Phytochemical Profiling and Antioxidant Activities of the Most Favored Ready-to-Use Thai Curries, Pad-Ka-Proa (Spicy Basil Leaves) and Massaman.

Food is one of the factors with the highest impact on human health. Today, attention is paid not only to food properties such as energy provision and palatability but also to functional aspects including phytochemical, antioxidant properties, etc. Massaman and spicy basil leaf curries are famous Thai food dishes with a good harmony of flavor and taste, derived from multiple herbs and spices, including galangal rhizomes, chili pods, garlic bulbs, peppers, shallots, and coriander seeds, that provide an array of health benefits. The characterization of phytochemicals detected by LC-ESI-QTOF-MS/MS identified 99 components (Masaman) and 62 components (spicy basil leaf curry) such as quininic acid, hydroxycinnamic acid, luteolin, kaempferol, catechin, eugenol, betulinic acid, and gingerol. The cynaroside and luteolin-7-O-glucoside found in spicy basil leaf curry play a key role in antioxidant activities and were found at a significantly higher concentration than in Massaman curry. Phenolic and flavonoid compounds generally exhibit a bitter and astringent taste, but all the panelists scored both curries higher than 7 out of 9, confirming their acceptable flavor. Results suggest that the Massaman and spicy basil leaves contain various phytochemicals at different levels and may be further used as functional ingredients and nutraceutical products.

Antioxidant and Nitric Oxide Inhibitory Activity of the Six Most Popular Instant Thai Curries.

All living organisms undergo molecular damage by free radical products. Disrupting the balance between antioxidants and free radicals leads to greater risks of diabetes, hypertension, stroke, and cancer. Consumption of curries containing various herbs and spices provides antioxidant and anti-inflammatory benefits which promote health. The antioxidant and nitric oxide (NO) inhibitory properties of six popular Thai curries, including green curry (G), Panang curry (P), Massaman curry (M), spicy basil leaf curry (SB), southern sour curry (SS), and southern spicy yellow curry (SY) were determined. All six curries contained phenolic and flavonoid compounds and provided antioxidant activity based on electron transfer and hydrogen atom donor properties, as well as having the ability to reduce oxidized metal. The highest antioxidant value was found in SB, followed by M, SS, and SY. The replacement of sugar with dried stevia powder at 50% (Re) improved antioxidant activity. The ORAC assay provided five times higher results than DPPH, ABTS, and FRAP. Extracts of all curries at 1 mg/mL on the macrophage cell line RAW 264.7 showed no cytotoxicity. The highest NO inhibition was found in SB (p < 0.05). All curry extracts contained quercetin, kaempferol, luteolin, and apigenin. The six selected popular Thai curries had antioxidant and anti-inflammatory health benefits. Nutraceuticals, functional foods, and the ingredients of each raw material and curry powder should be further investigated.

Trihydroxyxanthones from the heartwood of Maclura cochinchinensis modulate M1/M2 macrophage polarisation and enhance surface TLR4.

The anti-inflammatory actions of phytochemicals have attracted much attention due to the current state of numerous inflammatory disorders. Thai traditional medicine uses Maclura cochinchinensis (Lour.) Corner to treat chronic fever and various inflammatory diseases, as well as to maintain normal lymphatic function. Five flavonoids and five xanthones were isolated from the heartwood of M. cochinchinensis and we investigated the anti-inflammatory properties of the isolated compounds. All isolated compounds possessed an anti-inflammatory effect by decreasing prostaglandin E2 (PGE2) synthesis in lipopolysaccharide (LPS)-activated murine macrophages with varying degrees of potency. The greatest decrease in M1 inflammatory mediators, nitric oxide, PGE2, and proinflammatory cytokines was observed with 1,3,7-trihydroxyxanthone and 1,3,5-trihydroxyxanthone treatment of LPS-activated macrophages. The anti-inflammatory mechanism of the two xanthones is mediated by the suppression of inducible nitric oxide synthase, cyclooxygenase-2, and phosphatidylinositol 3-kinase/protein kinase B expression and the upregulation of M2 anti-inflammatory signalling proteins phosphorylated signal transducer and activator of transcription 6 and peroxisome proliferator-activated receptors-γ. 1,3,7-Trihydroxyxanthone exhibits superior induction of anti-inflammatory M2 mediator of LPS-activated macrophages by upregulating arginase1 expression. Following the resolution of inflammation, the two xanthones enhanced surface TLR4 expression compared to LPS-stimulated cells, possibly preserving macrophage function. Our research highlights the role of the two xanthones in modulating the M1/M2 macrophage polarisation to reduce inflammation and retain surface TLR4 once inflammation has been resolved. These findings support the use of xanthones for their anti-inflammatory effects in treating inflammatory dysregulation.

