Analysis of Antioxidant and Anti-Inflammatory Activities of Solvent Fractions from Rhynchosia nulubilis Cultivated with Ganoderma lucidum Mycelium (2025)

Keywords: Ganoderma lucidum mycelium, Rhynchosia nulubilis, antioxidant activity, anti-inflammatory activity

Fraction yields

The crude ethanol extract of Rhynchosia nulubilis cultivated with Ganoderma lucidum mycelium (RNGM) was prepared as follows: first the plant material was extracted with 10 times volume (v/w) of 80% ethanol, and then the crude extract was dissolved in water, followed by fractionation using a series of organic solvents, which were hexane, chloroform, and ethyl acetate, sequentially applied based on their polarity. Then, the RNGM extract, distilled water and hexane were transferred to a separatory funnel and shaken to ensure the mixture was well mixed. After shaking, the separatory funnel was fixed on a stand without further shaking to allow the partitioning of the organic solvent and water fractions to occur. The hexane solvent fraction was removed, and similar volumes of the other organic solvents were sequentially added to the water fraction to obtain the chloroform and ethyl acetate fractions as well. The fractions were evaporated under reduced pressure, freeze-dried, and stored at −10°C, and the yields of the four extract fractions were expressed as percentages of the dry weight of the crude RNGM extract.

Antioxidant activities of solvent fractions

The antioxidant activities of the solvent fractions were estimated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) (10), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) (11), and ferric reducing ability of plasma (FRAP) assays (12).

Anti-inflammatory activities of solvent fractions

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (10) was used to evaluate the cytotoxicity of the fraction samples against cultured RAW 264.7 cells. The nitrite in the culture medium was measured using the Griess assay and used as an indicator of NO synthesis in fraction-treated RAW 264.7 cells (13).

Determination of total polyphenol and flavonoid contents of solvent fractions

The total phenolic content was determined using the Folin-Denis method (14) with slight modifications. The total flavonoid content of the fractions was determined using the aluminum chloride test according to the method of Saiqa et al. (15).

Determination of β-glucan content

The β-glucan assay was conducted according to the β-D-glucan enzymatic assay kit (Megazyme International Ireland Ltd., Bray, Wicklow, Ireland).

Determination of isoflavones

The extraction of isoflavones from the solvent fractions was performed using the procedure described by Wang et al. (16) with slight modifications, and the stability of target isoflavones and complete conversion after hydrolysis were assessed. The isoflavones were determined acid hydrolysates. In brief, 1 g of the solvent fraction was dissolved in 10 mL 80% methanol, sonicated for 2 h at 25°C, centrifuged at 8,000 rpm for 15 min, diluted to obtain a final concentration of 100 μg/mL, filtered using a 0.45 μm filter, and then analyzed using high-performance liquid chromatography (HPLC). For the hydrolysis procedure, 500 mg of the solvent fraction was added to 5 mL 1 N HCl, and the mixture was heated at 100°C for 1 h. Then, the samples were neutralized with 40% NaOH, diluted to obtain a final concentration of 100 μg/mL, and filtered using 0.45 μm filters. Standard stock solutions at a concentration of 1 mg/mL were prepared by dissolving pure daidzein and genistein in 100% methanol. The concentrations of isoflavones were determined under the HPLC conditions shown in Table 1, and each experiment was repeated at least thrice.

Statistical analysis

Statistical analysis was performed with SPSS package program (version 10, Statistical Package for the Social Science, SPSS Inc., Chicago, IL, USA). The results are expressed as mean±standard deviation (SD), and a 2-tailed value of P<0.05 was considered statistically significant. A difference in the continuous variables according to different concentrations and samples with antioxidant activity were tested using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test.

Yield of solvent fractions

Natural products contain numerous bioactive compounds, which possess different polarities. Therefore, the solvent used for extraction has to complement the polarity of the target components. Additionally, some factors, should be considered when selecting the extraction solvents such as safety, cost effectiveness, and convenient recycling. Therefore, four solvents including n-hexane, chloroform, ethyl acetate, and water were chosen to investigate extraction yields. The yield of the four solvents were quite different, and the polarity-based fractionation revealed the following order: water (10.87%)> ethyl acetate (3.24%)> chloroform (2.61%)> n-hexane (0.86%). The yields of the polar solvents were higher than those of the non-polar solvents, and the lowest yield was from the n-hexane fraction.

