α-Glucosidase inhibitor produced by an endophytic fungus, Xylariaceae sp. QGS 01 from Quercus gilva Blume

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Original Research Paper

␣-Glucosidase inhibitor produced by an endophytic fungus, Xylariaceae sp. QGS 01 from Quercus gilva Blume Anastasia Wheni Indrianingsih a,∗ , Sanro Tachibana b a

Research Unit for Natural Products Technology, Indonesian Institute of Sciences, Gading, Playen, Gunungkidul, Yogyakarta, 55581, Indonesia b Department of Applied Biosciences, Faculty of Agriculture, Ehime University, 3-5-7 Tarumi Matsuyama, Ehime 790-8566, Japan Received 6 January 2017; received in revised form 18 April 2017; accepted 8 May 2017

Abstract Xylariaceae sp. QGS 01, an endophytic fungus isolated from the stem of Quercus gilva Blume showed high ␣-glucosidase inhibitory activity. ␣-Glucosidase inhibitor have the role as one of carbohydrate-hydrolyzing enzymes to postpone absorption of glucose in the digestive organs. The ␣-glucosidase inhibitor constituents were isolated from the ethyl acetate extract of the mycellium of endophytic fungi Xylariaceae sp. QGS 01 using a bioassay-guided fractionation technique. Further separation and purification of the active fraction led to the isolation of constituents with strong inhibitory activities against ␣-glucosidase: 8-hydroxy-6,7-dimethoxy-3-methylisocoumarine (1) with inhibitory concentration (IC50 ) values against ␣-glucosidase from Saccharomyces cerevisiae of 41.75 ␮g/mL, while quercetin as the standard had an IC50 value of 4.80 ␮g/mL. The results of the present study showed that the endophytic fungus Xylariaceae sp. QGS 01 is potentially a rich source of antidiabetic medicine. © 2017 Beijing Academy of Food Sciences. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Keywords: Endophytic fungi; Quercus qilva Blume; ␣-Glucosidase inhibitory activity; Xylariaceae sp.; Isocoumarine derivative

1. Introduction Diabetes mellitus (DM) is a serious global health program which is characterized by high blood glucose levels, which lead to complications such as retinopathy, hypertension, neuropathy and diabetic foot ulcers [1]. Type 2 DM caused by defects in insulin secretion or insulin resistance [2] is the most frequently encountered form of DM, accounting for more than 80% of all cases [3]. The ␣-glucosidase enzyme in the intestine is essential for carbohydrate degradation so that the resulted monosaccharides can be absorbed. The inhibition of ␣-glucosidase enzyme leads to a delay in the digestion of ingested carbohydrates [4]. Thus ␣-glucosidase inhibitors exhibit high promise as therapeutic agents for the treatment of type 2 of DM [5]. Most of DM treatments are based on the use of synthetic drugs, which are associated with several side effects [6]. Therefore, the development of natural compounds as alternatives pharmaceuticals

∗

Corresponding author. E-mail addresses: anastasia wheni [email protected], [email protected] (A.W. Indrianingsih).

for the treatment of DM without any side effects is urgently needed. Another advantage is that natural compounds may be safely consumed in the daily diet, thereby reducing the risk of DM [7]. Endophytic fungi are the microorganisms that spend all or part of their life cycles within plant tissue without causing harmful effects on the plant [8]. Endophytic fungi usually get nutrition and protection from their host plant and promote the growth of the plant by producing certain bioactive substances [9]. Endophytic fungi in plants are promising sources of bioactive metabolites. Recent studies show that endophytic fungi have an ability to produce many novel chemicals that could be directly use as drugs or source of bioactive natural products [10]. Several research indicate endophytic fungi have bioactive compounds that could potentially be applied in various applications such as antioxidant [11], antifungal [12], antiviral [13], antibacterial [14] and cyotoxic [15]. In our previous study, we found that several bioactive compounds such as catechin, epicatechin, tiliroside, ␤-sitosterol glucoside and condensed tannins were isolated from Quercus gilva Blume and Quercus phillyraeoides A. Gray [16,17]. Catechin and epicatechin