1,7­Bis(4­hydroxy­3­methoxyphenyl)­1,4,6­heptatrien­3­one alleviates lipopolysaccharide­induced inflammation by targeting NF­κB translocation in murine macrophages and it interacts with MD2 in silico.

Trienones are curcuminoid analogues and are minor constituents in the rhizomes of numerous Curcuma plant species. Studies investigating the biological activities of trienones, particularly their anti­inflammatory activities, are limited. In the present study, the trienone 1,7­bis(4­hydroxy­3­methoxyphenyl)­1,4,6­heptatrien­3­one (HMPH) was structurally modified from curcumin using a novel and concise method. HMPH was shown to exhibit potential anti­inflammatory effects on lipopolysaccharide (LPS)­activated RAW264.7 macrophages. Furthermore, LPS­induced nitric oxide secretion in RAW264.7 cells was markedly and dose­dependently inhibited by HMPH; in addition, HMPH had a greater efficacy compared with curcumin. This inhibition was accompanied by the suppression of inducible nitric oxide synthase and cyclooxygenase­2 expression, as well as pro­inflammatory cytokine secretion. To elucidate the molecular mechanism underlying the anti­inflammatory effects of HMPH, the effects of this compound on nuclear factor­κB (NF­κB) translocation were assessed. HMPH significantly inhibited the translocation of p65 NF­κB into the nucleus to a greater extent than curcumin, thus indicating that HMPH has more potent anti­inflammatory activity than curcumin. In addition, an in silico modelling study revealed that HMPH possessed stronger binding energy to myeloid differentiation factor 2 (MD2) compared with that of curcumin, and indicated that the anti­inflammatory effects of HMPH may be through upstream inhibition of the inflammatory pathway. In conclusion, HMPH may be considered a promising compound for reducing inflammation via targeting p65 NF­κB translocation and interfering with MD2 binding.

Diterpenoid trigonoreidon B isolated from Trigonostemon reidioides alleviates inflammation in models of LPS-stimulated murine macrophages and inflammatory liver injury in mice.

The roots of Trigonostemon reidioides, Thai medicinal plant, have long been used as an antidote, laxative, and antiasthmatic, and also used as folk remedy for relieving inflammatory symptoms from poisonous insect and snake bites as well as abscesses and sprains. Here, we studied anti-inflammatory effects of a major diterpenoid named trigonoreidon B (TR-B) isolated from T. reidioides roots in lipopolysaccharide (LPS)-activated RAW264.7 macrophages and D-galactosamine (D-GalN)/LPS-induced inflammatory liver injury in mice. RAW264.7 cells were treated with TR-B or other available minor diterpenoids, and cell viability was determined by AlamarBlue. The levels of inflammatory mediators were determined by nitrite assay, ELISA, and luminescence. NF-κB nuclear translocation was investigated by indirect immunofluorescence. Expression levels were determined by real-time PCR and Western blotting. Transaminases and caspase activities were determined by using assay kits. Our results showed that TR-B was able to suppress PI3K/Akt activation and inflammatory induction in LPS-activated macrophages. These events were concomitant with TR-B's ability to hamper activated generation of reactive oxygen species, nitric oxide, prostaglandin E2, and cytokines as well as NF-κB p65 nuclear translocation. In an in vivo model of inflammatory liver injury, an administration of TR-B protected mice from D-GalN/LPS-induced liver injury by suppressing the elevation of serum TNF-α, transaminase activities, and hepatocyte apoptosis as well as an improvement of liver histopathology. During protection against liver damage, TR-B also prevented the loss of Akt phosphorylation. Collectively, the results of this present study suggested that TR-B exerted an anti-inflammatory effect via attenuating macrophage-mediated inflammation and inflammatory liver injury in vivo. TR-B may represent a promising lead compound for anti-inflammatory drug development.

Ursodeoxycholyl lysophosphatidylethanolamide negatively regulates TLR-mediated lipopolysaccharide response in human THP-1-derived macrophages.

