Antioxidative and Antiinflammatory Activities of the Chloroform Extract of Ganoderma lucidum Found in South India

Antioxidative and anti-inflammatory activities of Ganoderma lucidum (Curt.: Fr.) P. Karst. (Aphyllophoromycetideae) from tropical South India were investigated. The chloroform extract of the mushroom showed marked free radical scavenging activities. The anti-inflammatory activity of the extract at concentrations of 100 and 50 mg/kg was evaluated in carrageenan induced acute and formalin induced chronic inflammatory models in mice. The extract showed remarkable antiinflammatory activity in both models, comparable to the standard reference drug diclofenac. The results suggest that anti-inflammatory activity of the chloroform extract of G. lucidum is possibly attributed to its free radical scavenging properties. This study also reveals the potent therapeutic uses of G. lucidum from South India.


Introduction
Mushrooms represent a major and as yet largely untapped source of potent pharmaceutical products.Nearly 10,000 mushroom species are known, of which 2000 are safe for humans and about 300 of them possess medicinal properties.Ganoderma lucidum, has been used in folk medicine in China and Japan for over 2000 years for a wide range of ailments.In Chinese folklore, fruiting bodies of Ganoderma have been regarded as a panacea for all types of diseases, perhaps due to its demonstrated efficacy as a popular remedy to treat a large number of diseases, namely chronic hepatitis, arthritis, hypertension, hyperlipidemia, insomnia, bronchitis, neoplasia, asthma, gastric ulcer, atherosclerosis, diabetes, debility due to prolonged illness etc [1].Almost all medicinal properties have been attributed to G. lucidum and thus, it is known as 'mushroom of immortality' in China, Japan and Korea.
Ganoderma spp.occur in morphological types such as black, light black, red, purple, yellow and white.Each type of Ganoderma has its own characteristic biological properties.The commonly used medicinal Ganoderma include G. lucidum, G. tsuge, G. capense and G. applanatum.The fruiting bodies of G. lucidum contain a variety of chemical substances, major components are terpenoids and polysaccharides.Currently 130 triterpenoids, and more than 100 types of polysaccharides are reported from G. lucidum [2].
In biological systems potentially harmful reactive oxygen species (ROS) are produced during the normal aerobic metabolism.Antioxidants are deployed to prevent generation of ROS or to scavenge those formed.Deficiency of antioxidative defenses may lead to oxidative stress, which might be associated with a variety of disorders such as coronary heart diseases, neural disorders, diabetes, arthritis and cancers [3,4].For chronic diseases, such as osteoarthritis and rheumatoid arthritis, life long dependency on antiinflammatory drugs is necessary.The most widely used non-steroidal antiinflammatory drugs (NSAID) suffer from several side effects.Hence, the search for effective antiinflammatory agents that could be safely used on a long-term basis is a priority [5].
G. lucidum has been found to occur widely in India particularly in the tropical areas.Previous studies at Amala Cancer Research Centre showed that the methanolic extract of G. lucidum occurring in tropical South India possessed significant antioxidative and antiinflammatory activities [6], [7].Some physiological effects and distinctive properties of Ganoderma are strain dependent and evidence for strain specific terpenoids has been reported in this mushroom [8].In this communication, we report the antioxidative and antiinflammatory activities of the chloroform extract of G. lucidum occurring in the tropical South India.

Plant collection and identification
Fruiting bodies of G. lucidum growing on gulmohar trees (Delonix regia Raf.) were collected from the out skirts of Thrissur, Kerala, South India.The specimen was identified by Prof. K.M. Leelavathi ( Dept. of Botany, Calicut University, Calicut, India).A voucher specimen is deposited in the herbarium of Centre for Advanced Studies in Botany, University of Madras, Chennai (HERB.MUBL-3172).Sci Pharm.2009; 77; 111-121.

Preparation of extract
Fruiting bodies of the mushroom were cut into small pieces, dried at 45-50°C for 48 h and powdered.The powdered material (200 g) was extracted with petroleum ether using a Soxhlet apparatus for 24 h.The defatted material was then extracted with chloroform by the same process.The chloroform extract was evaporated to dryness at 40°C using a rotary vacuum evaporator.The residue (3g) thus obtained was used for the experiments.

