Abstract
Gibellula scorpioides BCC 39989 is one of the species of spider-parasitic Gibellula (Hypocreales, Cordycipitaceae) discovered in Thailand. Investigation of the invertebrate-pathogenic fungus G. scorpioides BCC 39989 led to the isolation of eight known compounds (1–8). Their chemical structures were determined by employing extensive spectroscopic analysis. Among them, ergosterol peroxide (1) and methyl o-hydroxycinnamate (2) exhibited significant biological activities. Both compounds notably suppressed LPS-induced nitric oxide production and reduced IL-6 and TNF-α secretion in RAW264.7 macrophages. In addition, compounds 1, 2, and oleic acid (7) significantly inhibited lipid accumulation and reduced intracellular triglyceride content in 3T3-L1 adipocytes in a dose-dependent manner. Compound 1 also displayed potent antiproliferative activity against human colorectal adenocarcinoma (SW480) cells, with an IC50 value of 13.33 µg/mL, whereas compound 2 was less effective. Genotoxicity evaluation of ergosterol peroxide (1) using the cytokinesis-block micronucleus assay in V79 cells revealed no genotoxic effects at concentrations ranging from 3.125 to 50 µg/mL, both with and without metabolic activation. These findings highlight G. scorpioides as a promising source of bioactive compounds, particularly ergosterol peroxide (1), which exhibits potent anti-inflammatory, anti-adipogenic, and antiproliferative properties without genotoxicity.
Supplementary Information
The online version contains supplementary material available at 10.1038/s41598-026-35326-7.
Keywords: Gibellula scorpioides, Invertebrate-pathogenic fungus, Anti-inflammatory activity, Anti-adipogenic activity, Antiproliferative activity, Genotoxicity
Subject terms: Biochemistry, Cancer, Drug discovery, Microbiology
Introduction
Fungi are recognized as one of the potential sources of natural bioactive substances with a broad range of biological properties1. Fungal secondary metabolites have been reported to treat chronic infections as antibiotic agents, such as retapamulin, pleuromutilin, lefamulin, penicillins, and cephalosporins2. More recently, fungal secondary metabolites have entered clinical trials to treat chronic diseases such as cancer, autoimmune diseases, and hypercholesterolemia3. Gibellula is one of the promising sources of bioactive secondary metabolites. Gibellula species, such as Gibellula sp., are composed of bis(naphtho-α-pyrone) named pigmentosins A and B whereas Gibellula gamsii strain BCC 47868 has been found to contain 1,3-disubstituted β-carboline alkaloids named gibellamines A and B. These compounds exhibited anti-biofilm activity against Staphylococcus aureus4,5. Furthermore, Gibellula sp. BCC 36964 has been reported to contain several bioactive compounds, including gibellulins A and B, 7-deoxy-7,8-didehydrosydonic acid, sydowiol C and diorcinol, which displayed antibacterial activity and cytotoxicity against MCF-7, KB, and NCI-H187 cancer cells6. As part of our ongoing research on bioactive secondary metabolites from invertebrate-pathogenic fungi, we turned our attention to the fungal strain Gibellula scorpioides BCC 39989. This strain was recently reported as a new species in the spider-parasitic genus Gibellula from Thailand7, but its secondary metabolites production remained unexplored. Herein we report the isolation of secondary metabolites from the invertebrate-pathogenic fungus G. scorpioides BCC 39989. Chemical investigation on the broth and mycelial ethyl acetate extracts was carried out and led to the isolation of eight compounds, which included ergosterol peroxide (1)8, methyl o-hydroxycinnamate (2)9, coumarin (3)10, benzophenone (4)11, palmitic acid (5)12, methyl palmitate (6)13, oleic acid (7)12, and methyl stearate (8)14. Additionally, the isolated compounds available in sufficient quantities, ergosterol peroxide (1), methyl o-hydroxycinnamate (2), and oleic acid (7), were evaluated for their anti-inflammatory activity, ability to suppress lipid accumulation in 3T3-L1 adipocytes, antiproliferative effects against human colorectal adenocarcinoma, and in vitro genotoxicity in the V79 (CCL-93) cell line.
Materials and methods
Instrumentation and analytical methods
Specific rotations were recorded on a JASCO-1020 polarimeter (USA). UV spectra were measured on a Shimadzu UV-VIS spectrometer model 2450 (Shimadzu, Japan), while IR spectra were acquired with a PerkinElmer Spectrum FTIR spectrometer (Perkin Elmer, USA). EI-MS spectra were obtained on a GC-MS/MS, Agilent 7890B GC system (Agilent Technologies, USA) equipped with Triple Quadrupole mass spectrometer (Agilent Technologies Mass selective detector model 7000 D, USA). 1D and 2D NMR spectra were recorded on the Bruker AVANCE 400 MHz NMR spectrometer (Bruker, Switzerland) using tetramethylsilane (TMS) as an internal standard. Biological assays were performed using the following instruments: Multiskan Go microplate reader (Thermo Scientific, Finland), Haemocytometer (BOECO, Germany), Inverted microscope (Nikon, Japan), Allegra® X-I5R centrifuge (Beckman Coulter, USA), Clean Bio Calibration equipment (Thailand), Thermo/Heraeus HERA Cell 150 CO2 incubator (USA), and Metafer®, Axio Imager.Z2, (Zeiss, Germany). Thin-layer chromatography (TLC) and precoated TLC (PTLC) were performed on silica gel 60 GF254 (Merck). Column chromatography (CC) was carried out on Sephadex™ LH-20 (Cytiva, Sweden), silica gel (Merck) type 60 (230–400 mesh ASTM) or type 100 (70–230 mesh ASTM), and reverse phase C18 silica gel, 40–63 μm (Silicycle Inc, Canada).
