Effects of hypo-osmotic shock on osmoregulatory responses and expression levels of selected ion transport-related genes in the sesarmid crab Episesarma mederi (H. Milne Edwards, 1853).

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.

Thermal adaptation in the honeybee (Apis mellifera) via changes to the structure of malate dehydrogenase.

In honeybees there are three alleles of cytosolic malate dehydrogenase gene: F, M and S. Allele frequencies are correlated with environmental temperature, suggesting that the alleles have temperature-dependent fitness benefits. We determined the enzyme activity of each allele across a range of temperatures in vitro The F and S alleles have higher activity and are less sensitive to high temperatures than the M allele, which loses activity after incubation at temperatures found in the thorax of foraging bees in hot climates. Next, we predicted the protein structure of each allele and used molecular dynamics simulations to investigate their molecular flexibility. The M allozyme is more flexible than the S and F allozymes at 50°C, suggesting a plausible explanation for its loss of activity at high temperatures, and has the greatest structural flexibility at 15°C, suggesting that it can retain some enzyme activity at cooler temperatures. MM bees recovered from 2 h of cold narcosis significantly better than all other genotypes. Combined, these results explain clinal variation in malate dehydrogenase allele frequencies in the honeybee at the molecular level.

Antibacterial activity of a cardanol from Thai Apis mellifera propolis.

BACKGROUND: Propolis is a sticky, dark brown resinous residue made by bees that is derived from plant resins. It is used to construct and repair the nest, and in addition possesses several diverse bioactivities. Here, propolis from Apis mellifera from Nan province, Thailand, was tested for antibacterial activity against Gram(+ve) (Staphylococcus aureus and Paenibacillus larvae) and Gram(-ve) (Escherichia coli) bacteria. MATERIALS AND METHODS: The three bacterial isolates were confirmed for species designation by Gram staining and analysis of the partial sequence of 16S rDNA. Propolis was sequentially extracted by methanol, dichloromethane and hexane. The antibacterial activity was determined by agar well diffusion and microbroth dilution assays using streptomycin as a positive control. The most active crude extract was further purified by quick column and adsorption chromatography. The apparent purity of each bioactive fraction was tested by thin layer chromatography. The chemical structure of the isolated bioactive compound was analyzed by nuclear magnetic resonance (NMR). RESULTS: Crude methanol extract of propolis showed the best antibacterial activity with a minimum inhibition concentration (MIC) value of 5 mg/mL for S. aureus and E. coli and 6.25 mg/mL for P. larvae. After quick column chromatography, only three active fractions were inhibitory to the growth of S. aureus and E. coli with MIC values of 6.25 and 31.3 µg/mL, respectively. Further adsorption chromatography yielded one pure bioactive fraction (A1A) with an IC50 value of 0.175 µg/mL for E. coli and 0.683 µg/mL for P. larvae, and was determined to be cardanol by NMR analysis. Scanning and transmission electron microscopy analysis revealed unusual shaped (especially in dividing cells), damaged and dead cells in cardanol-treated E. coli. CONCLUSION: Thai propolis contains a promising antibacterial agent.

Anti-Malassezia globosa (MYA-4889, ATCC) activity of Thai propolis from the stingless bee Geniotrigona thoracica.

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.

In vitro cytotoxic activity on KATO-III cancer cell lines of mangiferolic acid purified from Thai Tetragonula laeviceps propolis.

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.

Expression of a secretory α-glucosidase II from Apis cerana indica in Pichia pastoris and its characterization.

