Antioxidant Activity and Cytotoxicity against Cancer Cell Lines of the Extracts from Novel Xylaria Species Associated with Termite Nests and LC-MS Analysis

Xylaria species associated with termite nests or soil have been considered rare species in nature and the few which have been reported upon have been found to act as a rich source of bioactive metabolites. This study evaluated 10 ethyl acetate extracts of five new Xylaria species associated with termite nests or soil for their antioxidant activity, and cytotoxicity against different cancer and normal cell lines. DPPH and ABTS radical scavenging activities of the extracts demonstrated strong capacity with low IC50 values. The highest observed activities belonged to X. vinacea SWUF18-2.3 having IC50 values of 0.194 ± 0.031 mg/mL for DPPH assay and 0.020 ± 0.004 mg/mL for ABTS assay. Total phenolic content ranged from 0.826 ± 0.123 to 3.629 ± 0.381 g GAE/g crude extract which correlated with antioxidant activities. The high total phenolic content could contribute to the high antioxidant activities. Cytotoxicity was recorded against A549, HepG2, HeLa and PNT2 and resulted in broad spectrum to specific activity depending on the cell lines. The highest activities were observed with X. subintraflava SWUF16-11.1 which resulted in 11.15 ± 0.32 to 13.17 ± 2.37% cell viability at a concentration of 100 µg/mL. Moreover, LC-MS fingerprints indicated over 61 peaks from all isolates. There were 18 identified and 43 unidentified compounds compared to mass databases. The identified compounds were from various groups of diterpenoids, diterpenes, cytochalasin, flavones, flavonoids, polyphenols, steroids and derivatives, triterpenoids and tropones. These results indicate that Xylaria spp. has abundant secondary metabolites that could be further explored for their therapeutic properties.


Introduction
Reactive oxygen species (ROS) is the most important class of free radicals normally generated in living systems and it plays essential roles resulting in positive or negative effects in cells. Low or suitable concentrations of ROS can stimulate signaling pathways in cells to respond to various physiological functions such as immune function (i.e., defense against pathogenic microorganisms), mitogenic response and redox regulation [1,2]. The anhydrous, dimethyl sulfoxide (DMSO), ethyl acetate (analytical grade) (EtOAc), methanol (analytical grade) (MeOH), methanol (MS grade), water (MS grade) (Fisher Scientific, UK), malt extract, peptone, potato dextrose agar (PDA), yeast extract (Himedia, Mumbai, India), and sodium carbonate (Na 2 CO 3 ) (Ajax, New Zealand) were used in the present study.

Fungal Cultivation and Extraction
The fungal isolates were cultured on PDA in 9 cm Petri dishes at 30 °C for 2 weeks. Mycelium on a PDA plate was cut into small pieces before being transferred into 200 mL of YM medium, and incubated at 30 °C for 6 weeks. Each cultural broth was filtrated and extracted with an equal volume of ethyl acetate three times. Then, the solvent was removed by evaporation under reduced pressure at 45 °C using a rotary evaporator. The crude extracts were stored in tightly caped glass bottles at −20 °C until used.

Fungal Cultivation and Extraction
The fungal isolates were cultured on PDA in 9 cm Petri dishes at 30 • C for 2 weeks. Mycelium on a PDA plate was cut into small pieces before being transferred into 200 mL of YM medium, and incubated at 30 • C for 6 weeks. Each cultural broth was filtrated and extracted with an equal volume of ethyl acetate three times. Then, the solvent was removed by evaporation under reduced pressure at 45 • C using a rotary evaporator. The crude extracts were stored in tightly caped glass bottles at −20 • C until used.

DPPH Scavenging Activity
The DPPH scavenging assay was modified from Xiang et al. [24]. Briefly, 55 microliters of each extract at different concentrations prepared by a two-fold dilution were added into 96-well plates. Then, 150 µL of 0.2 mM DPPH solution was mixed and incubated for 30 min in the dark. The absorbance was measured at 517 nm. Butylated hydroxytoluene (BHT) and Trolox were used as positive controls. All reactions were performed in triplicate (n = 3). The percentage of DPPH scavenging activity was calculated using the following equation: DPPH radical scavenging (%) = [(Abs control − Abs sample )/Abs control ] × 100 where Abs control is the absorbance of the control reaction and Abs sample is the absorbance of the extract. IC 50 value is the concentration of the extract required to inhibit 50% of DPPH radical scavenging. A scatter graph was plotted to obtain IC 50 value.

