Glycosaminoglycan isolated from Ganoderma lucidum exhibits protective effects on experimental ulcers in rats1
Shufeng Zhou2, Yihuai Gao1, Jianmin Hou3, Jin Lan4, Aiguo Ma5, He Gao6
1Division of Pharmacology and Clinical Pharmacology, Faculty of Medical and Health Sciences, Auckland University, Auckland, New Zealand; 2Landcare Research, Private Bag 92170, Auckland, New Zealand; 3Hospital of Qingdao, The Navy of China, Qingdao, China; 4The Institute of Nutriceuticals, Qingdao Ocean University, Qingdao, China; 4Department of Human Nutrition, Qingdao Medical College, Qingdao University, China; 5New Zealand Institute of Natural Medicine, Auckland, New Zealand; 6Division of Pharmacology and Clinical Pharmacology, Faculty of Medicine and Health Science, The University of Auckland, New Zealand.
*Correspondence Author: Dr Shufeng Zhou, MD, PhD.
Division of Medical and Health Sciences, Faculty of Medical and Health Sciences, Auckland University, Auckland, New Zealand.
Running title: Ulcer-healing effects of glycosaminoglycan from Ganoderma
Keywords: Glycosaminoglycan, Ganoderma, Experimental ulcer, Rat
Glycosaminoglycans (GAG), composed of monomers of glycosamine with amino group at C2 position, are present in various natural sources. Many of them have beneficial effects on a variety of diseases including cancer, liver disease and diabetes. The aims of this study were to investigate the protective effects of GAG from Ganoderma lucidum on experimental ulcers in rats using ranitidine as positive control. GAG was isolated from the fruiting body of G. lucidum using a multi-step procedure and the resulting extract identified by chromatographic methods and chemical analyses of hydrolysates. Rat experimental ulcers were induced by stress (swimming in cold water for 30 min), administration of acetic acid or indomethacin, and ligation of gastric pylorus. GAG (0.5 and 1.0 g/kg) was dissolved in sterile water and administered by force-feeding immediately before ulcer-inducer administration or 30 min before the start of swimming in cold water and ligation of gastric pylorus. Control animals received the same amount of vehicle. Ranitidine (100 mg/kg, p.o.) was used a positive control. The results indicate that GAG had a similar effects on the reduction of the ulcer formation and free gastric H+ levels (P < 0.001) compared to ranitidine-treated group. The preventative effects appear to be more marked in the acetic acid- and indomethacin-treated groups. These results indicate that GAG isolated from G. lucidum is an active polysaccharide component with ulcer-healing effects. Further studies are required to explore the mechanism of action of GAG.
Ganoderma (G.) lucidum (Curt.:Fr.) P.Karst. has been used extensively as a tonic and herbal medicine in Asian countries for 2000 years (Shiao et al., 1994; Wasser and Weis, 1999). Many components with biological and medicinal effects have been isolated from G. lucidum. These include polysaccharides, triterpenes, nucleosides, and steroids (Gao, 2000). Recent studies using animal models have indicated that these components extracted from the fruiting body, mycelia and spores of Ganoderma have a wide range of pharmacological actions, including inhibition of tumour growth, stimulation of immune system, scavenging of free radicals, antihypertension, anti-HIV, and wound-healing effects (Shiao et al., 1994; Wasser and Weis, 1999).
Many types of polysaccharides have been isolated from G. lucidum and their structures and activities further investigated (Su, 1991). One of these polysaccharides isolated from G. lucidum, glycosaminoglycan (GAG) has been partially isolated by (Sone et al., 1985; Tomoda et al., 1986). The crude extracts were dialysed and fractionated into ganoderan A, B and C by chromatography, the latter was further fractionated into subfractions, such as glycan V, which was found to be mainly composed of GAG, as identified by infra-red spectroscopy and X-ray diffraction analysis. GAG is composed of monomer of glycosamine with amino group at C2 position. Each glycosamine monomer is linked by ⠨14) linkage into linear polysaccharide unit (Figure 1). GAG is soluble in weak acid solution, but precipitates in alkaline. The biological effects of GAG from other natural sources rather than G. lucidum have been extensively studied (Ref ??). However, the biological activities of GAG from G. lucidum have yet to be investigated.
G. lucidum belongs to basidiomycotina class of fungi. Its cell walls consist of four layers: the outer layer, which contains shapeless glucans; the second layer, which is rich in glycoprotein; the third layer, which is composed of proteins; and the inner layer (up to 18 nm), composed of radial microfibres, which contain GAG and glucans with monosaccharide acetyl-glycosamine (Su et al., 1997). The aim of this study was to isolate GAG from G. lucidum by a simple extraction procedure and to investigate its protective effects on experimental ulcers in rats.
Indomethacin and ranitidine were obtained from Sigma-Aldrich Chemical Co. (Sydney, Australia). All other reagents were of analytical or HPLC grade as appropriate.
