Cellular responses to 8-methyl nonanoic acid, a degradation by-product of dihydrocapsaicin, in 3T3-L1 adipocytes.

BACKGROUND: Capsaicinoids, such as dihydrocapsaicin (DHC), exert the health-promoting effects of chili peppers on energy metabolism. The metabolic responses to capsaicinoids are primarily mediated through transient receptor potential cation channel subfamily V member 1 (TRPV1). However, the varying contributions of their metabolites to beneficial health outcomes remain unclear. 8-methyl nonanoic acid (8-MNA), a methyl-branched medium chain fatty acid (MCFA), is an in vivo degradation by-product of DHC. Since MCFAs have emerged as metabolic modulators in adipocytes, here we examined various cellular responses to 8-MNA in 3T3-L1 adipocytes. METHODS: The viability of 3T3-L1 adipocytes exposed to various concentrations of 8-MNA was assessed by the Calcein AM assay. Biochemical assays for lipid accumulation, AMP-activated protein kinase (AMPK) activity, lipolysis and glucose uptake were performed in 3T3-L1 adipocytes treated with 8-MNA during 48-h nutrient starvation or 5-day maturation. RESULTS: 8-MNA caused no impact on cell viability. During nutrient starvation, 8-MNA decreased lipid amounts in association with AMPK activation, a molecular event that suppresses lipogenic processes. Moreover, 3T3-L1 adipocytes that were treated with 8-MNA during 5-day maturation exhibited a reduced lipolytic response to isoproterenol and an increased glucose uptake when stimulated with insulin. CONCLUSIONS: These results suggest that 8-MNA derived from DHC modulates energy metabolism in adipocytes and also support the idea that the metabolic benefits of chili consumption are partly attributable to 8-MNA.

Antibacterial oral sprays from kaffir lime (Citrus hystrix DC.) fruit peel oil and leaf oil and their activities against respiratory tract pathogens.

BACKGROUND AND AIM: Kaffir lime fruit peel oil and Kaffir lime leaf oil have been reported for their activities against respiratory tract pathogens. The purpose of the study was to develop clear oral sprays to be used as a first-defense oral spray. EXPERIMENTAL PROCEDURE: Clear antibacterial oral sprays were prepared and analyzed for their respective active major compounds, using GC-MS. The sprays were tested against a Gr. A streptococcal clinical isolate and 3 standard respiratory tract pathogens, using Broth microdilution method. A 4-month stability test was carried out as well. RESULTS AND CONCLUSION: Six clear oral sprays, three formulae composed of Kaffir lime fruit peel oil (6, 10, 13%v/v KLO) and the other three formulae containing Kaffir lime leaf oil (4, 8, 12%v/v KLLO), were developed. The active compounds in KLO were α-terpineol and terpinene-4-ol whereas that in KLLO was citronellal. All oral sprays exhibited antibacterial activity against one Group A streptococcal clinical isolate and three respiratory pathogenic pathogens, Staphylococcus aureus ATCC 29213, Streptococcus pneumoniae ATCC 49619, and Haemophilus influenzae ATCC 49247, among which the strongest activity was against H. influenzae ATCC 49247. The antibacterial activity of all oral sprays remained unchanged in an accelerated stability test, at 4, 30, and 45 °C under 75% relative humidity, throughout the 4-month storage.

In vitro study of anti-coccidial activity of essential oils from indigenous plants against Eimeria tenella.