ABTS Scavenging Activity
The ABTS scavenging activity was evaluated according to the method of Almeida et al. [25] with some modifications. A stock of ABTS radical cation (ABTS •+ ) was prepared by the reaction of 7 mM ABTS solution (5 µL) with 140 mM potassium persulfate (88 µL), and incubated for 16 h in the dark. The ABTS •+ solution was diluted with 95% ethanol to obtain absorbance 1.0 at 734 nm. Ten microliters of different concentrations of the extracts was added into 96-well plate, mixed with the ABTS •+ solution (290 µL), and incubated in the dark. After 7 min, the absorbance was measured at 734 nm. BHT and Trolox were used as positive controls. All samples were evaluated in triplicate. The % ABTS radical scavenging activity was calculated using the following equation: where Abs control is the absorbance of the control reaction and Abs sample is the absorbance of the extract. The antiradical activity was expressed as IC 50 (mg/mL), which represented the extract concentrations scavenging 50% of ABTS radicals. IC 50 values were calculated using Prism 8.0 calculated from the plotted graph between % scavenging and concentration.

Total Phenolic Content
Total phenolic content (TPC) was quantified using Folin-Ciocalteu method according to Rusu et al. [26] with some modifications. The stock of Folin-Ciocalteu's reagent was prepared by Folin-Ciocalteu's reagent with water (1:10, v/v). The extract (25 µL) was added into 96-well plate and mixed with 75 µL of 7.5% (w/v) of sodium carbonate (Na 2 CO 3 ). After 3 min, 100 µL of the stock of Folin-Ciocalteu's reagent was added and incubated for 30 min in the dark. The absorbance was measured at 765 nm. The TPC was expressed as g gallic acid equivalent per g of crude extract (g GAE/g). The experiments were evaluated in triplicate. The cell lines of human lung carcinoma (A549), human liver carcinoma (HepG2) and human cervical adenocarcinoma (HeLa) were purchased from the American Type Culture Collection (ATCC), (LGC Standards, Middlesex, UK), and the immortalized human normal prostate cell line (PNT2) was obtained from the European Collection of Authenticated cell cultures (ECACC), (Public Health England, Salisbury, UK). All cells were cultured in 75 cm 2 cell culture flasks in complete medium (RPMI 1640 stable Glutamine Medium supplemented with 10% fetal bovine serum (FBS) and 1% Pen/strep) at 37 • C in an atmosphere of 95% humidity and 5% CO 2 . After reaching 70-80% confluence, the cells were washed with phosphate buffer saline (PBS) and the cells dislodged using trypsin (1× trypsin-EDTA solution in PBS). The cells were then resuspended with fresh complete medium and used to seed new cell culture flasks. For experimental purposes, the cells were seeded (100 µL/well) into 96-well plates at differing concentrations (5 × 10 3 cells/well for A549, HepG2 and HeLa cells, and 10 × 10 4 cells/well for PNT2 cells).

Assessment of the Cytotoxicity of Xylaria Species Crude Extracts against Cancer Cell Lines
The assessment of the cytotoxicity of the crude extracts was performed on cells growing in 96-well plates (as described above) using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay following Guetchueng et al. [27]. After seeding, the cells were first cultured overnight before treatment with the crude extracts. The cells were then treated with crude extracts (100 µg/mL (100 µL/well)) in complete medium with 0.2% (v/v) DMSO. In addition, cells were treated with complete medium (100 µL/well) as a negative control and doxorubicin (50 µg/mL (100 µL/well)) as a positive control. Both these controls also contained DMSO (0.2% (v/v)). After 48 hours' treatment, cell viability was determined using the (MTT) assay. The treatments were removed from the wells and MTT in complete medium (0.5 mg/mL (100 µL/well)) was added and the cells incubated for further 2 h at 37 • C. This mixture was removed and 200 µL/well DMSO added to solubilize the formazan crystals. The plate was shaken slowly for 20 min then the absorbance of each well was measured at 570 nm. All crude extracts (and controls) were assessed in triplicate. The effect of each crude extract on the proliferation of cancer cells was expressed as percentage of the absorbance of the average negative control absorbance reading, which were calculated using the equation: Cell viability (%) = (Abs sample/ Abs control ) × 100 where Abs control is the absorbance of the control reaction and Abs sample is the absorbance of the extract.

