Exploring the therapeutic potential of Thai medicinal plants: in vitro screening and in silico docking of phytoconstituents for novel anti-SARS-CoV-2 agents.

BACKGROUND: The high virulence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for coronavirus disease 2019 (COVID-19), has triggered global health and economic concerns. The absence of specific antiviral treatments and the side effects of repurposed drugs present persistent challenges. This study explored a promising antiviral herbal extract against SARS-CoV-2 from selected Thai medicinal plants based on in vitro efficacy and evaluated its antiviral lead compounds by molecular docking. METHODS: Twenty-two different ethanolic-aqueous crude extracts (CEs) were rapidly screened for their potential activity against porcine epidemic diarrhea virus (PEDV) as a surrogate using a plaque reduction assay. Extracts achieving ≥ 70% anti-PEDV efficacy proceeded to the anti-SARS-CoV-2 activity test using a 50% tissue culture infectious dose method in Vero E6 cells. Molnupiravir and extract-free media served as positive and negative controls, respectively. Potent CEs underwent water/ethyl acetate fractionation to enhance antiviral efficacy, and the fractions were tested for anti-SARS-CoV-2 performance. The fraction with the highest antiviral potency was identified using liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Molecular docking analyses of these compounds against the main protease (Mpro) of SARS-CoV-2 (6LU7) were performed to identify antiviral lead molecules. The top three hits were further evaluated for their conformational stability in the docked complex using molecular dynamics (MD) simulation. RESULTS: The water fraction of mulberry (Morus alba Linn.) leaf CE (WF-MLCE) exhibited the most potent anti-SARS-CoV-2 efficacy with low cytotoxicity profile (CC50 of ~ 0.7 mg/mL), achieving 99.92% in pre-entry mode and 99.88% in postinfection treatment mode at 0.25 mg/mL. Flavonoids and conjugates were the predominant compounds identified in WF-MLCE. Molecular docking scores of several flavonoids against SARS-CoV-2 Mpro demonstrated their superior antiviral potency compared to molnupiravir. Remarkably, myricetin-3-O-ß-D-galactopyranoside, maragrol B, and quercetin 3-O-robinobioside exhibited binding energies of ~ - 9 kcal/mol. The stability of each ligand-protein complex of these compounds with the Mpro system showed stability during MD simulation. These three molecules were pronounced as antiviral leads of WF-MLCE. Given the low cytotoxicity and high antiviral potency of WF-MLCE, it holds promise as a candidate for future therapeutic development for COVID-19 treatment, especially considering its economic and pharmacological advantages.

The synergistic effect of Cordyceps sinensis and Gymnema inodorum (Lour.) Decne. extracts on amelioration of glucose intolerance and dyslipidemia in high-fat diet-fed mice.

Cordyceps sinensis (C. sinensis) and Gymnema inodorum (Lour.) Decne. (G. inodorum) have been widely used for treating various illnesses. The study focused on exploring the effects of C. sinensis extract (CSE), G. inodorum extract (GIE), using alone and combined (COM) on ameliorating glucose intolerance, dyslipidemia, and obesity in mice fed with a high-fat diet (HFD). The results revealed that the oral glucose tolerance test (OGTT), total cholesterol (TC), triglycerides (TG), and LDL-cholesterol (LDL) exhibited a significant decrease in all groups treated with CSE, GIE, and COM compared to the control (p < 0.05). Obviously, CSE plus GIE exhibited a synergistic effect on amelioration of OGTT, TC, TG, and LDL, which is also the first report. Furthermore, the extracts showed no toxicity in the mice's vital organs. These results suggest that CSE, GIE, and their combined could have the potential as complementary therapeutic approaches for managing hyperglycaemia and dyslipidemia.

Free radical scavenging and anti-isolated human LDL oxidation activities of Butea superba Roxb. extract.

