This study characterized the phytochemical constituents and multi-faceted bioactivity of Heterotrigona apicalis propolis from East Kalimantan using UPLC–MS/MS, in vitro assays, gene-expression analysis, and in silico docking with ADMET predictions. Fifteen secondary metabolites were identified with predominantly phenolic acids, flavonoids, and prenylated chromans with some of them firstly reported. The extract displayed high total phenolic content (217.3 mg GAE/g), total flavonoid content (45.8 mg QE/g) and dose-dependent antioxidant activity (DPPH IC₅₀ = 227.1 µg/mL). In HepG2 cells, the crude extract exerted moderate selective cytotoxicity (MTT IC₅₀=32.6 µg/mL) and dose-dependent transcriptional effects: AKR1C3 was downregulated at low dose but upregulated at high dose, MAPK/ERK was suppressed at both doses, and NF-κB was mildly reduced. Molecular docking indicated that key constituents (kushenol B, sophoranodichromane D, kuwanon T) bind liver-relevant targets of glutathione reductase, AST/ALT, and HCV NS3/4A with favorable poses overlapping native ligands. In silico ADMET profiling largely met Lipinski rules of five, predicted good intestinal absorption, limited CNS exposure, and few hepatotoxicity alerts, though some compounds require further toxicity evaluation. Together, these results position H. apicalis propolis as a phenolic-rich source of multi-target hepatoprotective and chemopreventive candidates, meriting in vivo validation and detailed ADME/toxicity studies.

Background Type 2 Diabetes Mellitus (T2DM) is a rapidly increasing global health concern, closely associated with lifestyle-related factors such as obesity, dietary habits, and physical inactivity. Integrative approaches combining nutritional and life-style interventions may offer complementary strategies for improving metabolic outcomes under real-world clinical conditions. Objective This study aimed to evaluate the effects of a combined intervention including bee-derived products (propolis and bee bread), dietary modification, and structured physical activity on glycaemic control and metabolic parameters in patients with T2DM. Methods This pilot, practice-based observational study applied a within-subject design, in which each participant served as their own control. A total of 130 patients initiated the intervention, and 10 participants who completed the full protocol were included in the final analysis. The intervention consisted of daily supplementation with propolis (15 drops) and bee bread (10 g), a structured yoga programme (three sessions per week), and a progressive dietary intervention including timerestricted feeding over a three-month period. Primary outcome measures included fasting blood glucose (FBG), glycated haemoglobin (HbA1c), and liver function parameters (SGPT and SGOT). Results Significant improvements were observed in glycaemic and metabolic parameters. Mean fasting blood glucose decreased from 234.1 ± 71.5 mg/dL to 110.0 ± 23.7 mg/dL (p = .001), while HbA1c levels decreased from 9.5 ± 2.1% to 5.8 ± 0.6% (p = .001). Liver function parameters also improved, with reductions in SGPT and SGOT levels. Postprandial physical activity was associated with additional reductions in blood glucose levels. HPLC-DAD analysis of propolis identified bioactive compounds including caffeic acid phenethyl ester (CAPE) and chlorogenic acid. Conclusion The findings suggest that a combined intervention including bee-derived products, dietary modification, and structured physical activity may be associated with substantial improvements in metabolic parameters in patients with T2DM. These results support the potential relevance of integrative, multimodal approaches in real-world clinical settings. Further studies with larger sample sizes and optimised intervention protocols are required to confirm these findings.

Sesamolin, a lignan with known pharmacological benefits, has been extensively studied for its neuroprotective, antioxidative, and anticancer properties. This study builds upon previous investigations into the acute toxicity of sesamolin, presenting a detailed examination of its effects on zebrafish embryonic development through a proteomic approach. Zebrafish embryos were exposed to 25 and 50 μM concentrations of sesamolin for 72 h, resulting in significant developmental abnormalities, including yolk sac edema, spinal curvature, blood clots, and defects in erythropoiesis. Proteomic analysis revealed a dose-dependent downregulation of 162 proteins, including vital clusters such as vitellogenins, ribosomal proteins, cytoskeletal proteins, heat shock proteins, hemoglobin proteins, and ATP synthase. These alterations are linked to disrupted protein synthesis, impaired structural integrity, energy production deficits, and abnormal embryonic nutrient utilization. The study suggests that sesamolin exerts its toxic effects by interfering with key molecular mechanisms essential for normal embryogenesis, specifically affecting protein expression involved in development, cell structure, and metabolic processes. These findings provide novel insights into the potential adverse effects of sesamolin on embryonic development and highlight the need for further research to elucidate the underlying molecular pathways and assess the compound's broader toxicological risks, particularly in relation to human health.