BACKGROUND: α-glucosidase (HBGase) plays a key role in hydrolyzing α-glucosidic linkages. In Apis mellifera, three isoforms of HBGase (I, II and III) have been reported, which differ in their nucleotide composition, encoding amino acid sequences and enzyme kinetics. Recombinant (r)HBGase II from A. cerana indica (rAciHBGase II) was focused upon here due to the fact it is a native and economic honeybee species in Thailand. The data is compared to the two other isoforms, AciHBGase I and III from the same bee species and to the three isoforms (HBGase I, II and III) in different bee species where available. RESULTS: The highest transcript expression level of AciHBGase II was found in larvae and pupae, with lower levels in the eggs of A. cerana indica but it was not found in foragers. The full-length AciHBGase II cDNA, and the predicted amino acid sequence it encodes were 1,740 bp and 579 residues, respectively. The cDNA sequence was 90% identical to that from the HBGase II from the closely related A. cerana japonica (GenBank accession # NM_FJ752630.1). The full length cDNA was directionally cloned into the pPICZαA expression vector in frame with a (His)(6) encoding C terminal tag using EcoRI and KpnI compatible ends, and transformed into Pichia pastoris. Maximal expression of the rAciHBGase II-(His)(6) protein was induced by 0.5% (v/v) methanol for 96 h and secreted into the culture media. The partially purified enzyme was found to have optimal α-glucosidase activity at pH 3.5 and 45°C, with > 80% activity between pH 3.5-5.0 and 40-55°C, and was stabile (> 80% activity) at pH 4-8 and at < 25-65°C. The optimal substrate was sucrose. CONCLUSIONS: Like in A. mellifera, there are three isoforms of AciHBGase (I, II and III) that differ in their transcript expression pattern, nucleotide sequences and optimal enzyme conditions and kinetics.

Four new alkylamides from the fruits of Piper retrofractum and antityrosinase evaluation.

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.

Distinctions in bone matrix nanostructure, composition, and formation between osteoblast-like cells, MG-63, and human mesenchymal stem cells, UE7T-13.

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 lacks zebrafish embryotoxicity but exhibits evidence of anti-angiogenesis, anti-oxidant and anti-inflammatory activities.

Sesamin, the major lignan in sesame seeds (Sesamum indicum L.), is known to have several pharmaceutical activities. 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 significantly 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 fluorescent 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.

Chemical constituents and free radical scavenging activity of corn pollen collected from Apis mellifera hives compared to floral corn pollen at Nan, Thailand.

BACKGROUND: Bee pollen is composed of floral pollen mixed with nectar and bee secretion that is collected by foraging honey (Apis sp.) and stingless bees. It is rich in nutrients, such as sugars, proteins, lipids, vitamins and flavonoids, and has been ascribed antiproliferative, anti-allergenic, anti-angiogenic and free radical scavenging activities. This research aimed at a preliminary investigation of the chemical constituents and free radical scavenging activity in A. mellifera bee pollen. METHODS: Bee pollen was directly collected from A. mellifera colonies in Nan province, Thailand, in June, 2010, whilst floral corn (Zea mays L.) pollen was collected from the nearby corn fields. The pollen was then sequentially extracted with methanol, dichloromethane (DCM) and hexane, and each crude extract was tested for free radical scavenging activity using the DPPH assay, evaluating the percentage scavenging activity and the effective concentration at 50% (EC50). The most active crude fraction from the bee pollen was then further enriched for bioactive components by silica gel 60 quick and adsorption or Sephadex LH-20 size exclusion chromatography. The purity of all fractions in each step was observed by thin layer chromatography and the bioactivity assessed by the DPPH assay. The chemical structures of the most active fractions were analyzed by nuclear magnetic resonance. RESULTS: The crude DCM extract of both the bee corn pollen and floral corn pollen provided the highest active free radical scavenging activity of the three solvent extracts, but it was significantly (over 28-fold) higher in the bee corn pollen (EC(50) = 7.42 ± 0.12 µg/ml), than the floral corn pollen (EC(50) = 212 ± 13.6% µg/ml). After fractionation to homogeneity, the phenolic hydroquinone and the flavone 7-O-R-apigenin were found as the minor and major bioactive compounds, respectively. Bee corn pollen contained a reasonably diverse array of nutritional components, including biotin (56.7 µg/100 g), invert sugar (19.9 g/100 g), vitamin A and ß carotene (1.53 mg/100 g). CONCLUSIONS: Bee pollen derived from corn (Z. mays), a non-toxic or edible plant, provided a better free radical scavenging activity than floral corn pollen.

