Carbohydrate Polymers 77 (2009) 389–393

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Free radical scavenging of Ganoderma lucidum polysaccharides and its effect on

antioxidant enzymes and immunity activities in cervical carcinoma rats
Chen XiaoPing a,*, Chen Yan a, Li ShuiBing d, Chen YouGuo b, Lan JianYun c, Liu LanPing d
a
The Department of Obstetrics and Gynecology, YanCheng 1th People Hospital, YanCheng City, 224000 Jiangsu, China
b
Department of Obstetrics and Gynecology, First Affiliated Hospital of Soochow University, Suzhou 215006, China
c
Department of Pathology, YanCheng 1th People Hospital, YanCheng City, 224000, China
d
YanCheng 1th People Hospital, YanCheng City, 224000, China

a r t i c l e i n f o a b s t r a c t

Article history: Ganoderma lucidum are used as traditional edible and medicinal materials in China. In this study, antiox-
Received 16 December 2008 idant activities of polysaccharides from G. lucidum in China were investigated. The influence of G. lucidum
Accepted 12 January 2009 polysaccharides upon activities of serum antioxidant enzymes and immunity in rats with cervical cancer.
Available online 20 January 2009
The antioxidant activity was measured by DPPH , O , and OH free radicals scavenging. Results showed
that G. lucidum polysaccharides exhibited the higher DPPH , O , and OH free radicals scavenging activ-
Keywords: ities. The results still showed that G. lucidum polysaccharides could significantly enhance the antioxidant
Ganoderma lucidum polysaccharides
enzyme activities (SOD, CAT and GPx), and reduce levels of IL-1b, IL-6 and TNF-a in rats with cervical
Rat
Antioxidant activity
cancer.
Free radical Ó 2009 Elsevier Ltd. All rights reserved.
Immunity

1. Introduction Ganoderma polysaccharides (Tang & Zhong, 2002) and ganoderic

acid (i.e., GA) (Fang & Zhong, 2002; Tang & Zhong, 2003; Zhong &
Chinese herbal medicines have been widely used for thousands Tang, 2004).
years for the treatment of fractures and joint diseases. Ganoderma Carcinoma of the cervix is the second most common cancer to
lucidum is commonly used in traditional Chinese medicine. In the affect females worldwide and is the most common cause of can-
past, the development of herbal anti-osteoporosis formulas was cer-related death in developing countries (Pisani, Parkin, Bray, &
mainly pursued by scientists in Asian countries, including China, Ferlay, 1999). The purpose of this animal study was to examine
Japan and Korea (Hidaka, Okamoto, Yamada, Kon, & Kimura, in vitro free radical scavenging activities and the preventive effects
1999; Ke et al., 2009; Xu, Dick, Day, Randall, & Prince, 2003). G. of G. lucidum polysaccharides on oxidative injury and immunity
lucidum (Fr.) Krast (Polyporaceae), a mushroom-like higher fungus, activities in rats with cervical cancer. The free radical scavenging
has been a popular folk and an oriental medicine used to treat activities of G. lucidum polysaccharides was measured. The influ-
many diseases, such as hypertension, hypercholesterolemia, leuke- ence of G. lucidum polysaccharides upon activities of serum antiox-
mia, and gastric cancer (Paterson, 2006). The polysaccharides iso- idant enzymes and immunity in rats with cervical cancer were also
lated from G. lucidum have the antitumor activities (Li, Fang, & evaluated.
Zhang, 2007; Paterson, 2006; Zhang, Cui, Cheung, & Wang, 2007).
Recently, many new highly oxygenated triterpenes have been iso-
lated from the cultured mycelia of G. lucidum (Tang, Gu, & Zhong, 2. Materials and method
2006; Tang, Liu, Zhao, Wei, & Zhong, 2006; Zhou, Yang, & Yang,
2006), and their new biological functions such as inhibiting the 2.1. Extraction of polysaccharides
proliferation of lung cancer cell line 95-D, anti-HIV-1, and anti-
HIV-1 protease have been reported (El-Mekkawy et al., 1998; Lin, The fruiting bodies of G. lucidum were purchased from a local
Li, Lee, & Kan, 2003; Tang, Liu et al., 2006). In recent years, the sub- medicine shop in Yanchen city, china. Sporocarps were cut into
merged fermentation of G. lucidum has received great attention for small pieces, dried at 40–50 °C for 48 h and powdered. Polysaccha-
the efficient production of its valuable metabolites, especially rides were isolated by method of Mizuno (2000) and Pillai, Nair,
and Janardhanan (2008) and Yin and Dang (2008) with slight mod-
ification. The powdered sporocarps were defatted with petroleum
* Tel.: +86 515 88508820; fax: +86 515 88592872. ether and extracted with double distilled water at 80 °C for 8–
E-mail address: [email protected] (X. Chen). 10 h in several batches. The extract were combined, filtered, and