Melanin, synthesized by tyrosinase (TYR), is a natural pigment essential for skin protection and pigmentation. However, excessive melanin production can cause dermatological disorders. Safflospermidines, comprised of safflospermidine A and B isomers isolated from sunflower (Helianthus annuus L.) bee pollen, were shown to exhibit a strong in vitro TYR inhibitory activity against mushroom TYR. However, their anti-melanogenesis activity in cellular and in vivo models remains unexplored. This study firstly evaluated the effects of these safflospermidines on melanogenesis in α-melanocyte stimulating hormone-stimulated B16F10 cells, using kojic acid as a positive control. Cytotoxicity was evaluated using the MTT assay, while TYR activity and melanin content were measured to assess melanogenesis inhibition. The expression of key melanogenesis-related genes was analyzed through quantitative real-time reverse transcription (RT-q)PCR to elucidate the molecular mechanisms involved. Secondly, the melanogenic activity and potential toxicity of the compounds were confirmed in zebrafish embryos, with phenylthiourea (PTU) as a reference. The results revealed that a mixture of these two safflospermidines exhibited no cytotoxicity across the treated concentration range (0-500 µg/mL). At 62.5 µg/mL, safflospermidines significantly reduced the intracellular melanin content by 21.78 ± 4.01% and the TYR activity by 25.71 ± 3.08% in B16F10 cells through downregulation of TYR, TYR-related protein 1 (TRP-1), and TRP-2 gene expression. Additionally, the safflospermidines induced a noticeable reduction in dendritic cell structures, which likely contributed to the marked decrease in extracellular melanin levels. Kojic acid (250 μg/mL) significantly reduced the melanin content and TYR activity by suppressing all three melanogenesis-related genes. In zebrafish embryos, safflospermidines showed no toxicity or morphological abnormalities within a concentration range of 0-62.5 µg/mL, and at a concentration of 15.63 µg/mL the melanin production in zebrafish embryos was significantly reduced by 28.43 ± 9.17%. In comparison, 0.0015% (v/v) PTU decreased melanin production by 53.20 ± 3.75%. These findings suggest that safflospermidines are safe, effective melanin inhibitors with potential applications in pharmaceuticals and cosmetics for managing hyperpigmentation.

Propolis from H. apicalis is a potent natural antioxidant and anticancer agent, but its poor solubility, low bioavailability, and instability have limited its use in food and nutraceutical applications. Although propolis nanocarriers exist, no study has yet used East Kalimantan propolis combined with a novel reversed TPP mixing method in a food-compatible chitosan matrix. Here, we report a novel modified ionic gelation method by first mixing East Kalimantan propolis with varying concentrations of TPP prior to incorporation into a chitosan solution. The formulation using the lowest TPP concentration resulted the smallest particle size, approximately 240 nm as measured by dynamic light scattering (DLS) and 78 nm by transmission electron microscopy (TEM), along with a stable zeta potential of 42.8 mV and a highest encapsulation efficiency of 97 %. All formulations exhibited enhanced antioxidant activity, with the lowest-TPP formulation achieving the highest DPPH radical scavenging activity (77 % inhibition) and showed a stable antioxidant capacity after 21 days of storages at different temperature. Moreover, this formulation followed a sustained release profile consistent with the Higuchi model and showed selective cytotoxicity against breast cancer (BT474) and hepatocellular carcinoma (HepG2) cells while maintaining over 100 % viability in RAW 264.7 macrophages. This scalable and food-grade nanoparticle system significantly enhances the stability and bioavailability of propolis, offering a promising platform for the development of natural antioxidant and anticancer nutraceuticals.