Statistical Analysis
All the assays were carried out in triplicate on each occasion. The data were presented as the mean ± standard deviation (SD). Differences between the individual treatments were analyzed using one-way ANOVA with Tukey's method performed in SPSS programme version 26. Statistical significance was set at p-value < 0.05. IC 50 values were calculated by using Graph Pad Prism (Version 8.0)

LC-MS Chemical Profiles
The chemical constituents of the extracts were determined using liquid chromatography coupled with mass spectrometry (LC-MS) analysis according to the method previously described by Khan et al. [28] with modifications. The Alliance HPLC system 2695 (water) was used. Chromatography was performed using reversed-phase on a Phenomenex C18 column (150 mm × 4.6 mm × 5 µm). The conditions were set as follows; column temperature at 25 • C, UV-Vis detector at 200-400 nm, 1 mL/min of flow rate and 20 µL of injection volume. An elution gradient was used 0.1% acetic acid in water as mobile phase A and 0.1% acetic acid in MeOH as mobile phase B. The mobile phase composition was started from 30% B at 0 min and increased as linear gradient reaching 100% B at 30 min. The LC system was connected to a quadrupole time of flight (TOF) mass spectrometer (Water Micromass LCT) having an electrospray ion source. The response was recorded in real time by the mass spectrometer data system (Waters MassLynx version 4.1). The parameters were set as follows: electrospray interface 3000 V, sample cone 60 V, extraction cone 4 V, rangefinder lens 300 V, desolvation temperature 250 • C, source temperature 100 • C, nebulizer gas flow 20 L/h, desolvation gas flow 760 L/h, and TOF tube 4687 V. The Data acquisition method was set as follows: cycle time 1 s, scan duration 0.9 s, inter-scan delay 0.1 s, mass range 100-1500, and centroid mode. Positive ion mode was operated.

DPPH Free Radical Scavenging Activity
The DPPH free radical is a stable free radical, which has been widely used as a tool for estimating the free radical scavenging activities of antioxidants [29]. The DPPH scavenging activities of 10 Xylaria extracts with EtOAc are presented in Figure 2A (Table S1). All extracts represented moderate to high antioxidant activities demonstrated in term of IC 50 values ranged from 0.194 ± 0.031 to > 1.00 mg/mL. The extracts from different isolates were also varied within the same species. The highest significant activity belonged to X. vinacea SWUF18-2.3 (0.194 ± 0.031 mg/mL), while most extracts including X. siamensis SWUF17-20.2, X. thienhirunae SWUF17-44.1 and SWUF16-10.1 showed moderate activities ranging from 0.484 ± 0.017 to 0.541 ± 0.018 mg/mL (Table S1). In addition, nearly all extracts had antioxidant activity with IC 50 values less than 1 mg/mL except for X. chaiyaphumensis SWUF16-4.1 (>1.00 mg/mL), although they proved less effective than the standard BHT (0.105 ± 0.006) and Trolox (0.004 ± 0.000) examined.

ABTS Free Radical Scavenging Activity
The radical scavenging activities of 10 extracts determined by ABTS scavenging assays are presented in Figure 2B ( Table S1). The IC 50 of all extracts obtained was in a range of 0.020 ± 0.004 to 0.283 ± 0.021 mg/mL. Similar to DPPH assay, X. vinacea SWUF18-2.3 has the highest activity (IC 50 value 0.020 ± 0.004 mg/mL) from the other extracts, which were not different from the standard BHT (0.006 ± 0.000 mg/mL) and Trolox (0.003 ± 0.000 mg/mL). These indicated that SWUF18-2.3 had significantly high efficacy similar to the standards. Most extracts showed moderate antioxidant activity which ranged from 0.047 ± 0.003 to 0.119 ± 0.014 mg/mL, while the isolate of SWUF16-4.1 showed the lowest activity at 0.283 ± 0.021 mg/mL. However, the antioxidant efficacy of almost all the extracts showed a similar trend to the DPPH scavenging assay.

