A Cell-Based Systematic Review on the Role of Annexin A1 in Triple-Negative Breast Cancers.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that is often associated with a poorer prognosis and does not respond to hormonal therapy. Increasing evidence highlights the exploitability of Annexin A1 (AnxA1), a calcium dependent protein, as a precision medicine for TNBC. To systematically summarize the role of AnxA1 and its associated mechanisms in TNBC, we performed data mining using three main databases: PubMed, Scopus, and Ovid/Medline. The papers retrieved were based on two different sets of key words such as "Annexin A1" or "Lipocortin 1" and "Breast cancer" or "TNBC". A total of 388 articles were identified, with 210 chosen for comprehensive screening and 13 papers that met inclusion criteria were included. Current evidence from cell culture studies showed that AnxA1 expression is correlated with NF-κB, which promotes migration by activating ERK phosphorylation. AnxaA1 also activates TGF-ß signaling which upregulates MMP-9 and miR196a expression to enhance epithelial-mesenchymal transition and migratory capacity of TNBC cells. AnxA1 can steer the macrophage polarization toward the M2 phenotype to create a pro-tumor immune environment. Existing research suggests a potential role of AnxA1 in the metastasis and immune landscape of TNBC tumors. Preclinical and clinical experiments are warranted to investigate the feasibility and effectiveness of targeting AnxA1 in TNBC.

In Vitro Evaluation of the Antibacterial Activity of EndoSeal MTA, iRoot SP, and AH Plus against Planktonic Bacteria.

This study aimed to investigate the antibacterial activity of three endodontic sealers, AH Plus, iRoot SP, and EndoSeal MTA, against four planktonic bacteria species. The antibacterial activity of the three endodontic sealers was assessed using a modified direct contact test. Bacteria suspension of Actinomycoses viscosus, Enterococcus faecalis, Staphylococcus aureus, and Streptococcus mutans were left in contact with the sealers that were pre-set or set for 1, 3, 7, and l4 days for an hour. Freshly mixed AH Plus and EndoSeal MTA were highly effective against all four tested bacteria as no surviving bacteria were recovered after treatment. Meanwhile, freshly mixed iRoot SP was not able to kill all bacteria, regardless of the species, demonstrating a weak antibacterial effect. After 24 h, AH Plus lost its antibacterial activity. EndoSeal MTA showed a strong and extended bactericidal effect against S. aureus and S. mutans for 3 days and A. viscosus for 7 days. In conclusion, fresh AH Plus and EndoSeal MTA exhibited a potent effect against all four bacteria species. EndoSeal MTA remained effective after setting when tested against A. viscosus, S. aureus, and S. mutans. Among all tested sealers, iRoot SP demonstrates the weakest antibacterial activity.

Knockdown of Annexin A1 induces apoptosis, causing G2/M arrest and facilitating phagocytosis activity in human leukemia cell lines.

Annexin A1 (ANXA1) is an endogenous protein involved in the control of proliferation, cell cycle, phagocytosis, and apoptosis in several types of cancer. To investigate the effects of ANXA1 knockdown in leukemia cells, transfection with specific ANXA1 siRNA was performed. Cell cycle and apoptosis were analyzed using flow cytometry and a mechanism involving caspases and Bcl-2 was quantified using Western blotting. Phagocytosis activity was evaluated using hematoxylin & eosin staining. The ANXA1 expression was significantly downregulated after the knockdown and apoptosis was induced in tested cells. The expression of caspase-9 and -3 increased in U937 and Jurkat cells respectively. Bcl-2 expression was downregulated in K562 and Jurkat cells while upregulated in U937. The number of leukemic cells arrested at the G2/M phase and the phagocytosis index were significantly increased in transfected cells. This suggests that ANXA1 knockdown might be a potential approach in the therapeutic strategy for leukemia.

Immunomodulatory potential of Clinacanthus nutans extracts in the co-culture of triple-negative breast cancer cells, MDA-MB-231, and THP-1 macrophages.