Determination of antioxidant activity
In vitro antioxidant activity of the extract was determined by DPPH (1,1 diphenyl 2-picryl hydrazyl) scavenging activity using AEAC (Ascorbic acid equivalent antioxidant capacity) as standard.AEAC is the concentration of ascorbic acid required to give the same antioxidant capacity as test substance [12].Superoxide radical, lipid peroxidation and nitric oxide scavenging activities of the extract were determined by finding out IC 50 values.

Superoxide anion scavenging activity
Superoxide anion scavenging activity was determined according to the method of McCord and Fridovich (1969) [14].Quercetin was used as standard.

Inhibition of lipid peroxidation
Lipid peroxidation induced in rat liver homogenate [15] and its inhibition by the extract was determined by the method of Ohkawa et al. [16].Ascorbic acid was used as standard.

Determination of antiinflammatory activity
Acute and chronic antiinflammatory activities of the extracts were evaluated by carrageenan-induced acute and formalin-induced chronic inflammatory models in mice.The extract was administered orally.

Carrageenan-induced paw edema
Male Swiss albino mice were divided into four groups of six animals in each group.In all groups the inflammation was induced by single sub-plantar injection of 20 µl of freshly prepared 1% carrageenan suspension in normal saline [18].Group 1 treated with carrageenan alone served as control.Group 2 and 3 received G. lucidum extract at concentrations of 50 and 100 mg/kg body wt.orally 1 h before the carrageenan injection.The extract was presolubilized in 0.2% dimethyl sulfoxide (DMSO) and a fine suspension was prepared in phosphate buffered saline.Group 4 was administered with reference drug diclofenac (10 mg/kg body weight) also orally 1 h before carrageenan injection [18].The paw thickness of animals in all groups was measured using vernier calipers before and 3 h after carrageenan injection.

Formalin -induced paw edema
Experimental procedure was the same as described above except that single dose of 0.02 µl of formalin (2%) was used to induce inflammation [18].The extract was administered once daily for 6 consecutive days [19].
In the above two models, the degree of edema formation was determined as increase in paw thickness.In the case of acute anti-inflammatory activity, paw thickness was measured once daily for 6 days.The increase in paw thickness and percent inhibition were calculated as follows.
Increase in paw thickness in control/treatment Where P t is paw thickness at time t, P o is initial paw thickness, P c is the increase in paw thickness of the control group and P T is the increase in paw thickness of the treatment groups [16].

Animal Experiments
All animal experiments were carried out according to the guidelines of the Committee for the Purpose of Control of Experiments on Animals (Reg.No. 149/1999/CPCSEA) and approval of the Institutional Animal Ethics Committee was obtained.

Statistical analysis
Experimental data are expressed as mean ± SD.Student's t test was applied for expressing the significance and P value less than 0.05 was considered as significant.Sci Pharm.2009; 77; 111-121.

Phytochemical screening
The chloroform extract of G. lucidum was tested positive for terpenoids and alkaloids in preliminary phytochemical tests.The HPTLC fingerprint of the chloroform extract is shown in Fig. 1.

Antioxidant capacity of extract: DPPH radical scavenging activity
The results showed that the chloroform extract of G. lucidum showed potent radical scavenging activity.The activity of the extract at different concentrations is presented in Table 1.The result indicated free radical scavenging activity of the extract in a dose dependent manner.

Inhibition of lipid peroxidation
Chloroform extract of G. lucidum was effective in inhibiting the lipid peroxidation induced by Fe 2+ -ascorbate system in rat liver homogenate (Table 2).The generation of malondialdehyde (MDA) and related substances that react with thiobarbituric acid (TBARS) was found to be inhibited by the extract.This indicated lipid peroxidation inhibiting activity of the extract (IC 50 : 593.3 ± 25.1 μg/ml).

Nitric oxide radical scavenging activity
The chloroform extract of G. lucidum effectively reduced the generation of nitric oxide radicals from sodium nitroprusside solution (Table 2).This showed marked nitric oxide scavenging activity of the extract (IC 50 : 21.6 ± 1.5 µg /ml).