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isolated from Q. gilva Blume were having good antioxidant properties while tiliroside had strong ability as ␣-glucosidase inhibitor activity; therefore, in this study we conducted isolation of endophytic fungi from Quercus gilva Blume (Q. gilva). Endophytic fungus QGS 01 from Q. gilva was found to have a strong ␣-glucosidase inhibitory activity. This QGS 01 fungus was identified as a Xylariaceae sp. This present work highlighted the ␣-glucosidase inhibitory activity of constituents isolated from the mycelium extract of Xylariaceae sp. QGS 01. Extract of Xylariaceae sp. QGS 01 mycelium were obtained using ethyl acetate and the isolation of active constituent was conducted using bioassay-guided fractionation technique. An in vitro assay of ␣-glucosidase inhibitory activity was conducted using ␣glucosidase enzyme obtained from Saccharomyces cerevisiae (S. cerevisiae) yeast. This assay may be used for preliminary observations in the evaluation of pharmalogical activities and also to verify the medicinal effects of these active constituents isolated from endophytic fungus. 2. Materials and methods 2.1. General instrumentation and chemicals An analysis using gas chromatography (GC) was conducted on a GC-FID 2014 model (Shimadzu, Japan). The electron ionization mass spectra (EI-MS) of isolated constituents were recorded on a GC Mass Spectrometer (GC–MS QP 2010 Plus, Shimadzu, Japan). Single crystal analysis of isolated constituent was recorded on Rigaku Saturn 724 X-Ray Diffractometer. TLC was run on silica gel 60 F254 pre-coated plates (Merck 5554) and spots were detected using UV light. ␣-Glucosidase [(EC 3.2.1.20)] type I from S. cerevisiae, p-nitrophenyl ␣-d-glucopyranoside (p-NPG) and bis(trimethylsilyl) acetamide (BSA) were purchased from Wako Pure Chemicals, Ltd. (Osaka, Japan). Quercetin, palmitic acid, stearic acid, oleic acid, linoleic acid, and linolenic acid were purchased from Sigma-Aldrich Co., Ltd. (Tokyo, Japan). All solvents used in this study (methanol, ethanol, toluene, pyridine, ethyl acetate, chloroform, hexane, and acetone) were purchased from Wako Pure Chemicals, Ltd. 2.2. Isolation and culture of the endophytic fungus The stem of Quercus gilva Blume was collected from Ehime University Garden, Ehime Prefecture, Japan, in October 2014. Samples were cleaned in tap water and sterilized by consecutive washes in 75% EtOH (1 min), 1% NaOCl (2 min), 75% EtOH (30 s) and rinsed with sterile distilled H2 O three times. The surface sterilized material was cut into 0.4 × 0.4 cm pieces and the tissues were deposited on a Petri dish containing potato dextrose agar (PDA) medium and incubated at 25 ◦ C. The hyphal of was transferred to fresh PDA medium and purified for three times. 2.3. Molecular identification of fungus Fungal strains were maintained on PDA and incubated for 7 days. DNA was extracted from these fungi based on the Doyle

Table 1 ␣-Glucosidase inhibitory activity of Fraction 1 to Fraction 6 of mycelium extract from Xylariaceae sp. QGS 01. Fraction F1 F2 F3 F4 F5 F6

␣-Glucosidase inhibitory activity (IC50 ), ␮g/mL

Yield (g)

9.33a 24.97c 11.35b 9.56a 22.38c 9.67a

1.86 0.10 0.08 0.14 1.02 0.86

Different letters in the same column indicate significant differences (P < 0.05).