Triple-negative breast cancer is the main type of breast carcinoma that causes mortality among women because of the limited treatment options and high recurrence. Chronic inflammation has been linked with the tumor microenvironment (TME) in breast cancer progression. Clinacanthus nutans (CN) has gained much attention because of its anticancer properties, but its mechanism remains unclear. We aimed to study the qualitative phytochemical content and elucidate the cytotoxicity effects of CN on human triple-negative breast cancer (TNBC), MDA-MB-231 and human macrophage-like cells such as THP-1 by using sulforhodamine B (SRB) assay. As highly metastatic cells, MDA-MB-231 cells can migrate to the distal position, the effect of CN on migration were also elucidated using the scratch assay. The CN effects on ameliorating chronic inflammation in TME were studied following the co-culture of MDA-MB-231/THP-1 macrophages. The cytokine expression levels of IL-6, IL-1ß and tumor necrosis factor-alpha (TNF-α) were determined using ELISA assays. The results showed that both ethanolic and aqueous CN extracts contained alkaloid, phenol and tannin, flavonoid, terpenoid, glycoside and steroid. However, saponin was only found in the aqueous extract of CN. CN was not cytotoxic to both MDA-MB-231 and THP-1 cells. The ability of MDA-MB-231 to migrate was also not halted by CN treatment. However, CN ethanol extract decreased IL-6 at 25 µg/mL (p = 0.02) and 100 µg/mL (p = 0.03) but CN aqueous extract increased IL-6 expression at 50 µg/mL (p = 0.08) and 100 µg/mL (p = 0.02). IL-1ß showed decreased expression after treated with CN ethanol and CN aqueous both at 25 µg/mL (p = 0.03). TNF-α were significantly decreased after CN ethanol treatment at concentration 25- (p = 0.001), 50- (p = 0.000) and 100 µg/mL (p = 0.000). CN aqueous extract slightly inhibited TNF-α at all 25-50- and 100 µg/mL (p = 0.001, p = 0.000, p = 0.000, respectively). Overall, CN acts by ameliorating the pro-inflammatory condition in the TME and may be a potential strategy for its anticancer mechanism on highly metastatic breast cancer condition. The major pathways that link both cancer and inflammation were NF-κB and STATs thus further study on the upstream and downstream pathways is needed to fully understand the mechanism of CN extracts in cooling the inflamed TME in breast cancer.

Induction of cell death and modulation of Annexin A1 by phytoestrogens in human leukemic cell lines.

BACKGROUND: Phytoestrogens are polyphenolic plant compounds which are structurally similar to the endogenous mammalian estrogen, 17ß-estradiol. Annexin A1 (ANXA1) is an endogenous protein which inhibits cyclo-oxygenase 2 (COX-2) and phospholipase A2, signal transduction, DNA replication, cell transformation, and mediation of apoptosis. OBJECTIVE: This study aimed to determine the effects of selected phytoestrogens on annexin A1 (ANXA1) expression, mode of cell death and cell cycle arrest in different human leukemic cell lines. METHODS: Cells viability were examined by MTT assay and ANXA1 quantification via Enzyme-linked Immunosorbent Assay. Cell cycle and apoptosis were examined by flow cytometer and phagocytosis effect was evaluated using haematoxylin-eosin staining. RESULTS: Coumestrol significantly (p < 0.05) reduced the total level of ANXA1 in both K562 and U937 cells and genistein significantly (p < 0.05) reduced it in K562, Jurkat and U937 cells, meanwhile estradiol and daidzein induced similar reduction in U937 and Jurkat cells. Coumestrol and daidzein induced apoptosis in K562 and Jurkat cells, while genistein and estradiol induced apoptosis in all tested cells. Coumestrol and estradiol induced cell cycle arrest at G2/M phase in K562 and Jurkat cells with an addition of U937 cells for estradiol. Genistein induced cell cycle arrest at S phase for both K562 and Jurkat cells. However, daidzein induced cell cycle arrest at G0/G1 phase in K562, and G2/M phase of Jurkat cells. Coumestrol, genistein and estradiol induced phagocytosis in all tested cells but daidzein induced significant (p < 0.05) phagocytosis in K562 and Jurkat cells only. CONCLUSION: The selected phytoestrogens induced cell cycle arrest, apoptosis and phagocytosis and at the same time they reduced ANXA1 level in the tested cells. The IC50 value of phytoestrogens was undetectable at the concentrations tested, their ability to induce leukemic cells death may be related with their ability to reduce the levels of ANXA1. These findings can be used as a new approach in cancer treatment particularly in leukemia.

The influence of P-glycoprotein expression in the standard treatment of Helicobacter pylori infection in Sprague Dawley rats.