Antioxidant activities of solvent fractions

Anti-inflammatory activities of solvent fractions

The RAW 264.7 cells were incubated in the presence of the solvent fractions at wide concentration range (50, 100, 200, 300, and 400 μg/mL), and then the cell viability was evaluated using the MTT assay. We selected the entire concentration range for the subsequent experiment related to the inhibition of NO production because the cell viability was over 90% (Fig. 3A).

Inhibition of NO production by lipopolysaccharide (LPS)-stimulated RAW 264.7 cells

NO is formed as a natural byproduct of the normal metabolism of oxygen. However, stimulation of cells by environmental stress such as ultraviolet radiation or heat exposure can lead to an inordinate increase in the accumulation of nitrite. This may cause the damage to cellular structures and further induce the inflammation by the oxidative stress (18).

In the present study, the cells were simultaneously treated with l g/mL LPS and different concentrations of the fractions. The RAW 264.7 cells were stimulated by LPS, and the nitrite that subsequently accumulated in the culture medium was determined. The results showed that the pre-treated cells induced with LPS released a higher level of NO in the medium, compared to the control. The results indicate that the inhibition of LPS-induced NO was concentration-dependent within most of the assayed fractions. The LPS-treated cells with ethyl acetate and water fractions generally decreased NO production as the each fraction concentration increased (Fig. 3B). This study confirmed that the level of concentration-dependent NO secretion was significantly different among the fractions. The ethyl acetate extract showed higher inhibition of NO production than the other fractions including the chloroform and hexane fractions. These results are consistent with the reports by Moro et al. (2), which described the anti-inflammatory activity of the ethanolic and methanolic extracts of Agaricus bisporus, Cantherellus cibarius, Lactarius deliciosus, and Pleurotus ostreatus in RAW 264.7 cells. In addition, Moro et al. (2) reported that the methanolic extracts of Boletus edulis inhibited NO production at 500 μg/mL by 10%. Previous studies have reported positive correlations between phenolic compounds and anti-inflammatory effects (3). In addition, Kim et al. (4) reported that cancer is related to inflammatory responses. In this study, the solvent fractions of RNGM were considered to possess excellent potential as materials for preventing related diseases based on their anti-inflammatory effects.

Total polyphenol and flavonoid contents

The total polyphenols in the solvent fractions were determined using the Folin-Ciocalteu’s reagent. The total polyphenol content was determined using a linear gallic acid standard curve, and the results are presented in Table 3. The ethyl acetate fraction possessed the highest amount of total polyphenols, followed by the chloroform, water, and n-hexane fractions, which had the lowest [65.33, 41.08, 38.74, and 25.92 mg gallic acid equivalent (GAE)/g, respectively]. Furthermore, the ethyl acetate fraction, which showed the highest yield from RNGM, is considered to have a variety of biological activities. The major factors responsible for the antioxidant activity of phenolic compounds are their chemical structure and redox properties, which allow them to scavenge free radical, chelate transitional metals, and inhibit lipoxygenase (4, 20).

In previous studies, some widely consumed common edible mushrooms in Asia have been found to contain strong antioxidant effects due to their abundant phenolic compound content (18). It is well known that the antioxidant potential of mushroom extracts usually correlates with their phenolic content (3,4). Cheung et al. (3) reported that the antioxidant activities of mushroom extracts were related to their total polyphenol content. In addition, Xiao et al. (21) demonstrated that DPPH and ABTS radical scavenging activities are strongly correlated with antioxidant compounds. The observed high polyphenol content of the fractions could be responsible for the increased antioxidant activities. Based on a previous study (18), the antioxidant capacity of mushrooms is normally associated with phenolic compounds. Therefore, the antioxidant activity of Ganoderma lucidum mycelium is also recognized as the effect of contained phenolic compound. These observations led us to infer that the radical scavenging and reducing property of the ethyl acetate fraction might be partially attributed to its phenolic compounds.