and Doyle [18] method with a slight modification. The extracted DNA was used as a template for PCR to amplify the ITS1-F and ITS4-B regions. Products were then sequenced using two PCR primers and an automated ABI Prism DNA sequence. The result of sequencing was compared with the National Centre for Biotechnology Information (NCBI) GenBank database. A phylogenetic tree was constructed using MEGA software (version 5.2.2). The endophytic fungus was identified as Xylariaceae sp. QGS 01 (GenBank accession number: KU764517). 2.4. Fermentation and extraction The endophytic fungus Xylariaceae sp. QGS 01 was inoculated into the Erlenmeyer flasks (500 ml) at 25 ◦ C for 3 weeks, each flask containing 200 ml potato dextrose broth (PDB). Mycelium and culture broth were separated and extracted with equal volume of ethyl acetate at room temperature. The extract of endophytic fungus was condensed in a rotating evaporator under reduced pressure. The obtained crude extracts of the endophytic fungus (mycelium and culture broth extracts) were screened for the ␣-glucosidase activity and had the inhibitory concentration (IC50 ) of 12.50 and 21.70 ␮g/mL, respectively. The results indicated that the mycelium extract of Xylariaceae sp. QGS 01 had higher activity; therefore further research was carried out on the fungal strain Xylariaceae sp. QGS 01. Large scale fermentation of Xylariaceae sp. QGS 01 was conducted with total volume of 30 l which were resulting of 4.12 g of mycelium extract and 1.92 g of culture broth extract. 2.5. Isolation procedures of active constituents The ethyl acetate extract (4.12 g) was separated using silica column chromatography using solvents with increasing polarity from n-hexane, chloroform, ethyl acetate (EtOAc), and methanol (MeOH) to obtain 6 fractions (F1-F6). All fractions (F1-F6) were screened for ␣-glucosidase inhibitory activity (Table 1), with the F1 fraction exhibiting the highest activity. The active F1 fraction was separated again by silica column chromatography to get 4 fractions (F1.1-F1.4). Compound 1 (5.1 mg) was isolated from F1.1 as a colorless needle crystal by using eluent hexaneethyl acetate (4:1) and further purified by recrystallization using chloroform.

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2.6. Single crystal analysis of compound 1 A block of crystal was mounted on a glass fiber. All measurements were made on a Rigaku Saturn 724 X-Ray diffractometer using multi-layer mirror monochromated Mo-K␣ radiation. The crystal to detector distance was 44.96 mm. The data were collected at a temperature of −172 ± 1oC to a maximum 2␪ value of 63.0o. A total of 1440 oscillation images were collected. Cell constants and an orientation matrix for data collection corresponded to a primitive orthorhombic cell with dimensions: a = 7.086(3) Å, b = 13.885(5) Å, c = 21.390(8) Å and V = 2104.5(13) Å3. The data were corrected for Lorentz and polarization effects. The structure was solved by direct methods [19] and expanded using Fourier techniques. The values used for the mass attenuation coefficients were those of Creagh and Hubbell, [20]. All calculations were performed using the Crystal Structure crystallographic software package except for refinement, which was performed using SHELXL Version 2014. 2.7. GC/GC–MS analysis of fatty acid mixture Fatty acids mixture was isolated from the n-hexane fraction using alkali solution. The methylation of fatty acids was conducted using methanol acidified with sulphuric acid at 64 ◦ C for 30 min to form Fatty Acid Methyl Ester (FAME) for determination of fatty acid content. Chemical analysis was conducted using gas chromatography coupled with mass spectrometry (GC–MS QP-2010) equipped with a SPB-50 column (30 m × 0.25 mm ID, 0.25 ␮m film thickness). The analysis was performed according to Yamamoto et al., 2008 [21]; column temperature, 235 ◦ C, carrier gas helium linear gas velocity, 30 cm/sec; split ratio, 1/30, ion source temperature, 200 ◦ C and interface temperature, 280 ◦ C. The identification of chemicals was performed in comparison with database (NIST08 library) and confirmed using authentic standard samples.

Fig. 1. Xylariaceae sp. QGS 01 grown on PDA medium.

experiments were carried out in triplicate and the results are expressed as the mean ± SD of three determinations. 2.9. Statistical analysis All assays were conducted in triplicate. Statistical analyses were performed with SPSS 16.0 for an analysis of variance (ANOVA) followed by Duncan’s test. Differences at p < 0.05 were considered significant. 3. Results and discussion 3.1. Identification of endophytic fungi

2.8. Alpha-glucosidase inhibitory assay The inhibitory activity of ␣-glucosidase was evaluated as reported by Kim et al., 2005 [22]. Samples were dissolved in dimethyl sulfoxide at various concentrations (10 ␮l) and then treated with p-NPG (250 ␮l, 3 mM) in phosphate buffer solution (490 ␮l, 100 mM, pH 7). The solution was pre-incubated at 37 ◦ C for 5 min. Two hundred and fifty microliters of ␣-glucosidase (0.065 U/ml) was then added and the reaction continued for 15 min. The reaction was stopped by the addition of 1 ml of 0.2 M Na2 CO3 . The mixtures were measured at 400 nm using a UV–vis spectrophotometer. The percentage inhibition of ␣-glucosidase inhibitory activity was calculated using Eq. (1). ␣-Glucosidase inhibitory activity (%) = (