BACKGROUND: P-glycoprotein (P-gp) is an Adenosine triphosphate (ATP) dependent drug-efflux pump which is located abundantly in the stomach and protects the gut mucosa from xenobiotic. OBJECTIVE: The purpose of this study was to investigate the influence of P-gp modulation on the efficacy of treatment regimen. METHOD: P-gp modulation in rats was performed by using P-gp inducer (150 mg/kg rifampicin) and P-gp inhibitor (10 mg/kg cyclosporine A) for 14 days prior to be infected with Helicobacter pylori (H. pylori). The rats were further divided into groups, which were normal control, vehicle control, antibiotics and omeprazole, antibiotics only and omeprazole only for another 2 weeks of treatment. The ulcer formation and P-gp expression were determined by using macroscopic evaluation and western blot analysis, respectively. RESULTS: The highest P-gp expression was shown in the induced P-gp rats (2.00 ± 0.68) while the lowest P-gp expression was shown in the inhibited P-gp rats (0.45 ± 0.36) compared to the normal P-gp rats. In all groups, the rats which were infected with H. pylori, had a significant increase (p < 0.05) in P-gp expression level and a more severe ulcer formation compared to the healthy rats. The ulcer developed at different levels in the rats with inhibited, induced, or normal P-gp expression. After receiving the standard therapy for H. pylori, it was observed that the healing rate for ulcer was increased to 91% (rats with inhibited P-gp expression), 82% (rats with induced P-gp expression) and 75% in rats with normal P-gp. The use of rifampicin to induce P-gp level was also shown to be effective in eradicating the H. pylori infection. CONCLUSION: The synergism in the standard therapy by using two antibiotics (clarithromycin and amoxicillin) and proton pump inhibitor (omeprazole) have shown to effectively eradicate the H. pylori infection. Thus, P-gp expression influenced the effectiveness of the treatment.

Extracts of Andrographis paniculata (Burm.f.) Nees Leaves Exert Anti-Gout Effects by Lowering Uric Acid Levels and Reducing Monosodium Urate Crystal-Induced Inflammation.

Andrographis paniculata (Burm.f.) Nees has been found to have anti-inflammatory and immunostimulatory effects. This study was to investigate antihyperuricemic and anti-inflammatory effects of A. paniculata leaf extracts. Andrographolide, 14-deoxy-11,12-didehydroandrographolide, and neoandrographolide were quantified in 80% ethanol (EtOH80) and water extracts using High Performance Liquid Chromatography (HPLC) analysis. Antihyperuricemic activity was evaluated using a spectrophotometric in vitro inhibitory xanthine oxidase (XO) assay. The most active extract and andrographolide were further investigated in a hyperuricemic rat model induced by potassium oxonate to determine serum uric acid levels, liver XO activity, followed by Western blot analysis for renal urate transporter URAT1, GLUT9, and OAT1 to investigate the excretion of uric acid via kidney. Anti-inflammatory activity was assessed by in vitro interleukin assay for interleukin (IL-1α, IL-1ß, IL-6, IL-8), and tumor necrosis factor (TNF-α) in monosodium urate (MSU) crystal-induced human fibroblast-like synoviocyte (HFLS) cells using ELISA-kits, followed by Western blot analysis for the expression of MyD88, NLRP3, NF-κB p65, and caspase-1 proteins to investigate the inflammation pathway. In vivo assay of the most active extract and andrographolide were performed based on the swelling rate and inhibition of pro-inflammatory mediator release from synovial fluid of a rat knee joint induced by MSU crystals. The results showed that the EtOH80 extract had a greater amount of andrographolide (11.34% w/w) than the water extract (1.38% w/w). In the XO inhibitory activity, none of the samples exhibited greater than 50% inhibition. However, in a rat model, EtOH80 extract (200 mg/kg/day) and andrographolide (30 mg/kg/day) decreased serum uric acid levels and reduced liver XO activity, reduced the protein expression levels of URAT1 and GLUT9, and restored the decrease in OAT1 levels. In the in vitro anti-inflammatory study, EtOH80 extract and andrographolide significantly decreased production of IL-1α, IL-1ß, IL-6, and TNF-α, as well as inhibited the synthesis of MyD88, NLRP3, NF-κB p65, and caspase-1 in a concentration-dependent manner, almost comparable to dexamethasone. The EtOH80 extract (200 mg/kg/day) and andrographolide (30 mg/kg) significantly decreased swelling rate and IL-1α, IL-1ß, IL-6, and TNF-α in the synovial fluid of rat models in a time-dependent manner, comparable to indomethacin (3 mg/kg/day). In conclusion, the findings show that EtOH80 extract has a substantial anti-gout effect by lowering uric acid levels and suppressing pro-inflammatory mediator production due to the andrographolide content, that might be beneficial in the treatment of gouty-inflammation.

Anti-hyperuricemic and Anti-inflammatory Effects of Marantodes pumilum as Potential Treatment for Gout.

