellsthan PSP at 1 g/kg BW/d. GLPP also dose-dependently increased phagocytic index, phagocytic coefficient, and 50%hemolysin value in the EAC tumor-bearing mice, indicating its potential immunostimulating property. In addition,GLPP at 300 mg/kg BW/d was comparable to PSP at 1000 mg/kg BW/d in preventing the decrease of thymus index,spleen index, white blood cells, and bone marrow karyote numbers induced by Cy treatment and 60Co radiation.These data demonstrated the potential utilization of GLPP as an adjuvant to conventional treatments of cancersand its use for cancer prevention.

Keywords: antitumor activity, cancer prevention, Ganoderma lucidum, immunomodulation, polysaccharidepreparation

Introduction

Cancer is a leading cause of death for people less than 65 yold in Western countries, and there is no indication that the

mortality rates caused by cancer are decreasing (Lavillonniere and

Bougnoux 1999). Recently, many alternative approaches such

as developing novel polysaccharides and polysaccharopeptides

with anticancer and/or immunomodulating capacities have been

shown to be effective in cancer prevention and treatment (Cui and

Chisti 2003; Yoo and others 2004; Lindequist and others 2005; Sul-

livan and others 2006). It is widely accepted that cancer cells may

survive from host immune systems through multimechanisms and

remain highly proliferative (Sullivan and others 2006). It is also

noted that chemotherapy and radiotherapy, 2 conventional treat-

ments for cancer, may suppress host immune system function. The

immunosuppressive properties of cancer and its treatments have

led to the effort to discover and develop novel immunostimulat-

ing agents, and to investigate their potential in cancer prevention

and possible utilization as adjuvants for conventional cancer treat-

ments to improve the survival rate of these therapies (Lindequist

and others 2005; Sullivan and others 2006).

MS 20070314 Submitted 4/27/2007, Accepted 5/16/2007. Authors Pang, Gao,Liu, and Yao are with School of Life Science and Technology, China Phar-maceutical Univ., Nanjing, MD 210009, PR China. Author Chen is withSchoolof Pharmacy,China PharmaceuticalUniv., Nanjing, MD 210009, PRChina. AuthorsSlavin and Yuare withDept. of Nutrition andFood Science,Univ. of Maryland, College Park, MD 20742, U.S.A. Direct inquiries to au-thor Yao (E-mail: [email protected]).

Authors Pang and Chen contributed equally to this study.

Ganoderma lucidum (Fr.) Karst is an edible medicinal mush-

room. Its fruit body, which is well known as Lingzhi in Chinaand Reishi or Manetake in Japan, has been traditionally used for

health promotion and longevity in the China and other East Asian

countries for more than 2000 y. A fewG. lucidum polysaccharides

and polysaccharopeptides, including the commercially available

Ganopoly, have been shown to exhibit antitumor and immunos-

timulating activities (Miyazaki and Nishijima 1981; Maruyama and

others 1989; Lin 2001; Bao and others 2002; Wang and others 2002;

Gao and others 2003; Shao and others 2004; Yuen and Gohel 2005;

Yue and others 2006). Growing evidence suggests that the anti-

cancer or antitumor capacity of these mushroom polysaccharides

and polysaccharopeptides is strongly associated with and may be

mediated through their immunostimulating activities (Ooi and Liu

2000; Wasser 2002; Sullivan and others 2006; Zhang and others

2007). These bioactive mushroom polysaccharides and polysac-

charopeptideshave very diverse molecularand chemicalstructures

(Bao andothers 2002; Wasser 2002; Sullivan andothers 2006; Zhang

and others 2007) as well as different physical properties (Wasser

2002;Sullivan and others 2006). It is widely recognized that polysac-

charides or polysaccharopeptides prepared from different sources

and parts ofG. lucidum or by different methods may significantly

differ in their physicochemical properties and biological activities

(Maruyama and others 1989; Cui and Chisti 2003; Yue and others

2006). Scientific data are required for individual G. lucidumderived

nutraceutical preparations to optimize their utilization in health

promotion and disease prevention.

The present research was conducted to develop novel polysac-

charide preparations from G. lucidum grown in Anhui province

C 2007 Institute of Food Technologists Vol. 72, Nr. 6, 2007JOURNAL OF FOOD SCIENCE S435doi: 10.1111/j.1750-3841.2007.00431.xFurther reproduction without permission is prohibited

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A bioactive G. lucidum polysaccharide extract . . .

of China, and to investigate their possible antitumor and

immunostimulating properties as well as their potential in reduc-

ing toxicity and side effects of chemotherapy and radiotherapy in

mice with and without 3 selected transplanted experimental tu-

mors. Anhui is a developing province in central China, and alter-

native agriculture is in high demand to enhance farm profitability

and the agricultural economy. Information from this research will

be used to promote the commercial production and utilization of

G. lucidumfrom Anhui for cancer prevention and treatment while

enhancing the local agricultural economy.

Materials and Methods

MaterialsThe fruit bodiesofG. Lucidum(Fr.) Karst were obtained from the

Jingde Huangshan Lingzhi Industry Co. Ltd. Anhui, China. These

fruit bodies were confirmed for identity and deposited by the Inst.

of Microbiology, Chinese Academy of Sciences. The polysaccha-

ropeptide PSP from the extraction of Coriolus versicolor mycelia,

one of the most widely used mushroom polysaccharopeptides to

treat cancer andother diseases in China, was a gift from the Jiangsu

Suzhong Pharmaceutical Co. Ltd. China, Norm: 0.5 g/capsule, Lot:

021011. PSP was used as a positive control in the present study.

PSP and GLPP were dissolved in normal saline for administration.