Fungal material
Gibellula scorpioides (BBH 30255) was collected from Pu Kaeng Waterfall, Doi Luang National Park, Phan District, Chiang Rai Province, Thailand (19° 32′ 33″ N 19°46′ 03″ E) in 2009. The sample was isolated in pure culture following the techniques described by Kuephadungphan et al. (2022)7. The pure culture and voucher specimen were deposited at BIOTEC Culture Collection (BCC) and BIOTEC Bangkok Herbarium (BBH), Pathum Thani, Thailand, respectively. Five nuclear DNA regions of this strain were amplified, including internal transcribed spacer regions (ITS), partial regions of the nuclear large subunit (LSU) of the ribosomal DNA, translation elongation factor 1-alpha (tef), and the largest and second-largest subunits of RNA polymerase II (RPB1 and RPB2). The protocols for PCR amplifications used in this study followed Kuephadungphan et al. (2022)7. The generated sequence data were submitted to GenBank (LSU = PP993895, tef = PP998048, RPB1 = PP998049 and RPB2 = PP998050), as shown in Table 1.
Table 1.
List of species and GenBank accession number of sequences used in this study.
Fermentation and extraction
Gibellula scorpioides strain BCC 39989 was grown on liquid yeast, malt, and glucose medium (YMGB, 10 g of malt extract, 4 g of D-glucose, 4 g of yeast extract and 1,000 mL of distilled water, pH 6.3) by cutting five mycelial plugs using a cork borer (7 × 7 mm) from a well grown culture into fifty of 500 mL Erlenmeyer flasks containing 200 mL of YMGB, and incubated at 23 °C on a shaker at 140 rpm. The free glucose content of each fermented broth was tentatively monitored using Urine Test Strip For 2 Items, DIRUI®. The fermentation was prolonged for half of the time each strain had taken for glucose consumption. The mycelia and supernatant were separated by vacuum filtration. For the culture filtrate, it was transferred into a separation funnel, ethyl acetate (EtOAc) was added in a 1:1 ratio, mixed, and the resulting solution was separated into two phases; the upper phase (EtOAc phase) and the lower phase (water phase), followed by filtration through filter paper with anhydrous sodium sulfate (anh. Na2SO4). The solution was then evaporated at 40 °C to obtain a dry extract, which was dissolved in acetone and methanol, and further evaporated to obtain the BE extract. The mycelia were extracted with acetone (1:1) in ultrasonic bath sonicator at 40 °C for 30 min. The acetone was removed from the mycelium by filtration, followed by evaporation at 40 °C to remove residual acetone. The mycelial extract was then dissolved in 100 mL of distilled water and transferred to a separation funnel. The extraction process was followed the same method as for the culture filtrate extraction, culminating in evaporation to obtain the CE extract.
Isolation of metabolites (1–8)
The mycelial extract, CE (2.47 g), was separated by CC over silica gel using a gradient of EtOAc/hexane (10:90 → 100:0) to afford five fractions (CE1-CE5). Fraction CE1 (672.8 mg) was purified by CC over silica gel using a gradient of MeOH/CH2Cl2 (1:99 → 100:0) as a mobile phase to yield compound 6 (1.1 mg). Fraction CE2 (874.3 mg) was separated by CC over Sephadex LH-20 using MeOH to give two subfractions (CE21-CE22). Subfraction CE21 (751.8 mg) was subjected to CC over silica gel using a gradient of EtOAc/hexane (20:80 → 100:0) as a mobile phase to yield three subfractions (CE211-CE213). Separation of subfraction CE211 (372.0 mg) by CC over Sephadex LH-20 using MeOH gave two subfractions. The first subfraction contained compound 5 (15.0 mg). The second subfraction (311.9 mg) was further purified by CC over Sephadex LH-20 using MeOH to afford compound 7 (20.0 mg). Subfraction CE212 (168.2 mg) was chromatographed by CC over silica gel using a gradient of CH2Cl2/hexane (40:60 → 100:0) as an eluent to yield compound 8 (4.5 mg). Fraction CE3 (807.2 mg) was applied to CC over Sephadex LH-20 using MeOH as an eluent, and subsequent PTLC using MeOH/CH2Cl2 (1:24) as a mobile phase (2 runs) to afford compound 2 (8.2 mg). Fraction CE4 (162.5 mg) was submitted by CC over reverse phase C18 silica gel using H2O/MeOH/ACN (10:45:45) as an eluent to yield compound 1 (32.4 mg). The broth extract, BE (270.1 mg), was fractionated by CC over Sephadex LH-20 using MeOH to afford six fractions (BE1-BE6). Fraction BE5 (27.1 mg) was purified by PTLC using MeOH/CH2Cl2 (2:23) as a mobile phase (2 runs) to afford compounds 2 (7.6 mg), 3 (4.2 mg) and 4 (5.1 mg). Additionally, compound 2 (1.0 mg) was obtained from fraction BE6 (10.3 mg) after purification by PTLC using MeOH/CH2Cl2 (1:24) as a mobile phase (2 runs).
Biological assay
Cytotoxic activity
Cell culture
3T3-L1 cells, mouse embryo preadipocytes; RAW 264.7 cells, murine macrophages; V79(CCL-93) cell line, Chinese hamster lung fibroblast, were purchased from the American Type Culture Collection (ATCC, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, USA), supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% antibiotic-antimycotic in 75 cm2 plates. They were maintained at 37 °C in a humidified cell incubator with 5% CO2 − 95% O2. Sub-culturing was performed when the cells reached approximately 80% confluence.