Moreover, the results of the total flavonoid content analysis of each RNGM fraction showed that the ethyl acetate fraction of RNGM had the highest content [18.50 mg quercetin equivalent (QE)/g], followed by the chloroform (10.32 mg QE/g), n-hexane (3.76 mg QE/g), and water (2.12 mg QE/g), fractions. Therefore, the total flavonoid content appeared to be related to the antioxidant activities measured for all the fractions because the solvent fraction which has highest flavonoid content showed highest DPPH radical scavenging activity. In addition, the fractions with a neutral polarity contained more total flavonoid content than the other fractions. The result of the phytochemical analysis indicated that considerable quantities of common bioactive components such as terpenoids, alkaloids, phenolics, flavonoids, and saponins were concentrated in the medium polar and polar fractions such as the chloroform, ethyl acetate, and butanol fractions (15). Other study showed that the total polyphenol content was higher in the ethyl acetate fraction than that in the water fractions of Flammulina velutipes (3.17~3.50 mg GAE/g) and Lyophyllum decastes (1.52~2.92 mg GAE/g) (22). Based on these results, ethyl acetate fractions of RNGM can be considered as potential candidates for the development as functional materials with antioxidant effects.

β-Glucan content

Fibrous structural extracellular matrix within the cell walls of yeast plants are formed by a group of glucose polymers, the β-glucans (8,9). Previous studies have demonstrated that various β-glucans act as immunostimulants that nonspecifically activate cellular and humoral components of the host immune system, which increase the functional activity of macrophages, mononuclear cells, and neutrophils, leading to protection against infectious diseases and cancer (23,24). In this study, the ethyl acetate fraction (13.02%) possessed the highest amount of total β-glucan, followed by the water (6.32%), chroloform (1.43%), and n-hexane fractions (0.85%) (Table 3). Previous studies reported that high levels of β-glucans also occur naturally in edible mushrooms such as Lentinus edodes, Grifola frondosa, Sparassis crispa Fr., and Tremella fuciformis (24,25). Jung et al. (26) reported that the β-glucan content of the Hypsizigus marmoreus water extract was 9.32%, and Lee and Lee (27) reported the β-glucan content of brown rice cultured with mushroom mycelia extract was 18.4%. In addition, San-Blas and San-Blas (28) demonstrated the generation of high β-glucan in the membrane fraction of the mycelium phases. Beneficial health effects such as antioxidant (29), immunomodulatory, antitumor, antiviral, and anti-inflammatory (24) of the mushroom β-glucan have been documented in previous reviews. Therefore, based on these previous studies, the high antioxidant and anti-inflammatory activity of the ethyl acetate fraction of RNGM might be related to β-glucans.

Isoflavone contents

Soy isoflavones are beneficial in preventing both prostate and breast cancers, reducing the risk of cardiovascular diseases, and alleviating menopausal symptoms (16). Soybeans contain the three isoflavone glucosides: daidzin, genistin, and glycitin as well as their aglycone forms daidzein and genistein, which are biologically active compounds. To measure the isoflavones, each fraction was dissolved in 80% methanol and hydrolyzed with HCl. The isoflavone content of the solvent fractions of RNGM was measured and is shown in Table 4. The results showed that the amount of daidzein and genestin of the methanol extract was found in the ethyl acetate fraction at 19.36 and 11.51 mg/g, respectively. After hydrolysis, the amount of daidzein and genestin was 27.60 and 39.38 mg/g, respectively, in the ethyl acetate fraction. Wang et al. (16) reported that the aglycone content in soybean was less than 2%. Since soybean isoflavones typically contains 80.00~98.37% of biologically inactive glucosides, numerous studies have been conducted to convert glucosides to aglycones by removing the glucose combined with the glucosides. Previous studies reported that the aglycones showed a higher biological activity than the glucosides in the ethyl acetate fraction of soy after digestion (8,9). Jung et al. (30) reported the genistein has an antioxidant effect by scavenging reactive oxygen species. Therefore, the antioxidant effects of the solvent fractions of RNGM might be related to isoflavones, which is consistent with the findings of other studies (31).

Based on these results, it is suggested that the ethyl acetate fraction of Rhynchosia nulubilis cultivated with Ganoderma lucidum mycelium may be a potential natural sources of antioxidant and immunomodulatory and other useful products for nutritional and pharmaceutical applications. Therefore, further studies regarding the components and structure of ethyl acetate fraction are needed.

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