The bile acid-phospholipid conjugate ursodeoxycholyl oleoyl-lysophophatidylethanolamide (UDCA-18:1LPE) is an anti-inflammatory and anti-fibrotic agent as previously shown in cultured hepatocytes and hepatic stellate cells as well as in in vivo models of liver injury. We hypothesize that UDCA-18:1LPE may directly inhibit the activation of immune cells. We found that UDCA-18:1LPE was capable of inhibiting the migration of phorbol ester-differentiated human THP-1 cells. We examined anti-inflammatory activity of UDCA-18:1LPE during activation of THP1-derived macrophages. Treatment of these macrophages by bacterial lipopolysaccharide (LPS) for 24 h induced the release of pro-inflammatory cytokines TNF-α, IL-6 and IL-1ß. This release was markedly inhibited by pretreatment with UDCA-18:1LPE by ~ 65-90%. Derivatives with a different fatty-acid chain in LPE moiety also exhibited anti-inflammatory property. Western blotting and indirect immunofluorescence analyses revealed that UDCA-18:1LPE attenuated the expression of phosphorylated p38, MKK4/MKK7, JNK1/2, and c-Jun as well as nuclear translocation of NF-κB by ~ 22-86%. After LPS stimulation, the Toll-like receptor adaptor proteins, myeloid differentiation factor 88 and TNF receptor associated factor 6, were recruited into lipid rafts and UDCA-18:1LPE inhibited this recruitment by 22% and 58%, respectively. Moreover, LPS treatment caused a decrease of the known cytoprotective lysophosphatidylcholine species containing polyunsaturated fatty acids by 43%, and UDCA-18:1LPE co-treatment reversed this decrease. In conclusion, UDCA-18:1LPE and derivatives inhibited LPS inflammatory response by interfering with Toll-like receptor signaling in lipid rafts leading to an inhibition of MAPK and NF-κB activation. These conjugates may represent a class of lead compounds for development of anti-inflammatory drugs.

2',4-Dihydroxy-3',4',6'-trimethoxychalcone from Chromolaena odorata possesses anti-inflammatory effects via inhibition of NF-κB and p38 MAPK in lipopolysaccharide-activated RAW 264.7 macrophages.

CONTEXT: Immune dysregulation has been implicated in the pathogenesis of many diseases. Macrophages play a crucial role contributing to the onset, progression, and resolution of inflammation. Macrophage inflammatory mediators are of considerable interest as potential targets to treat inflammatory diseases. OBJECTIVE: The present study was conducted to elucidate the anti-inflammatory mechanism of 2',4-dihydroxy-3',4',6'-trimethoxychalcone (1), the major chalcone isolated from Chromolaena odorata (L.) R.M.King & H.Rob, against lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages. MATERIALS AND METHODS: Cell viability, nitric oxide (NO), and proinflammatory cytokines of LPS-activated RAW 264.7 cells were measured by MTT, Griess, and ELISA assays, respectively. Cell lysates were subjected to Western blotting for investigation of protein expression. RESULTS AND DISCUSSION: Treatment with the major chalcone 1 significantly attenuated the production of NO and proinflammatory cytokines, tumor necrosis factor-α, interleukin-1ß, and interleukin-6 in a dose-dependent manner. The chalcone suppressed nuclear factor-κB (NF-κB) stimulation by preventing activation of inhibitor κB kinase (IKK) α/ß, degradation of inhibitor κB (IκB) α, and translocation of p65 NF-κB into the nucleus. Additionally, the chalcone markedly repressed the phosphorylation of p38 mitogen-activated protein kinase (MAPK), but no further inhibition was detected for c-Jun N-terminal activated kinases or extracellular regulated kinases. Thus, suppression of NF-κB and p38 MAPK activation may be the core mechanism underlying the anti-inflammatory activity of 2',4-dihydroxy-3',4',6'-trimethoxychalcone (1). CONCLUSION: These findings provide evidence that 2',4-dihydroxy-3',4',6'-trimethoxychalcone (1) possesses anti-inflammatory activity via targeting proinflammatory macrophages. This anti-inflammatory chalcone is a promising compound for reducing inflammation.

Isomahanine induces endoplasmic reticulum stress and simultaneously triggers p38 MAPK-mediated apoptosis and autophagy in multidrug-resistant human oral squamous cell carcinoma cells.