Malassezia globosa, a lipophilic pathogen, is known to be involved in various chronic skin diseases. Unfortunately, the available treatments have unwanted side effects and microbial drug resistance is evolving. As the antimicrobial activity of propolis is outstanding, this study aimed to examine the potential of propolis from the stingless bee Geniotrigona thoracica against the yeast. Anti-M. globosa growth activity was ascertained in agar well diffusion and broth microdilution assays and the inhibitory concentration value at 50% (IC50) was determined. Since the yeast cannot synthesize its own fatty acids, extracellular lipase is important for its survival. Here, anti-M. globosa extracellular lipase activity was additionally investigated by colorimetric and agar-based methods. Compared to the crude hexane and crude dichloromethane extracts, the crude methanol partitioned extract (CMPE) exhibited the best anti-M. globosa growth activity with an IC50 of 1.22 mg/mL. After CMPE was further enriched by silica gel column chromatography, fraction CMPE1 (IC50 of 0.98 mM or 184.93 μg/mL) presented the highest activity and was later identified as methyl gallate (MG) by nuclear magnetic resonance analysis. Subsequently, MG was successfully synthesized and shown to have a similar activity, and a minimal fungicidal concentration of 43.44 mM or 8.00 mg/mL. However, lipase assay analysis suggested that extracellular lipase might not be the main target mechanism of MG. This is the first report of MG as a new anti-Malassezia compound. It could be a good candidate for further developing alternative therapeutic agents.

The essential function of melanin is to protect our skin against harmful environmental factors. However, excessive melanin production can cause undesirable hyperpigmentation issues, such as freckles and melasma. Although several compounds are used to control melanin production by inhibiting tyrosinase (TYR), their efficacy is limited by skin-related adverse effects and cytotoxicity concerns. Consequently, searching for new natural compounds with an effective TYR inhibitor (TYR-I) activity but less harmful effects continues. Plant-based natural extracts are an alternative that are in great demand due to their safety and diverse biological properties. This study assessed ten isolated plant compounds for their TYR-I activities using an in vitro mushroom TYR inhibition assay. Among these compounds, piperine (400 μM) demonstrated the highest TYR-I activity, with a potency of 36.27 ± 1.96 %. Hence, this study examined the effect of piperine on melanogenesis in melanocyte stimulating hormone-treated B16F10 melanoma cells and using kojic acid as a positive reference. Cell viability was evaluated through the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Measurements of cellular TYR activity and melanin content were performed and related to changes in the transcriptional expression levels of melanogenesis-related genes, assessed via quantitative real-time reverse transcriptase (RT-q)PCR analysis. The results revealed that piperine at a concentration of 44 μM significantly reduced cellular TYR activity by 21.51 ± 2.00 % without causing cytotoxicity. Additionally, at the same concentration, piperine significantly decreased the intracellular melanin content by 37.52 ± 2.53 % through downregulating transcription levels of TYR and TYR-related protein 1 (TRP-1) but not TRP-2. Kojic acid, at a concentration of 1407 μM, induced a significant decrease in the melanin content and cellular TYR activity by suppressing all three melanogenesis-related genes. These findings suggest that piperine has potential as a potent depigmenting agent.

Gastric cancer is a global health concern, but current treatment with chemotherapy and surgery is often inadequate, prompting the exploration of alternative treatments. Propolis is a natural substance collected by bees known for its diverse properties linked to floral sources. The Dichloromethane Partitioned Extract (DPE) from Tetragonula laeviceps propolis, in Bankha district, Thailand was previously shown to possess significant cytotoxicity against KATO-III gastric cancer cells, while showing lower cytotoxicity toward WI-38 normal fibroblast cells. Here, the DPE was further fractionated by column chromatography, identified active fractions, and subjected to structural analysis using nuclear magnetic resonance spectroscopy. Cytotoxicity against KATO-III cells was reevaluated, and programmed cell death was analyzed using flow cytometry. Expression levels of cancer-related genes were measured using quantitative real-time reverse transcriptase PCR. Cardol C15:2 (compound 1) and mangiferolic acid (MF; compound 2) were discovered in the most active fractions following structural analysis. MF exhibited strong cytotoxicity against KATO-III cells (IC50 of 4.78–16.02 μg/mL), although this was less effective than doxorubicin (IC50 of 0.56–1.55 μg/mL). Morphological changes, including decreased cell density and increased debris, were observed in KATO-III cells treated with 30 μg/mL of MF. Significant induction of late-stage apoptosis and necrosis, particularly at 48 and 72 h, suggested potential DNA damage and cell cycle arrest, evidenced by an increased proportion of sub-G1 and S-phase cells. Doxorubicin, the positive control, triggered late apoptosis but caused more necrosis after 72 h. Furthermore, MF at 30 μg/mL significantly increased the expression level of COX2 and NFκB genes linked to inflammation and cell death pathways. This upregulation was consistent at later time points (48 and 72 h) and was accompanied by increased expression of CASP3 and CASP7 genes. These findings suggest MF effectively induces cell death in KATO-III cells through late apoptosis and necrosis, potentially mediated by upregulated inflammation-related genes.

