A mutation is a change in genetic information due to DNA sequence changes. Mutations can occur naturally during the DNA repair process in humans and can also be caused by environmental factors, such as chemicals, metals, radiation, stress, diet, and tobacco. Changes in the DNA sequence due to mutations affect the structure or function of the encoded protein, leading to changes in genetic traits. Mutations are one of the leading causes of cancer, and more than 90% of cancers are reported to be caused by mutations due to the environmental factors (Anand et al., 2008). Reactive oxygen species are also generated by the same environmental factors that cause mutations; the generation of excess reactive oxygen species causes oxidative stress. Oxidative stress refers to a state in which the balance of the body’s antioxidant system is disrupted. Reactive oxygen species also react with DNA and proteins in the cell to induce or promote mutations, thereby causing cancer (Pisoschi and Pop, 2015).
Antimutagenic activity that inhibits mutagenesis, includes preventing mutation-causing events, inactivating mutagens through chemical interactions before they affect the genes, and stopping the mutation process of genes damaged by mutagens (Mitscher et al., 1996). Several studies have been actively conducted on natural products to identify antimutagens because they exhibit various physiological activities while being less toxic. Recently, the antimutagenic activity of various natural materials, such as cat's claw and guava, have been reported (Almeida et al., 2017; da Costa et al., 2015; Di Sotto et al., 2015; Gontijo et al., 2018; Nag et al., 2015; Todorova et al., 2015; Zahin et al., 2017).
Ganoderma lucidum, common name Lingzhi, is a medicinal mushroom from the family Ganodermataceae distributed in China, Korea, and Tropics. In traditional Chinese medicine, G. lucidum has been used to improve health and prolong life (Zhang et al., 2003). Ganoderma lucidum has also been reported to have various physiological properties, such as anti-cancer, anti-inflammation, anti-diabetes, antioxidant, antibacterial, anti-aging, and immunity enhancement effects (Bhardwaj et al., 2014; Boh, 2013; Cai et al., 2016; Ferreira et al., 2015; Ma et al., 2015; Socala et al., 2015; Wang et al., 2014). It has been reported that G. lucidum contains more than 400 physiologically active ingredients, including triterpenoids, polysaccharides, nucleotides, sterols, steroids, fatty acids, and proteins. Among them, the major physiologically active compound is known to be a polysaccharide-type β-glucan (Boh et al., 2007; Wong et al., 2014).
Meanwhile, Lakshmi et al. (2003; 2006) reported that when the Ames test was carried out with G. lucidum collected from the Thrissur region in southern India, antimutagenic activity was found against benzo[a]pyrene-induced mutations in the Salmonella typhimurium TA98 and TA100 strains. However, these studies were conducted with G. lucidum collected in the tropics and from in ethanol and methanol extracts; additionally, there have been no other reports on the antimutagenic activity of G. lucidum.
In the present study, G. lucidum native to Korea was used. The polysaccharide-type β-glucan, which is known as the physiologically active component of G. lucidum, was partially purified and used as a sample and not as an extract. The mutagenic, antimutagenic, and antioxidant effects of the β-glucan from G. lucidum were investigated.
MATERIALS AND METHODS
Materials and extraction
In this study, dried fruiting bodies of Ganoderma lucidum were used (Fig. 1). Fruiting bodies of G. lucidum were collected from Mt. Songni, located in Boun-gun, Chungcheongbuk-do, Korea. The Fruiting bodies powder (100 g) was added to distilled water (20 l) and subjected to hot-water extraction at 100°C for 3 hours. This process was carried out three times, individually. Each hot-water extracted mixture was filtered and vacuum-filtered (8 mm Advantec) in a rotary vacuum evaporator, and fully concentrated.
|Figure 1. Dried fruiting body of G. lucidum and its powder.|
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Three concentrates obtained by hot-water extraction of G. lucidum were partially purified as the G. lucidum water extract (GLW), G. lucidum glycoside extract (GLG), and G. lucidum polysaccharide extract (GLP) (Fig. 2).