This study was designed to evaluate the in vitro anticoccidial properties against Eimeria tenella of different essential oils and their major active components. Efficacy of ten essential oils from different Thai indigenous plants were preliminarily screened and only those with potential were further tested for effective concentrations and identifying their active compounds. Oocysticidal property was evaluated in term of sporulation inhibition of oocysts and the percentage of unsporulated, sporulated and degenerated oocysts, after treatment with 125µg/ml of the selected essential oil, the sample was enumerated by haemocytometer, while coccidiocidal activity was assessed by the inhibition of sporozoite invasion in MDBK cell lines. Results showed that only Boesenbergia pandurata and Ocimum basilicum essential oils had strong sporulation inhibition activity by providing a higher ratio of degenerated oocysts and their IC50 were 0.134 and 0.101mg/ml, respectively. GC-MS analysis of B. pandurata essential oil found trans-b-ocimene, camphor, 1,8-cineole, geraniol, camphene, methyl cinnamate, l-limonene and linalool as the major components, while methyl chavicol, α-bergamotene, 1,8-cineole and trans-ß-ocimene were the main compounds of O. basilicum essential oil. Methyl cinnamate and camphor were the active components of B. pandurata oil, whereas methyl chavicol was the active component of O. basilicum oil by exhibiting the oocysticidal effect against E. tenella with IC50 values of 0.008, 0.023 and 0.054mg/ml, respectively. Furthermore, B. pandurata and O. basilicum oils also showed a strong cytotoxic property against coccidia with more than 70% inhibition of sporozoite invasion in MDBK cell lines, and their IC50 were 0.004 and 0.004mg/ml, respectively. Methyl cinnamate as well as camphor from B. pandurata and methyl chavicol from O. basilicum were also effective with IC50 values of 0.029, 0.023, and 0.022mg/ml, respectively.

Development of molecular approach based on PCR assay for detection of histamine producing bacteria.

Histamine fish poisoning becomes highly concern not only in public health but also economic aspect. Histamine is produced from histidine in fish muscles by bacterial decarboxylase enzyme. Several techniques have been developed to determine the level of histamine in fish and their products but the effective method for detecting histamine producing bacteria is still required. This study was attempted to detect histamine producing bacteria by newly developed PCR condition. Histamine producing bacteria were isolated from scombroid fish and determined the ability to produce histamine of isolated bacteria by biochemical and TLC assays. PCR method was developed to target the histidine decarboxylase gene (hdc). The result showed that fifteen histamine producing bacterial isolates and three standard strains produced an amplicon at the expected size of 571 bp after amplified by PCR using Hdc_2F/2R primers. Fifteen isolates of histamine producing bacteria were classified as M. morganii, E. aerogenes, and A. baumannii. The lowest detection levels of M. morganii and E. aerogenes were 10(2) and 10(5) Cfu/mL in culture media and 10(3) and 10(6) Cfu/mL in fish homogenates, respectively. The limit of detection by this method was clearly shown to be sensitive because the primers could detect the presence of M. morganii and E. aerogenes before the histamine level reached the regulation level at 50 ppm. Therefore, this PCR method exhibited the potential efficiency for detecting the hdc gene from histamine producing bacteria and could be used to prevent the proliferation of histamine producing bacteria in fish and fish products.

Biological Activity of Dolichandrone serrulata Flowers and Their Active Components.

Dolichandrone serrulata (DC.) Seem flowers are widely used as vegetables in northern and eastern Thailand. Biological studies of the methanolic extract of these flowers have shown promising antioxidant activity. Biological-guided separation of D. serrulata flowers yielded six compounds, identified as hallerone, protocatechuic acid, rengyolone, cleroindicin B, ixoside, and isomaltose. This is the first report on hallerone, protocatechuic acid, rengyolone, cleroindicin B, and isomaltose in D. serrulata. Protocatechuic acid was the most potent scavenger of 2,2-diphenyl-l-picrylhydrazyl and hydroxyl radicals with IC50 values of 25.6 +/- 0.6 and 29.6 +/- 0.4 microM, respectively. Hallerone and rengyolone showed moderate scavenging action on superoxide radicals and inhibited H202 induced reactive oxygen species production in HEK-293 cell. In addition, the other isolated compounds showed weak activity.

Bioactivity-guided Separation of the Active Compounds in Acacia pennata Responsible for the Prevention of Alzheimer's Disease.

The objective of this study was to evaluate the health benefits of plants used in Thai food, specifically Acacia pennata Willd., in Alzheimer's prevention. A. pennata twigs strongly inhibited ß-amyloid aggregation. Bioactivity-guided separation of the active fractions yielded six known compounds, tetracosane (1), 1-(heptyloxy)-octadecane (2), methyl tridecanoate (3), arborinone (4), confertamide A (5) and 4-hydroxy-1-methyl-pyrrolidin-2-carboxylic acid (6). The structures were determined by spectroscopic analysis. Biological testing revealed that tetracosane (1) was the most potent inhibitor of ß-amyloid aggregation, followed by 1-(heptyloxy)-octadecane (2) with IC50 values of 0.4 and 12.3 µM. Methyl tridecanoate (3), arborinone (4) and 4-hydroxy-1-methyl-pyrrolidin-2-carboxylic acid (6) moderately inhibited ß-amyloid aggregation. In addition, tetracosane (1) and methyl tridecanoate (3) weakly inhibited acetylcholinesterase (AChE). These results suggested that the effect of A. pennata on Alzheimer's disease was likely due to the inhibition of ß-amyloid aggregation. Thus A. pennata may be beneficial for Alzheimer's prevention.