BACKGROUND: Butea superba Roxb. (B. superba), is an herbal plant traditionally used for rejuvenation. Additionally, there have been reports on its antioxidant properties. Low-density lipoproteins (LDL) oxidation is the leading cause of cardiovascular diseases (CVDs). Natural products with antioxidant properties have the potential to inhibit LDL oxidation. However, no work has been done about the anti-isolated human LDL oxidation of B. superba extract (BSE). This study aimed to investigate the antioxidant potential of BSE and its ability to prevent isolated human (LDL) oxidation induced by free radical agents. METHODS: The antioxidant properties were investigated by antioxidant assays, including 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), ferric reducing ability power (FRAP), nitric oxide (NO) and peroxynitrite scavenging assay. More so, anti-isolated human LDL oxidation activities were evaluated by 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) and 3-morpholinosydnonimine hydrochloride (SIN-1) induced LDL oxidation assay. RESULTS: BSE exhibited a significant (p < 0.05) antioxidant activity in all the test systems, demonstrating its potential as a potent free radical scavenger. It displayed scavenging effects on DPPH (p < 0.05; IC50 = 487.67 ± 21.94 µg/ml), ABTS (p < 0.05; IC50 = 30.83 ± 1.29 µg/ml). Furthermore, it generated significantly (p < 0.05) increased antioxidant capacity in a dose-dependent manner in FRAP assay and exhibited significantly (p < 0.01) higher percent NO scavenging activity than gallic acid. Besides, BSE at 62.5 µg/ml exhibited a considerable percent peroxynitrite scavenging of 71.40 ± 6.59% after a 2 h period. Moreover, BSE demonstrated anti-isolated human LDL oxidation activity induced by AAPH and SIN-1 (p < 0.05) and revealed scavenging activity similar to ascorbic acid (p > 0.05). Identifying the main constituents of BSE revealed the presence of genistein, daidzein, and biochanin A through Liquid Chromatography-Mass Spectrometer/Mass Spectrometer (LC-MS/MS) analysis. CONCLUSION: This is the first report that the presence of isoflavones in BSE could play an important role in its antioxidation and isolated human LDL oxidation scavenging properties. These findings suggest the potential for developing antioxidant herbal supplements. However, further studies must be investigated, including efficacious and safe human dosages.

Effects of Heliotropium indicum L. on Uterine Involution and Its Underlying Mechanisms: an in vivo and in vitro Study.

OBJECTIVE: To investigate the effect of Heliotropium indicum L. (H. indicum L.) on uterine involution and its underlying mechanisms in both in vivo and in vitro study. METHODS: For in vivo studies, postpartum rats were randomly divided into 2 groups (n=24 for each): control group and treated group which were orally and daily administered with ethanolic extract of H. indicum L. (250 mg/kg body weight) until day 5 of postpartum. Uteri were collected for analysis of weight, cross-sectional area, collagen cross-sectional area, and collagen content on postpartum day 1, 3, and 5 (n=8 for each) from both groups. Blood samples were collected for hepatotoxicity and 17ß-estradiol (E2) measurement. For in vitro studies, the extract effects on uterine contraction at half maximum effective concentration of 2.50 mg/mL were studied in organ bath system for at least 20 min. RESULTS: Uterine parameters were significantly decreased after treated with extract of H. indicum L. (P<0.05). H. indicum L. extract significantly accelerated the reduction of those parameters and significantly decreased E2 (P<0.05). The extract facilitated uterine involution with no hepatotoxicity. H. indicum L. extract significantly stimulated uterine contraction (P<0.05) and synergized with oxytocin, prostaglandin and its precursor, linoleic acid. By investigating the different sequencing of the extract with the additional stimulants (added before or after), the two showed antagonistic effects, but still showed potentiated force when compared with control (without the stimulants). CONCLUSIONS: The underlying mechanisms by which H. indicum L. facilitated uterine involution might be due to reducing E2 which induces collagenase activity, leading to decreases in uterine weight and size and stimulating uterine contraction. Our study provides new findings for future drug development for facilitating uterine involution with H. indicum L.

Antiproliferative, Antiangiogenic, and Antimigrastatic Effects of Oroxylum indicum (L.) Kurz Extract on Breast Cancer Cell.