GLW was prepared by lyophilizing the G. lucidum concentrate and cooling to −70°C.
GLG was prepared by adding 99% ethanol to the G. lucidum concentrate four times and allowing the mixture to stand at 4°C for 24 hours to precipitate the carbohydrates (Zhao et al., 2010). The precipitate was then recovered and lyophilized. The final result was used as GLG.
|Figure 2. Partial purification scheme of polysaccharides from G. lucidum.|
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The purification process of GLP is as follows: the G. lucidum concentrate was adjusted to pH 3 by adding a 20% trichloroacetic acid solution and allowed to stand at 4°C for 12 hours. Next, centrifugation (1,700 × g, 10 minutes) was performed, and only the supernatant (deproteinization solution) was collected. This procedure was repeated twice (Huang et al., 2010). The carbohydrates from the supernatant were precipitated in the same manner as in the GLG purification procedure. The precipitate was collected and dialyzed (to remove low-molecular-weight carbohydrates) and then lyophilized. GLP was obtained as the final product. The yield (%) of each extract was determined as follows: weight (g) of each extract obtained per 100 g of the dry G. lucidum.
Determination of carbohydrate and β-glucan content
The total carbohydrate content was determined by using the phenol-sulfuric acid method (Dubois et al., 1956) with glucose as the standard. To each sample (250 μl), 5% phenol (250 μl), and H2SO4 (750 μl) were added and incubated at 22°C for 30 minutes. Then, the absorbance was measured at 470 nm with a spectrophotometer.
β-glucan content was measured using a Mushroom and Yeast β-glucan Assay kit (Megazyme, Wicklow, Ireland). Glucose was used as the standard, and β-glucan content was expressed as total glucan content minus α-glucan.
The total carbohydrate and β-glucan content were calculated as milligrams (mg) of glucose equivalent (GE) per gram (g) of extract using the calibration curve equation of glucose.
Mutagenicity was assessed in Salmonella typhimurium strains according to the methods described by Maron and Ames (1983) and in the Organization for Economic Cooperation and Development (OECD) (1997) Guideline (Test no. 471). Salmonella typhimurium TA98 (frameshift mutation) and TA100 (base pair mutation) strains, which are used for the Ames test, were purchased from Korea Biotechnology Research Institute. Prior to the mutagenic and antimutagenic tests, the strains were routinely subjected to genetic analysis with histidine and biotin-dependence, biotin-dependence, histidine-dependence, ultraviolet radiation (uvr) B mutation, rfa mutation, R-factor, and spontaneous return tests. 2-aminoanthracene (2-AA) (1.0 μg/plate), an indirect mutagen (in the presence of the liver metabolizing system, S-9), was used as the positive control. DMSO was added to the agar plate as a negative control. The concentration of the G. lucidum extracts was 500–5,000 μg/plate.
Agar (2 ml) was placed in a sterile tube at 45°C. Then, the S-9 mixture (500 μl), sample (100 μl) dissolved in DMSO, and S. typhimurium (100 μl, 1–2 × 109 CFU/ml) were added sequentially. The tube was then vortexed for 2–3 seconds and plated on a glucose agar plate. After incubation for 48 hours in a 37°C incubator, the number of revertant colonies on each plate was counted using an automatic colony counter.
An antimutagenic activity test was performed according to the method described by Maron and Ames (1983). Agar (2 ml) was placed in a sterile tube at 45°C. Then, the S–9 mixture (500 μl), sample (50 μl), S. typhimurium strain (100 μl, 1–2 × 109 CFU/ml), and indirect-mutagen, 2-AA (50 μl), were added sequentially. The tube was then vortexed for 2–3 seconds and plated on a glucose agar plate. After incubation for 48 hours in a 37°C incubator, the number of revertant colonies on each plate was counted using an automatic colony counter. At the same time, positive and negative controls were used for all tests, and all tests were performed in triplicate at each test concentration.
The inhibition activity was calculated as follows:
where M = number of revertant colonies by the mutagen, A = number of spontaneous revertant colonies, and B = number of revertant colonies when the sample and mutagen were both added to the test strains.