Determination of Total Phenolic Content
The content of total phenolic compounds of all extracts is presented in Figure 2D ( Table S1). The results varied from 0.826 ± 0.123 to 3.629 ± 0.381 g GAE/g crude extract according to the equation using gallic acid as the standard curve (Y = 0.05946 * X + 0.07322 and R 2 = 0.9928) ( Figure 2C). The highest total phenolic content of all Xylaria extracts belonged to X. vinacea SWUF18-2.3 (3.629 ± 0.381 g GAE/g extract) followed by X. siamensis SWUF17-20.2 (2.232 ± 0.176 g GAE/g extract). The results showed correlation between antioxidant activities (DPPH and ABTS scavenging activities) and total phenolic contents. The high total phenolic content contributed to the high antioxidant activities. Xylaria vinacea SWUF18-2.3 was the highest both total phenolic content and antioxidant activities, while X. chaiyaphumensis SWUF16-4.1 was the lowest both total phenolic content and antioxidant activities.

Cytotoxicity against Cancer Cell Lines
Cytotoxicity of Xylaria extracts against different human cancer cell lines including A549, HepG2, HeLa and normal cell line PNT2-was determined using the MTT assay. The extracts showed varying cytotoxicity exhibited in the different cell lines at a concentration of 100 µg/mL ( Figure 3 and Table S2). The ranges of % cell viability was 13.16 ± 3.59 -87.26 ± 7.45% for A549, 11.15 ± 0.32 -104.12 ± 6.55% for HepG2, 11.97 ± 1.28 -91.82 ± 1.91% for HeLa and 13.17 ± 2.37 -99.68 ± 4.67% for PNT2. Doxorubicin was used as the positive control having % cell viability of all cell lines ranging 15.99 ± 0.55 to 20.32 ± 1.09% at concentration of 50 µg/mL. The results revealed that most extracts exhibited cytotoxicity against at least one kind of the cancer cell lines. Xylaria subintraflava SWUF16-11.1 displayed the most significant inhibition to all cell lines with % cell viability ranged from 11.15 ± 0.32 to 13.17 ± 2.37. Some extracts revealed broad spectrum to inhibit several cancer cell lines, but some extracts were specific to only one cell lines. The extracts of X. subintraflava SWUF16-11.1 and SWUF17-24.2 showed highly significant activity against A549, HepG2 and HeLa while the extract of X. vinacea SWUF18-2.3 showed high cytotoxic activity against only HeLa cell (42.65 ± 3.32% cell viability). The extracts of X. subintraflava SWUF17-24.2 and X. chaiyaphumensis SWUF17-49.2 showed high cytotoxicity against cancer cell lines but low cytotoxicity against normal cell lines of PNT2. Xylaria subintraflava SWUF17-24.2 extract exhibited cytotoxicity against A549, HepG2 and HeLa ranged from 28.58 ± 0.95 to 38.54 ± 1.90% cell viability, while low cytotoxicity against PNT2 at 59.94 ± 6.76% cell viability. Xylaria chaiyaphumensis SWUF17-49.2 extract exhibited cytotoxicity against A549 cell lines at 45.14 ± 2.53 and 48.89 ± 7.51% cell viability, respectively, but showed low cytotoxicity against PNT2 at 73.10 ± 2.45 and 70.37 ± 12.46% cell viability, respectively.