In vitro antiproliferative/cytotoxic activity on cancer cell lines of a cardanol and a cardol enriched from Thai Apis mellifera propolis.

BACKGROUND: Propolis is a complex resinous honeybee product. It is reported to display diverse bioactivities, such as antimicrobial, anti-inflammatory and anti-tumor properties, which are mainly due to phenolic compounds, and especially flavonoids. The diversity of bioactive compounds depends on the geography and climate, since these factors affect the floral diversity. Here, Apis mellifera propolis from Nan province, Thailand, was evaluated for potential anti-cancer activity. METHODS: Propolis was sequentially extracted with methanol, dichloromethane and hexane and the cytotoxic activity of each crude extract was assayed for antiproliferative/cytotoxic activity in vitro against five human cell lines derived from duet carcinoma (BT474), undifferentiated lung (Chaco), liver hepatoblastoma (Hep-G(2)), gastric carcinoma (KATO-III) and colon adenocarcinoma (SW620) cancers. The human foreskin fibroblast cell line (Hs27) was used as a non-transformed control. Those crude extracts that displayed antiproliferative/cytotoxic activity were then further fractionated by column chromatography using TLC-pattern and MTT-cytotoxicity bioassay guided selection of the fractions. The chemical structure of each enriched bioactive compound was analyzed by nuclear magnetic resonance and mass spectroscopy. RESULTS: The crude hexane and dichloromethane extracts of propolis displayed antiproliferative/cytotoxic activities with IC(50) values across the five cancer cell lines ranging from 41.3 to 52.4 µg/ml and from 43.8 to 53.5 µg/ml, respectively. Two main bioactive components were isolated, one cardanol and one cardol, with broadly similar in vitro antiproliferation/cytotoxicity IC(50) values across the five cancer cell lines and the control Hs27 cell line, ranging from 10.8 to 29.3 µg/ml for the cardanol and < 3.13 to 5.97 µg/ml (6.82 - 13.0 µM) for the cardol. Moreover, both compounds induced cytotoxicity and cell death without DNA fragmentation in the cancer cells, but only an antiproliferation response in the control Hs27 cells However, these two compounds did not account for the net antiproliferation/cytotoxic activity of the crude extracts suggesting the existence of other potent compounds or synergistic interactions in the propolis extracts. CONCLUSION: This is the first report that Thai A. mellifera propolis contains at least two potentially new compounds (a cardanol and a cardol) with potential anti-cancer bioactivity. Both could be alternative antiproliferative agents for future development as anti-cancer drugs.

Phytochemical content, especially spermidine derivatives, presenting antioxidant and antilipoxygenase activities in Thai bee pollens.

Background: Bee pollen (BP) is full of useful nutrients and phytochemicals.Its chemical components and bioactivities depend mainly on the type of floral pollen. Methods: Monofloral BP from Camellia sinensis L., Mimosa diplotricha, Helianthus annuus L., Nelumbo nucifera, Xyris complanata, and Ageratum conyzoides were harvested. Crude extraction and partition were performed to yield solvent-partitioned extracts of each BP. Total phenolic content (TPC) was assayed by the Folin-Ciocalteu method, while the flavonoid content (FC) was measured by the aluminium chloride colorimetric method. Antioxidant capacity was measured by the (i) 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, (ii) 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) scavenging activity and its Trolox equivalent antioxidant capacity (TEAC), and (iii) ferric reducing antioxidant power (FRAP). All samples were tested for lipoxygenase inhibitory (LOXI) activity. The most active sample was enriched by silica gel 60 column chromatography (SiG60-CC) and high performance liquid chromatography (HPLC), observing the chemical pattern of each fraction using thin layer chromatography. Chemical structure of the most active compound was analyzed by proton nuclear magnetic resonance and mass spectrometry. Results: Dichloromethane (DCM)-partitioned BP extracts of H. annuus L. and M. diplotricha (DCMMBP) showed a very high TPC, while DCMMBP had the highest FC. In addition, DCMMBP had the strongest DPPH and ABTS radical scavenging activities (as a TEAC value), as well as FRAP value. Also, DCMMBP (60 µg/mL) gave the highest LOXI activity (78.60 ± 2.81%). Hence, DCMMBP was chosen for further enrichment by SiG60-CC and HPLC. Following this, the most active fraction showed higher antioxidant andLOXI activities with an EC50 for DPPH and ABTS of 54.66 ± 3.45 µg/mL and 24.56 ± 2.99 µg/mL (with a TEAC value of 2,529.69 ± 142.16 µmole TE/g), respectively, and a FRAP value of 3,466.17 ± 81.30 µmole Fe2+/g and an IC50 for LOXI activity of 12.11 ± 0.36 µg/mL. Triferuloyl spermidines were revealed to be the likely main active components. Conclusions: TPC, FC, and spermidine derivatives played an important role in the antioxidant and antilipoxygenase activities in M. diplotricha bee pollen.