Cytotoxic activity on RAW 264.7, 3T3-L1 and V79(CCL-93) cells
RAW 264.7, 3T3-L1 and V79(CCL-93) cells viability was analyzed using a 3-(4,5-dimethylthiazol-2-yl)−2,5-diphenyltetrazolium bromide (MTT) assay modified from ISO 10993-5:2009(E)15. Cells were cultured in DMEM supplemented with 10% FBS at 37 °C (5% CO2) for 2 days (90% confluency, density of 1 × 104 cells/well) and then treated with various concentrations of samples for 48 h. After incubation, 0.5 mg/mL of MTT solution was administered and cells were incubated for 2 h in the incubator at 37 °C. After the reaction, the medium was removed, MTT formazan crystals were dissolved in 1 mL of dimethyl sulfoxide (DMSO), and 200 µL were transferred to 96-well plates. The optical density was measured at 570 nm using a microplate reader (Multiskan Go, Thermo/Scientific). Percent viability was calculated by equation: percentage of cell viability = 100 x (OD570s/OD570m), when OD570s is optical density of sample and OD570m is optical density of medium.
Anti-inflammatory activity
Anti-inflammatory activity was performed as described by Zhang et al. (2019)16.
Inhibition of nitric oxide (NO) production
RAW 264.7 cells were seeded at a density of 5 × 105 cells/well in 24-well plates and then incubated for 24 h at 37 °C with 5% CO2. After incubation, the cells were cultured in DMEM supplemented with various concentrations of the samples and Lipopolysaccharide (LPS, 1 µg/mL), and then further incubated for an additional 24 h. At the end of the incubation period, each culture supernatant was mixed with Griess reagent (Promega, USA) to determine NO production by RAW 264.7 cells. The optical density at 540 nm of the mixture was measured using a microplate reader. The nitrite concentration was calculated using the equation: Inhibition percentage = (OD of LPS – OD of sample)/(OD of LPS – OD of control) x 100.
Proinflammatory cytokines (Interleukin6 (IL6) and tumor necrosis factor alpha (TNF-α))
RAW 264.7 cells were seeded in 96-well plates at the density of 5 × 105 cells/well. After 24 h of incubation, the adhered cells were treated with various concentrations of samples. Twenty-four hours later, the concentrations of IL-6 and TNF-α in the cell supernatant were determined using an ELISA assay. ELISA kits were obtained from AlphaLISA Research reagent of PerkinElmer Co., Ltd., and each sample was measured three times according to the manufacturer’s instructions.
Lipid accumulation of 3T3-L1 adipocytes
The method was performed with modifications based on Zeng et al. (2012)17.
Cell culture
3T3-L1 cell line was cultured in DMEM, supplemented with 10% FBS, 1% penicillin-streptomycin (P/S) (10,000 unit/mL penicillin and 10,000 µg/mL streptomycin) (Gibco, USA) at 37 °C (5% CO2). At two days post-confluence (day 0), cells were treated with DMEM containing 10% fetal bovine serum, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 1 µM dexamethasone and 10 µg/mL insulin. After two days incubation (day 2), the medium was replaced with DMEM containing 10% FBS and 1 µg/mL insulin, and from the fourth day (day 4), the medium was replaced with DMEM containing only 10% FBS (control) and various concentration of samples and then incubated for 6 h.
Oil red O staining assay
After differentiation, cells were washed with Dulbecco’s Phosphate Buffered Saline (DPBS) and fixed with 2 mL of 10% formalin at 4 °C for 1 h. After fixation, cells were washed with DPBS and stained with 2 mL of 0.35% Oil red O at 25 ◦C for 30 min. After washing with distilled water, isopropanol was added to dissolve the precipitate. The optical density at a wavelength of 500 nm was determined by a microplate reader. Percent inhibition of new droplet was calculated by comparing to a positive control.
Triglyceride (TG) assay
Differentiated adipocytes were treated with DMEM supplemented with 10% FBS and various concentrations of samples at 37 °C (5% CO2) for 6 days, the cell washing by DPBS for 2 times. The cells were added lipid extraction solution of assay kit to dissolved lipid crystal and then incubated at 90–100 °C for 30 min. Measurement of the TG level in 3T3-L1 adipocyte extracts was carried out by Adipogenesis Assay Kit (MAK040) which was performed as per protocol. The % TG reduction was calculated using the equation: C = Sa/Sv, when Sa is TG level of sample from calibration curve, Sv is volume of sample and C is TG concentration of sample. TG reduction percentage was compared to a positive control.
Statistical analysis
All experiments were repeated at least three times. Data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by post hoc multiple comparison tests (Tukey, Duncan, Dunnett) to evaluate differences among treatment groups. LPS served as the reference control for the anti-inflammatory assays, whereas Simvastatin was used as the reference control for the 3T3-L1 lipid accumulation assay. Comparisons between two groups were conducted using Student’s t-test. A value of p < 0.05 was considered statistically significant.
Antiproliferative activity
Cell culture
SW480 cells, human colorectal adenocarcinoma, were purchased from American Type Culture Collection (ATCC, USA). The cells were cultured in DMEM medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and 1% P/S (Gibco, USA) at 37 °C, 5% CO2 atmosphere. The cells were further sub-cultured when they reached approximately 80% confluence.
Cytotoxicity of ergosterol peroxide (1) and methyl o-hydroxycinnamate (2) on SW480 cells
SW480 cells were seeded approximately 5 × 103 cells into each well of 96-well plates. After 24 h, the cells were treated with compounds 1 and 2. Both compounds were initially reconstituted in dimethyl sulfoxide (DMSO) and diluted at designated concentrations by using a complete medium containing DMSO. The treatment lasted for 48 h. Then cell viability was determined by MTS assay using CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit (Promega, USA) and IC50 was calculated using GraphPad Prism 5 (GraphPad, USA). The percentage of cell viability was calculated by the following equation: percentage of cell viability = (ODtest – ODblank)/(ODcontrol – ODblank) × 100%. ODtest is the optical density of the sample-treated cells, ODcontrol is the optical density of cells cultured in a DMSO-containing medium, and ODblank is the optical density of the wells containing only culture medium.
Statistical analysis
All data were represented as the mean ± SD of at least triplicate samples. Comparison between control and study groups was performed with Student’s t-test. Statistical significance was defined as *P < 0.05.