Propolis is a natural product produced by bees and has antioxidant properties that depend on the local flora and the bee species. Propolis from two dominant stingless bee species in Thailand, Tetragonula laeviceps and Tetragonula pegdeni were collected from four locations in Ratchaburi province and one location in Chiangmai province, with different dominant plants at each location. Samples were extracted in methanol (MeOH) and sequentially partitioned with hexane, dichloromethane (DCM), and MeOH. All partitioned extracts were examined by three antioxidant methods: 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2 & PRIME;-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and ferric reducing ability (FRAP) assays. For the DPPH, the MeOH-partitioned extract of T. laeviceps from Bankha district (MPE7) was the most active [50% effective concentration (EC50) of 75.62 & mu;g/mL), while the DCM-partitioned extract of T. pegdeni from Suan phueng district (DPE2) was the most active in the ABTS and FRAP assays (EC50 of 22.06 & mu;g/mL and 10.23 mM/g, respectively). DPE2 also showed the highest phenolic and flavonoid contents (98.94 & mu;g gallic acid equivalents/mg dry weight and 286.07 & mu;g quercetin equivalents/mg, respectively). High performance liquid chromatography analysis revealed the identification and quantification of 12 phenolic compounds, with the highest concentration of vitexin in MPE7 and MPE5. Interestingly, kaempferol (5.16 & mu;g/mg) was only detected in DPE2. The different antioxidant properties among sites are expected due to the specific flora in each location. Furthermore, the bioactivity of propolis extracts differs significantly between bee species in a similar location, suggesting that they have different flora preferences. These results verify the potential of extracts to effectively scavenge radicals.

Four new alkylamides named retroframides A-D (1-4) together with twenty-two known compounds were isolated from the fruits of Piper rectrofractum. The structures of new compounds were elucidated on the basis of spectroscopic data including 2D NMR and chemical derivatization followed by GC-MS analysis. Of isolated compounds, piperine (25) and pellitorine (26) revealed moderate inhibition against tyrosinase with percentage inhibition of 36.1 and 40.7.

This study examined the osmoregulatory responses to hypo-osmotic shock in the commercially and ecologically important crab Episesarma mederi (H. Milne Edwards, 1853). After the acclimation for one week at a salinity of 25 PSU, Adult males E. mederi were immediately exposed to salinities of 5 PSU and 25 PSU (the control group). The time course of changes in haemolymph osmolality, gill Na+/K+ ATPase (NKA) activity, oxygen uptake rates, and mRNA expression levels of ion-transport related genes, including the NKA-α subunit, V-type H+ATPase (VT) and Na+/K+/2Cl-(NKCC), were determined. The results showed that E. mederi was a strong hyperosmoregulator after exposure to 5 PSU, achieved by modulations of NKA activity in their posterior gills rather than the anterior gills. The crabs acclimated to 5 PSU increased oxygen uptake, especially during the initial exposure, reflecting increased energetic costs for osmotic stress responses. In the posterior gills, the NKA activities of the crabs acclimated to 5 PSU at 3, 72 and 168 h were significantly higher than those in the control group. Elevated NKA-α subunit expression levels were detected at 6 h and 12 h. Increased expression levels of VT and NKCC were identified at 6 h and 12 h, respectively. Our results indicate that elevated gill NKA activity at 3 h could result from enzyme activity and kinetic alterations. On the other hand, the gill NKA activity at 72 and 168 h was sustained by elevated NKA-α subunit expression. Hence, these adaptive responses in osmoregulation enable the crabs to withstand hypo-osmotic challenges and thrive in areas of fluctuating salinity in mangroves and estuaries.