First report of fatty acids in Mimosadiplotricha bee pollen with in vitro lipase inhibitory activity.

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 (IC50). The DCM partitioned extract of X. complanata BP (DCMXBP) had the highest active α-amylase inhibitory activity with an IC50 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 IC50 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 (IC50 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 included one saturated acid (palmitic acid) and two polyunsaturated acids (linoleic and linolenic acids). In contrast, the pooled fraction of DCMMBP2-2 was inactive but pure, and was identified as naringenin, which has previously been reported to be present in M. pigra L. Thus, it can be concluded that naringenin was compound marker for Mimosa BP. The fatty acids in BP are nutritional and pose potent PPLI activity.

Developmental effects of sesamolin on zebrafish (Danio rerio) embryos.

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 vitro antiproliferative activity of partially purified Trigona laeviceps propolis from Thailand on human cancer cell lines.

BACKGROUND: Cancers are some of the leading causes of human deaths worldwide and their relative importance continues to increase. Since an increasing proportion of cancer patients are acquiring resistance to traditional chemotherapeutic agents, it is necessary to search for new compounds that provide suitable specific antiproliferative affects that can be developed as anticancer agents. Propolis from the stingless bee, Trigona laeviceps, is one potential interesting source that is widely available and cultivatable (as bee hives) in Thailand. METHODS: Propolis (90 g) was initially extracted by 95% (v/v) ethanol and then solvent partitioned by sequential extractions of the crude ethanolic extract with 40% (v/v) MeOH, CH2Cl2 and hexane. After solvent removal by evaporation, each extract was solvated in DMSO and assayed for antiproliferative activity against five cancer (Chago, KATO-III, SW620, BT474 and Hep-G2) and two normal (HS27 fibroblast and CH-liver) cell lines using the MTT assay. The cell viability (%) and IC50 values were calculated. RESULTS: The hexane extract provided the highest in vitro antiproliferative activity against the five tested cancer cell lines and the lowest cytotoxicity against the two normal cell lines. Further fractionation of the hexane fraction by quick column chromatography using eight solvents of increasing polarity for elution revealed the two fractions eluted with 30% and 100% (v/v) CH2Cl2 in hexane (30DCM and 100DCM, respectively) had a higher anti-proliferative activity. Further fractionation by size exclusion chromatography lead to four fractions for each of 30DCM and 100DCM, with the highest antiproliferative activity on cancer but not normal cell lines being observed in fraction# 3 of 30DCM (IC50 value of 4.09 - 14.7 µg/ml). CONCLUSIONS: T. laeviceps propolis was found to contain compound(s) with antiproliferative activity in vitro on cancer but not normal cell lines in tissue culture. The more enriched propolis fractions typically revealed a higher antiproliferative activity (lower IC50 value). Overall, propolis from Thailand may have the potential to serve as a template for future anticancer-drug development.

Betulinic acid decreases lipid accumulation in adipogenesis-induced human mesenchymal stem cells with upregulation of PGC-1α and UCP-1 and post-transcriptional downregulation of adiponectin and leptin secretion.