Breast cancer recurrence continues to pose a major clinical problem, despite significant advancements in early diagnosis and an aggressive mode of treatment. This study aimed at investigating the anticancer activity of Oroxylum indicum extract (OIE) by assessing cell proliferation, cell migration, and angiogenesis in metastatic breast cancer MDA-MB-231 cell lines. This study also estimated the phytochemical profiles of OIE by LC-QTOF-MS. The extract was found to contain six identified flavonoid substances, and baicalein was the most abundant substance in the extract. Cell proliferation capacity was performed by cell counting kit-8 (CCK-8) and colony formation assays. The effect of OIE on cell migration was determined using wound healing and transwell assays. Meanwhile, MDA-MB-231-induced angiogenesis on chick chorioallantoic membrane (CAM) was applied to investigate the ex vivo antiangiogenesis activity of the extracts. OIE at concentrations lower than 600 µg/mL had no cytotoxic effects against MDA-MB-231 cells. OIE was found to inhibit the long-term colony formation ability of MDA-MB-231 cells in a concentration-dependent manner. Antimigration and antiangiogenesis activities were further investigated using noncytotoxic concentrations of OIE ranging from 25 to 150 µg/mL. OIE greatly reduced the migration of MDA-MB-231 breast cancer cells in a dose-dependent manner. OIE significantly suppressed the MDA-MB-231-induced angiogenesis, and there was no substantial toxic effect on natural angiogenesis. Interestingly, the concentration of OIE at 150 µg/mL was as practically potent as pazopanib, the positive anticancer drug, at 4.37 µg/mL in inhibiting MDA-MB-231 cell migration and angiogenesis induced by these cells. Therefore, the inhibitory effects of OIE in cell proliferation and cell migration, together with antiangiogenesis in MDA-MB-231 breast cancer cells, suggesting that OIE has the potential to be a novel adjunct candidate for breast cancer chemotherapeutic agents.

Synergistic Interaction between Boesenbergia rotunda (L.) Mansf. Essential Oil and Cloxacillin on Methicillin-Resistant Staphylococcus aureus (MRSA) Inhibition.

Currently, antibiotic resistance is widespread among bacteria. This problem requires greater awareness because bacterial resistance increases, reducing antibiotic use effectiveness. Consequently, new alternative treatments are needed because the treatment options for these bacteria are limited. This work aims to determine the synergistic interaction and mechanism of action of Boesenbergia rotunda essential oil (BREO) against methicillin-resistant Staphylococcus aureus (MRSA). Gas chromatography-mass spectrometry identified 24 BREO chemicals (GC-MS). The main components of BREO were ß-ocimene (36.73%), trans-geraniol (25.29%), camphor (14.98%), and eucalyptol (8.99%). BREO and CLX inhibited MRSA DMST 20649, 20651, and 20652 with a minimum inhibitory concentration (MIC) of 4 mg/mL and 512 µg/mL, respectively. The checkerboard method and the time-kill assay revealed synergy between BREO and CLX with fractional inhibitory concentration (FIC) <0.5 and log reduction >2log10 CFU/mL at 24 hours compared to the most effective chemical. BREO inhibited biofilm formation and increased membrane permeability. Exposure alone to BREO or in combination with CLX inhibited biofilm formation and increased cytoplasmic membrane (CM) permeability. The scanning electron microscopy (SEM) and transmission electron microscopy (TEM) results revealed that alterations in the cell walls, cytoplasmic membrane, and leakage of intracellular components of MRSA DMST 20651 after treatment with BREO alone and in combination with CLX were observed. These results indicate that BREO synergizes and could reverse the antibacterial activity of CLX against MRSA strains. The synergy of BREO may lead to novel drug combinations that increase the effectiveness of antibiotics against MRSA.

Stephania suberosa Forman extract synergistically inhibits ampicillin- and vancomycin-resistant Enterococcus faecium.