Genotoxicity assay
The method was evaluated on Chinese hamster lung fibroblasts (V79 cell line) according to the OECD guidelines for testing of chemicals no. 487 (2016)18 and M. Fenech (2007)19 (in vitro micronucleus assay).
Cell culture
V79 cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin. The cells were maintained in 75 cm² culture flasks at 37 °C in a 5% CO₂ atmosphere. Cells were harvested from the flasks by trypsinization using 0.25% trypsin-EDTA and then seeded into 6-well plates at a density of approximately 1 × 10⁵ cells/mL, with 3 mL of culture medium per well.
Cytokinesis-block micronucleus (CBMN) assay
In the absence of a metabolic activation system (-S9, without liver microsomes: S9 pooled from rat (Sprague-Dawley)), the cells were treated with 3 mL/well of compound 1 dissolved in culture medium at concentrations of 3.125 µg/mL, 6.25 µg/mL, 12.5 µg/mL, 25 µg/mL, and 50 µg/mL. Additionally, 3 mL/well of Mitomycin C (MMC) at a concentration of 1.25 µg/mL was used as the positive control, and 3 mL/well of culture medium was used as the negative control. This treatment was carried out for both short-term (4 h) and long-term (24 h) exposures. In the presence of a metabolic activation system (+ S9), Benzo(a)pyrene (BP) at a concentration of 5 µg/mL was used as the positive control, and the treatment was performed for long-term (24 h).
After a 4-hour exposure, 3 mL/well of cytochalasin B (Cyt B, 6 µg/mL) solution was added to each well. For the 24-hour exposure, 30 µL/well of cytochalasin B (Cyt B, 600 µg/mL) solution was added to each well, and the plate was gently swirled to ensure thorough mixing. After both exposure times, all plates were incubated at 37 °C in a 5% CO2 atmosphere for 18–20 h. At harvest, the cells were trypsinized using 0.25% trypsin-EDTA and washed twice with DPBS (1 mL/well). A hypotonic solution (0.56% w/v KCl) was then added, and the mixture was thoroughly mixed and allowed to settle at room temperature for 10 min, followed by centrifugation. The cells were resuspended in a fixation solution (300 mL of 0.9% w/v NaCl solution, 250 mL methanol, and 50 mL acetic acid) and incubated at 4 °C for 10 min. The slides were then stained with Fluoroshield™ containing 4′,6-diamidino-2-phenylindole (DAPI). The frequency of micronuclei (MN), defined as the number of micronucleated cells per 2,000 mononucleate cells (MNC) and binucleate cells (BNC), as well as the Cytokinesis-block proliferation index (CBPI) for each treatment, was microscopically examined using the Metafer scanning and imaging platform (Metafer®) according to Metafer procedures.
Cytokinesis-block proliferation index (CBPI) was calculated from the number of MNC and BNC within the same area on a slide which at least 500 cells per culture were scored. CBPI was calculated using following equation: CBPI = [(No. of MNC) + (2 x No. of BNC)]/Total No. of viable cells.
All experiments were performed in triplicate. MN frequencies and CBPI values were expressed as mean ± SD. The data were analyzed by one-way ANOVA and Tukey’s honestly significant difference test using the SPSS program. Significance of the data was considered at p < 0.05.
Results
Gibellula scorpioides BCC 39989 and its natural products
Gibellula scorpioides BCC 39989 (Fig. 1) was isolated as an invertebrate-pathogenic fungus from Portia sp. Phylogenetic relationships among BCC 39989 and other Gibellula species were inferred based on the analyses of ITS, LSU, tef, RPB1 and RPB2 sequences (Fig. 2). Gibellula scorpioides BCC 39989 was fermented using YMGB medium and the fermentation broth and mycelia were extracted with ethyl acetate. Purification of the broth and mycelial ethyl acetate extracts by successive chromatographic procedures yielded eight known metabolites (1–8) (Fig. 3). Their structures were elucidated by analysis of spectroscopic data, including IR, UV, NMR (Fig S1-S16) and MS (Fig S17-S24). All structures were further confirmed by comparison of the 1H and 13C NMR data (Table S1-S8) with those previously reported8–14. The relative configuration was assigned according to NOEDIFF data and/or coupling constants while the absolute configuration was determined by comparison of the specific rotation with those of the known compound.
Fig. 1.
Gibellula scorpioides (BBH 30255): a Fungus growing on Portia sp., b Colonies obverse on PDA at 25 °C after 20 days in white light/dark cycles in the laboratory, c Colonies reverse on PDA at 25 °C after 20 days in white light/dark cycles in the laboratory. Scale bars: a, b, c = 10 mm.
Fig. 2.
The phylogenetic tree based on RAxML analyses of combined ITS, LSU, tef, RPB1 and RPB2 sequence data. Bootstrap proportions/Bayesian posterior probabilities ≥ 70% are provided above corresponding nodes; nodes with 100% support are shown as thick lines.
Fig. 3.
Chemical structures of compounds 1–8 isolated from Gibellula scorpioides BCC 39989.