BACKGROUND: Controlling cellular functions, including stem cell growth and differentiation, can be the key for the treatment of metabolic disorders, such as type II diabetes mellitus (T2DM). Previously identified as peroxisome proliferator-activated receptor gamma (PPARγ) antagonist, betulinic acid (BA) may have the capability to control stem cell homeostasis, benefiting T2DM treatment. In this study, the effects of BA on osteogenesis and adipogenesis mechanisms of human mesenchymal stem cells (hMSCs) were investigated. RESULTS: We observed that BA increased hMSC osteogenesis by enhancing the alkaline phosphatase activity, calcium deposition, and mRNA expressions of osteogenic markers, namely, runt-related transcription factor 2, osteocalcin, and osteopontin. In addition, BA decreased hMSC adipogenesis with the decrease in glycerol-3-phosphate dehydrogenase activity, reduced intracellular lipid accumulations, down-regulated CCAAT-enhancer-binding protein alpha, and suppressed post-transcriptional adiponectin and leptin secretion. BA increased the brown adipocyte characteristics with the increase in the ratio of small lipid droplets and glucose uptake. Furthermore, the mRNA expressions of brown adipocyte markers, namely, PPARγ coactivator one alpha, uncoupling protein 1, and interleukin-6 increased. CONCLUSIONS: Our results uncovered the mechanisms of how BA improved glucose and lipid metabolisms by decreasing white adipogenesis and increasing brown adipogenesis. Altogether, BA may be used for balancing glucose metabolisms without the potential side effects on bone loss or weight gain.

Safflospermidines from the bee pollen of Helianthus annuus L. exhibit a higher in vitro antityrosinase activity than kojic acid.

BACKGROUND: Ozone deterioration in the atmosphere has become a severe problem causing overexposure of ultraviolet light, which results in humans in melanin overproduction and can lead to many diseases, such as skin cancer and melasma, as well as undesirable esthetic appearances, such as freckles and hyperpigmentation. Although many compounds inhibit melanin overproduction, some of them are cytotoxic, unstable, and can cause skin irritation. Thus, searching for new natural compounds with antityrosinase activity and less/no side effects is still required. Here, bee pollen derived from sunflower (Helianthus annuus L.) was evaluated. MATERIALS AND METHODS: Sunflower bee pollen (SBP) was collected from Apis mellifera bees in Lopburi province, Thailand in 2017, extracted by methanol and sequentially partitioned with hexane and dichloromethane (DCM). The in vitro antityrosinase activity was evaluated using mushroom tyrosinase and the half maximal inhibitory concentration (IC50) is reported. The antioxidation activity was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and reported as the half maximal effective concentration. Two pure compounds with antityrosinase activity were isolated by silica gel 60 column chromatography (SG60CC) and high performance liquid chromatography (HPLC), and their chemical structure deduced by Nuclear Magnetic Resonance (NMR) analysis. RESULTS: The DCM partitioned extract of SBP (DCMSBP) had an antityrosinase activity (IC50, 159.4 µg/mL) and was fractionated by SG60CC, providing five fractions (DCMSBP1-5). The DCMSBP5 fraction was the most active (IC50 = 18.8 µg/mL) and further fractionation by HPLC gave two active fractions, revealed by NMR analysis to be safflospermidine A and B. Interestingly, both safflospermidine A and B had a higher antityrosinase activity (IC50 of 13.8 and 31.8 µM, respectively) than kojic acid (IC50 of 44.0 µM). However, fraction DCMSBP5 had no significant antioxidation activity, while fractions DCMSBP1-4 showed a lower antioxidation activity than ascorbic acid. CONCLUSION: Safflospermidine A and B are potential natural tyrosinase inhibitors.

Molecular dynamics reveals insight into how N226P and H227Y mutations affect maltose binding in the active site of α-glucosidase II from European honeybee, Apis mellifera.