Osteoblast-like cells and human mesenchymal stem cells (hMSCs) are frequently employed as osteoprogenitor cell models for evaluating novel biomaterials in bone healing and tissue engineering. In this study, the characterization of UE7T-13 hMSCs and MG-63 human osteoblast-like cells was examined. Both cells can undergo osteogenesis and produce calcium extracellular matrix; however, calcium nodules produced by MG-63 lacked a central mass and appeared flatter than UE7T-13. The absence of growing calcium nodules in MG-63 was discovered by SEM-EDX to be associated with the formation of alternating layers of cells and calcium extracellular matrix. The nanostructure and composition analysis showed that UE7T-13 had a finer nanostructure of calcium nodules with a higher calcium/phosphate ratio than MG-63. Both cells expressed high intrinsic levels of collagen type I alpha 1 chain, while only UE7T-13 expressed high levels of alkaline phosphatase, biomineralization associated (ALPL). High ALP activity in UE7T-13 was not further enhanced by osteogenic induction, but in MG-63, low intrinsic ALP activity was greatly induced by osteogenic induction. These findings highlight the differences between the two immortal osteoprogenitor cell lines, along with some technical notes that should be considered while selecting and interpreting the pertinent in vitro model.

Sesamin, the major lignan in sesame seeds (Sesamum indicum L.), is known to have several pharmaceutical ac-tivities. However, its toxicological profile is still limited, especially regarding embryotoxicity. This study aimed to evaluate the developmental toxicity of sesamin in zebrafish embryos. After 72 h exposure, sesamin did not affect the survival and hatching rates, nor did it cause malformation in zebrafish embryos. Cardiotoxicity was also evaluated by monitoring embryo heartbeats and erythrocyte staining using o-dianisidine. The results showed that sesamin did not affect heart morphology, heart rate, or cardiac output in zebrafish embryos. The present study also evaluated sesamin's anti-angiogenesis, antioxidant and anti-inflammation activities. Sesamin signifi-cantly decreased the sub-intestinal vessel plexus as revealed by alkaline phosphatase staining indicating the compound exhibited anti-angiogenesis activity. For the antioxidant and anti-inflammatory assays, oxidative stress and inflammation in zebrafish embryos were induced by hydrogen peroxide and lipopolysaccharide, respectively. The reactive oxygen species (ROS) and nitric oxide (NO) production were detected using a fluo-rescent dye. Sesamin significantly decreased ROS and NO production in zebrafish embryos. In addition, the transcription examination by qRT-PCR of oxidative-and inflammation-related genes showed that sesamin affected the genes in a manner that correlated with results from the efficacy assays. In conclusion, the present study revealed that sesamin did not cause embryotoxicity and cardiotoxicity in zebrafish embryos. In addition, it exhibited evidence of anti-angiogenesis, antioxidant and anti-inflammatory activities.

Propolis is one of the economic bee products with biological activities, but these activities can vary according to the local plants and bee species. This study aimed to evaluate the cytotoxic and antityrosinase activity of the methanol-, hexane-, and dichloromethane-partitioned propolis extracts (MPE, HPE, and DPE, respectively) of two dominant stingless bee species in Thailand (Tetragonula laeviceps and Tetragonula pegdeni) sourced from four locations in Ratchaburi province and one location in Chiangmai province. Their antiproliferative/cytotoxic activity, as the relative cell viability, was screened against the liver (Hep-G2) and gastric carcinoma (KATO-III) cancer cell lines in comparison to the untransformed lung fibroblast (WI-38) cell line using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Interestingly, DPE7 was the extract that showed great potential anticancer properties because it was significantly higher cytotoxic to cancer cell lines (Hep-G2 and KATO-III, with IC50 values of 36.40 and 35.15 mg/mL, respectively) than to normal cell lines (WI-38, with IC50 values of 46.52 mg/mL). Although DPE7 did not exhibit the highest antityrosinase activity, its moderate antityrosinase activity (IC50 of 1.388 mg/mL) considered it possible for further purification though not as effective as Kojic acid (IC50 of 0.0432 mg/mL). Besides, the different bioactivities in propolis from different sites were expected due to the different flora in each location. However, further studies are needed to better understand the properties and safety aspects of selected partitioned extracts.