Isolation and purification of glycosaminoglycan from G. lucidum
GAGA was isolated as an acidic polysaccharide from the fruiting body of G. lucidum. The fruiting bodies (5 kg) were cut into small pieces, which were then boiled in 100oC hot water fro 3 hr. After cooling at room temperature, the mixtures were filtered using a 100 ?m filter. The resulting residues were hydrolysed by 1M NaOH. The hydrolysates were filtered, and the residues collected, and acidified by addition of 0.1 M HCl. The resulting mixture was filtered, and supernatant collected. The supernatant was treated with 1M NaOH and 0.1 M HCl twice as above described. The final collection of supernatant was precipitated by ethanol, resulting in a powder form of GAG.
Chemical analysis by acidic hydrolysis and chromatography has indicated that the monosaccharide is glycosamine. The infrared spectral data for the monosaccharide glycosamine formed by acid hydrolysis of glycosamine is shown in Figure 3. A spectrometric method has been developed to determine the content of GAG in G. lucidum (Zhang and Zhang, 1997), and the method had acceptable accuracy (80-120% recovery) and precision (<20% coefficient variation). The content of GAG in G. lucidum determined by this method is 0.26 ? 0.03%.
Experiments were performed on male Wistar Kyoto rats (190-220 g) were housed under constant temperature, lighting and humidity according to institutional guidelines. Sterile food and water was available ad libitum. The animals were fasted for 24 h before experiments with free access to tap water until 1 h before testing. All animal procedures were approved by the Institutional Animals Ethics Committee.
Experimental ulcer models
Four types of rat experimental ulcers were induced by stress (30 min swimming in cold water), administration of 0.1% acetic acid by force-feeding, indomethacin administration and ligation of pylorus. Indomethacin (60 mg/kg) was administered by means of Teflon-fitted metal tubing attached to 1 2-ml syringe (Tanaka et al., 1996).
GAG (0.5 and 1.0 g/kg) was dissolved in sterile water and administered by force-feeding immediately before ulcer-inducer administration or 30 min before the start of swimming in cold water and ligation of gastric pylorus. Control animals received the same amount of vehicle. Ranitidine (100 mg/kg, p.o.) was used a positive control.
Gastric lesion assessment
Two hours later the animals were killed by decapitation, the stomach was removed, opened along the greater curvature and examined under 3-fold magnification.
Data are mean ? SD. Test results for treatment group effects were considered statistically significant if P < 0.05. An unpaired Student’s t-test was performed to compared the treatment and control groups.
In four types of rat experimental ulcers induced by stress, acetic acid, indomethacin and ligation of pylorus, GAG (0.5 and 1.0 g/kg) had a similar effects on the reduction of the ulcer formation and free gastric H+ levels (P < 0.001, unpaired t-test), compared to ranitidine. The preventative effects appear to be more remarkable in the acetic acid- and indomethacin-treated groups.
This study has indicated that GAG is present in Chinese strain of G. lucidum. Since GAG is insoluble in water, but soluble in weak acid solution, its extraction was more difficulty and involved multiple steps. The isolated GAG was structurally confirmed by chemical hydrolysis and chromatographic methods, as GAG is composed of monomers of glycosamine with amino group at C2 position.
To the best of our knowledge, the biological activity of purified GAG from G. lucidum has not been reported. In a previous study, Ganoderan A, B, C and E isolated by hot water from G. applautum had an inhibition rate of 64.9, 61.9, 19.3 and 1.5% respectively in mouse sarcoma 180 model (Sasaki et al., 1971). The residues after hot water extraction were hydrolysed by 5% acetic acid, resulted in Ganoderan D and F, which inhibited mouse sarcoma growth by 78.1-95.3%. It is supposed that the major component in Ganoderan D and F should be GAG. Purified GAG from other fungi has been shown to have anti-tumour, immnuo-modualting, anti-radiation, scavenging of free radicals, and would-healing effects, which have been observed with other glucan (such as /font>-D-glucan). It appears that different glucans from Ganoderma may have identical activity.
Like polysaccharides from other sources, GAG from Ganoderma has shown significant ulcer-preventative effects in rat models. It is known that the GAG level reduced in gastric ulcers. Direct administration of GAG into the stomach may protect the gastric tissue from exposure to H+ ions by forming a layer of artificial mucus. As increased tumour necrosis factor (TNF)-a levels have been observed in the formation of experimental ulcers induced by drug (such as indomethacin), acid (such as acetic acid), and stress, administration of drugs inhibiting TNFa secretion can accelerate gastric ulcer healing (Tanaka et al., 1996; Li and Cho, 1999; Shimizu et al., 2000). Therefore, anti-TNF-a could be a mechanism of action of GAG.
In conclusion, this study has indicated that GAG isolated from G. lucidum had preventative effects on experimental ulcers in the rat. The possible synergistic interaction between these different glucans should be investigated. As GAG isolated by weak acid solution appears to be an active polysaccharide component of G. lucidum in animal model, further experimental are required to provide the evidence for the efficacy and safety of GAG for future clinical studies.
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Figure 1. The basic unit of glycosaminoglycan.