0144-8617/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carbpol.2009.01.009
390 X. Chen et al. / Carbohydrate Polymers 77 (2009) 389–393

concentrated to about one third of the original volume and chilled

ethanol about five times the original volume was added and kept at
4 °C for 48 h. The precipitate was collected after centrifugation,
redissolved in distilled water and treated with Sevag’s reagent
(Staub, 1999) several times to remove protein and then dialyzed
against deionised water for 48 h at 4 °C. The polysaccharides
(crude polysaccharide) were again precipitated with ethanol and
the precipitate thus obtained was lyophilized. The crude polysac-
charide was dissolved in water and reprecipitated with equal vol-
ume of cetyl trimethyl ammonium hydroxide and kept for
overnight. The supernatant obtained was precipitated with chilled
ethanol. After centrifugation, the precipitate obtained was run
through DEAE cellulose column and eluted with deionised water.
The precipitate thus obtained was lyophilized to get a light brown
powder, (neutral polysaccharide).

2.2. Isolation and purification of Ganoderma lucidum polysaccharides

An aliquot was then applied to an anion-exchange column

(5  50 cm) of DEAE-Sepharose Fast flow (Pharmacia), and eluted
stepwise as two fractions (F1 and F2) (Fig. 1) with 0.1, 0.3, 0.5,
0.7 and 0.9 M NaCl in Tris–HCl buffer (pH 8.5).

2.3. Thin-layer chromatography (TLC)

Thin-layer chromatography (TLC) was performed on a silica gel

plate (5  20 cm, Silica gel GF254, Qingdao Haiyang Chemical Co.). Fig. 2. Thin-layer chromatography.
An aliquot of each sample was spotted onto the silica gel plate with
a developing solvent system of chloroform/methanol (10:1, v/v) or
pertroleum ether/ethyl acetate (2:1, v/v). The spots were visualised and absorbance at 560 nm was measured as a blank value. After
by spraying the plates with spraying solutions of 1% solution of a 10 min incubation at ambient temperature with 0.025 ml of
phenylamine- diphenylamine-phosphate in water. Result from 0.03 mM PMS, the absorbance was again measured.
thin-layer chromatography (TLC) indicated that F1 and F2 were
both composed of mannose (Fig. 2). 2.4.2. Hydroxyl radical-scavenging activity
The hydroxyl radical-scavenging activity was assayed according
2.4. Free radical scavenging of Ganoderma lucidum polysaccharides to the method of Lopes, Schulman, and Hermes-Lima (1999).
Briefly, the polysaccharides sample was mixed with a solution con-
2.4.1. Superoxide anion radical-scavenging activity taining 5 mM 2-deoxyribose, 100 mM H2O2, and 20 mM PBS (pH
Superoxide anion radical-scavenging activity was measured by 7.2). Then, reaction was started by the addition of Fe2+ (6 lM final
a non-enzymatic method (Nishikimi, Rao, & Yagi, 1972) modified concentration) to this mixture. The reaction was carried out for
slightly (Kuda, Hishi, & Maekawa, 2006). Sample solution 15 min at room temperature and stopped by adding 4% phosphoric
(0.025 ml) was treated with 0.1 ml of 25 mM phosphate buffer acid (v/v) and 1% thiobarbituric acid (TBA, w/v, in 50 mM NaOH).
(pH 7.2), 2 mM NADH (0.025 ml) and 0.5 mM NBT (0.025 ml), After boiling for 15 min at 95° C, sample was cooled to room tem-
perature and the absorbance was read at 532 nm.