Cyclophosphamide (Cy), a clinical medicine for tumor treatment

was a gift from Jiangsu Hengrui Medicament Co. Ltd. China. Dex-

trans with different molecular weights were gifts from Natl. Inst. for

the Control of Pharmaceutical and Biological Products, China. All

other chemicals were of analytical purity and used without further

purification.

AnimalsICRspeciesmice, 18 to 22 g, half male andhalffemale, purchased

from the Experimental Animal Center, China Pharmaceutical Univ.

Animal quality certificate number is SCXK (HU) 2002-0010. Ani-

mals were kept at 18 to 24 C with a relative humidity of 70%. Ani-

mal experiments were carried out under protocols approved by the

China Pharmaceutical Univ. Animal Care and Use Committee.

Experimental tumorsSarcoma 180 (S180) cells, Ehrlichi Ascites Carcinoma (EAC) cells,

and Hepatoma solidity (Heps) cells were provided by Jiangsu Inst.

of Cancer Research.

Preparation ofG. lucidumpolysaccharide (GLPP)The air-dried G. lucidumfruit bodies (6 kg) were ground and ex-

tracted with 120 L of hot water for 14 h. After filtration, the solid

residues were extracted with hot water for another 2 times. All fil-

trates were combined and concentrated to a final volume of 60 L.

This concentrated solution was mixed with 90 L of ethanol to pre-cipitate polysaccharides. After being kept at 4 C for 4 h,theprecip-

itated polysaccharides with higher molecular weight were removed

by centrifugation and the supernatant was treated with ethanol at

a ratio of 1:3 (v/v, solution/ethanol) at 4 C for another 4 h. The

later precipitates were collected by centrifugation, and washed se-

quentially with ethanol, acetone, and diethyl ether, followed by dry-

ing at 40 C under a reduced pressure to obtain a brownish pow-

der named GLPP. The GLPP was subjected to chemical analysis and

used in mouse feeding studies.

Chemical analysisTotal carbohydrates were determined by the phenol-sulfuric

acid assay using glucose as a standard (Dubois and others 1956).

Protein content of GLPP was analyzed using the Kjeldahl method

with a converting factor of 6.25 (James 1995). Amino acid compo-

sition of the protein was analyzed as described previously and the

protein moiety was released byhydrolysiswith 6 M HCl at 110Cfor

22 h in a sealed reaction tube (Mazumder and others 2002). Mois-

tureand ash contents were determined using the method described

in the Chinese Pharmacopoeia (2005). Crude fat content was an-

alyzed according to the protocol described in 1993 edition of the

NSPRC. The specific rotation of GLPP solution (0.2%, H 2O) was de-

termined on a WZZ-2S automatic polarimeter (Shanghai PhysicalOptics Instrument Co., China) at 20 1 C.

Determination of molecular weightMolecular weight of GLPP was determined by gel permeation

chromatography (GPC, Agilent 1100, Agilent Technologies, Palo

Alto, Calif., U.S.A.) using a BioSep-SCE-S 2000 column (300 7.8

mm, Phenomenex, Torrance, Calif., U.S.A.). Ten milligrams of the

GLPP were dissolved in 1 mL of the mobile phase containing 0.7%

Na2SO4 and 0.02% NaN3 to obtain the testing sample solution. The

chromatography was conducted at 35 C using the mobile phase

at a flow rate of 0.5 mL/min and a refractive index detector. Pre-

liminary calibration of the column was conducted using dextrans

with different molecular weights. Multichrom with GPC softwaredesigned for polysaccharide was employed to acquire and analyze

the molecular weight data.

Determination of monosugar compositionGLPP was converted to volatile derivatives and analyzed by gas

chromatography (GC, HP-6890, Agilent Technologies) (Honda and

others 1981). Briefly, GLPP was hydrolyzed with 2 M trifluoroacetic

acid (TFA) at 100 C for 10 h and reduced with NaBH4 to obtain

alditols. The alditols were acetylated with Ac 2O to prepare corre-

sponding alditol acetates. Rhamnose, fucose, mannose, glucose,

and galactose were also converted to their alditol acetates, which

were used as GC standards. Acetyl inositol was used as internal

standard for monosaccharide quantification. Gas chromatographicanalysis was performed with a capillary column (HP-5.5% phenyl

methyl siloxane)and a flame ionization detector (FID). N2 was used

as the carrier gas at a flow rate of 1 mL/min. The injector tempera-

ture was kept at 260 C and the detector was maintained at 300 C.

The column temperature was held at 150 C for 2 min, increased

to 220 C at a rate of 2 C /min, and from 220 to 280 C at a rate of

30 C/min.

Infrared spectraGLPP was analyzed using an 8400 s SHIMADZU FT-IR in-

strument (Kyoto, Japan) with KBr pellets for detecting functional

groups.