LC-MS Chemical Profiles
LC-MS is one of the most effective analytical tools for organic compound analysis. The EtOAc extracts of secondary metabolites from Xylaria spp. were analysed to obtain LC chromatograms and MS spectrum using LC-ESI-QTOF MS method in positive mode. The gradient and condition of LC-MS analysis were optimized to separate and derive the highest peak numbers and the highest capacity of spectrum of the extracts. The results of total ion chromatograms (TIC) displayed more over 61 peaks of all extracts as shown in Figure 4 (Table S3) . The details of m/z spectra of each peak no. are described in Table S3. In this study, four major peaks of no. 1, 2, 3 and 60 were found in all Xylaria extracts, which presented at the same m/z spectrum and retention time. Peak no. 1 had a major ion at m/z 227.1 and minor peaks at m/z 197.1 and 261.1, which matched with prednosone in mass bank library. Peak no. 2 was predicted as xylaropyrone because of the highest [M+H] + ion peak at m/z 211.1, which was similar to previous description by Siriwach et al. [30]. For peaks no. 3

LC-MS Chemical Profiles
LC-MS is one of the most effective analytical tools for organic compound analysis. The EtOAc extracts of secondary metabolites from Xylaria spp. were analysed to obtain LC chromatograms and MS spectrum using LC-ESI-QTOF MS method in positive mode. The gradient and condition of LC-MS analysis were optimized to separate and derive the highest peak numbers and the highest capacity of spectrum of the extracts. The results of total ion chromatograms (TIC) displayed more over 61 peaks of all extracts as shown in Figure 4 (Table S3) . The details of m/z spectra of each peak no. are described in Table S3. In this study, four major peaks of no. 1, 2, 3 and 60 were found in all Xylaria extracts, which presented at the same m/z spectrum and retention time. Peak no. 1 had a major ion at m/z 227.1 and minor peaks at m/z 197.1 and 261.1, which matched with prednosone in mass bank library. Peak no. 2 was predicted as xylaropyrone because of the highest [M + H] + ion peak at m/z 211.1, which was similar to previous description by Siriwach et al. [30]. For peaks no. 3 and 60, their major peaks did not match any compounds in database including the previous reports. They were then noted as unidentified compounds, which might be new or mixed compounds representing in one peak.