1.4 2.4.3. Measurement of DPPH free radical-scavenging activity

The DPPH free radical-scavenging activities of the G. lucidum
polysaccharides extract, fractions, and subfractions derived from
1.2
Rhodemela confervoides were measured using the method in a liter-
ature report (Yen & Chen, 1995) as well as our previous publication
1.0
(Duan, Zhang, Li, & Wang, 2006).

0.8 2.5. Animal experiment

A (240)

0.6 2.5.1. Treatment of animals

Thirty-two rats of Wistar strain weighing 170–190 g were pur-
0.4 chased from the Central Animal House, Suzhou University. The ani-
mals were housed in polypropylene cages and maintained under
controlled conditions of 12 h light/12 dark cycle and 50% relative
0.2
humidity at 25–30 °C. The animals were fed pellet diet and water
ad libitum. The study was approved by Institutional Animal Ethics
0.0
Committee, Yanchen 1th Hospital, Suzhou University and the ani-
0 10 20 30 40 50 60
mals were maintained in accordance with the Guide for the Care
Number
and Use of Laboratory Animals. After a period of 1 week, Twenty-
Fig. 1. Isolation and purification of Ganoderma lucidum polysaccharides by an four rats were induced cervical cancer according to the reference
anion-exchange column. (Gao, Shi, Di, & Sun, 2008). Then, the animals with cervical cancer
 X. Chen et al. / Carbohydrate Polymers 77 (2009) 389–393 391

were divided into three groups of eight rats each and maintained poly Vc
as follows:
100

OH- scavenging rate (%)

Group I (normal control) received the same volume of physio- 90
logical saline twice daily. 80
Group II (model control) received the same volume of physio- 70
logical saline twice daily. 60
Group III (low dose of polysaccharides treatment) received 50
polysaccharides (dissolved in 2 ml distilled water) at a dosage 40
of 150 mg/kg body weight twice daily. 30
Group IV (high dose of polysaccharides treatment) received
20
polysaccharides (dissolved in 2 ml distilled water) at a dosage
10
of 300 mg/kg body weight twice daily.
0
Food and water were fed ad libitum to all groups. At the end of the 0 0.5 1 1.5 2 2.5 3
experimental period of 40 days, body weights of the rats were content (mg/ml)
measured. The rats were sacrificed. Blood was collected in heparin-
Fig. 4. Hydroxyl radical-scavenging activity of Ganoderma lucidum polysaccharides.
ised tubes and plasma was separated. The blood was immediately
homogenized in 0.1 M Tris–HCl, pH 7.4. Plasma homogenate was
used for various analyses. Fig. 3, the superoxide anion radical scavenging effects of the crude
polysaccharides increased with increasing concentration. The
2.5.2. Biochemical analysis highest scavenging activity of the crude extracts was 80.4% at the
SOD, GSH-Px and CAT activities were assessed using commer-
concentration of 2.2 mg/ml, after that, the value no longer in-
cially available kits (Sigma, America) according to the manufac- creased (Fig. 3).
turer’s instructions. One unit of enzyme activity was defined as the
amount of protein needed to decrease the reference rate to 50% of 3.2. Effect on hydroxyl radical scavenging activities
maximum inhibition. IL-1b, IL-6 and TNF-a were measured by stan-
dard enzyme-linked immunosorbent assay (ELISA) using commer-
The hydroxyl radical is one of representative reactive oxygen
cially available BD OptEIA ELISA kits (BD Biosciences, San Diego,
species generated in the body. In this work, the antioxidant activ-
CA), as described previously (Stapp, Polis, Sánchez Piñero, 1999).
ities of the crude G. lucidum polysaccharides were low at the tested
concentration range of 0.2–2.6 mg/ml determined by DPPH free
2.5.3. Statistical analysis
radical-scavenging assay. They were 48.4%, 54.8% and 58.2%,
All data were given as means ± standard deviation (SD). Com-
respectively at the tested concentration of 2.2, 2.4 and 2.6 mg/ml.
parisons between the means of various treatment groups were
The polysaccharides showed lower antioxidant activities than did
analyzed using Dunnett’s t-test followed by analysis of variance
vitamin C, their scavenging effects increased with increasing con-
(ANOVA). P < 0.05 was considered to be significant.
centration (Fig. 4).