A0 − A 1 x 100) A0 (1)

where A0 is the absorbance of the control and A1 is absorbance in the presence of the sample. The inhibitory concentration (IC50 ) of the samples was calculated using a regression analysis from the graph plotting scavenging activity against concentration. All

Fig. 1. shows the endophytic fungus of QGS 01 isolated from the stem of Q. gilva. According to a molecular gene analysis strain QGS 01 was similar (99–100%) to Xylariaceae sp. (GenBank accession number: KU764517). A phylogenetic tree of Xylariaceae sp. QGS 01 is shown in Fig. 2. The Xylariaceae is well known ascomycete family that has wide biological diversity and has been found to be the source of many metabolites with novel structures. Several alkaloids and a new pyridine derivative had been isolated from the culture broth of a marine-derived fungus Xylariaceae sp. SCSGAF0086 [23]. This is the first report of a Xylariaceae sp. isolated from Q. gilva. 3.2. Isolation of α-glucosidase inhibitory active compounds Between the mycelium and the culture broth extracts, the higher ␣-glucosidase inhibitory activity was the mycelium of QGS 01 fungus (IC50 = 12.50 ␮g/mL), therefore, the mycelium of QGS 01 fungus was further separated to obtain the most active constituents on ␣-glucosidase inhibitory activity. The

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Colletotrichum gloeosporioides strain CMUBE1852 |31789255| Colletotrichum gloeosporioides strain CMUBE1851 |31789254| Colletotrichum gloeosporioides strain CMUBE1814 |31789241| Colletotrichum gloeosporioides strain PDC113 |AY266394.1| Nigrospora sphaerica isolate CY202 |312434508| Nigrospora sp. LH4 |325563908| Nigrospora sphaerica strain xsd08093 |218454110| Xylariaceae sp. LCC28 |591389397| Xylariaceae sp. 4Y-Rc6-1 |401782386| QGS01 Xylariaceae sp. TA1-4-1 |432140632| 15

10

5

0

Fig. 2. Phylogenetic tree for endophytic fungus QGS 01 based on ITS sequences.

ethyl acetate extract of QGS 01 fungus mycelium was fractionated using silica gel chromatography to obtain the F1 fraction as the most active fraction (Table 1). The active F1 fraction was separated again by silica column chromatography to get 4 fractions (F1.1-F1.4). Compound 1 was isolated from F1.1 (IC50 = 8.25 ␮g/mL) as the most active constituents. Its spectral data were compared with reported data and the structure was identified as isocoumarin derivative (1). Compound 1: EI-MS, m/z (I, %): 236 [M]+ (100), 221 [M-CH3]+ (96), 193 [M-CH3-CO]+ (70); m.p: 196–198 ◦ C. Crystal data: C12 H12 O5 , Mr = 236.19, a colorless needle crystal, a primitive orthorhombic, approximate crystal dimensions 0.100 × 0.020 × 0.010 mm3 . Space group, a = 7.086(3) Å, b = 13.885(5) Å, c = 21.390(8) Å and V = 2104.5(13) Å. Based on this information and search of the literature [24], compound 1 was identified as 8-hydroxy-6,7-dimethoxy-3methylisocoumarine. The X-ray diffractogram of 1 is shown in Fig. 3. 8-Hydroxy-6,7-dimethoxy-3-methylisocoumarine (6-Omethylreticulol) had been reported as a metabolite of fungus Streptomyces mobaraensis [25] and liverwort Wettsteinia schusterana [26]. Reticulol and its derivatives had been proved to be a potential antitumor agent. Reticulol showed antitumor activity against melanoma B16F10, it had the ability to inhibit topoisomerase I (Topo I), an enzyme involved in the melanoma metastatis mechanism [27]. Dihydroisocoumarins, secondary metabolites produced by Xylariaceae family have also been isolated and identified [28]. However, this is the first report about compound 1 isolated from Xylariaceae sp. QGS 01 having antidiabetic activity on inhibiting ␣-glucosidase enzyme. There has been no report yet about isocoumarin derivative isolated from Q. gilva Blume as a host plant of Xylariaceae sp QGS01. Fatty acid mixture was major constituents of Xylariaceae sp. QGS 01. Analysis using GC–MS and comparison with authentic standards showed that fatty acid mixture had two saturated fatty acids, palmitic acid (C16:0) and stearic acid (C18:0) and three unsaturated fatty acids, namely oleic acid (C18:1), linoleic acid (C18:2) and linolenic acid (C18:3) (Fig. 4). The major