Sesamolin is one of the major active compounds found in sesame seeds (Sesamum indicum L.) that are commonly and increasingly used as an ingredient in cuisines and various food products. The compound has been reported to have several pharmaceutical activities such as antioxidant, antimicrobial, neuroprotective, and anticancer. However, the toxicological profile of sesamolin does not currently include developmental toxicity. In this study, we assessed sesamolin toxicity to embryonic development of zebrafish by exposure for 72 h at concentrations ranging from 10 to 50 μM. The evaluation revealed that sesamolin did not affect survival and hatching rates. However, it did induce embryo malformations and reduced embryonic heart rates in a dose-dependent manner. By qRT-PCR analysis, it downregulated the expression of oxidative stress-related genes, including superoxide dismutase 1 (sod1), catalase (cat), and glutathione S-transferase pi 2 (gstp2). Alkaline phosphatase staining of embryos revealed that sesamolin inhibited the development of subintestinal vessels, and hemoglobin staining revealed a negative impact on embryonic erythropoiesis. These findings showed that sesamolin affected genes related to angiogenesis and erythropoiesis. The risks of sesamolin to embryonic development found in this study may imply similar effects in humans and other mammals.

In the western honey bee (Apis mellifera) populations, cytosolic malate dehydrogenase (cMDH) allele frequencies are correlated with the environmental temperatures.The Slow and Fast alleles predominate in hot climates; the Medium allele predominates in cold climates.We wondered whether natural selection has had anything to do with the Medium allele in the imported exotic A. mellifera population in Thailand.We genotyped workers (n = 1032) from 86 commercial colonies from three regions.Three alleles: Fast, Medium, and Slow, were detected.Over 96% of the Thai A. mellifera are either homozygous for the Slow or heterozygous for the Fast allele; and the Medium allele has the lowest frequency (c.a.10%) in all sampled population.This might be indicative of selection against the Medium allele.However, as the cMDH allele frequencies in the Thai A. mellifera are similar to those seen in the source populations in the United States and Northern Italy, it might also be that the observed frequencies are reflective of frequent imports from the United States, and that natural selection is unable to overcome the effects of migration.

Bee pollen (BP) is full of nutrients and phytochemicals, and so it is widely used as a health food and alternative medicine.Its composition and bioactivity mainly depend on the floral pollens.In this work, BP collected by Apis mellifera with different monoculture flowering crops (BP1-6) were used.The types of floral pollen in each BP were initially identified by morphology, and subsequently confirmed using molecular phylogenetic analysis.Data from both approaches were consistent and revealed each BP to be monofloral and derived from the flowers of Camellia sinensis L., Helianthus annuus L., Mimosa diplotricha, Nelumbo nucifera, Xyris complanata, and Ageratum conyzoides for BP1 to BP6, respectively.The crude extracts of all six BPs were prepared by sequential partition with methanol, dichloromethane (DCM), and hexane.The crude extracts were then tested for the in vitro (i) α-amylase inhibitory, (ii) acetylcholinesterase inhibitory (AChEI), and (iii) porcine pancreatic lipase inhibitory (PPLI) activities in terms of the percentage enzyme inhibition and half maximum inhibitory concentration (IC 50 ).The DCM partitioned extract of X. complanata BP (DCMXBP) had the highest active α-amylase inhibitory activity with an IC 50 value of 1,792.48 ± 50.56 μg/mL.The DCM partitioned extracts of C. sinensis L. BP (DCMCBP) and M. diplotricha BP (DCMMBP) had the highest PPLI activities with an IC 50 value of 458.5 ± 13.4 and 500.8± 24.8 μg/mL, respectively), while no crude extract showed any marked AChEI activity.Here, the in vitro PPLI activity was focused on.Unlike C. sinensis L. BP, there has been no previous report of M. diplotricha BP having PPLI activity.Hence, DCMMBP was further fractionated by silica gel 60 column chromatography, pooling fractions with the same thin layer chromatography profile.The pooled fraction of DCMMBP2-1 was found to be the most active (IC 50 of 52.6 ± 3.5 μg/mL), while nuclear magnetic resonance analysis revealed the presence of unsaturated free fatty acids.Gas chromatography with flame-ionization detection analysis revealed the major fatty acids