3. Results and discussion 3.3. Effect on the DPPH radical-scavenging activity

3.1. Superoxide anion radical-scavenging Because DPPH can be kept indefinitely with little decomposition
and because it neither dimerizes nor reacts with oxygen (Yu, Che,
Superoxide is generated in biological systems during the normal Ma, & He, 2009; Yu, Yin, Yang, & Liu, 2009), it proved to be quite
catalytic function of certain enzymes, and in the case of fresh meat, useful in a variety of investigations, such as polymerization inhibi-
the oxidation of myoglobin (Naguib, 2000). In the present study, tion or radical chemistry (Wang & Luo, 2007), the determination of
we investigated the scavenging or preventive capacity of G. lucidum antioxidant properties of amines, phenols or natural compounds
extracts against the superoxide anion free radicals. As illustrated in

poly Vc
poly Vc
100
90 100
DPPH- scavenging rate (%)
O- scavenging rate (%)

80 90
70 80
60 70
50 60
40 50
30 40
20 30
10 20
0 10
0
0 0.5 1 1.5 2 2.5 3
0 0.5 1 1.5 2 2.5 3
content (mg/ml)
content (mg/ml)
Fig. 3. Superoxide anion radical-scavenging activity of Ganoderma lucidum
polysaccharides. Fig. 5. DPPH radical-scavenging activity of Ganoderma lucidum polysaccharides.
392 X. Chen et al. / Carbohydrate Polymers 77 (2009) 389–393

Table 1
Effect of Ganoderma lucidum polysaccharides on antioxidant enzyme and immunity activities in rats with cervical cancer.

Indexes Normal control Model control Ganoderma lucidum polysaccharides (I) Ganoderma lucidum polysaccharides (II)
a b
SOD (U/ml) 143.5 ± 11.8 67.4 ± 3.2 87.8 ± 5.1 115.4 ± 7.3b
CAT (U/ml) 15.54 ± 1.13 7.82 ± 0.541a 9.48 ± 0.45b 13.91 ± 1.17b
GPx (U/ml) 25.85 ± 1.43 10.42 ± 0.84a 17.34 ± 1.05b 26.39 ± 1.33b
IL-1b (mg/ml) 1.932 ± 1.107 2.981 ± 0.084a 2.471 ± 0.048b 2.215 ± 0.131b
IL-6 (mg/ml) 2.765 ± 0.093 3.772 ± 0.068a 3.472 ± 0.081b 3.185 ± 0.092b
TNF-a (mg/ml) 1.231 ± 0.089 1.827 ± 0.104a 1.613 ± 0.057b 1.437 ± 0.071b
a
P < 0.01; compared with normal control.
b
P < 0.01, compared with model control.

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the DPPH radical-scavenging assay system was successfully used the field of pharmaceutical and functional foods.
for the evaluation on the antioxidant activity of the crude extract
from G. lucidum. The DPPH assay method is based on the reduction
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