Radical scavenging activity
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity assay was performed by modifying the method described by Blois (1958). A sample (200 μl) was added to a DPPH solution (0.4 mM, 800 μl), diluted in methanol, and allowed to react at the room temperature for 30 minutes. The absorbance was measured at 520 nm using a spectrophotometer.
The 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay for total antioxidant capacity was carried out by modifying the method of Re et al. (1999). When potassium persulfate (2.6 mM) was added to the ABTS solution (7.4 mM), the ABTS cation was formed after 12–18 hours. One hundred microliter of the sample was added to the activated ABTS solution (1 ml), and the absorbance was measured at 735 nm after 10 minutes.
The radical scavenging activity was calculated as the rate of decrease in sample absorbance relative to the blank absorbance (100%). The percentage of scavenging activity of radicals was calculated using the following equation:
where A blank and A sample are the absorbances of the control and extract, respectively. All samples were analyzed in triplicate.
For both DPPH and ABTS assays, L-ascorbic acid was used as a standard. The RC50 (concentration required for a 50% reduction of radicals) was calculated using the graph of radical scavenging activity versus extract concentration.
Total polyphenolic content determination
The total polyphenolic content was determined by the method described by Dewanto et al. (2002). The sample (100 μl, 1.0 mg/ml) was added to a 2% Na2CO3 solution (2 ml) and reacted with the 50% Folin–Ciocalteu reagent (100 μl) for 30 minutes at the room temperature.
Gallic acid was used as a standard and the absorbance at 750 nm was measured using a spectrophotometer. The total polyphenolic content was calculated as milligrams (mg) of gallic acid equivalent (GAE) per gram (g) of extract using the calibration curve equation of gallic acid.
|Table 1. Yield, total carbohydrate, and β-glucan content of G. lucidum extracts.|
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|Table 2. Evaluation of mutagenic activity of G. lucidum extracts in S. typhimurium TA 98 and TA 100 with S9 enzyme system.|
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Data obtained from the experiments are presented as means ± standard deviations (SD) of three replicates. IBM Statistical Package for the Social Sciences 22 (SPSS Inc., Chicago, IL) was used as a statistical program. A one-way analysis of variance was conducted to compare the results obtained from testing each type of G. lucidum extracts. Tukey’s multiple comparison test was used to determine the significance of the differences between the groups. Differences at p < 0.05 were considered significant.
RESULTS AND DISCUSSION
Ganoderma lucidum has long been used in traditional Chinese medicine for health promotion and life extension. Ganoderma lucidum has many bioactive ingredients, but polysaccharide-type β-glucan is known to be the main active ingredient (Boh et al., 2007). The purpose of this study was to partially purify polysaccharide, the major physiologically active ingredient of G. lucidum, and to examine its antimutagenic and antioxidant activities. Previous studies have reported various physiological activities for polysaccharides, but the results were diverse due to various factors such as differences in the extraction method of polysaccharides and differences in the content of active ingredients depending on the country of origin. In this study, polysaccharides were purified using hot water extraction and the antimutagenic activity of the polysaccharides was first reported.
The yield, carbohydrate, and β-glucan content
Table 1 shows the yield, total carbohydrate content, and β-glucan content of GLW, and the partially purified GLG and GLP from wild G. lucidum. The yields of GLW, GLG, and GLP were 7.63%, 1.38%, and 1.04%, respectively. The total carbohydrate content of GLW, GLG, and GLP was 32 ± 7, 53 ± 4, and 80 ± 9 g GE/100 g, respectively, and the ratio increased significantly as the partial purification proceeded. Moreover, the percentage of β-glucan in the total carbohydrate content was 15.2% ± 2.2% for GLW, 29.3% ± 2.3% for GLG, and 48.0% ± 0.5% for GLP. The percentage increased greatly as the partial purification progressed. When the yield of β-glucan according to the solvent was compared, the percentage of β-glucan in the water extract obtained by Kozarski et al. (2012), the ethanol extract obtained by VeljoviÄ‡ et al. (2017), and the GLP obtained in this study was 41.4% ± 1.4%, 15.64% ± 0.01%, and 48.0% ± 0.5%, respectively. Among them, the GLP we obtained in this study had the highest amount of β-glucan.