Increasing antibiotic resistance in enterococci is among the most serious public health problems worldwide. The new naturally occurring antibacterial agents were explored. This study, therefore, investigated the antibacterial potential of Stephania suberosa extract (SSE) and its synergism with ampicillin (AMP) or vancomycin (VAN) against AMP- and VAN-resistant Enterococcus faecium. Disc diffusion assay revealed that SSE inhibited E. faecium DMST 12829, 12852, 12970, and a reference strain of Enterococcus faecalis ATCC 29,212 in a dose-dependent manner. The minimum inhibitory concentration (MIC) of SSE against all E. faecium isolates was 0.5 mg/mL. E. faecium DMST 12,829 and 12,852 were highly resistant to AMP, as indicated by high MIC values, and E. faecium DMST 12,829 and 12,970 were resistant to VAN. Enterococcus spp. were killed by SSE at the minimum bactericidal concentrations (MBCs) ranging from 0.5 to 4 mg/mL. Checkerboard determination showed that SSE plus AMP and SSE plus VAN combinations exhibited synergistic interaction against E. faecium isolates. The killing curve assay of E. faecium isolates confirmed the antibacterial and synergistic activities of combined agents by dramatically reducing the viable counts compared to a single agent. Scanning electron microscope elucidated the cell damage and abnormal cell division. Enterococcal proteases were also inhibited by SSE. These findings support that SSE could reverse the activity of AMP and VAN. Moreover, it can synergistically inhibit AMP- and VAN-resistant E. faecium. Our combined agents could be attractive candidates for developing new combinatorial agents to resurrect the efficacy of antibiotics for treating AMP- and VAN-resistant E. faecium infections.

Butea superba Roxb. Extract Ameliorates Scopolamine-Induced Cognitive and Memory Impairment in Aged Male Rats.

Butea superba Roxb. (B. superba) is a herb that has been used for rejuvenation, to improve sexual performance, or to prevent erectile dysfunction function. Alzheimer's disease (AD) is a chronic neurodegenerative disorder that is the main cause of progressive dementia. This study aimed to investigate the amelioration for cognitive and memory dysfunction of B. superba ethanolic extract (BSE), a possible mechanism of action, and its toxicity. The results from the Y-maze test, novel object recognition test, and passive avoidance test exhibited that the administration of BSE at 50 mg/kg (BSL) and 200 mg/kg (BSH) could ameliorate scopolamine-induced cognitive impairment in all behavior testing. Moreover, BSE could prevent the cognitive deficit in a dose-dependent manner in a passive avoidance test. Furthermore, BSE inhibited acetylcholinesterase's (AChE) ex vivo activity in the cerebral cortex and hippocampus. Also, the in vitro and ex vivo antioxidative effects of BSE revealed that BSE had free radical scavenging activities in both DPPH and FRAP assay. Furthermore, male rats treated with BSE at 200 mg/kg/day for two weeks could significantly increase serum testosterone compared with control (P < 0.05). The GC-MS analysis and previous studies revealed that BSE contained propanoic acid, 3,3'-thiobis-, didodecyl ester, oleic acid, gamma-sitosterol, and stigmasterol which may play an important role in cognitive and memory impairment prevention. The toxicity test of BSE in rats at 50 and 200 mg/kg/day for two weeks showed that relative organ weight, serum creatinine, ALT, ALP, and CBC levels of both treated groups were not significantly different compared to the CON (P > 0.05). These results suggest that BSE may not be toxic to the vital organ and blood. In conclusion, BSE has the potential to be developed as a health supplement product or medicine for AD prevention and treatment.

The Effect of Pluchea indica (L.) Less. Tea on Blood Glucose and Lipid Profile in People with Prediabetes: A Randomized Clinical Trial.