Effect of GLPP on growth of inoculatedtumor S180, Heps, and EAC in mice

GLPP was investigated for its antitumor activity against inocu-

lated Sarcoma 180 (S180), Hepatoma solidity (Heps), or Ehrlichi As-

cites Carcinoma (EAC) tumor cells using ICR mice according to the

protocol previously described (Xu and others 2002; Yoo and others

2004). S180, Heps, and EAC tumor cells were inoculated at doses of

2 106 cells, respectively, on day 0. Twenty-four hours after the in-

oculation, mice were weighed, and randomly divided into 6 groups

for each tumor type, with 10 mice in each group. Each group had 5

male and 5 female mice. The 6 groups included 3 GLPP treatment

groups at dosage levels of 300, 100, and 33.3 mg/kg body weight,

and the PSP (1000mg/kg bodyweight)and Cyclophosphamide(Cy)

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(25 mg/kg body weight) groups as the positive controls, respec-

tively, and a vehicle group (the control) that received the same vol-

ume of normal saline. Treatment began on day 1 and was orally

given once a day for 7 incessant d. On the 2nd d after the last treat-

ment (day 8), mice were weighed and killed, and tumors were seg-

regated and weighed. Antitumor activity was evaluated by tumor

growth inhibitory ratio (IR%)= [(TWcontrol TWtreatment )/TWcontrol]

100%, where TWcontrol is the average tumor weight for mice in

the control group (g/animal) and TWtreatment is the average tumor

weight of treatment group (g/animal) (Yoo and others 2004). Trip-licate mice feeding studies were conducted, and results from each

replicate study were reported separately.

Effect of GLPP on the phagocytosisfunction of reticuloendothelial system(RES) of the EAC tumor-bearing mice

ICR mice were weighed and randomlydividedinto 6 groupswith

10 (5 male and 5 female) mice each. The 6 groups included the nor-

mal group without inoculation of EAC tumor cells and any treat-

ment, the control group with EAC cells inoculated but no treat-

ment, the PSP group at a dose level of 1000 mg/kg bodyweight, and

3 GLPP treatment groups at dose levels of 300, 100, and 33.3 mg/kg

body weight, respectively. Treatments began on day 1, and wereorally administered once a day for 13 d. On day 6, EAC tumor cells

were inoculated to mice in the 5 groups according to protocol pre-

viously described (Xu and others 2002). Twenty-four hours after the

last administration, each mouse was given the India ink (1:5 dilu-

tion by normal saline) at a dose of 0.05 mL/10 g body weight by tail

intravenous injection. Twenty microliters of blood were taken from

the orbital vein at 1 (t1) and 5 (t5) min after injection, and placed

in 2 mL of 0.1% Na2CO3 immediately. After mixing, the solution

was measured for absorbance at 680 nm against the 0.1% Na2CO3solution blank. Then, mice were weighed and killed, and the total

weights of the liver and spleen were estimated. The phagocytic in-

dex(k) andthe phagocytic coefficient () were calculated according

to the following equations (Chen and others 2005):

k= (logA1 logA5)/(t5 t1) = log(A1/A5)/4

= body weight (g)/thetotalweight of liver andspleen(g) k1/3

where A1 and A5 are the absorbance values of the blood-0.1%

Na2CO3 mixtures at 680 nm for the blood samples collected at 1

(t1) and 5 (t5) min after injection of India ink, respectively.

Effect of GLPP on the humoral immunityof the EAC tumor-bearing mice

ICR mice were weighed and randomly divided into 6 groups,

each of which had 10 mice, 5 male and 5 female. The 6 groups in-

cluded the normal group without inoculation of EAC tumor cellsand any treatment, the control group with EAC cells inoculated but

no treatment, the PSP group at a dose level of 1000 mg/kg body

weight, and 3 GLPP treatment groups at dose levels of 300, 100, and

33.3 mg/kg body weight, respectively. Treatment was orally given

once a day for 13 d. On the 6th d after the 1st administration, EAC

tumor cells were inoculated in mice in the 5 groups except the con-

trol without EAC tumor-bearing, according to the protocol previ-

ously described (Xu and others 2002). On the 9th d after the 1st ad-

ministration of treatment, mice in all groups were sensitized by 0.5

mL of 20% sheep red blood cell (SRBC) suspension. Four days af-

ter being sensitized by SRBC, mice were bled and serum samples

were diluted with normal saline at a blood to saline ratio of 1:500

(v/v). Hemolysin concentration was determined by measuring the

absorbance at 540 nm. The 50% hemolysin value (HC50) was calcu-

lated as: pt/

HC50 of the sample = (Asample/HC50 of SRBC) 500

where Asample is the absorbance value of sample at 540 nm, and

HC50 of SRBC is the absorbance value of 0.25 mL of 20% SRBC un-

der the same experimental conditions.

Effect of GLPP on toxicity of cyclophosphamide(Cy) therapy in Heps tumor-bearing miceAccording to the protocol of previously described (Xu and oth-

ers 2002), ICR mice with tumor Heps inoculated were used in this

experiment. Twenty-four hours after the inoculation, mice were

weighed and randomly divided into 6 groups, each of which had

10 mice, 5 male and 5 female. The 6 groups included the control

group without Cy and any treatment, the Cy group, 3 GLPP with Cy

treatment groups at GLPP levels of 300, 100, and 33.3 mg/kg body

weight, and the PSP with Cy group (positive control) at a PSP dose

of 1000 mg/kg body weight. GLPP and PSP were orally given once a

day for 9 d. On the 6th d after their administration, Cy was contin-

uously given once a day for 2 d at a level of 100 mg/kg body weight

to mice in the 5 groups but not those in the control group. On the9th d after the 1st administration of GLPP and PSP, the mice were

weighed and blood sample was collected from the orbital vein. The

number of the peripheral white blood cells wascounted with an in-

verted microscope XSZ-D (Chongqing Optical Instrument Factory,

China). After takingthe blood sample, the mice were killed, and the

total weight of thymus and spleen was measured and used to cal-

culate the thymus index and the spleen index. The entire femoral

bone of 1 thigh was segregated and the number of bone marrow

karyotes was measured.