Discussion
The discovery of novel secondary metabolites from insect-associated fungi has been of interest to researchers in recent years [23,[31][32][33]. Lately, twelve new species of Xylaria associated with termite nests or growing on soil were described by our previous study [19]. Ten isolates of 5 species, X. chaiyaphumensis, X. siamensis, X. subintraflava, X. thienhirunae and X. vinacea, were selected to assess their biological activities and secondary metabolite analysis. The antioxidant capacity of all EtOAc extracts from Xylaria species was determined by using the DPPH and ABTS radical scavenging assays. The results showed that all extracts had antioxidant potentials, especially X. vinacea SWUF18-2.3, which had the lowest IC 50 values (0.020 ± 0.004 to 0.194 ± 0.031 mg/mL) and they were not significantly different from the standard BHT (0.006 ± 0.000 mg/mL) and Trolox (0.003 ± 0.000 mg/mL) by ABTS scavenging assay. The IC 50 values of DPPH scavenging assay of all extracts were slightly higher than ABTS assay. It is because the DPPH method has more limitations and is characterised by a lower sensitivity [34]. The antioxidant activity results in this study are better than those reported for X. nigripes, which is a traditional medicinal fungus and is a Xylaria species associated with termite nests [35]. The extracts of X. nigripes obtained from various solid-state fermentations with ethanol solvent had IC 50 values of DPPH radical scavenging activity ranged from 2.0 ± 0.1 to 2.7 ± 0.1 mg/mL. In addition, Ko et al. [21] reported that the DPPH activity of X. nigripes extracts from culture mycelium using different extraction solvents of aqueous and ethanol had IC 50 62.07 µg/mL and 73.49 µg/mL, respectively, while the commercial mycelium from a drug store in China known as "Wu Ling Shen" were extracted with the same solvents and showed the IC 50 487.68 µg/mL and >500 µg/mL, respectively. The extracts of all Xylaria species in this study showed strong antioxidant activities compared with other reports of Xylaria associated from termite nests, and they were noted as good sources of antioxidant activities for further study. However, the antioxidant capacities of fungal extracts are strongly dependent on the culture media and the extracting solvent, due to the presence of different antioxidant compounds of varied chemical characteristics and polarities that may or may not be soluble in a particular solvent.
The phenolic compounds present in several fungi and relate to biological functions including antioxidant activity. The total phenolic compounds of the Xylaria extracts in this study were high levels and varied from 0.826 ± 0.123 to 3.629 ± 0.381 g GAE/g crude extract. The high total phenolic contents and the strong antioxidant activities were from the same species X. vinacea SWUF18-2.3. It might be that of the different phenolic compounds contributed to the antioxidant activity. In a previous study, it has been reported that antioxidant activity from Xylaria spp. could be affected mainly by the presence of phenolic compounds. As the total phenolic compounds increase, the antioxidant potential of the fungi increases. This study was supported by previous studies which found that the levels of phenolic compounds resulted in their antioxidant capacity or that they correlated [35][36][37].
Fungal secondary metabolites have recently attracted great interest, owing to their versatile applications including cytotoxicity against cancer cells [38,39]. In our previous study, several active compounds extracted from Xylaria culture broth with EtOAc showed high cytotoxicity against different cancer cell lines [40]. McCloskey et al. [40] demonstrated that the cytotoxicity of chaxine C derived from EtOAc extract of X. allantoidea SWUF76 had strong effects against HeLa, HT29 colon cancer cell, HCT116 colon cancer cell, MCF-7 breast cancer cell and normal Vero cells. Moreover, two new tetralone derivatives named xylariol A and B derived from X. hypoxylon AT-028 exhibited cytotoxicity against HepG2 cell lines with IC 50 of 22.3 and 21.2 µg/mL, respectively [41]. In this study, the cytotoxicity of Xylaria extracts showed different potential to cancer cell lines, although within the same species. The extracts of X. subintraflava SWUF16-11.1 exhibited the strongest cytotoxicity against all cancer cell lines at the concentration of 100 µg/mL, but SWUF17-24.2 showed less activity than those isolates within the same species. For X. chaiyaphumensis isolates, only SWUF17-49.2 showed % cell viability against A549 less than 50%, while other isolates of SWUF16-4.1 and SWUF16-11.4 showed higher % cell viability. These results could be because of difference in responsiveness and sensitivity of different cancer cells to different compounds [42]. Liu et al. [43] reported that different phenanthroindolizidine alkaloids (PAs) revealed difference of one ethoxyethyl on molecule resulting in different results of inhibition which showed greater effects on HepG2 compared with HCT116 and HT29 cancer cells. The extracts of this study were crude extracts containing several compounds, which may have been the results of synergistic or antagonistic effects to different cancer cell lines. In addition, the extracts of some isolates revealed relationships between cytotoxicity and antioxidant activities. The high cytotoxicity of X. subintraflava SWUF16-11.1 extract had strong antioxidant activities and high content of total phenolic compounds. In contrast, the extract of X. vinacea SWUF18-2.3 showed low toxicity but it contained high antioxidant activity. Therefore, the increasing of antioxidant activities of some extracts did not affect to the cytotoxicity against cancer cells. However, until now, only one study has proved that the extracts of Xylaria species from termite nests have anticancer activity [31]. Regarding their interesting biological activities and presence of highly cytotoxic metabolites, which may be a potential source of leads for drug development after more extensive pharmacological screening of the isolated compounds e.g., target identification and validation, preclinical studies and clinical development.
The results of TIC profiles in our study shared four main peaks (no. 1, 2, 3 and 60) within all Xylaria extracts. One main peak, no. 2 was identified as xylaropyrone, which was previously reported from endophytic X. feejeensis [30] and xylaropyrone derivatives (B and C) from endophytic Xylaria sp. SC1440 [52]. These compounds showed moderate antimicrobial activity against Saccharomyces cerevisiae, Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus at different concentrations [30]. For the other 3 main peaks, peak no. 1 matched to prednisone, which is a group of steroids having anti-inflammatory and immunomodulation properties, while other 2 main peaks no. 3 and 60 were unidentified compounds that may be new or complex compounds and need to be purified to determine their chemical structure by analysis in a further study. In addition, each Xylaria isolate provided differences in their metabolic characteristics or spectrum profiles. This may be because of their genetic variations combined with the different locations and soil properties of each isolate. The previous studies by Tang et al. [53] and Li et al. [54] reported that the metabolic compounds obtained from microorganism were complex and the metabolic accumulation was variable due to the great influences of genetic and environmental factors. Most spectra of mass patterns obtained from Xylaria EtOAc extracts in the present study exhibited the different peaks of mass at the same retention times, which implied that those peaks cannot be representative in discriminating the different species. Due to the complex of ion spectrum and the limitation of fungal metabolites in mass databases, many peaks were noted as unidentified compounds. In addition, all extracts obtained from novel Xylaria species associated with termite nests (subgenus Pseudoxylaria), which have revealed a separate lineage from other Xylaria species associated with different habitats analyzed by phylogenetic analysis [18,19]. Therefore, the metabolites from such a termite-associated species have only received a few reports and proved to be rather unique cyclic tetrapeptides [55], which were different from the regular Xylaria species living as saprophytes or endophytes [56]. The highest number of peaks belonged to X. subintraflava SWUF16-11.1 followed by X. vinacea SWUF18-2.3, which exhibited the highest antioxidant activities and cytotoxicity against all cancer cell lines. The results may be related to the most peak numbers of the extracts from LC-MS fingerprints. The various groups of identified compounds found in the present study have been reported as processing biological activities including antimicrobial, anti-viral, antioxidant, anti-inflammatory, anticancer, anti-tumor and anti-insecticidal activities [10,13,22,56]. For example, bassianolide (peak no. 59) from X. chaiyaphumensis SWUF16-4.1 and SWUF17-49.2 was firstly reported from a wood-rot fungus Xylaria sp. BCC1067 and contained antiplasmodial, antimycobacterial and antitumor activities [57]. A 19,20-epoxy cytochalasin C (peak no. 9, 17) from X. subintraflava SWUF16-11.1 and SWUF17-24.2 has been previously found from X. obovata [58], endophyte Diaporthe sp. RJ-47 [59] and Nemania sp. UM10M, which has antimalarial activity and cytotoxicity against SK-MEL cell lines [60]. Some identified compounds andrographolide (peak no. 22) [61], naringin (peak no. 34) [62], rottlerin (peak no. 29) [63,64] tiliroside (peak no. 49) [65,66] have been reported from plants and contained anti-inflammatory, anti-cancer, anti-hepatotoxicity, anti-atherosclerosis, anti-diabetes and antioxidant activity. In addition, tofisopam (peak no. 28) from the extracts of X. vinacea SWUF18-2.3 was reported as synthetic compounds and has anxiolytic activity [67] and recently reported as post COVID neuro-psychiatric sequelae [68]. Isolation of new species of fungal metabolites may lead to the development of a new class of anticancer drugs through further study. Nevertheless, there were no report of LC-MS fingerprints derived from Xylaria spp. including Xylaria species associated from termite nests or soil. This is the first preliminary study of untargeted metabolic profiles which have contributed some important data about secondary metabolite compositions from the novel species of Xylaria from termite nests or soil.