Polyphenolic compounds and antioxidant activities of the leaves of Glochidion hypoleucum.

Bioassay-guided fractionation of the methanol extract of Glochidion hypoleucum (Miq.) Boerl leaves led to the isolation of five polyphenolic compounds, methyl gallate, gallic acid, apigenin-8-C-ß-D-glucopyranoside (vitexin), luteolin-8-C-ß-D-glucopyranoside (orientin), and luteolin-6-C-ß-D-glucopyranoside (isoorientin). The chemical structures of the isolated compounds were determined using spectroscopic (NMR, UV-Vis, IR) and mass spectrometric techniques. The antioxidative properties of the methanol extract and isolated polyphenols were evaluated using 1,1-diphenyl-2-picrylhydrazyl (DPPH) for radical scavenging activity and 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) to measure the level of reactive oxygen species (ROS). With the exception of vitexin, the crude methanol extract and the polyphenolic compounds inhibited DPPH radicals with IC50 values ranging from 2.46 ± 0.05 to 40.0 ± 0.3 µg/mL. In addition, the crude methanol extract attenuated H202-induced intracellular ROS production in a dose-dependent manner in HEK-293 cells. Gallic acid and isoorientin significantly reduced the ROS level in HEK-293 cells at a concentration of 20 µM.

Synergistic interaction and mode of action of Citrus hystrix essential oil against bacteria causing periodontal diseases.

CONTEXT: Citrus hystrix de Candolle (Rutaceae), an edible plant regularly used as a food ingredient, possesses antibacterial activity, but there is no current data on the activity against bacteria causing periodontal diseases. OBJECTIVE: C. hystrix essential oil from leaves and peel were investigated for antibiofilm formation and mode of action against bacteria causing periodontal diseases. MATERIALS AND METHODS: In vitro antibacterial and antibiofilm formation activities were determined by broth microdilution and time kill assay. Mode of action of essential oil was observed by SEM and the active component was identified by bioautography and GC/MS. RESULTS AND DISCUSSION: C. hystrix leaves oil exhibited antibacterial activity at the MICs of 1.06 mg/mL for P. gingivalis and S. mutans and 2.12 mg/mL for S. sanguinis. Leaf oil at 4.25 mg/mL showed antibiofilm formation activity with 99% inhibition. The lethal effects on P. gingivalis were observed within 2 and 4 h after treated with 4 × MIC and 2 × MIC, respectively. S. sanguinis and S. mutans were completely killed within 4 and 8 h after exposed to 4 × MIC and 2 × MIC of oil. MICs of tested strains showed 4 times reduction suggesting synergistic interaction of oil and chlorhexidine. Bacterial outer membrane was disrupted after treatment with leaves oil. Additionally, citronellal was identified as the major active compound of C. hystrix oil. CONCLUSIONS: C. hystrix leaf oil could be used as a natural active compound or in combination with chlorhexidine in mouthwash preparations to prevent the growth of bacteria associated with periodontal diseases and biofilm formation.

Neurite Outgrowth and Neuroprotective Effects of Quercetin from Caesalpinia mimosoides Lamk. on Cultured P19-Derived Neurons.