Effect of GLPP on toxicity of cobalt-60 (60Co)radiation in Heps tumor-bearing mice

ICR mice with tumor Heps inoculated were used in this exper-

iment (Xu and others 2002). Twenty-four hours after the inocu-

lation, mice were weighed, and randomly divided into 6 groups,

each of which had 10 mice, 5 male and 5 female. The 6 groups in-

cluded the untreatedcontrol group (normal saline), the 60Co group,

3 GLPP plus 60Co treatment groups (300, 100, 33.3 mg GLPP/kg

body weight), and the PSP plus 60Co group (1000 mg PSP/kg body

weight). Mice in all 5 groups except the control group were treated

with 60Co radiation at the dose of 5 Gray (Gy). PSP and GLPP were

orally given to each group once a day for 8 d. On the 2nd d after the

last administration of PSP and GLPP, the mice were weighed, and

blood was collected from the orbital vein. The number of the pe-

ripheral white blood cells was measured using an inverted micro-

scope XSZ-D (Chongqing Optical Instrument Factory, China). Then

the mice were killed, and the total weight of thymus andspleen wasmeasured andused to calculatethe thymus index andspleen index.

The entire femoral bone of 1 thigh was segregated and its number

of bone marrow karyotes was measured.

Statistical analysisThedatawerereported as mean standard deviation (SD). Anal-

ysis of variance (ANOVA) and Students t-tests were conducted to

identify differences among means. Statistical significance was de-

clared at P< 0.05.

Results and Discussion

There could be about 140000 mushrooms available on Earth,

and approximately 14000 of them are known (Wasser 2002).

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Among these known mushrooms, about 2000 are considered safe

for consumption and about 700 species are classified as medici-

nal mushrooms, which contain significant levels of bioactive com-

ponents, including antitumor and immunostimulating polysaccha-

rides with or without a peptide moiety. In addition to their tradi-

tional use for health promotion and disease prevention and treat-

ment in China, Korea, Japan, and eastern Russia, consumption of

medicinal mushroom in Western countries has recently arisen be-

cause of consumer desire for foods with health beneficial proper-

ties, awareness of complementary medicines, cost of medical treat-ment, increase of aging population, and scientific evidence show-

ing the efficacy and safety of these mushroom and their compo-

nents. This has triggered the recent research and development of

nutraceuticals and functional foods from these medicinal mush-

rooms, such as G. lucidum and Coriolus versicolor. In the present

research, a novel low molecular weight polysaccharide (GLPP)

was prepared from G. lucidum grown in Anhui province, China.

GLPP was characterized for its carbohydrate and protein con-

tents, monosaccharide profile, configuration of glycosidic linkages,

amino acid profile of the peptide moiety, and selected physical

properties. GLPP was also evaluated and compared to a commer-

cially available PSP, an antitumor and immunostimulating polysac-

charide from C. versicolor, for its potential in suppressing thegrowth of transplanted tumor cells, its possible immunomodulat-

ing property, and its capacity in reducing the toxicity and side ef-

fects induced by chemotherapy and radiation.

Characterization of GLPPThe polysaccharide (GLPP) isolated from the fruiting body ofG.

lucidum was a brownish powder, which was odorless and taste-

less. The overall yield of GLPP was 3.7% of the starting G. lu-

cidum fruit body powder on a dry weight basis. GLPP was solu-

ble in water but insoluble in commonly used organic solvents such

as ethanol, acetone, chloroform, and ethyl ether. Its 2% aqueous

solution exhibited a specific optical rotation of+25.6, which is

different from that of+129.6, 52.7, and 38.3 observed for 3

polysaccharides isolated from G. lucidumbyBao andothers (2002).

GLPP showed a positive result when tested with sulphuric acid-

anthrone reagent, confirming the presence of a carbohydrate moi-

ety. Furthermore, GLPP was notable to reactwith eitherFehlings or

iodinepotassium iodide reagents, suggesting that it did not con-

tain significant level of reducing sugar or polysaccharide compo-

nents capable of forming complex with iodine. The Lowry assay

was positive, which confirmed the presence of a peptide moiety

in the GLPP preparation. Further proximate examination showed

that GLPP contained 61.2% carbohydrate and 10.6% protein along

with 12.5% inorganic components and minor amounts of crude

fat (2.9%) and moisture (1.6%). This ratio of carbohydrate to pro-

tein is much lower than that of 93.5:6.5% observed for a G. lu-

cidum polysaccharide preparation with immunopotentiating re-ported by Shao and others (2004). This ratio is also different from

that of 55.2:41.3% reported for an antitumor extract with a molec-

ular weight of 10000 and above prepared from G. lucidum by hot

water extraction and dialysis using a PM-10 membrane (Maruyama

and others 1989).

Protein isolated from GLPP was analyzed for its amino acid pro-

file. The results showed that the GLPP protein contained 17 differ-

entkinds of amino acids. Their name andmolar ratiowereAsp 0.68,

Thr 0.50, Ser 0.58, Glu 0.46, Gly 0.41, Ala 0.44, Cys 0.05, Val 0.30, Met

0.10, Ile0.74,Leu 0.14, Tyr 0.10, Phe0.21,Lys 0.28, His 0.05, Arg0.24,

Pro 0.36. Acidic and basic aminoacids accountedfor about22% and

10% of the total amino acids, respectively. Nonpolar amino acids

consisted of over 40% of the total amino acids, whereas polar neu-

tral amino acids were about 26% of that. Acidic and neutral amino

acids accounted for approximately 90% of all the 17 amino acids

present.This ratio is higherthan that of 70%detectedin thepeptide

moieties ofC. versicolorpolysaccharopeptides, which exhibited an-

ticancer and immunostimulating activities (Cui and Chisti 2003).