European honeybee, Apis mellifera, produces α-glucosidase (HBGase) that catalyzes the cleavage of an α-glycosidic bond of the non-reducing end of polysaccharides and has potential applications for malt hydrolysis in brewing industry. Characterized by their substrate specificities, HBGases have three isoforms including HBGase II, which prefers maltose to sucrose as a substrate. Previous study found that the catalytic efficiency of maltose hydrolysis of N226P mutant of HBGase II was higher than that of the wild type (WT), and the catalytic efficiency of maltose hydrolysis of WT was higher than those of H227Y and N226P-H227Y mutants. We hypothesized that N226P mutation probably caused maltose to bind with better affinity and position/orientation for hydrolysis than WT, while H227Y and N226P-H227Y mutations caused maltose to bind with worse affinity and position/orientation for hydrolysis than WT. Using this hypothesis, we performed molecular dynamics on the catalytically competent binding conformations of maltose/WT, maltose/N226P, maltose/H227Y, and maltose/N226P-H227Y complexes to elucidate effects of N226P and H227Y mutations on maltose binding in HBGase II active site. Our results reasonably support this hypothesis because the N226P mutant had better binding affinity, higher number of important binding residues, strong and medium hydrogen bonds as well as shorter distance between atoms necessary for hydrolysis than WT, while the H227Y and N226P-H227Y mutants had worse binding affinities, lower number of important binding residues and strong hydrogen bonds as well as longer distances between atoms necessary for hydrolysis than WT. Moreover, results of binding free energy and hydrogen bond interaction of residue 227 support the role of H227 as a maltose preference residue, as proposed by previous studies. Our study provides important and novel insight into how N226P and H227Y mutations affect maltose binding in HBGase II active site. This knowledge could potentially be used to engineer HBGase II to improve its efficiency.

Water-dispersible unadulterated α-mangostin particles for biomedical applications.

α-Mangostin, the extract from pericarp of Garcinia mangostana L . or mangosteen fruit, has been applied in various biomedical products because of its minimal skin irritation, and prominent anti-inflammatory, antimicrobial and immune-modulating activities. Owing to its low water solubility, the particle formulations are necessary for the applications of α-mangostin in aqueous media. The particle formulations are usually prepared using surfactants and/or polymers, usually at a larger amount of these auxiliaries than the amount of α-mangostin itself. Here, we show the self-assembly of α-mangostin molecules into water-dispersible particles without a need of any polymers/surfactants. Investigations on chemical structure, crystallinity and thermal properties of the obtained α-mangostin particles, in comparison to the conventional α-mangostin crystalline solid, confirm no formation of the new compound during the particle formation and suggest changes in intermolecular interactions among α-mangostin molecules and significantly more hydroxyl functionality positioned at the particles' surface. The ability of the water suspension of the α-mangostin to inhibit the growth of Propionibacterium acnes, the acne-causing bacteria, is similar to that of the solution of the conventional α-mangostin in 5% dimethyl sulfoxide. Moreover, at 12.7 ppm in an aqueous environment of RAW 264.7 cell culture, α-mangostin suspension exhibits five times higher anti-inflammatory activity than the conventional α-mangostin solution, with the same acceptable cytotoxicity of less than 20% cell death.

Genetic Characterization of Exotic Commercial Honey Bee (Hymenoptera: Apidae) Populations in Thailand Reveals High Genetic Diversity and Low Population Substructure.

Domestication of animal species is often associated with a reduction in genetic diversity. The honey bee, Apis mellifera Linnaeus, 1758, has been managed by beekeepers for millennia for both honey and wax production and for crop pollination. Here we use both microsatellite markers and sequence data from the mitochondrial COI gene to evaluate genetic variation of managed A. mellifera in Thailand, where the species is introduced. Microsatellite analysis revealed high average genetic diversity with expected heterozygosities ranging from 0.620 ± 0.184 to 0.734 ± 0.071 per locus per province. Observed heterozygosities were generally lower than those expected under Hardy-Weinberg equilibrium, both locally and across the population as a whole. Mitochondrial sequencing revealed that the frequency of two evolutionary linages (C-Eastern European and O-Middle Eastern) are similar to those observed in a previous survey 10 yr ago. Our results suggest that Thai beekeepers are managing their A. mellifera in ways that retain overall genetic diversity, but reduce genetic diversity between apiaries.