Quercetin has been isolated for the first time from ethyl acetate extract of Caesalpinia mimosoides Lamk. C. mimosoides Lamk. (Fabaceae) or Cha rueat (Thai name) is an indigenous plant found in mixed deciduous forest in northern and north-eastern parts of Thailand. Thai rural people consume its young shoots and leaves as a fresh vegetable, as well as it is used for medicinal purposes.The antioxidant capacity in terms of radical scavenging activity of quercetin was determined as IC50 of 3.18 ± 0.07 µg/mL, which was higher than that of Trolox and ascorbic acid (12.54 ± 0.89 and 10.52 ± 0.48 µg/mL, resp.). The suppressive effect of quercetin on both purified and cellular acetylcholinesterase (AChE) enzymes was investigated as IC50 56.84 ± 2.64 and 36.60 ± 2.78 µg/mL, respectively. In order to further investigate the protective ability of quercetin on neuronal cells, P19-derived neurons were used as a neuronal model in this study. As a result, quercetin at a very low dose of 1 nM enhanced survival and induced neurite outgrowth of P19-derived neurons. Furthermore, this flavonoid also possessed significant protection against oxidative stress induced by serum deprivation. Altogether, these findings suggest that quercetin is a multifunctional compound and promising valuable drugs candidate for the treatment of neurodegenerative disease.

Bioautography-guided isolation of antibacterial compounds of essential oils from Thai spices against histamine-producing bacteria.

The outbreak of histamine fish poisoning has been being an issue in food safety and international trade. The growth of contaminated bacterial species including Morganella morganii which produce histidine decarboxylase causes histamine formation in fish during storage. Histamine, the main toxin, causes mild to severe allergic reaction. At present, there is no well-established solution for histamine fish poisoning. This study was performed to determine the antibacterial activity of essential oils from Thai spices against histamine-producing bacteria. Among the essential oils tested, clove, lemongrass and sweet basil oils were found to possess the antibacterial activity. Clove oil showed the strongest inhibitory activity against Morganella morganii, followed by lemongrass and sweet basil oils. The results indicated that clove, lemongrass and sweet basil oils could be useful for the control of histamine-producing bacteria. The attempt to identify the active components using preparative TLC and GC/MS found eugenol, citral and methyl chavicol as the active components of clove, lemongrass and sweet basil oils, respectively. The information from this study would be useful in the research and development for the control of histamine-producing bacteria in fish or seafood products to reduce the incidence of histamine fish poisoning.

Effect of lipid types on physicochemical characteristics, stability and antioxidant activity of gamma-oryzanol-loaded lipid nanoparticles.

In the present study gamma-oryzanol, an antioxidant, was incorporated into three different types of solid lipid: wax, triglycerides, a mixture of glycerides as solid lipid nanoparticles (SLN) and liquid lipid (Miglyol 812) as nanoemulsion (NE). Instability was found only from NE due to its significant increase in particle size and decreased entrapment efficiency (%EE) at a storage temperature of 45 degrees C. Solid lipid type in SLN plays an important role only on %EE, but not chemical stability. A decrease in crystallinity of SLN was observed with the incorporation of gamma-oryzanol and low recrystallization index were found with two glycerides-based SLN. The in vitro release studies demonstrated that a biphasic release pattern fitted well with the Higuchi model of SLN formulations. In comparison, nearly constant release was observed in NE comprised of similar composition. Wax-based SLN demonstrated the lowest cytotoxicity. NE, wax-based SLN and a mixture of glycerides-based SLN were considered to enhance the antioxidant activity of gamma-oryzanol.

Physicochemical characteristics, cytotoxicity, and antioxidant activity of three lipid nanoparticulate formulations of alpha-lipoic acid.

Exogenously supplied alpha-lipoic acid (LA) has proven to be effective as an antioxidant. In an effort to develop a water-soluble formulation for topical administration, LA was formulated in the form of solid lipid nanoparticles (SLN), nanostructure lipid carriers (NLC), and nanoemulsion (NE) and characterized in terms of physical and biological properties. Mean particle size of 113, 110, and 121 nm were obtained for NE, NLC, and SLN, respectively, with narrow size distribution. Zeta potential was approximately in the range of -25 to -40 mV. Disc and spherical structures of nanoparticles were observed by cryo-scanning electron microscopy. Entrapment efficiency of LA in three formulations was found to be more than 70%. After 120 days of storage at 25 degrees C, physical stability of all formulations remained unchanged whereas the entrapment efficiency of SLN and NLC could be maintained, suggesting relative long-term stability. Prolonged release of LA formulation following the Higuchi model was found where a faster release was observed from NE compared with that of SLN and NLC. More than 80% of cell survivals were found up to 1 microM of LA concentrations. Antioxidant activity analysis demonstrated that all LA-loaded formulations expressed antioxidant activity at a similar magnitude as pure LA. These results suggest that chosen compositions of lipid nanoparticles play an important role on drug loading, stability, and biological activity of nanoparticles. Both SLN and NLC demonstrated their potential as alternative carriers for aqueous topical administration of LA.