The average molecular weight of GLPP was about 6600 dalton

by a gel permeation chromatographic (GPC) analysis. The polysac-

charide moiety of GLPP appeared heterogeneous and contained 3

kinds of monosaccharides: mannose, glucose, and galactose at a

molar ratio of 0.9:15.0:1.0. This monosaccharide composition is dif-ferent from those reported before for G. lucidum polysaccharides

and polysaccharopeptides (Bao and others 2002; Shao and others

2004). No fucose was detected in the GLPP under the current ex-

perimental conditions.

The FT-IR spectra of the polysaccharide GLPP had a broad O-

H stretching peak at 3379 per cm, a C-H stretching peak at 2921

per cm, a carbonyl (C = O) stretching peak at 1652 per cm, an un-

symmetrical carbonyl stretching peak at 1612 per cm, and a C-O

stretching peak at 1411 per cm. The peak at 894 per cm in the FT-IR

spectra of GLPP indicated the presence of-type glycosidic link-

ages in the polysaccharide. These peaks were characteristic peaks

of carbohydrates (Barker and others 1954). The 1200 to 1000 per

cm region, which is referred to as the finger print region, is gen-erally unique to each carbohydrate preparations. Taking all these

physical, chemical, and molecular properties into account, GLPP is

a novel low molecular polysaccharopeptide from G. lucidum fruit

body powder.

Inhibition of tumor growth in miceThe results in Table 1 to 3 showed that GLPP at levels of 300 and

100 mg/kg body weight (BW)/d, PSP at a dose of 1 g/kg BW/d, and

Cyat 25 mg/kgBW/d were able to significantly inhibit the growth of

S180, Heps, and EAC tumor cells in mice, whereas GLPP at a dose of

33.3 mg/kg WB/d could only inhibit the growth of EAC in mice un-

der the experimental conditions. These data indicated the poten-

tial antitumor property of GLPP and suggested that its antitumor

potency may be altered by the type of tumor. Also noted was that

GLPP at 300, 100, and33.3 mg/kg BW/d exhibitedinhibitory rates of

36.09% to 39.61%, 30.39% to 32.37%, and 19.19% to 24.77% against

the inoculated S180 tumor (Table 1), respectively, suggesting that

GLPP may dose-dependently inhibit the growth of transplanted tu-

mor. This statement was supported by the observations that GLPP

also dose-dependently suppressed the growth of inoculated Heps

andEAC tumor cells in mice (Table 2 and3). Interestingly, GLPP at a

dose of 100 mg/kg BW/d had same growth inhibitory effect against

all 3 tested inoculated tumor cells as that of the PSP at a dose of 1

g/kg BW/d. Furthermore, 300 mg/kg BW/d GLPP showed stronger

growth inhibition against all 3 tested tumor cells than 1 g/kg BW/d

PSP under the same experimental conditions, suggesting that GLPP

may be a stronger antitumor agent than PSP. In addition, GLPP andPSP treatments did not result in notable changes of body weight

gains in mice, whereas Cy treated mice had lower body weight gain

during the experimental periods. The results of replicated mouse-

feeding experiments were consistent for all tested tumor types (Ta-

ble 1 to 3). Taking together, these data indicate the possible utiliza-

tion of GLPP in tumor treatment and prevention.

Immunomodulating potentialof GLPP in EAC tumor-bearing mice

The phagocytosis function of RES and the humoral immunity

were determined to investigate the possible immunomodulating

capacity of GLPP in EAC tumor-bearing mice. EAC tumor-bearing


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mice had significantly lower phagocytic index, phagocytic coeffi-

cient, and 50% hemolysin value than that detected in the normal

mice (Table 4), indicating tumor-induced suppression of immune

function. This is similar to the previous observation that immune

function progressively decreased with the persistent growth of tu-

mor, andthe immunodeficiency of tumor patients during theirlater

periods was very obvious.

Both PSP andGLPPwere able to significantly increasethe phago-

cytic index, phagocytic coefficient, and 50% hemolysin value in the

EAC tumor-bearing mice. Furthermore,a higher intake of GLPPwasassociated to a greater immunostimulating potential in EAC tumor-

bearing mice. No difference was observed in the immunostimu-

lating potential between GLPP at a level of 300 mg/kg BW/d and

PSP at 1000 mg/kg BW/d dosage. These results are supported by

previous observations that G. lucidum polysaccharides may pro-

mote phagocyte system, enhance both humoral and cellular immu-

nities, and stimulate the function of antigen-presenting cells (Bao

and others 2002; Shao and others 2004; Lin 2005). The cellular and

molecular mechanisms involved in the immunostimulating activ-

Table 1 --- The inhibitory activity of GLPP on the growth of transplanted S 180 tumor in micea

Experiments1 2 3

Dose (mg/Groups kg/day) BW (g) TW (g) IR(%) BW (g) TW (g) IR (%) BW (g) TW (g) IR (%)

Control N/A 19.59/27.37 1.40a 0.38 19.59/26.42 1.52a 0.31 19.75/26.40 1.63a 0.33GLPP 300 19.56/27.68 0.90b 0.17 36.09 19.31/26.11 0.93b 0.32 38.56 19.50/26.30 0.99b 0.28 39.61

100 19.55/27.18 0.98b 0.14 30.39 19.28/26.25 1.03b 0.30 31.97 19.65/26.55 1.10b 0.27 32.3733.3 19.17/27.06 1.13ab 0.60 19.19 19.82/26.53 1.16b 0.30 23.73 19.80/26.10 1.23b 0.30 24.77

Cy 25 19.95/24.70 0.54c 0.18 61.20 19.84/24.71 0.56c 0.23 63.15 19.95/24.70 0.65c 0.24 60.39PSP 1000 19.01/27.90 0.97b 0.30 30.74 19.69/26.03 1.04b 0.24 31.64 19.80/26.00 1.08b 0.28 33.54

aData are reported as mean SD. (n= 10). 1, 2, and 3 represent 3 repeated experiments. GLPP, Cy, and PSP stand for a polysaccharide from G. lucidum,Cyclophosphamide, Polysaccharopeptide PSP, respectively, while the control received the same volume of normal saline. BW: body weight, TW: tumor weight(average per animal in each group), IR: inhibitory rate, which was calculated as: [(TW control TWtreatment)/TWcontrol] 100%, where TWcontrol is the tumor weight ofthe control group and TW treatment is the tumor weight of treatment group. Means with different letters (ac) within a column are significantly different ( P< 0.05).