Biological assay
Anti-inflammatory activity
The isolated secondary metabolites, ergosterol peroxide (1) and methyl o-hydroxycinnamate (2), were evaluated for cytotoxicity, inhibition of nitric oxide (NO) production, and suppression of IL-6 and TNF-α secretion in LPS-stimulated RAW 264.7 macrophages. Cells were co-treated with LPS (1 µg/mL) and various concentrations of each compound. The results are shown in Figs. 4, 5 and 6. Compounds 1 and 2 were non-cytotoxic at concentrations of 15, 30, and 62.5 µg/mL, with RAW 264.7 cell viability remaining above 80% (Fig. 4). At higher concentrations (125–250 µg/mL), both compounds significantly reduced cell viability compared with the LPS control and the positive control (diclofenac), although the responses to LPS and diclofenac differed. Compound 1 was the most cytotoxic, decreasing viability to 57.5% at 250 µg/mL. Accordingly, only non-cytotoxic concentrations (15–62.5 µg/mL) were used in subsequent anti-inflammatory assays, including NO suppression and inhibition of IL-6 and TNF-α release. Within this concentration range, compound 2 at 62.5 µg/mL produced the greatest inhibition of NO production (83.88%), followed by compound 1 at 62.5 µg/mL (72.30%) and compound 2 at 30 µg/mL (67.66%) (Fig. 5). All three treatments showed significantly greater NO inhibition than diclofenac (40 µg/mL). Compound 2 at 62.5 µg/mL exhibited the highest activity (p < 0.001), significantly exceeding all other tested conditions. At 30 µg/mL, compound 1 showed moderate inhibition, which was statistically indistinguishable from diclofenac (p < 0.01), whereas compound 2 demonstrated a statistically significant effect at the same dose (p < 0.001). Both compounds at 15 µg/mL showed significantly lower inhibition than diclofenac (p < 0.001). IL-6 secretion displayed a similar trend. The lowest IL-6 level was observed with compound 1 at 62.5 µg/mL (3048.33 pg/mL), which was comparable to diclofenac (3060 pg/mL) and significantly lower than in LPS-stimulated cells. Compound 2 at 62.5 µg/mL also significantly reduced IL-6 relative to both the LPS control and diclofenac (p < 0.001), although its effect remained less potent than that of compound 1 at the same concentration (Fig. 6A). TNF-α secretion exhibited a distinct trend. Compound 2 produced the strongest inhibition across all concentrations examined, reducing TNF-α levels to 50.91–62.30 pg/mL, significantly lower than diclofenac (102.17 pg/mL), compound 1, and the LPS control (p < 0.001). In contrast, compound 1 exhibited only weak suppression of TNF-α, with levels remaining close to those of LPS-stimulated cells (Fig. 6B). In summary, compound 1 at 62.5 µg/mL was the most effective in suppressing IL-6 secretion, while compound 2 exhibited the strongest inhibitory effects on NO and TNF-α production. The superior activity of compound 2 across multiple inflammatory markers suggests a broader regulatory effect on LPS-induced inflammatory pathways. These findings underscore the distinct, concentration-dependent mechanisms by which the two compounds modulate inflammatory responses.
Fig. 4.
Cytotoxicity of compounds 1 and 2 in RAW 264.7 murine macrophage cells compared with vehicle control (DMSO), LPS (1 µg/mL), and the positive control diclofenac (40 µg/mL). Cell viability was assessed using the MTT assay. All values represent the mean ± SEM (n = 3). Statistical significance was evaluated using one-way ANOVA followed by Dunnett’s post hoc test. ***P < 0.001 versus diclofenac (40 µg/mL), LPS (1 µg/mL), and DMSO. ns = not significant versus diclofenac (40 µg/mL) and LPS (1 µg/mL).
Fig. 5.
Percentage inhibition of nitric oxide (NO) production in LPS-stimulated RAW 264.7 macrophages following pretreatment with various concentrations of compounds 1 and 2. Diclofenac (40 µg/mL) was used as the positive control. Data are expressed as the mean ± SEM (n = 3). Statistical significance was determined using one-way ANOVA followed by Dunnett’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. ns = not significant versus diclofenac (40 µg/mL). ###P < 0.001 indicates significantly lower inhibitory activity compared with diclofenac (40 µg/mL).
Fig. 6.
Concentrations of IL-6 [A] and TNF-α [B] secreted into the culture media of RAW 264.7 macrophages following pretreatment with various concentrations of compounds 1 and 2. Cytokine expression levels were quantified using ELISA kits. All values are presented as the mean ± SEM (n = 3). Statistical significance was assessed using one-way ANOVA followed by Dunnett’s post hoc test. ***P < 0.001 indicates significantly higher activity compared with diclofenac (positive control), whereas ###P < 0.001 indicates significantly lower activity. ns = not significant compared with diclofenac.
Lipid accumulation of 3T3-L1 adipocytes
The inhibitory effects of the tested compounds on lipid storage in adipocytes were evaluated using Oil Red O staining and a triglyceride (TG) reduction assay in differentiated 3T3-L1 adipocytes. Ergosterol peroxide (1), methyl o-hydroxycinnamate (2), and oleic acid (7) exhibited high cell viability across most concentrations tested, with survival values ranging from 99.85 to 113.41%, significantly higher than that of the Simvastatin control (93.53%, p < 0.001). Only compound 2 at 100 µg/mL showed reduced viability (86.60%), indicating a tendency toward cytotoxicity at the highest dose (Fig. 7). The effects of compounds 1, 2, and 7 on lipid accumulation, assessed using Oil Red O staining and TG content, are presented in Fig. 8. At concentrations of 20, 50, and 100 µg/mL, compounds 1 and 7 inhibited lipid deposition more effectively than Simvastatin in a dose-dependent manner, with compound 7 at 100 µg/mL producing the strongest inhibition among all treatments (p < 0.001). Compound 2 exhibited only moderate activity and was consistently less potent than compounds 1 and 7. At the lowest concentration (10 µg/mL), compound 7 was significantly less effective than Simvastatin, whereas compound 1 at 10 µg/mL showed no significant difference. Across all treatment groups, compound 7 at 10 µg/mL demonstrated the lowest activity relative to the positive control (Fig. 8A). All three compounds also produced dose-dependent reductions in intracellular TG content (Fig. 8B). Compound 1 displayed a clear dose-responsive increase in TG inhibition, with 50 µg/mL (28.78%) and 100 µg/mL (32.72%) producing significantly greater inhibition than Simvastatin (24.97%, p < 0.001). Compound 2 showed modest TG-lowering activity at all concentrations; although inhibition increased to 20.12% at 100 µg/mL, it remained significantly lower than that of Simvastatin (p < 0.01). Compound 7 exhibited a biphasic response: minimal inhibition at 10 µg/mL (5.14%), significantly lower than Simvastatin (p < 0.001), but strong inhibition at 100 µg/mL, exceeding the positive control (p < 0.001).