Antibacterial activity of Thai medicinal plants against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major nosocomial pathogen which causes severe morbidity and mortality worldwide. Seventeen Thai medicinal plants were investigated for their activity against MRSA. Garcinia mangostana was identified as the most potent plant, and its activity was traced to the prenylated xanthone, alpha-mangostin (MIC and MBC values of 1.95 and 3.91 microg/ml, respectively).

The effect of cetyl palmitate crystallinity on physical properties of gamma-oryzanol encapsulated in solid lipid nanoparticles.

This present study was aimed at investigating the effect of the crystallinity of cetyl palmitate based solid lipid nanoparticles (SLNs) on the physical properties of γ-oryzanol-loaded SLNs. SLNs consisting of varying ratios of cetyl palmitate and γ-oryzanol were prepared. Their hydrodynamic diameters were in the range 210-280 nm and the zeta potentials were in the range -27 to -35 mV. The size of SLNs increased as the amount of cetyl palmitate decreased whereas no significant change of zeta potentials was found. Atomic force microscopy pictures indicated the presence of disc-like particles. The crystallinity of SLNs, determined by differential scanning calorimetry and powder x-ray diffraction, was directly dependent on the ratio of cetyl palmitate to γ-oryzanol and decreased with decreasing cetyl palmitate content in the lipid matrix. Varying this ratio in the lipid mix resulted in a shift in the melting temperature and enthalpy, although the SLN structure remained unchanged as an orthorhombic lamellar lattice. This has been attributed to a potential inhibition by γ-oryzanol during lipid crystal growth as well as a less ordered structure of the SLNs. The results revealed that the crystallinity of the SLNs was mainly dependent on the solid lipid, and that the crystallinity has an important impact on the physical characteristics of active-loaded SLNs.

Identification and functional characterization of the moss Physcomitrella patens delta5-desaturase gene involved in arachidonic and eicosapentaenoic acid biosynthesis.

The moss Physcomitrella patens contains high levels of arachidonic acid and lesser amounts of eicosapentaenoic acid. Here we report the identification and characterization of a delta5-desaturase from P. patens that is associated with the synthesis of these fatty acids. A full-length cDNA for this desaturase was identified by data base searches based on homology to sequences of known delta5-desaturase cDNAs from fungal and algal species. The resulting P. patens cDNA encodes a 480-amino acid polypeptide that contains a predicted N-terminal cytochrome b5-like domain as well as three histidine-rich domains. Expression of the enzyme in Saccharomyces cerevisiae resulted in the production of the delta5-containing fatty acid arachidonic acid in cells that were provided di-homo-gamma-linolenic acid. In addition, the expressed enzyme generated delta5-desaturation products with the C20 substrates omega-6 eicosadienoic and omega-3 eicosatrienoic acids, but no products were detected with the C18 fatty acid linoleic and alpha-linolenic acids or with the C22 fatty acid adrenic and docosapentaenoic acids. When the corresponding P. patens genomic sequence was disrupted by replacement through homologous recombination, a dramatic alteration in the fatty acid composition was observed, i.e. an increase in di-homo-gamma-linolenic and eicosatetraenoic acids accompanied by a concomitant disappearance of the delta5-fatty acid arachidonic and eicosapentaenoic acids. In addition, overexpression of the P. patens cDNA in protoplasts isolated from a disrupted line resulted in the restoration of arachidonic acid synthesis.

Bruguiesulfurol, a new sulfur compound from Bruguiera gymnorrhiza.

A new cyclic 4-hydroxy-dithiosulfonate, bruguiesulfurol (1), as well as two known 4-hydroxydithiolane 1-oxides, brugierol (2) and isobrugierol (3) were isolated from the flowers of Bruguiera gymnorrhiza. With stably-transfected HepG2 cells, the three isolates activated antioxidant response element (ARE) luciferase activation with (EC(50)) values of 56.7, 3.7 and 1.8 microM, respectively. Compounds 2 and 3 also inhibited phorbol ester-induced NF-kappaB (nuclear factor-kappaB) luciferase activity with IC (50) values of 85.0 and 14.5 microM, respectively. In addition, compound 2 inhibited enzyme cyclooxygenase-2 (COX-2) activity with an IC(50) value of 6.1 microM. The structures of these isolates were determined by spectral data, and that of compound 1 was confirmed by X-ray crystallographic analysis.