Table 2 --- The inhibitory activity of GLPP on the growth of transplanted Heps tumor in micea

Experiments

1 2 3Dose (mg/

Groups kg/day) BW (g) TW (g) IR(%) BW (g) TW (g) IR (%) BW (g) TW (g) IR (%)

Control N/A 19.62/27.65 1.62a 0.42 19.85/28.05 1.58a 0.36 19.60/27.65 1.62a 0.35GLPP 300 19.87/27.67 1.05b 0.42 34.98 20.20/27.75 1.01b 0.25 35.89 19.98/27.90 1.05b 0.34 35.41

100 19.33/27.81 1.11b 0.19 31.39 19.95/28.20 1.08b 0.28 31.58 19.65/27.30 1.10b 0.34 31.7733.3 19.60/27.51 1.33ab 0.45 17.77 19.95/27.65 1.27ab 0.32 19.66 19.45/27.75 1.26b 0.28 22.13

Cy 25 19.73/23.40 0.64c 0.20 60.31 20.50/24.50 0.58c 0.18 63.22 19.85/24.60 0.60c 0.20 62.98PSP 1000 19.59/26.49 1.10b 0.24 32.20 19.65/27.90 1.05b 0.34 33.61 19.95/27.45 1.10b 0.28 32.14

aData are reported as mean SD. (n= 10). 1, 2, and 3 represent 3 repeated experiments. GLPP, Cy, and PSP stand for a polysaccharide from G. lucidum,Cyclophosphamide, Polysaccharopeptide PSP, respectively, while the control received the same volume of normal saline. BW: body weight, TW: tumor weight(average per animal in each group), IR: inhibitory rate, which was calculated as: [(TW control TWtreatment)/TWcontrol] 100%, where TWcontrol is the tumor weight ofthe control group and TW treatment is the tumor weight of treatment group. Means with different letters (ac) within a column are significantly different ( P< 0.05).

Table 3 --- The inhibitory activity of GLPP on the growth of transplanted EAC tumor in micea

Experiments

1 2 3Dose (mg/

Groups kg/day) BW (g) TW (g) IR(%) BW (g) TW (g) IR (%) BW (g) TW (g) IR (%)

Control N/A 19.60/27.00 1.54a 0.24 19.65/26.23 1.66a 0.35 19.80/26.20 1.67a 0.36GLPP 300 19.10/26.99 1.01c 0.21 34.11 19.65/26.92 1.07b 0.30 35.73 19.85/26.60 1.03b 0.31 38.31

100 19.50/26.69 1.05bc 0.28 31.51 19.75/26.61 1.14b 0.31 31.50 19.95/26.06 1.11b 0.27 33.3933.3 19.40/26.85 1.25b 0.21 18.68 19.71/26.28 1.26b 0.31 24.08 19.65/26.25 1.25b 0.30 25.24

Cy 25 19.80/24.40 0.54d 0.20 64.58 19.93/24.35 0.60c 0.24 63.55 19.70/24.04 0.62c 0.22 62.83PSP 1000 19.70/26.24 1.07bc 0.29 30.27 19.95/26.80 1.12b 0.30 32.35 19.90/26.35 1.14b 0.27 31.59

aData are reported as mean SD (n= 10). 1, 2, and 3 represent 3 repeated experiments. GLPP, Cy, and PSP stand for a polysaccharide from G. lucidum,Cyclophosphamide, Polysaccharopeptide PSP, respectively, while the control received the same volume of normal saline. BW: body weight, TW: tumor weight(average per animal in each group), IR: Inhibitory Rate, which was calculated as: [(TW control TWtreatment)/TWcontrol] 100%, where TWcontrol is the tumor weight of

the control group and TW treatment is the tumor weight of treatment group. Means with different letters (ad) within a column are significantly different ( P< 0.05).

ity ofG. lucidumpolysaccharides were summarized and discussed

in detail by Lin (2005) and Chen and others (2004). It is very im-

portant to improve the immunity of tumor patients, because tu-

mors formation and development is closely linked with host im-

mune state. G. lucidum (Fr.) Karst is known to have a wide range

of health benefits, and its polysaccharide components are impor-

tant for its antitumor and immunostimulating activities (Lei and

Lin 1993; Zhang and Lin 1999). Taken together, our findings sug-

gest the potential application of GLPP in cancer treatment and

immunostimulation.