Table 2 ␣-Glucosidase inhibitory activity of constituents from Xylariaceae sp. QGS 01. Constituents

␣-Glucosidase inhibitory activity (IC50 ), ␮g/mL

1 (isocoumarin) Fatty acids mixture Fraction 1.1 Palmitic acid (std) Stearic acid (std) Oleic acid (std) Linoleic acid (std) Linolenic acid (std) Quercetin (std)

41.75e 5.95c 8.25d 95.27g 88.22f 2.15a 2.79a 4.31b 4.81b

Different letters in the same column indicate significant differences (P < 0.05).

constituent in fatty acids mixture were linoleic acid (33.7%) followed by oleic acid (29.8%), palmitic acid (24.1%), stearic acid (7.5%) and linolenic acid (3.6%). The mass spectra of methylated fatty acids are shown in Fig. 5. The ␣-glucosidase activity of compound 1, fatty acid mixture, and standard are shown in Table 2. This resulted suggested that compound 1 was confirmed as the most active constituent as ␣-glucosidase inhibitor with IC50 of 41.75 ␮g/mL, meanwhile the fatty acid mixture from F1.1 also exhibit strong activity with IC50 of 5.95 ␮g/mL. These results indicated that fatty acids with a double bond showed stronger inhibition than fatty acids without double bond. The chemical structure of the isolated fatty acids fractions are shown in Fig. 6. It is suggested that the existence of a double bond was important factor of inhibition ␣-glucosidase acitivity [29]. Other fatty acids were also reported to have ␣-glucosidase activity, such as fatty acids from endophytic fungus Colletotrichum sp. [30], fatty acids from sea cucumber [31], and fatty acids from wheat germ [32]. ␣-Glucosidase inhibitor assay were performed as antidiabetic properties capability in the terms of the ability of the constituents to inhibit an intestinal carbohydrate-digesting enzyme, namely ␣-glucosidase. The ␣-glucosidase inhibitors delay intestinal carbohydrate absorption by blocking the activity of glucosidase

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Fig. 3. X-ray structure of compound 1.

Fig. 4. Gas chromatogram profile of the standard (a) and isolated fatty acids (b): (1) palmitic acid methyl ester, (2) stearic acid methyl ester, (3) oleic acid methyl ester, (4) linoleic acid methyl ester, (5) linolenic acid methyl ester.

enzyme, and consequently reduce the concentration of postprandial blood glucose and slow down the rise in blood sugar level; therefore, they play a significant role as chemotherapeutic agents for non-insulin-dependent DM. Acarbose, miglitol, and voglibose are some examples of ␣-glucosidase inhibitors presently being used in the primary treatment of type 2 DM [33]. However, since the currently available ␣-glucosidase inhibitors for clinical use are causing several side effects, safer antidiabetic agent are needed. Effective and safe ␣-glucosidase inhibitors from nature

have been sought in the development of physiological functional food or constituents for antidiabetic therapy [34]. Our study showed that constituents from ethyl acetate extract of Xylariaceae sp. QGS 01 are potent inhibitor of ␣-glucosidase activity, and therefore suggests that endophytic fungus Xylariaceae sp. QGS 01 as a rich source of natural antidiabetic medicine. This report is the first report about secondary metabolites (isocoumarine derivative) produced by Xylariaceae sp. having antidiabetic activity on inhibiting ␣-glucosidase enzyme.

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Fig. 5. MS spectra of fatty acid derivatives (a) Palmitic acid methyl ester (m/z 270), (b) Stearic acid methyl ester (m/z 298), (c) Oleic acid methyl ester (m/z 296), (d) Linoleic acid methyl ester (m/z 294), and (e) Linolenic acid methyl ester (m/z 292).

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Fig. 6. The chemical structure of the isolated fatty acids fractions; saturated fatty acids: (a) palmitic acid (C16:0), (b) stearic acid (C18:0) and unsaturated fatty acids, (c) oleic acid (C18:1), (d) linoleic acid (C18:2), (e) linolenic acid (C18:3).