Marantodes pumilum (Primulaceae) has been used in Malaysian folk medicine to help women regain strength after delivery and for "sickness in the bones." It was previously revealed that its extracts inhibited xanthine oxidase (XO) activity in vitro. The leaves and roots of M. pumilum var. alata (MPA), var. pumila (MPP), and var. lanceolata (MPL) were individually extracted in ethanol (80%). The anti-hyperuricemic activity was initially assessed by XO inhibition with a spectrophotometric in vitro assay. The most active extract was further investigated on hyperuricemic rat model induced by potassium oxonate to determine serum uric acid levels and liver XO effect. The in vitro anti-inflammatory activity was carried out on monosodium urate (MSU) crystal-induced pro-inflammatory cytokines (i.e., interleukin (IL)1α, IL-1ß, IL-6, IL-8, and tumor necrosis factor (TNF)-α) secretion using human peripheral blood mononuclear cells and ELISA technique, and prostaglandin E2 (PGE2)secretion using radioimmunoassay. The active extract was then investigated on gout-induced inflammation with MSU crystals to determine pro-inflammatory cytokines and PGE2 secretion levels in the synovial fluid of rat knee joint. Quantitative analysis using validated HPLC was performed on the extracts to determine presence of bioactive flavonoids. The findings revealed that extract of MPP leaves gave the highest inhibitory activity on XO (IC50 130.5 µg/mL) compared to other extracts tested. However, all extracts possessed significantly lower activity compared to allopurinol (IC50 0.13 µg/mL). Oral administration of MPP leaf extract (200 mg/kg) significantly reduced serum uric acid level in hyperuricemic rats in time-dependent manner to the baseline level and it was as effective as allopurinol (5 mg/kg). The extract also inhibited liver XO activity (25%) compared to allopurinol (45%). In vitro anti-inflammatory assay showed that extract of MPP roots inhibited MSU crystals-induced secretion of IL-1α, IL-1ß, IL-8, TNF-α, and PGE2 with IC50 values of 36, 25, 38, 18, and 46 µg/mL, respectively. Oral administration of the MPP root extract (200 mg/kg) significantly decreased IL-1α, IL-1ß, IL-6, TNF-α, and PGE2 levels in rat's synovial fluid as effective as indomethacin. There were no significant body weight changes of all experimental animals. MPP extracts showed presence of myricetin, quercetin and kaempferol. Myricetin was detected with values of 0.2 and 0.6 mg/g for root and leaf extracts, respectively. The anti-hyperuricemic of MPP leaf and anti-inflammatory of MPP root indicated that MPP may be promising for complementary therapy of gout.

Evaluation of phytoestrogens in inducing cell death mediated by decreasing Annexin A1 in Annexin A1-knockdown leukemia cells.

BACKGROUND: Phytoestrogens are plant compounds that are structurally similar to estrogen and that possess anti-cancer properties. Previous studies have reported that coumestrol, daidzein and genistein could induce cell death by reducing Annexin A1 protein in leukemic cell lines. Annexin A1 (ANXA1) is involved in cell progression, metastasis, and apoptosis in several types of cancer cells. The present study sought to investigate if the effects of phytoestrogens on apoptosis, cell cycle arrest and phagocytosis in ANXA1-knockdown leukemic cells are mediated through ANXA1 or occurred independently. METHODS: Transfection of ANXA1 siRNA was conducted to downregulate ANXA1 expression in Jurkat, K562 and U937 cells. Apoptosis and cell cycle assays were conducted using flow cytometry. Western blot was performed to evaluate ANXA1, caspases and Bcl-2 proteins expression. Phagocytosis was determined using hematoxylin and eosin staining. RESULTS: The expression of ANXA1 after the knockdown was significantly downregulated in all cell lines. Genistein significantly induced apoptosis associated with an upregulation of procaspase-3, -9, and - 1 in Jurkat cells. The Bcl-2 expression showed no significant difference in Jurkat, K562 and U937 cells. Treatment with phytoestrogens increased procaspase-1 expression in Jurkat and U937 cells while no changes were detected in K562 cells. Flow cytometry analysis demonstrated that after ANXA1 knockdown, coumestrol and genistein caused cell cycle arrest at G2/M phase in selected type of cells. The percentage of phagocytosis and phagocytosis index increased after the treatment with phytoestrogens in all cell lines. CONCLUSION: Phytoestrogens induced cell death in ANXA1-knockdown leukemia cells, mediated by Annexin A1 proteins. Graphical abstract.

In vitro anti-allergic activity of Moringa oleifera Lam. extracts and their isolated compounds.