Potential of GLPP in reducing the toxicityof chemotherapy and radiation

GLPP was evaluated and compared with PSP for its potential in

reducingthe toxicity of chemotherapyand radiationusing the Heps

tumor-bearing mice. Chemotherapy using cyclophosphamide (Cy)

decreased thymus index, spleen index, peripheral white blood cell

number, and bone marrow karyote count in the Heps tumor-

bearing mice (Table 5). GLPP at a dose of 300 mg/kg BW/d was as


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effective as PSP at a daily dose of 1000 mg/kg BW in prevention of

Cy induced decrease of thymus index, spleen index, and bone mar-

row karyote numbers (Table 5). Also noted was that GLPP at a dose

of 300 mg/kg BW/d and PSP at 1000 mg/kg BW/d exhibited similar

protective effects against 60Co radiation induced loss of white blood

and bone marrow karyotes, and the decrease of thymus and spleen

indexes (Table 6). Furthermore, a higher dose of GLPP was associ-

ated with a stronger preventive effect for all measured parameters

during Cy treatment and 60Co radiation. These data suggest the po-

tential application of GLPP in reducing the toxicity of chemother-apy and radiotherapy.

The results from the present research are supported by findings

from previous in vitro, animal, and pilot human studies. In 2006,

an in vitro study was conducted to investigate the antitumor and

immunomodulating activities of selected Ganoderma species in-

cludingG. lucidum, andfound that hot waterextracts ofG. lucidum

could significantly suppress the proliferation of MCF-7 and MDA-

MB-231 human breast cancer cells in a dose-dependent manner,

while showing no significant cytotoxic effect on human normal

mammary epithelial cells (Yue and others 2006). Earlier in 2003,

Gao and others showed that Ganopoly, a commercial G. lucidum

polysaccharide extract, at a daily oral dose of 1800 mg for 12 wk,

was able to significantly increase mean plasma levels of IL-2, IL-

Table 4 --- The effect of GLPP on the phagocytic function of RES and the HC50 of the EAC tumor-bearing micea

Dose Phagocytic index PhagocyticGroups (mg/kg/d) k 101 coefficient 101 HC50

Control without EAC bearing 7.08a 1.28 8.27a 0.79 344.68a 101.23Control with EAC bearing 3.96b 1.33 6.33c 1.08 156.84d 39.25GLPP 33.3 5.91ab 2.77 6.68bc 1.16 173.25cd 50.75

100 5.78a 2.32 7.69ab 1.20 176.75bd 41.58300 6.60a 2.10 7.67ab 1.55 190.58bc 31.34

PSP 1000 6.62a 2.73 7.64ab 1.60 211.40bc 68.11

aData are reported as mean SD. (n= 10). GLPP and PSP stand for a polysaccharide from G. lucidum and Polysaccharopeptide PSP, respectively. The controlwithout EAC bearing is normal mice that received the same volume of normal saline, while the control with EAC bearing is EAC tumor-bearing mice that receivedthe same volume of normal saline. Phagocytic index, k, was calculated as: (logA1 logA5)/(t5 t1) = log(A1/ A 5)/4, phagocytic coefficient was calculated as:body weight (g)/the total weights of the liver and spleen (g) k1/3. HC 50 of the sample was calculated as: (Asample/HC50 of SRBC) 500, where HC50 is 50%hermolysin value, Asample is the absorbance value of sample,HC 50 of SRBC is the absorbance value of 0.25 mL of 20% SRBC. Means with letters (ad) within acolumn are significantly different (P< 0.05).

Table 5 --- Effect of GLPP on toxicity to the Cy chemotherapy in Heps tumor-bearing micea

Dose Thymus index Spleen index Peripheral Wbc Number of bone marrowGroups (mg/kg body weight/d) (g/kg body weight) (g/kg body weight) (109 /L) karyote (105)

Control 2.52a 0.85 4.03a 1.16 14.46a 2.44 68.99a 33.25Cy 1.70b 0.66 2.28c 0.96 0.52e 0.15 18.08c 5.22GLPP + Cy 300 2.32a 0.24 3.08b 0.63 0.80be 0.39 24.51b 7.24

100 2.23a 0.29 2.87bc 0.73 0.72bc 0.25 23.26b 5.5633.3 1.85b 0.43 2.31c 0.68 0.64cde 0.35 22.46bc 16.96

PSP + Cy 1000 2.23a 0.37 3.11b 0.52 0.67bd 0.16 22.58b 4.08

aData are reported as mean SD. (n= 10). Cy, GLPP + Cy, and PSP + Cy stand for mice groups treated with Cy, Cy with GLPP, and Cy with PSP, respectively.The control received the same volume of normal saline. Dose for Cy was 100 mg/kg body weight/d for mice in Cy, GLPP plus Cy, and PSP plus Cy groups. Thymusindex = Thymus weight/body weight; Spleen index = spleen weight/body weight; Wbc: white blood cell. Means with different letters (ae) within a column aresignificantly different (P< 0.05).

Table 6 --- Preventive effect of GLPP on toxicity to the Cobalt-60 (60Co) radiation in Heps tumor-bearing micea

Dose Thymus index Spleen index Peripheral Wbc No. of bone marrowGroups (mg/kg/d) (g/kg body weight) (g/kg body weight) (109 /L) karyote (105)

Control 5.15a 1.26 2.58a 1.33 11.24a 3.30 79.58a 20.9160Co 1.78d 0.60 0.85c 0.28 1.52d 0.49 18.50c 8.89GLPP + 60Co 300 2.28bc 0.41 1.47b 0.34 2.12c 0.40 39.68b 17.20

100 2.03bd 0.31 1.37b 0.50 2.07c 0.50 39.08b 8.5433.3 1.91cd 0.42 1.23b 0.31 1.61cd 0.87 20.48c 12.51

PSP + 60Co 1000 2.27b 0.25 1.84a 0.42 2.90b 0.61 37.21b 14.69

aData are reported as mean SD. (n= 10). 60Co, GLPP + 60Co, and PSP + 60Co stand for mice treated with 60Co only, 60Co with GLPP, and 60Co with PSP,respectively. The control received the same volume of normal saline. Thymus Index was calculated as: Thymus weight/body weight; Spleen Index was calculated

as: spleen weight/body weight; Wbc: white blood cell. Means with different letters (ad) within a column are significantly different ( P< 0.05).