Advanced oral squamous cell carcinoma (OSCC) is typically aggressive and closely correlated with disease recurrence and poor survival. Multidrug resistance (MDR) is the most critical problem leading to therapeutic failure. Investigation of novel anticancer candidates targeting multidrug-resistant OSCC cells may provide a basis for developing effective strategies for OSCC treatment. In the present study, we investigated the cytotoxic mechanism of a carbazole alkaloid, namely isomahanine, in a multidrug­resistant OSCC cell line CLS-354/DX. We demonstrated that CLS-354/DX cells overexpressing multidrug resistance-associated protein 1 (MRP1) were resistant to anticancer drugs cisplatin and camptothecin. Isomahanine effectively induced cytotoxicity against CLS-354/DX cells regardless of resistance. Apoptosis as determined by FITC­Annexin V/PI staining and western blot analysis of cleaved caspase-3 and cleaved poly(ADP­ribose) polymerase (PARP) was significantly induced in a time-dependent manner upon isomahanine treatment. Isomahanine-induced caspase­dependent apoptosis was determined using z-VAD­fmk. The effects on autophagy in isomahanine-treated cells were investigated via conversion of LC3B and degradation of p62/SQSTM1 (p62). Isomahanine obviously induced autophagic flux as shown by an increase in punctate GFP-LC3B and the LC3B-II/LC3B-I ratio with a concomitant decrease in p62 levels. Autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) protected isomahanine-induced cell death, indicating the activation of autophagic cell death. Endoplasmic reticulum (ER) stress and MAPK activation were examined to elucidate the mechanism underlying cell death. The expression levels of PERK, CHOP and phosphorylated MAPK (p38, ERK1/2 and JNK1/2) were upregulated following isomahanine treatment. We found that p38 MAPK inhibitor (SB203580) significantly attenuated isomahanine-induced apoptosis and autophagic flux and this prevented cell death. Collectively, the present study demonstrated that isomahanine was able to induce ER stress and trigger p38 MAPK-mediated apoptosis and autophagic cell death in multidrug-resistant OSCC cells. The potential cytotoxic action of isomahanine may provide the development of anticancer candidates for treating multidrug-resistant cancer.

Ursodeoxycholyl Lysophosphatidylethanolamide Protects Against CD95/FAS-Induced Fulminant Hepatitis.

Increased activation of CD95/Fas by Fas ligand in viral hepatitis and autoimmunity is involved in pathogenesis of fulminant hepatitis and liver failure. We designed a bile-acid phospholipid conjugate ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE with LPE containing oleate at the sn-1) as a hepatoprotectant that was shown to protect against fulminant hepatitis induced by endotoxin. We herein further assessed the ability of UDCA-LPE to prevent death receptor CD95/Fas-induced fulminant hepatitis. C57BL/6 mice were intravenously administered with CD95/Fas agonistic monoclonal antibody (Jo-2) with or without 1 h pretreatment with 50 mg/kg UDCA-LPE. Jo-2 administration caused massive hepatocyte damage as seen by histology, and this was associated with a significant decrease in hepatic phosphatidylcholine (PC), lysoPC, and lysophosphatidylethanolamine levels. By histology, UDCA-LPE pretreatment improved hepatocyte damage and restored the loss of these phospholipids in part by a mechanism involving an inhibition of cytosolic phospholipaseA2 expression. Accordingly, Jo-2 treatment increased hepatic expression of cleaved caspase 8, caspase 3, and poly (ADP-Ribose) polymerase-1, and on the other hand decreased that of anti-apoptotic cellular FLICE-inhibitory protein. UDCA-LPE pretreatment was able to reverse all these changes. Moreover, UDCA-LPE attenuated inflammatory response by lowering the levels of Jo-2-induced proinflammatory cytokines TNF-α, IL-6, and IL-1ß in liver and serum. UDCA-LPE was also able to decrease the levels of stimulated Th1/Th17 cytokines in Jo-2-primed isolated splenocytes. Taken together, UDCA-LPE exhibited potent anti-inflammatory effects against CD95/Fas-induced fulminant hepatitis.

Carbazole alkaloids from Murraya koenigii trigger apoptosis and autophagic flux inhibition in human oral squamous cell carcinoma cells.