The Ames test was performed to investigate whether the metabolites of the G. lucidum extracts cause mutations. According to the OECD guidelines, mutagenicity is considered to be present if the number of revertant colonies treated with an unknown sample is more than twice the number of spontaneous revertant colonies. Table 2 shows the number of revertant colonies of S. typhimurium TA98 and TA100 mutants when treated with G. lucidum extracts (500–5,000 μg/plate) in the presence of the liver metabolizing enzyme (S-9 fraction), which digests the extract into its metabolites. The number of spontaneous revertant colonies was 25 ± 3 in TA98 and 118 ± 16 in TA100. When treated with 2-AA (1.0 μg/plate), which is a representative mutagenic compound, the number of revertant colonies was significantly higher, 531 ± 31 in TA98 and 657 ± 15 in TA100. On the other hand, when the TA98 culture was treated with G. lucidum extracts, regardless of the increased concentration of the extract, the number of revertant colonies did not deviate from the OECD guideline standard and showed no statistically significant increase. In addition, the TA100 strain culture showed no significant difference in the number of revertant colonies, even when the concentration of the G. lucidum extracts was increased. Thus, we concluded that the metabolites of G. lucidum extracts, produced by the S-9 fraction, did not induce mutagenesis.
|Table 3. Evaluation of antimutagenic activity of G. lucidum extracts against 2-AA induced mutation in S. typhimurium TA 98 and TA 100 with S9 enzyme system.|
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After confirming that G. lucidum extracts did not induce mutations, the Ames test was performed to investigate the antimutagenic activity of GLW against 2-AA-induced mutations in S. typhimurium TA98 and TA100 strains. The antimutagenic effect of G. lucidum extracts is shown in Table 3. In the presence of the S-9 fraction, the number of spontaneous reversion mutations was 25 ± 3 in the TA98 strain and 122 ± 6 in the TA100 strain. On the other hand, the number of revertant mutations after 2-AA treatment was 583 ± 14 in TA98 and 679 ± 19 in TA100, which was significantly higher than that of spontaneous revertant mutations. Ganoderma lucidum extracts (50–500 μg/plate) showed antimutagenic activity in both TA98 and TA100 strains in a dose-dependent manner for mutations induced by 2-AA (p < 0.05). At the highest treatment concentration (500 μg/plate), the mutagenic-inhibition percentage of GLW, GLG, and GLP was 20.6%, 32.8%, and 38.5%, respectively, in the TA98 strain, and 27.6%, 33.0%, and 50.4%, respectively, in the TA100 strain. GLP showed a statistically significant difference from GLW and GLG at the concentration (500 μg/plate) at which the antimutagenic activity was the highest (p < 0.05).
In a study on the antimutagenic effect of G. lucidum, Lakshmi et al. (2003) reported that the G. lucidum ethanol extract (5,000 μg/plate) inhibited mutations induced by benzo[a] pyrene by 65.0% in the TA98 strain and 70.3% in the TA100 strain. Lakshmi et al. (2006) also reported that the methanol extract of G. lucidum (3,000 μg/plate) inhibited mutations induced by benzo[a] pyrene by 60.7% in the TA98 strain and 59.6% in the TA100 strain. 2-AA and benzo[a]pyrene are both carcinogenic precursors, which are metabolized by the cytochrome (CY)P450 enzyme in the liver. Their metabolites (N-hydroxyaryl amine, benzo[a] pyrene diol epoxide) covalently link to DNA to form adducts, leading to mutations (Murata and Kawanishi, 2011). Therefore, it is suggested that G. lucidum inhibited the (CY)P450 enzyme involved in metabolizing 2-AA and benzo[a]pyrene producing the antimutagenic activity. In addition, antimutagenic and anti-cancer effects of certain β-glucans (Mantovani et al., 2008), and antimutagenic effects of specific β-glucan derivatives due to their radical scavenging ability (Krizková et al., 2003) have been reported. Therefore, it is suggested that β-glucan, in the form of the G. lucidum polysaccharide used in this study, can directly or indirectly contribute to the antimutagenic activity. In this study, all three extracts of G. lucidum showed antimutagenic activity in a dose-dependent manner, and GLP, which had the highest β-glucan content, showed the highest antimutagenic activity. Based on the results of this and previous studies, it was concluded that β-glucan of the GLW extract acted as a major contributor to its antimutagenic activity. Therefore, we suggest that studies to identify the β-glucan components of G. lucidum, and studies on the inhibitory effect of β-glucan on the CYP450 enzymatic activity required for bioactivity of 2-AA should be carried out in the future.