BACKGROUND: Moringa oleifera Lam. is a commonly used plant in herbal medicine and has various reported bioactivities such as antioxidant, antimicrobial, anticancer and antidiabetes. It is rich in nutrients and polyphenols. The plant also has been traditionally used for alleviating allergic conditions. This study was aimed to examine the anti-allergic activity of M. oleifera extracts and its isolated compounds. METHOD: M. oleifera leaves, seeds and pods were extracted with 80% of ethanol. Individual compounds were isolated using a column chromatographic technique and elucidated based on the nuclear magnetic resonance (NMR) and electrospray ionisation mass spectrometry (ESIMS) spectral data. The anti-allergic activity of the extracts, isolated compounds and ketotifen fumarate as a positive control was evaluated using rat basophilic leukaemia (RBL-2H3) cells for early and late phases of allergic reactions. The early phase was determined based on the inhibition of beta-hexosaminidase and histamine release; while the late phase was based on the inhibition of interleukin (IL-4) and tumour necrosis factor (TNF-α) release. RESULTS: Two new compounds; ethyl-(E)-undec-6-enoate (1) and 3,5,6-trihydroxy-2-(2,3,4,5,6-pentahydroxyphenyl)-4H-chromen-4-one (2) together with six known compounds; quercetin (3), kaempferol (4), ß-sitosterol-3-O-glucoside (5), oleic acid (6), glucomoringin (7), 2,3,4-trihydroxybenzaldehyde (8) and stigmasterol (9) were isolated from M. oleifera extracts. All extracts and the isolated compounds inhibited mast cell degranulation by inhibiting beta-hexosaminidase and histamine release, as well as the release of IL-4 and TNF-α at varying levels compared with ketotifen fumarate. CONCLUSION: The study suggested that M. oleifera and its isolated compounds potentially have an anti-allergic activity by inhibiting both early and late phases of allergic reactions.

Phyllanthus amarus protects against spatial memory impairment induced by lipopolysaccharide in mice.

Phyllanthus amarus Schumach. and Thonn. is a wide spread medicinal herb with various traditional uses. It is well documented for its antioxidant, anti-inflammatory, and hepatoprotective activities. Therefore, it is of interest to evaluate the 80% ethanol extract of Phyllanthus amarus (PA) on spatial memory using the 8-radial arm maze (8-RAM) in mice after induction of neuro inflammation by lipopolysaccharide (LPS) in a 14- and 28-days treatment study. LC-MS/MS was performed to profile the chemical composition in PA extract. Mice were treated orally with 5% v/v tween 20, PA extract (100, 200 and 400 mg/kg), or ibuprofen (IBF 40 mg/kg) for 14 and 28 days. All groups were challenged with LPS (1 mg/kg) via intraperitoneal (i.p.) injection a day prior to the 8-RAM task except for the negative control group which received an i.p. injection of saline. Data obtained were analyzed with one-way ANOVA followed by post hoc Dunnett's test (comparison of all groups against vehicle control). Analysis of LC-MS/MS data revealed the presence of 16 compounds in the PA extract. Administration of PA extract at 200 and 400 mg/kg for 14 and 28 days significantly (*P<0.05) decreased the working and reference memory errors against LPS-induced spatial memory impairment. The observed protective action is possibly due to the putative antineuroinflammatory effects of PA. In conclusion, PA extract possess neuroprotective effects against spatial memory impairment mediated by LPS.

Effects of annexin A1 on apoptosis and cell cycle arrest in human leukemic cell lines.

Recent studies suggest that annexin A1 (ANXA1) promotes apoptosis in cancerous cells. This study aims to investigate the effects of ANXA1 on apoptosis and cell cycle arrest in K562, Jurkat and U937 cells and peripheral blood mononu-clear cells (PBMC). Cells were treated with ANXA1 and cyclophosphamide prior to flow cytometry analysis for apoptosis and cell cycle arrest induction. At 2.5µM, ANXA1 induced significant apoptosis in K562 (p ≤ 0.001) and U937 (p ≤ 0.05) cells, with EC50 values of 3.6 and 3.8 µM, respectively. In Jurkat cells, induction was not significant (EC50, 17.0 µM). No significant apoptosis induction was observed in PBMC. ANXA1 caused cycle arrest in the G0/G1 phase in K562 and U937 cells with p ≤ 0.001 for both, and (p ≤ 0.01) for Jurkat cells. ANXA1 induced apoptosis and cycle arrest in the G0/G1 phase in K562 and U937 cells, causing only cell cycle arrest in Jurkat cells.

Protective Effects of Phyllanthus amarus Against Lipopolysaccharide-Induced Neuroinflammation and Cognitive Impairment in Rats.