Fig. 7.
Cytotoxicity of compounds 1, 2, and 7 in 3T3-L1 preadipocytes. Cell viability following treatment with various concentrations of compounds 1, 2, and 7 was evaluated using the MTT assay. Data are expressed as the mean ± SEM (n = 3). Statistical significance was determined using one-way ANOVA followed by Dunnett’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 indicate significantly higher viability compared with Simvastatin (positive control), whereas #P < 0.05 indicates significantly lower viability. ns = not significant.
Fig. 8.
Effects of compounds 1, 2, and 7 on lipid accumulation in 3T3-L1 adipocytes. Percentage of lipid deposit inhibition [A] and triglyceride (TG) reduction [B] in 3T3-L1 cells treated with various concentrations of compounds 1, 2, and 7. All values are presented as the mean ± SEM (n = 3). Statistical significance was determined using one-way ANOVA followed by Dunnett’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 indicate significantly higher activity compared with Simvastatin (positive control), whereas #P < 0.05, ##P < 0.01, ###P < 0.001 indicate significantly lower activity. ns = not significant.
In the anti-inflammatory and anti-adipogenic assays, the tested compounds exhibited clear dose-dependent effects; however, evaluation at higher concentrations for IC₅₀ determination was not feasible due to cytotoxicity at elevated doses.
The effect of compounds 1 and 2 on SW480 cells
The effect of compounds 1 and 2 on SW480 cells was investigated by assessing their cytotoxicity after pretreatment. The cells were treated with these two compounds at designated concentrations for 48 h, followed by assessment of cell viability using the MTS assay. The results indicated that both compounds affected the cell viability of SW480 cells in a dose-dependent manner. The IC50 values of compounds 1 and 2 on SW480 cells were determined to be 13.33 µg/mL and 169.87 µg/mL, respectively, suggesting that compound 1 rather than compound 2 effectively inhibited the proliferation of SW480 cells. Interestingly, the percentage of cell viability was greater than 100% for SW480 cells treated with compound 1 at concentrations of 2.50 µg/mL and 5.00 µg/mL, and for cells treated with compound 2 at concentrations of 31.13 µg/mL and 62.25 µg/mL.
Genotoxicity
The effects of compound 1, negative, and positive controls were assessed on the V79 cell line in the presence (+ S9) and absence (-S9) of metabolic activation for both 4-hour and 24-hour treatments. MTT assay results showed no cytotoxic effects of compound 1 on V79 cell viability after a 4-hour treatment at concentrations ranging from 3.125 to 25 µg/mL. The concentration range was selected in accordance with OECD guideline recommendations. However, after 24 h, concentrations of 3.125 and 6.25 µg/mL reduced V79 cell viability, and a concentration of 12.5 µg/mL resulted in less than 70% viability, suggesting that higher concentrations and longer exposure times may impact cell survival, as shown in Table 2. The effect of compound 1 on V79 cells was assessed in the absence of a metabolic activation system (-S9) by measuring micronucleus (MN) frequencies and cytokinesis-block proliferation index (CBPI), as shown in Table 3. MN frequencies for compound 1 at concentrations of 3.125, 6.25, 12.5, 25, and 50 µg/mL were not significantly different from the negative control at both exposure times and were lower than the positive control. In addition, CBPI values for all concentrations of compound 1 were greater than 1, similar to the negative control. These results suggest that compound 1 is non-cytotoxic and non-genotoxic in the in vitro genotoxicity assay. In the presence of a metabolic activation system (+ S9) for a 4-hour treatment, MN frequency and CBPI were reported in Table 4. At all concentrations, MN frequencies for compound 1 were not significantly different from the negative control but were significantly lower than the positive control (BP 5 µg/mL). The CBPI values for compound 1 did not differ significantly from either the negative or positive control. Based on these results, compound 1 at concentrations ranging from 3.125 to 50 µg/mL did not exhibit a genotoxic effect in V79 cells, both with and without the metabolic activation system, as indicated by CBPI values greater than 118.
Table 2.
Cell survival percentages obtained after exposure of V79 cells to various concentrations of ergosterol peroxide (1) and their respective controls, as determined by MTT assay.
| Treatment (µg/mL) | cell viability of V79 (%) (Mean ± SEM) | |
|---|---|---|
| 4 h | 24 h | |
| Negative control | 100 | 100 |
| Positive control; MMC | 90.04 ± 2.26 | 83.11 ± 4.87 |
| Positive control; BP + S9 fraction | 81.35 ± 2.13 | - |
| 3.125 | 98.25 ± 1.48 | 95.87 ± 2.58 |
| 6.25 | 91.81 ± 0.91 | 84.74 ± 1.55 |
| 12.5 | 87.19 ± 1.73 | 53.26 ± 2.38 |
| 25 | 76.62 ± 0.89 | 18.19 ± 1.84 |
| 50 | 68.07 ± 1.23 | 10.93 ± 0.55 |
Table 3.
Micronucleus (MN) frequency and cytokinesis-block proliferation index (CBPI) observed in V79 cells treated with different concentrations of ergosterol peroxide (1) and their respective controls, without metabolic activation (-S9), at 4 and 24 h.
| Treatment (µg/mL) | MN frequency (cells/2,000 BNC, Mean ± SEM) |
CBPI (Mean ± SEM) |
||
|---|---|---|---|---|
| 4 h | 24 h | 4 h | 24 h | |
| Negative control | 25.08 ± 1.96 | 26.74 ± 3.42 | 1.67 ± 0.07 | 1.81 ± 0.08 |
| Positive control; MMC | 138.07 ± 0.55 | 158.96 ± 0.19 | 1.50 ± 0.09 | 1.28 ± 0.02 |
| 3.125 | 23.48 ± 1.58 | 24.25 ± 1.89 | 1.47 ± 0.16 | 1.87 ± 0.15 |
| 6.25 | 24.59 ± 1.18 | 23.81 ± 1.51 | 1.70 ± 0.06 | 1.61 ± 0.15 |
| 12.5 | 24.95 ± 0.30 | 24.60 ± 1.12 | 1.57 ± 0.11 | 1.88 ± 0.05 |
| 25 | 25.17 ± 3.94 | ND | 1.89 ± 0.04 | ND |
| 50 | 27.90 ± 2.79 | ND | 1.89 ± 0.01 | ND |
Table 4.