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4. Conclusion Endophytic fungi Xylariaceae sp. QGS 01 was isolated from the stem of Quercus gilva Blume and showed high activity of ␣-glucosidase inhibitory activity. One active constituent: 8hydroxy-6,7-dimethoxy-3-methylisocoumarine (1) was isolated from endophytic fungi Xylariaceae sp. QGS 01. Compound 1 had inhibitory concentration (IC50 ) values against ␣-glucosidase from Saccharomyces cerevisiae of 41.75 ␮g/mL. Acknowledgement The authors are deeply thankful to Dr. Shigeki Mori of the Integrated Center for Sciences, Ehime University for obtaining the data of X-ray crystallography. References [1] S.O. Oyedemi, G. Bradley, A.J. Afolayan, Ethnobotanical survey of medicinal plants used for the management of diabetes mellitus in the Nkonkobe municipality of South Africa, J. Med. Plant Res. 3 (2009) 1040–1044. [2] R.P. Lanza, D.M. Ecker, W.M. Kuhtreiber, J.P. Marsh, J. Ringeling, W.L. Chick, Transplantation of islets using microencapsulation: studies in diabetic rodents and dogs, J. Mol. Med. 77 (1999) 206–210. [3] B. Mlinar, J. Marc, A. Janez, M. Pfeifer, Molecular mechanisms of insulin resistance and associated diseases, Clin. Chim. Acta 375 (2007) 20–35. [4] P.M. Heacock, S.R. Hertzler, J.A. Williams, B.W. Wolf, Effects of a medical food containing an herbal ␣-glucosidase inhibitor on postprandial glycemia and insulinemia in healthy adults, J. Am. Diet Assoc. 105 (2005) 65–71. [5] A.D. Baron, Postprandial hyperglycaemia and alpha-glucosidase inhibitors, Diabetes Res. Clin. Pract. 40 (1998) S51–S55. [6] B.B. Zhang, D.E. Moller, New approaches in the treatment of type 2 diabetes, Curr. Opin. Chem. Biol. 4 (2000) 461–467. [7] C. Coman, O.D. Rugina, C. Socaciu, Plants and natural compounds with antidiabetic action, Not. Bot. Horti. Agrobot. 40 (2012) 314–325. [8] D. Dutta, K.C. Puzari, R. Gogoi, P. Dutta, Endophytes: exploitation as a tool in plant protection, Braz. Arch. Biol. Technol. 57 (2014) 621–629. [9] R. Tan, W. Zou, Endophytes: a rich source of functional metabolites, Nat. Prod. Rep. 18 (2001) 448–459. [10] A.A.I. Gunailaka, Natural products from plant-associated microorganism: distribution, structural diversity, bioactivity, and implications of their occurrence, J. Nat. Prod. 69 (2006) 509–526. [11] J.T. Zhao, Y.J. Fu, M. Luo, Y.G. Zu, W. Wang, C.J. Zhao, C.B. Gu, Endophytic fungi from pigeon pea [Cajanus cajan (L.) millsp.] produce antioxidant cajaninstilbene acid, J. Agric. Food Chem. 60 (2012) 4314–4319. [12] S.S. Gao, X.M. Li, C.S. Li, P. Proksch, B.G. Wang, Penicisteroids A and B, antifungal and cytotoxic polyoxygenated steroids from the marine algaderived endophytic fungus Penicillium chrysogenum QEN-24S, Bioorg. Med. Chem. Lett. 21 (2011) 2894–2897. [13] G. Zhang, S. Sun, T. Zhu, Z. Lin, J. Gu, D. Li, Q. Gu, Antiviral isoindolone derivatives from an endophytic fungus Emericella sp. associated with Aegiceras corniculatum, Phytochemistry 72 (2011) 1436–1442. [14] Y. Igarashi, H. Ogura, K. Furihata, N. Oku, C. Indananda, Thamchaipenet A Maklamicin, an antibacterial polyketide from an endophytic Micromonospora sp, J. Nat. Prod. 74 (2011) 670–673.

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Please cite this article in press as: A.W. Indrianingsih, S. Tachibana, ␣-Glucosidase inhibitor produced by an endophytic fungus, Xylariaceae sp. QGS 01 from Quercus gilva Blume, Food Sci. Hum. Wellness (2017), http://dx.doi.org/10.1016/j.fshw.2017.05.001