Background: Phyllanthus amarus (PA) is widely studied for its hepatoprotective properties but has recently received increasing attention due to its diverse anti-inflammatory effects. However, the effects of PA in modulating immune responses in the central nervous system leading to protection against functional changes remain unexplored. Therefore, we sought to examine the protective effects of 80% v/v ethanol extract of PA on lipopolysaccharide (LPS)-induced non-spatial memory impairment and neuroinflammation. Methods: Selected major phytoconstituents of PA extract were identified and quantified using high-performance liquid chromatography. Subchronic neurotoxicity was performed in male Wistar rats given daily oral administration of 100, 200, and 400 mg/kg of the PA extract. Their neurobehavioral activities (functional observation battery and locomotor activity) were scored, and the extracted brains were examined for neuropathological changes. Rats were treated orally with vehicle (5% Tween 20), PA extract (100, 200, and 400 mg/kg), or ibuprofen (IBF; 40 mg/kg) for 14 and 28 days before being subjected to novel object discrimination test. All groups were challenged with LPS (1 mg/kg) given intraperitoneally a day prior to the behavioral tests except for the negative control group. At the end of the behavioral tests, the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, nitric oxide (NO), inducible nitric oxide synthase (iNOS), CD11b/c integrin expression, and synaptophysin immunoreactivity were determined in the brain tissues. Results: Gallic acid, ellagic acid, corilagin, geraniin, niranthin, phyllanthin, hypophyllanthin, phyltetralin, and isonirtetralin were identified in the PA extract. Subchronic administration of PA extract (100, 200, and 400 mg/kg) showed no abnormalities in neurobehavior and brain histology. PA extract administered at 200 and 400 mg/kg for 14 and 28 days effectively protected the rodents from LPS-induced memory impairment. Similar doses significantly (p < 0.05) decreased the release of proteins like TNF-α, IL-1ß, and iNOS in the brain tissue. NO levels, CD11b/c integrin expression, and synaptophysin immunoreactivity were also reduced as compared with those in the LPS-challenged group. Conclusion: Pre-treatment with PA extract for 14 and 28 days was comparable with pre-treatment with IBF in prevention of memory impairment and alleviation of neuroinflammatory responses induced by LPS. Further studies are essential to identify the bioactive phytochemicals and the precise underlying mechanisms.

Effects of MAO-A and CYP450 on primaquine metabolism in healthy volunteers.

Eliminating the Plasmodium vivax malaria parasite infection remains challenging. One of the main problems is its capacity to form hypnozoites that potentially lead to recurrent infections. At present, primaquine is the only drug used for the management of hypnozoites. However, the effects of primaquine may differ from one individual to another. The aim of this work is to determine new measures to reduce P. vivax recurrence, through primaquine metabolism and host genetics. A genetic study of MAO-A, CYP2D6, CYP1A2 and CYP2C19 and their roles in primaquine metabolism was undertaken of healthy volunteers (n = 53). The elimination rate constant (Ke) and the metabolite-to-parent drug concentration ratio (Cm/Cp) were obtained to assess primaquine metabolism. Allelic and genotypic analysis showed that polymorphisms MAO-A (rs6323, 891G>T), CYP2D6 (rs1065852, 100C>T) and CYP2C19 (rs4244285, 19154G>A) significantly influenced primaquine metabolism. CYP1A2 (rs762551, -163C>A) did not influence primaquine metabolism. In haplotypic analysis, significant differences in Ke (p = 0.00) and Cm/Cp (p = 0.05) were observed between individuals with polymorphisms, GG-MAO-A (891G>T), CT-CYP2D6 (100C>T) and GG-CYP2C19 (19154G>A), and individuals with polymorphisms, TT-MAO-A (891G>T), TT-CYP2D6 (100C>T) and AA-CYP2C19 (19154G>A), as well as polymorphisms, GG-MAO-A (891G>T), TT-CYP2D6 (100C>T) and GA-CYP2C19 (19154G>A). Thus, individuals with CYP2D6 polymorphisms had slower primaquine metabolism activity. The potential significance of genetic roles in primaquine metabolism and exploration of these might help to further optimise the management of P. vivax infection.

Suppressive Effects of the Standardized Extract of Phyllanthus amarus on Type II Collagen-induced Rheumatoid Arthritis in Sprague Dawley Rats.

BACKGROUND: Standardized extract of Phyllanthus amarus has been shown to possess inhibitory effects on cellular and humoral immune responses in Wistar-Kyoto rats and Balb/c mice. OBJECTIVE: In the present study, the standardized extract of P. amarus was investigated for its suppressive effects on type II collagen-induced rheumatoid arthritis (TCIA) in Sprague Dawley rats. METHOD: The major components of the extracts, lignans and phenolic compounds were analysed by using a validated reversed phase HPLC and LC-MS/MS. A rheumatoid arthritis rat model was induced by administering a bovine type II collagen emulsion subcutaneously at the base of tail, on day 0 and 7 of the experiment. Effects of the extract on severity assessment, changes in the hind paw volume, bone mineral density, body weight and body temperature were measured. Concentrations of cytokines (TNF-α, IL-1ß, IL-1α, IL-6) released, matrix metalloproteinases (MMP-1, MMP-3 MMP-9) and their inhibitor (TIMP-1), haematological and biochemical changes were also measured. ELISA was used to measure the cytokines and proteinases in the rat serum and synovial fluid according to manufacturer's instructions. RESULTS: The extract dose-dependently modulated the progression in physical parameters (i.e. decrease in body weight, increase in body temperature, reduced hind paw volume, reduced the severity of arthritis), bone mineral density, haematological and biochemical perturbations, serum cytokines production and levels of matrix metalloproteinases and their inhibitor in the synovial fluid. Histopathological examination of the knee joint also revealed that the extract effectively reduced synovitis, pannus formation, bone resorption and cartilage destruction. CONCLUSION: The results suggest that the oral administration of a standardized extract of P. amarus was able to suppress the humoral and cellular immune responses to type II collagen, resulting in the reduction of the development of TCIA in the rats.