Micronucleus (MN) frequency and cytokinesis-block proliferation index (CBPI) observed in V79 cells treated with different concentrations of ergosterol peroxide (1) and their respective controls, with metabolic activation (+ S9), at 4 h.
| Treatment (µg/mL) | MN frequency (cells/2,000 BNC, Mean ± SEM) |
CBPI (Mean ± SEM) |
|---|---|---|
| Negative control | 19.59 ± 0.43 | 1.85 ± 0.07 |
| Positive control; BP | 67.05 ± 4.11 | 1.77 ± 0.08 |
| 3.125 | 17.59 ± 1.67 | 1.79 ± 0.18 |
| 6.25 | 19.47 ± 2.56 | 1.40 ± 0.02 |
| 12.5 | 19.79 ± 1.28 | 1.41 ± 0.13 |
| 25 | 18.32 ± 0.63 | 1.80 ± 0.12 |
| 50 | 19.40 ± 1.84 | 1.95 ± 0.01 |
Discussion
The effects of compounds 1 and 2 on RAW264.7 cells showed cytotoxicity at concentrations of 125 µg/mL and 250 µg/mL, where cell survival is less than 80%. However, at concentrations ranging from 15 µg/mL to 62.5 µg/mL, both 1 and 2 exhibited significant inhibition of LPS-induced NO, IL-6, and TNF-α production secreted from the cells in a dose-response manner. Notably, at concentrations of 30 µg/mL and 62.5 µg/mL, these compounds demonstrated greater inhibition of NO production than the positive control (Diclofenac). Additionally, at all concentrations, both compounds significantly suppressed LPS-induced TNF-α secretion and IL-6 expression in RAW264.7 cells. Remarkably, compound 2 displayed more potent inhibition of TNF-α cytokine secretion than compound 1 and the positive control (Diclofenac). Ergosterol peroxide (1) is a common fungal sterol previously reported from numerous edible and medicinal mushrooms, including Pleurotus ferulae20, Hericium erinaceum21, Fomitopsis dochmius22, Ganoderma lucidum, Gymnopus dryophilus23, Cordyceps cicadae24, and Cordyceps sinensis25, as well as several microscopic fungi such as marine-derived Phoma sp26. and Acrophialophora jodhpurensis27. It is recognized as a major anti-tumor sterol and has been shown to suppress LPS-induced TNF-α secretion and IL-1α/β expression in RAW 264.7 macrophages without affecting viability. Mechanistic studies indicate that ergosterol peroxide inhibits LPS-induced NF-κB p65 and C/EBPβ DNA-binding activity and dose-dependently reduces phosphorylation of the MAPKs p38, JNK, and ERK, leading to reduced TNF-α production via suppression of MAPK signaling and downstream transcription factors28. It also attenuates LPS-induced iNOS expression and has been reported to suppress TNF-α secretion29. Ergosterol peroxide isolated from Cryptoporus volvatus has also shown potent antiviral activity against PDCoV, inhibiting viral infection in LLC-PK1 cells and downregulating NF-κB and p38/MAPK signaling. This activity was accompanied by decreased expression of IL-1β, IL-6, IL-12, TNF-α, IFN-β, Mx1, and PKR compared with poly(I: C) treatment30, further highlighting its broad anti-inflammatory and antiviral potential. In contrast, the anti-inflammatory activity of methyl o-hydroxycinnamate (2) has not been previously reported. Only its structural analogue, methyl p-hydroxycinnamate, has been shown to inhibit pro-inflammatory mediators and cytokines, suppress iNOS and COX-2 expression31, and attenuate LPS-induced inflammation in vivo by reducing inflammatory cell recruitment, lowering TNF-α and IL-6 levels in bronchoalveolar lavage fluid, inhibiting p38 MAPK/NF-κB activation, and upregulating HO-132. Collectively, these findings indicate that compounds 1 and 2 suppress LPS-induced NO production and reduce IL-6 and TNF-α secretion, supporting their potential as anti-inflammatory agents.
In a previous study, oleic acid (7) and linoleic acid were found to enhance changes in morphology and lipid accumulation in 3T3-L1 preadipocytes during the differentiation process33. This effect was attributed to methylation of the C/Ebpα and Pparγ promoters, particularly when combined with insulin, thereby modulating lipid accumulation in adipogenesis in 3T3-L1 cells. Conversely, ergosterol peroxide (1) extracted from Ganoderma lucidum significantly inhibited the expression of C/EBPα and PPARγ, key factors in pre-adipocyte differentiation. Furthermore, it suppressed the activation of MAPK pathway-related genes in 3T3-L1 cells, inhibiting adipocyte differentiation34. Based on these studies, the effects of compounds 1, 2 and 7 on lipid accumulation in 3T3-L1 adipocytes were observed. All compounds effectively suppressed intracellular lipid deposition and intracellular triglyceride (TG) levels in combination with insulin induction, at high concentration (100 µg/mL), significantly more than the positive control (Simvastatin), suggesting potential involvement in the same pathway.