Bioactive dammarane triterpenes from the mangrove plant Bruguiera gymnorrhiza.

Three new dammarane triterpenes, bruguierins A-C (1-3), were isolated from a petroleum ether extract of the flowers of Bruguiera gymnorrhiza. Their structures were determined on the basis of physical and spectroscopic data interpretation. With stably transfected HepG2 cells, the three isolates activated antioxidant response element (ARE luciferase activation) with EC(50) values of 7.8, 9.4, and 15.7 microM, respectively. Bruguierin A (1) also inhibited phorbol ester-induced NFkappaB (nuclear factor-kappaB) luciferase activation with an IC(50) value of 1.4 microM and selectively inhibited cyclooxygenase-2 (COX-2) activity with an IC(50) value of 0.37 microM. Compounds 2 and 3 were not active in these bioassays.

Hypocholesterolemic and antioxidant effects of aqueous extracts from the dried calyx of Hibiscus sabdariffa L. in hypercholesterolemic rats.

The present study was designed to investigate the hypolipidemic effects and antioxidant effects of Hibiscus sabdariffa L. (roselle) with regard to protection of LDL oxidation in vivo and ex vivo in rats made hypercholesterolemic by continuous cholesterol feeding. Administering the dried calyx extracts of roselle at doses of 500 and 1,000 mg/kg together with continuous cholesterol feeding to hypercholesterolemic rats for 6 weeks significantly decreased serum cholesterol level by 22% and 26%, respectively (p<0.001); serum triglycerides level by 33% and 28%, respectively (p<0.05); serum LDL level by 22% and 32%, respectively (p<0.05). However, serum HDL level was not affected. LDL was extracted from plasma of the hypercholesterolemic rats and the effects of the dried calyx extracts of roselle on the oxidation of LDL in vivo and ex vivo were examined. Six-week treatment with 250, 500 and 1,000 mg/kg of the extracts significantly decreased thiobarbituric acid reactive substances (TBARs) formation (p<0.05) while the formation of conjugated dienes during the oxidation of LDL induced by CuSO(4) was reduced, but not significantly different. These lines of evidence suggest that the aqueous extracts from the dried calyx of roselle possess both antioxidant effects against LDL oxidation and hypolipidemic effects in vivo. However, its mechanism(s) of action remains to be elucidated.

Bioactive constituents from roots of Bursera tonkinensis.

Bioassay directed-fractionation led to isolation of 12 compounds from the roots of Bursera tonkinensis Guillaum (Burseraceae), including burselignan, bursephenylpropane, and burseneolignan. Of the 12 compounds, only 4'-demethyldesoxypodophyllotoxin exhibited significant cytotoxic activities against KB, Col2 and LNCaP cell lines.

Quinone reductase inducers in Azadirachta indica A. Juss flowers, and their mechanisms of action.

We have previously shown that the flowers of neem tree (Azadirachta indica A. Juss, family Meliaceae), Thai variety, strongly induced the activity of glutathione S-transferase (GST) while resulting in a significant reduction in the activities of some cytochrome P(450)-dependent monooxygenases in rat liver, and possess cancer chemopreventive potential against chemically-induced mammary gland and liver carcinogenesis in rats. In the present study, 2 chemicals possessing strong QR inducing activity were fractionated from neem flowers using a bioassay based on the induction of QR activity in mouse hepatoma Hepa 1c1c7 cultured cells. Spectroscopic characteristics revealed that these compounds were nimbolide and chlorophylls, having CD (concentration required to double QR specific activity) values of 0.16 and 3.8 mug/ml, respectively. Nimbolide is a known constituent of neem leaves, but was found for the first time here in the flowers. Both nimbolide and chlorophylls strongly enhanced the level of QR mRNA in Hepa 1c1c7 cells, as monitored by northern blot hybridization, indicating that the mechanism by which these constituents of neem flowers induced QR activity is the induction of QR gene expression. These findings may have implication on cancer chemopreventive potential of neem flowers in experimental rats previously reported.