Phyllanthin from Phyllanthus amarus inhibits LPS-induced proinflammatory responses in U937 macrophages via downregulation of NF-κB/MAPK/PI3K-Akt signaling pathways.

Phyllanthin, a lignan from Phyllanthus species, has been reported to possess potent immunosuppressive properties on immune cells and on adaptive and innate immune responses in animal models. Herein, we investigated the inhibitory effects of phyllanthin isolated from Phyllanthus amarus on nuclear factor-kappa B (NF-κB), mitogen-activated protein kinase (MAPK), and PI3K-Akt signal transducing pathways in LPS-activated U937 cells. The lipopolysaccharide-stimulated excess production of prostaglandin was significantly suppressed by phyllanthin via the mechanisms linked to the modulatory effects of cyclooxygenase 2 protein and gene expression. Phyllanthin also significantly inhibited the release and mRNA expression of proinflammatory cytokines (interleukin-1 beta and tumor necrosis factor-alpha). Phyllanthin also significantly downregulated the phosphorylation of IκBα, NF-κB (p65), and IKKα/ß and suppressed the activation of JNK, ERK, p38MAPK, and Akt in a concentration-dependent manner. Additionally, phyllanthin downregulated the expression of upstream signaling molecules including MyD88 and toll-like receptor 4 that are essential for the activation of NF-κB, MAPKs, and PI3K-Akt signal transducing pathways. Based on these observations, phyllanthin may exert their suppressive effects on inflammatory process by mediating the release of inflammatory signaling molecules via the NF-κB, MAPKs, and PI3K-Akt signal transducing pathways. Thus, phyllanthin holds a great promise as a potential anti-inflammatory agent to treat various inflammatory diseases.

Anti-inflammatory effects of Phyllanthus amarus Schum. & Thonn. through inhibition of NF-κB, MAPK, and PI3K-Akt signaling pathways in LPS-induced human macrophages.

BACKGROUND: Phyllanthus amarus has been used widely in various traditional medicines to treat swelling, sores, jaundice, inflammatory diseases, kidney disorders, diabetes and viral hepatitis, while its pharmacological and biochemical mechanisms underlying its anti-inflammatory properties have not been well investigated. The present study was carried out to investigate the effects of 80% ethanolic extract of P. amarus on pro-inflammatory mediators release in nuclear factor-kappa B (NF-кB), mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase/Akt (PI3K-Akt) signaling activation in lipopolysaccharide (LPS)-induced U937 human macrophages. METHODS: The release of prostaglandin E2 (PGE2) and pro-inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1ß in a culture supernatant was determined by ELISA. Determination of cyclooxygenase-2 (COX-2) protein and the activation of MAPKs molecules (JNK, ERK and p38 MAPK), NF-κB and Akt in LPS-induced U937 human macrophages were investigated by immunoblot technique. The relative gene expression levels of COX-2 and pro-inflammatory cytokines were measured by using qRT-PCR. The major metabolites of P. amarus were qualitatively and quantitatively analyzed in the extract by using validated reversed-phase high performance liquid chromatography (HPLC) methods. RESULTS: P. amarus extract significantly inhibited the production of pro-inflammatory mediators (TNF-α, IL-1ß, PGE2) and COX-2 protein expression in LPS-induced U937 human macrophages. P. amarus-pretreatment also significantly downregulated the increased mRNA transcription of pro-inflammatory markers (TNF-α, IL-1ß, and COX-2) in respective LPS-induced U937 macrophages. It downregulated the phosphorylation of NF-κB (p65), IκBα, and IKKα/ß and restored the degradation of IκBα, and attenuated the expression of Akt, JNK, ERK, and p38 MAPKs phosphorylation in a dose-dependent manner. P. amarus extract also downregulated the expression of upstream signaling molecules, TLR4 and MyD88, which play major role in activation of NF-κB, MAPK and PI3K-Akt signaling pathways. The quantitative amounts of lignans, phyllanthin, hypophyllahtin and niranthin, and polyphenols, gallic acid, geraniin, corilagin, and ellagic acid in the extract were determined by HPLC analysis. CONCLUSION: The study revealed that P. amarus targeted the NF-κB, MAPK and PI3K-Akt signaling pathways to exert its anti- inflammatory effects by downregulating the prospective inflammatory signaling mediators.