Ergosterol peroxide (1) has been reported to have anti-tumor activities, such as inhibiting the proliferation of HT29 colon adenocarcinoma cells28. It also exhibited anti-colon cancer effects by inhibiting cell proliferation and suppressing clonogenic colony formation in HCT116, HT-29, SW620, and DLD-1 CRC cell lines35. In this study, the antiproliferation of compounds 1 and 2 on SW480 cells was investigated. The results revealed that both compounds affected the cell viability of SW480 cells in a dose-dependent manner. Compound 1 displayed significantly greater antiproliferative activity against SW480 cells. In contrast, compound 2 exhibited reduced activity. Notably, treatment of SW480 cells with compounds 1 and 2 at certain low concentrations resulted in a percentage of cell viability greater than 100% suggesting that these two compounds exhibit a protective effect as they are non-genotoxic to the cells36. This is further supported by the finding that ergosterol peroxide (1) did not induce genotoxic effects on V79 cells, as indicated by CBPI values greater than 118, when tested with and without a metabolic activation system at concentrations ranging from 3.125 µg/mL to 50 µg/mL. Similarly, compounds 1 and 2 were administered at concentrations ranging from 15 µg/mL to 62.25 µg/mL on RAW 264.7 cells and at concentrations from 10 µg/mL to 50 µg/mL on the 3T3-L1 cell line, the percentage of cell viability was greater than 80%. However, their cytotoxic effects become dominant at higher concentrations. Importantly, this study provides the first report of the genotoxicity profile of ergosterol peroxide, offering critical insights for future in vivo investigations, including potential implications for acute oral and subchronic toxicity and supporting broader risk assessment. While the genotoxicity findings indicate cellular safety of compound 1, comprehensive in vivo evaluations remain essential to establish the margin of safety and determine whether the in vitro biological activities translate into pharmacologically and toxicologically meaningful effects.
In comparison with previously reported natural or pharmacological anti-inflammatory agents, the potencies observed for the isolated compounds, particularly ergosterol peroxide, are moderate but noteworthy, especially given their structural simplicity and natural abundance. While their in vitro activities suggest potential biological relevance, further optimization or formulation would likely be required to achieve clinically meaningful potency. Importantly, both ergosterol peroxide (1) and methyl o-hydroxycinnamate (2) have not been reported from any Gibellula species to date. Their identification expands the chemical diversity of the genus and underscores its value as an underexplored source of bioactive metabolites. Variations in metabolite profiles among Gibellula species may reflect differences in cultivation conditions, including culture medium composition and the use of epigenetic modifiers. Such differences are likely to contribute to the divergent chemical outputs observed across studies. Although ergosterol peroxide demonstrated a favorable safety profile in vitro, its translational potential remains preliminary. Key hurdles include the need for detailed in vivo toxicological assessment, pharmacokinetic characterization, and evaluation of therapeutic windows. Nonetheless, the consistently high levels of ergosterol peroxide in G. scorpioides highlight this species as a promising biological source for large-scale compound production, offering opportunities for future industrial or pharmaceutical development.
Conclusion
Eight known metabolites (1–8) were isolated from the Thai spider-parasitic fungus Gibellula scorpioides BCC 39989 and structurally characterized using spectroscopic techniques. The major metabolites, ergosterol peroxide (1) and methyl o-hydroxycinnamate (2) emerged as potent bioactive compounds. Both compounds exhibited potent anti-inflammatory activity by suppressing NO production and reducing IL-6 and TNF-α cytokine levels in LPS-stimulated RAW264.7 macrophages, with compound 2 demonstrating superior TNF-α inhibition. Additionally, compounds 1, 2, and oleic acid (7) significantly reduced lipid accumulation and intracellular triglyceride content in 3T3-L1 adipocytes in a dose-dependent manner. Compounds 1 and 2 also demonstrated antiproliferative effects against SW480 human colorectal adenocarcinoma cells at low concentrations, with compound 1 displaying stronger activity and minimal cytotoxicity. Notably, ergosterol peroxide (1) showed no genotoxic effects on V79 cells, both with and without metabolic activation, indicating a favorable safety profile. These findings highlight the potential of these metabolites as promising lead candidates for development as anti-inflammation, anti-obesity, and anti-cancer agents. This study represents the first comprehensive investigation of secondary metabolites from the Thai spider-parasitic fungus Gibellula scorpioides and underscores the rich biodiversity of Thai fungi as a valuable source of natural products with therapeutic potential.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
We are grateful to the Thailand Science Research and Innovation (TSRI) and Thammasat University Research Fund for a research grant. The Center of Excellence for Innovation in Chemistry (PERCH-CIC), Ministry of Higher Education, Science, Research and Innovation and Thammasat University are gratefully acknowledged for partial support.
Author contributions
U. Rerk-am: compound isolation, structure elucidation, writing-original draft preparation; B. Soontornworajit, Y. Tantirungrotechai: review and editing; P. Rotkrua: antiproliferation assay, review and editing; P. Kaemchantuek: compound isolation, structure elucidation; S. Laovitthayanggoon, P. Kengkwasingh: anti-inflammation, lipid accumulation of 3T3-L1 adipocytes and genotoxicity assays; A. Khonsanit, D. Thanakitpipattana, W. Kuephadungphan: isolation, phylogenetic analyses, review and editing; J.J. Luangsa-ard, J. Arunpanichlert: conceptualization, supervision, writing-original draft preparation, resources, review and editing.
Funding information
This work was supported by the Thailand Science Research and Innovation Fundamental Fund, Contract No. TUFF 20/2565; Thammasat University Research Fund, Contract No. TUFT-FF 16/2565; the Southeast Asia-Europe Joint Funding Scheme (SEA-Europe Grant No. JFS20ST-127, Acronym: Antiviralfun) Grant no. P21-50844, from the National Science and Technology Development Agency (NSTDA).
Data availability
All sequence data generated in this study (Table 1) are available in GenBank (https://www.ncbi.nlm.nih.gov/genbank/).
Declarations
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
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Contributor Information
Janet Jennifer Luangsa-ard, Email: jajen@biotec.or.th.
Jiraporn Arunpanichlert, Email: jira_550@tu.ac.th.
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Associated Data
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Supplementary Materials
Data Availability Statement
All sequence data generated in this study (Table 1) are available in GenBank (https://www.ncbi.nlm.nih.gov/genbank/).