6, INF-, and NK cell activity while significantly reducing the con-

centration of IL-1 and TNF-, supporting the potential immunos-

timulating effect of G. lucidum polysaccharide in cancer treat-

ment. Several animal feeding studies have demonstrated that G.

lucidumcontains antitumor components that may inhibit inocu-

lated tumor cell growth in experimental animals and reduce tu-

mor metastasis, and suggested that the antitumor activity ofG. lu-

cidum components involve the hosts immunity (Yuen and Gohel

2005).

It has been well accepted that polysaccharopeptides with highermolecular weight may appear to exhibit stronger antitumor and

immunomodulatory activities (Ooi and Liu 2000). The molecular

weight of GLPP is much smaller than that of 53000 to 100000 and

8300 to 200000 for most reported G. lucidum polysaccharopep-

tides and polysaccharides with antitumor and immunostimulat-

ing properties, respectively (Bao and others 2002; Shao and others

2004; Yuen and Gohel 2005; Zhang and others 2007). The molecu-

lar weight of GLPP is also much smaller than that of approximately

100000 for PSP and PSK; both are well known C. versicolorpolysac-

charopeptides with antitumor and immunomodulating activities.

In the present study, no difference was observed between GLPP

at 300 mg/kg BW/d and a commercial PSP preparation at 1000

mg/kg BW/d in their antitumor and immunostimulating activities,


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suggesting that GLPP may be more effectivethan PSPin cancerpre-

vention and treatment on a per weight dosage basis.

It is also interesting that GLPP without detectable amounts of fu-

cose had both antitumor and immunomodulating activities under

the experimental conditions. This observation was in contrast to

the finding by Wang and others that terminal fucose residues with

(12) linkages are essential for the immunostimulating and an-

titumor activities of a polysaccharopeptide from G. lucidum(Wang

and others 2002). In addition to fucose, glucose, galactose, man-

nose, xylose, arabinose, ribose, and glucuronic acid have been de-tected in the mushroom antitumor polysaccharides (Wasser 2002;

Zhang and others 2007). The predominant monosaccharide in

GLPP was glucose, whichconsisted of about89% of the total mono-

sugars. This level is higher than that of 73% glucose observed in an

immunomodulating polysaccharidefrom G. lucidum(Bao and oth-

ers 2002). Mannose contributed a little more than 5% and galac-

tose contributed a little less than 6% of the rest monosaccharides

in GLPP. Furthermore, -glycosidic linkage and higher content of

acidic and neutral amino acids in GLPP may also play important

roles in its antitumor and immunostimulating activities (Ooi and

Liu 2000; Wasser 2002; Zhang and others 2007). It has been widely

accepted that chemical and molecular structures are important for

biological activitiesof nondigestiblepolysaccharides. More dataarerequired to clearly understand the relationships between mush-

room polysaccharide structures and their antitumor properties.

It should be pointed out that there were about 11% other

organic compounds present in GLPP. They could well be the

low-molecular-weight secondary metabolites such as terpenoids

andphenolics in G. lucidum, andmay also contribute to the overall

antitumor and immunostimulating capacity of GLPP. This is sup-

ported by the finding that a G. lucidum extract rich in triterpenes

inhibited human hepatoma Hup-7 cell growth (Lin and others

2003). Further study is required to characterize these components

and investigate their role in the health beneficial activities of GLPP.

In addition, results from this study suggested the potential use

of GLPP to reduce the toxic effects of chemotherapy and radiother-

apy. Both chemotherapy and radiotherapy are commonly used with

or without surgery for tumor treatment. Similar as other clinically

used cytotoxic agents, cyclophosphamide (Cy) may cause cell dam-

age and suppress immune functions in the tumor patients. These

antitumor medicines may also damage hematopoietic stem cells

and interfere in cellular DNA biosynthesis and lead to dysfunction

during the conversion of stem cells into blood cells in bone mar-

row (Zorzet and others 1998). The radiation during tumor radio-

therapy induces differential sublethal damage repair in tumor cells

compared with normal tissue (Jacques andothers 2005). The radia-

tion during tumor radiotherapy may also induce cellular damage

and impair the integrity of immune function in the hosts. These

side effects significantly hinder the survival rate of chemotherapy

and radiotherapy because the suppression of immunologic func-tion may lead to tumor worsening and complication, and the de-

pression of the hemopoietic system may cause patient exhaustion.

The present study showed that GLPP may also prevent the possi-

ble atrophy of spleen and thymus, the abnormality of peripheral

blood, and anemia induced by Cy and 60Co radiation, indicating

their potential to enhance the survival rate of chemotherapy and

radiotherapy.

Conclusions

The present study demonstrated the antitumor, immunostimu-lating, leukogenic, and antianemic activities of GLPPin tumor-bearing mice. These data suggest the potential utilization of GLPP

as an adjuvant to conventional treatments of cancers and its use

for cancer prevention. Clinical trial or plot human study is required

to confirm its potential in cancer treatment and prevention. Addi-

tional studies are essential to investigate the mechanisms involved

in their beneficial actions and their fate in experimental animals

and human beings.

AcknowledgmentsThis research was supported by a grant from the Natl. Natural

Science Foundation of China (Grant nr 30472061), a grant from

the Teaching and Research Award Program for Outstanding YoungTeachers of Univ., China (Grant nr 2002-383), and the Foundation

of Science and Technology for High Technology Research Project of

Jiangsu province, China.

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