Carbazole alkaloids, a major constituent of Murraya koenigii (L.) Sprengel (Rutaceae), exhibit biological effects such as anticancer activity via the induction of apoptosis, and they represent candidate chemotherapeutic agents. Oral squamous cell carcinoma (OSCC) is the most prevalent cancer of the oral cavity and a growing and serious health problem worldwide. In this study, we investigated the anticancer properties and mechanisms of action of two carbazole alkaloids derived from M. koenigii leaves, mahanine and isomahanine, in the OSCC cell line CLS-354. At 15 µM, mahanine and isomahanine were cytotoxic to CLS-354 cells, triggering apoptosis via caspase-dependent and -independent mechanisms. Autophagosomes, visualised using monodansylcadaverine (MDC) labelling, were numerous in carbazole alkaloid-treated cells. Mahanine and isomahanine markedly induced the expression of the autophagosome marker microtubule-associated protein 1 light chain 3, type II (LC3B-II). Genetic and chemical inhibition of autophagy via silencing of the Autophagy protein 5 gene and exposure to bafilomycin A1 (BafA1), respectively, did not arrest carbazole alkaloid-induced apoptosis, indicating that it occurs independently of autophagic activation. Surprisingly, both carbazole alkaloids caused increased accumulation of p62/sequestosome1 (p62/SQSTM1), with coordinated expression of LC3B-II and cleaved caspase-3, suggesting inhibition of autophagic flux. Our results suggest that inhibition of autophagic flux is associated with carbazole alkaloid-induced apoptosis. Our findings provide evidence of a novel cytotoxic action of natural carbazole alkaloids and support their use as candidate chemotherapeutic agents for the treatment of OSCC.

iPLA2ß deficiency attenuates obesity and hepatic steatosis in ob/ob mice through hepatic fatty-acyl phospholipid remodeling.

PLA2G6 or GVIA calcium-independent PLA2 (iPLA2ß) is identified as one of the NAFLD modifier genes in humans, and thought to be a target for NAFLD therapy. iPLA2ß is known to play a house-keeping role in phospholipid metabolism and remodeling. However, its role in NAFLD pathogenesis has not been supported by results obtained from high-fat feeding of iPLA2ß-null (PKO) mice. Unlike livers of human NAFLD and genetically obese rodents, fatty liver induced by high-fat diet is not associated with depletion of hepatic phospholipids. We therefore tested whether iPLA2ß could regulate obesity and hepatic steatosis in leptin-deficient mice by cross-breeding PKO with ob/ob mice to generate ob/ob-PKO mice. Here we observed an improvement in ob/ob-PKO mice with significant reduction in serum enzymes, lipids, glucose, insulin as well as improved glucose tolerance, and reduction in islet hyperplasia. The improvement in hepatic steatosis measured by liver triglycerides, fatty acids and cholesterol esters was associated with decreased expression of PPARγ and de novo lipogenesis genes, and the reversal of ß-oxidation gene expression. Notably, ob/ob livers contained depleted levels of lysophospholipids and phospholipids, and iPLA2ß deficiency in ob/ob-PKO livers lowers the former, but replenished the latter particularly phosphatidylethanolamine (PE) and phosphatidylcholine (PC) that contained arachidonic (AA) and docosahexaenoic (DHA) acids. Compared with WT livers, PKO livers also contained increased PE and PC containing AA and DHA. Thus, iPLA2ß deficiency protected against obesity and ob/ob fatty liver which was associated with hepatic fatty-acyl phospholipid remodeling. Our results support the deleterious role of iPLA2ß in severe obesity associated NAFLD.

Demethoxycurcumin from Curcuma longa rhizome suppresses iNOS induction in an in vitro inflamed human intestinal mucosa model.

BACKGROUND: It is known that inducible nitric oxide synthase (iNOS)/nitric oxide (NO) plays an integral role during intestinal inflammation, an important factor for colon cancer development. Natural compounds from Curcuma longa L. (Zingiberaceae) have long been a potential source of bioactive materials with various beneficial biological functions. Among them, a major active curcuminoid, demethoxycurcumin (DMC) has been shown to possess anti-inflammatory properties in lipopolysaccharide (LPS)-activated macrophages or microglia cells. However, the role of DMC on iNOS expression and NO production in an in vitro inflamed human intestinal mucosa model has not yet been elucidated. This study concerned inhibitory effects on iNOS expression and NO production of DMC in inflamed human intestinal Caco-2 cells. An in vitro model was generated and inhibitory effects on NO production of DMC at 65 µM for 24-96 h were assessed by monitoring nitrite levels. Expression of iNOS mRNA and protein was also investigated. DMC significantly decreased NO secretion by 35-41% in our inflamed cell model. Decrease in NO production by DMC was concomitant with down-regulation of iNOS at mRNA and protein levels compared to proinflammatory cytokine cocktail and LPS-treated controls. Mechanism of action of DMC may be partly due to its potent inhibition of the iNOS pathway. Our findings suggest that DMC may have potential as a therapeutic agent against inflammation-related diseases, especially in the gut.