|Table 4. DPPH and ABTS radical scavenging ability and total polyphenolic content of G. lucidum extracts.|
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Antioxidant activity of G. lucidum extracts
Reactive oxygen species are important as indirect and direct initiators of mutagenesis. Some studies suggest that antioxidant properties may have an inhibitory effect on DNA mutagenesis induced by oxidative stress (Jena, 2012). Given that 2-AA mediated genetic toxicity contributes to release of reactive oxygen species, the free radical scavenging activity of the G. lucidum extracts may play an important role in antimutagenic activity (Hasnat et al., 2013).
Ganoderma lucidum is reported to have various antioxidative effects. These effects include the inherent antioxidative effect of triterpenoids, antioxidative effect from an increase of specific enzyme activity, antioxidative effect from the formation of a β-glucan-protein complex, and antioxidant effect from an increase in β-glucan activity (Hasnat et al., 2013; Jia et al., 2009; Smina et al., 2011; XiaoPing et al., 2009; Zhu et al., 1999). However, there have been no reports on the ability of β-glucan to have its own radical scavenging activity. Therefore, DPPH and ABTS assay were carried out to investigate the antioxidant effect of β-glucan in G. Lucidum extracts.
The results are shown in Table 4. The results of the radical scavenging ability measurement are shown in Table 4. At the concentration of 1,000 μg/ml, DPPH radical scavenging activity (RC50 = 2,067.47 ± 51.18 μg/ml) and ABTS radical scavenging activity (RC50 = 1,017.98 ± 35.62 μg/ml) were the highest in GLW. Thus, the results in this study suggested that the β-glucan is not directly related to radical scavenging activity.
Total polyphenolic content
Polyphenolic compounds exhibit anti-cancer and antioxidant properties (Balasundram et al., 2006). The evidence accumulated by the in vitro tests showed that the antioxidant capacity of the plant extract was positively correlated with the total polyphenolic content (Canadanovic-Brunet et al., 2005; Oboh et al., 2008). The total polyphenolic content of G. lucidum extracts is shown in Table 4. Total polyphenolic content was highest in GLW (33.1 ± 1.9 mg GAE/g), while GLP (5.8 ± 0.6 mg GAE/g) showed the lowest polyphenolic content. The reason for the total polyphenolic content of the GLP low is thought to be due to loss of valuable compounds, such as polyphenols in the purification process of polysaccharide. As a result of radical scavenging ability and total polyphenolic content of G. lucidum extracts, it was concluded that the antioxidant effect of G. lucidum was due to the polyphenolic compound rather than the polysaccharide-type β-glucan.
Overall, we analyzed the antimutagenic and antioxidant activities of G. lucidum by partially purifying polysaccharide-type β-glucan from G. lucidum and confirming its contributing factors. Ganoderma lucidum contains many polysaccharides with anti-cancer activity. In this study, purified polysaccharide-type β-glucan showed antimutagenic activity. Therefore, it can be suggested that G. lucidum is excellent for not only preventing cancer but also in preventing mutations. Further studies on the toxicity, isolation, and characterization of β-glucan in G. lucidum should be performed. We suggest that purified β-glucan may be used as a raw material for future herbal medicines and health supplements.
FINANCIAL SUPPORT AND SPONSORSHIP
CONFLICT OF INTERESTS
The authors declare that they have no conflicts of interest.
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