Immunomodulatory effects of Tinospora crispa extract and its major compounds on the immune functions of RAW 264.7 macrophages.

The in vivo immunomodulatory activities of Tinospora crispa have been reported but its molecular mechanisms underlying its immunomodulatory properties remains obscure and the active constituents contributing to the activities have not been identified. The present study was aimed to investigate the immunomodulatory effects of T. crispa extract (TCE) and its chemical constituents on RAW 264.7 macrophages. Six known compounds including magnoflorine and syringin were isolated by various chromatographic techniques from TCE and their structures were determined spectroscopically. A validated HPLC method was used to quantify magnoflorine and syringin in the extract. The immunomodulatory effects of TCE and its isolated compounds on chemotaxis, phagocytosis, production of inflammatory mediators including reactive oxygen species (ROS), nitric oxide (NO), prostaglandin E2 (PGE2) and pro-inflammatory cytokines which include tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, IL-6 and monocyte chemoattractant protein-1 (MCP-1) on macrophages were assessed. TCE increased the chemotaxis and phagocytic activity of macrophages and significantly enhanced the production of ROS, NO and pro-inflammatory cytokines. All alkaloids isolated, specifically magnoflorine showed remarkable inducing effects on the chemotaxis, phagocytic activity, ROS and NO productions and the secretions of IL-1ß, TNF-α, IL6, PGE2 and MCP-1. In contrast, syringin potently reduced the chemotaxis, phagocytic activity, ROS and NO productions and secretions of IL-1ß, TNF-α, IL6, PGE2 and MCP-1. TCE showed strong immunostimulant effects on various components of the immune system and these activities were possibly contributed mainly by the alkaloids specifically magnoflorine. TCE has potential to be developed as an effective natural immunostimulant for improvement of immune-related disorders.

Moringa Genus: A Review of Phytochemistry and Pharmacology.

Moringa is a genus of medicinal plants that has been used traditionally to cure wounds and various diseases such as colds and diabetes. In addition, the genus is also consumed as a source of nutrients and widely used for purifying water. The genus consists of 13 species that have been widely cultivated throughout Asia and Africa for their multiple uses. The purpose of this review is to provide updated and categorized information on the traditional uses, phytochemistry, biological activities, and toxicological research of Moringa species in order to explore their therapeutic potential and evaluate future research opportunities. The literature reviewed for this paper was obtained from PubMed, ScienceDirect, and Google Scholar journal papers published from 1983 to March 2017. Moringa species are well-known for their antioxidant, anti-inflammatory, anticancer, and antihyperglycemic activities. Most of their biological activity is caused by their high content of flavonoids, glucosides, and glucosinolates. By documenting the traditional uses and biological activities of Moringa species, we hope to support new research on these plants, especially on those species whose biological properties have not been studied to date.

Anti-Inflammatory Effects of Hypophyllanthin and Niranthin Through Downregulation of NF-κB/MAPKs/PI3K-Akt Signaling Pathways.

Hypophyllanthin (HYP) and niranthin (NIR) are major lignans in Phyllanthus spp. and have been shown to possess strong anti-inflammatory activity. In this study, we investigated the anti-inflammatory effects and the underlying molecular mechanisms of HYP and NIR in in vitro cellular model of LPS-induced U937 macrophages. The effects of HYP and NIR on the production of prostaglandin E2 (PGE2), cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α), and interleukin-1ß (IL-1ß) were measured by using ELISA, Western blot, and qRT-PCR. The expressions of signaling molecules related to nuclear factor-kappa B (NF-κB), mitogen-activated protein kinases (MAPKs), and phosphatidylinositol 3'-kinase-Akt (PI3K-Akt) signaling pathways were examined. The role of NF-κB, MAPKs, and Akt signaling pathways was confirmed by using specific inhibitors (BAY 11-7082, U0126, SB202190, SP600125, and LY294002) mediated suppression of TNF-α and COX-2 production. HYP and NIR significantly inhibited the protein and gene levels of COX-2 as well as the downstream signaling products of PGE2, TNF-α, and IL-1ß. HYP and NIR also suppressed the inhibitors of kappa B (IκB), IkB kinases (Ikkα/ß), NF-κB phosphorylation, and IκB degradation. HYP suppressed the phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 while NIR only suppressed JNK and ERK but did not have effect on p38. These results demonstrate that HYP and NIR downregulated COX-2, TNF-α, and IL-1ß gene expressions in U937 macrophages by interfering with the activation of NF-κB, MAPKs, and Akt. In conclusion, these lignans have potential to be developed as anti-inflammatory agents targeting the NF-κB, MAPK, and PI3K-Akt pathways.