Anti-inflammatory activity
The chloroform extract of G. lucidum showed significant inhibitory effect against induced inflammation in both the experimental models.The carrageenan induced acute and formalin induced chronic inflammation were significantly inhibited by the extract.The effect was evident from the inhibition of the paw edema ( Fig. 2).
The effect was significantly high in 100 mg/kg body wt.extract treated group compared with standard reference drug, diclofenac.The carrageenan induced paw edema was reduced by 73.4% and 63.2% with treatment of chloroform extract of G. lucidum at concentrations of 100 and 50 mg/kg body weight respectively compared to that of the control group.Where as the inhibitory effect was 63.4% and 53.4% for formalin induced paw edema with the treatment of the extract at 100 and 50 mg/kg body weight respectively.Standard reference drug diclofenac showed an inhibition of 53.0% and 40.2% of carrageenan and formalin induced inflammation respectively (Fig. 2).Inhibition of acute and chronic inflammation by chloroform extract of G.lucidum (50 and100 mg/kg) administered orally.

Discussion
The results of the present investigation reveal that chloroform extract of G. lucidum possesses significant capacity to inhibit free radical formation and scavenging activity.The extract acts at two different levels as primary antioxidant.Antioxidants show activities at different level of protection [20].Although organisms are bestowed with antioxidant and repair systems that have evolved to protect them against oxidative damage, these systems are insufficient to prevent the damage totally [21].Hence antioxidants in diet are of great importance as possible protective agents to help human body to reduce oxidative damage.
Recently a large number of natural antioxidants have been isolated from different plants.[22][23][24].Mushrooms are functional food and are traditionally used in folk medicine in several countries.Human diet containing medicinal mushrooms possessing antioxidative properties would be potentially useful to help human body to reduce oxidative damage.The broad-spectrum medicinal property of G. lucidum might be due its significant antioxidantive activity.Our earlier reports also confirm this conclusion [6,25].
In the DPPH assay, the ability of antioxidant to scavenge stable purple-colored primary radical DPPH is tested by its depolarization spectrophotometrically at 515 nm [13].This shows the ability of the extract to scavenge stable free radicals.
The chloroform extract of G. lucidum shows significant superoxide anion, nitric oxide scavenging and lipid peroxidation inhibiting activity in a dose dependent manner.Simultaneous generation of NO an O 2 − favours the production of a toxic reaction product, peroxynitrite (ONOO − ) [26].The scavenging of the superoxide anion generated from the photoreduction of the riboflavin and nitric oxide from sodium nitroprusside indicate the possibility of preventing the peroxynitrite formation in the cell.Reducing the nitric oxide Sci Pharm.2009; 77; 111-121.generation in the digestive tract was found to be effective in preventing the reactions of nitrate with amines and amides to form carcinogenic nitrosamines and nitrosamides [27].Hence the NO scavenging activity of G. lucidum extract could play a preventive role against nitrosamine mediated carcinogenesis.
The anti-inflammatory activity of chloroform extract of G. lucidum is dose dependent.Carrageenan induced acute inflammation in animals is one of the most suitable test procedures to screen anti-inflammatory agents.The carrageenan induced edema is mediated by activation of platelet activating factor (PAF), prostaglandins and other inflammatory mediators [28].The first phase is attributed to the release of histamine 5-HT and kinins.The second phase is related to the release of prostaglandins [29][30][31].Carrageenan also induces a protein rich exudate containing large number of neutrophills [32].Formalin induced paw edema is also one of the most suitable test procedure to screen chronic anti-inflammatory agents as it closely resembled human arthritis [33].The nociceptive effect of formalin is also biphasic, an early neurogenic component followed by tissue mediated response [34].
The preliminary phytochemical analysis reveals that the major chemical constituents of the chloroform extract of G. lucidum are terpenoids.The major chemical components of G. lucidum are known to be polysaccharides and triterpenoids [35].Thus triterpenoids of chloroform extract might be responsible for the antioxidant and inflammatory activities [36].Kim et al (1999) [37] identified ganoderic acid A, B, G, and H, the triterpene component of commercial strain of G. lucidum, for its antiinflammatory activity.
In conclusion, the chloroform extract of South Indian G. lucidum exhibited significant antiinflammatory activity in mice.The chloroform extract also possessed significant antioxidative activity.However the findings suggest the therapeutic potentials of the chloroform extract of this mushroom for the prevention and the control of inflammation and diseases mediated through oxidative stress.
Fig. 2.Inhibition of acute and chronic inflammation by chloroform extract of G.lucidum (50 and100 mg/kg) administered orally.