Conclusions
Ethyl acetate extracts of Xylaria species associated with termite nests or soil possessed strong antioxidant activities and cytotoxicity against human cancer cell lines. The antioxidant ability may be from the action of phenolic compounds confirmed by total phenolic content experiment. The cytotoxicity exhibited a broad spectrum to specific activity against cancer cell lines. Moreover, LC-MS fingerprints presented over 61 peaks from ten isolates from six species which indicated that there were a many groups of secondary metabolites produced. The current study provided valuable information regarding the novel Xylaria species associated with termite nests or soil which have received limited attention concerning their biological activities and LC-MS fingerprints. Isolation and elucidation of bioactive chemical structures are now required for further study. However, the interesting activities may lead to those Xylaria associated with termite nests or soil becoming promising species of natural product sources for future therapeutic agents.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/antiox10101557/s1, Table S1: IC 50 values of antioxidant activities, DPPH and ABTS radical scavenging and total phenolic content of ethyl acetate extracts of Xylaria spp. and standard BHT and Trolox. Data expressed as means ± standard deviation. Means within each graph with different letters (a-g) differ significantly (p < 0.05), Table S2: Cytotoxicity of EtOAc extracts of Xylaria spp. against four different cell lines, A549, HepG2, HeLa and PNT2, at concentration of 100 µg/mL. Data expressed as means ± standard deviation. Means within each graph with different letters (a-g) differ significantly (p < 0.05), Table S3: Mass spectra data of peaks from Xylaria species and identified compounds.