Objective: Diabetes mellitus and dyslipidemia are currently increasing dramatically, and conventional medicine in the treatment of them has limited efficacies and serious adverse effects. Pluchea indica (L.) Less. tea (PIT) is widely consumed as a health-promoting drink in Southeast Asia. This study aimed to investigate whether P. indica tea has antidyslipidemic and antihyperglycemic effects and toxicity in humans. Design: A randomized clinical trial. Setting/Location: Nakhonratchasima, Thailand. Participants: Forty-five participants with prediabetes. Interventions: Participants were randomized to receive placebo tea, 1.5 g of PIT, and 1.5 g Camellia sinensis tea (green tea, CST) once daily for 12 weeks. Outcome Measurements: The oral glucose tolerance test (OGTT), total cholesterol, triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), blood urea nitrogen (BUN), creatinine, alanine transaminase (ALT), alkaline phosphatase (ALP), and complete blood count (CBC) before and after treatment were investigated. Results: The results showed that PIT significantly ameliorated hyperglycemia and significantly lower serum TG (109.22 ± 5.21 mg/dL) and LDL-C (122.20 ± 3.67 mg/dL) than placebo (145.56 ± 8.18 and 142.07 ± 8.58 mg/dL, respectively) (p < 0.05). Moreover, PIT exhibited serum TG (109.22 ± 5.21 mg/dL) significantly lower than CST (124.38 ± 4.70 mg/dL) (p < 0.05). In addition, the serum HDL-C of PIT (57.56 ± 3.05 mg/dL) was significantly higher than the placebo (46.44 ± 2.47 mg/dL) (p < 0.05). Furthermore, the toxicity testing showed that no significant difference in BUN, creatinine, ALT, ALP, and CBC of PIT-treated group compared with the placebo (p > 0.05). Conclusions: These results suggest that PIT may ameliorate hyperglycemia and dyslipidemia in prediabetes people. It may not be toxic to the kidney, liver, and blood. So, PIT has the potential to develop to be a health-promoting tea or herbal medicine for hyperglycemia and dyslipidemia prevention.

Gymnema inodorum (Lour.) Decne. Extract Alleviates Oxidative Stress and Inflammatory Mediators Produced by RAW264.7 Macrophages.

Gymnema inodorum (Lour.) Decne. (G. inodorum) is widely used in Northern Thai cuisine as local vegetables and commercial herb tea products. In the present study, G. inodorum extract (GIE) was evaluated for its antioxidant and anti-inflammatory effects in LPS plus IFN-γ-induced RAW264.7 cells. Major compounds in GIE were evaluated using GC-MS and found 16 volatile compounds presenting in the extract. GIE exhibited antioxidant activity by scavenging the intracellular reactive oxygen species (ROS) production and increasing superoxide dismutase 2 (SOD2) mRNA expression in LPS plus IFN-γ-induced RAW264.7 cells. GIE showed anti-inflammatory activity through suppressing nitric oxide (NO), proinflammatory cytokine production interleukin 6 (IL-6) and also downregulation of the expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and IL-6 mRNA levels in LPS plus IFN-γ-induced RAW264.7 cells. Mechanism studies showed that GIE suppressed the NF-κB p65 nuclear translocation and slightly decreased the phosphorylation of NF-κB p65 (p-NF-κB p65) protein. Our studies applied the synchrotron radiation-based FTIR microspectroscopy (SR-FTIR), supported by multivariate analysis, to identify the FTIR spectral changes based on macromolecule alterations occurring in RAW264.7 cells. SR-FTIR results demonstrated that the presence of LPS plus IFN-γ in RAW264.7 cells associated with the increase of amide I/amide II ratio (contributing to the alteration of secondary protein structure) and lipid content, whereas glycogen and other carbohydrate content were decreased. These findings lead us to believe that GIE may prevent oxidative damage by scavenging intracellular ROS production and activating the antioxidant gene, SOD2, expression. Therefore, it is possible that the antioxidant properties of GIE could modulate the inflammation process by regulating the ROS levels, which lead to the suppression of proinflammatory cytokines and genes. Therefore, GIE could be developed into a novel antioxidant and anti-inflammatory agent to treat and prevent diseases related to oxidative stress and inflammation.

The Free Radical Scavenging and Anti-Isolated Human LDL Oxidation Activities of Pluchea indica (L.) Less. Tea Compared to Green Tea (Camellia sinensis).

Tea is one of the most popular beverages in the world. Camellia sinensis tea (CST) or green tea is widely regarded as a potent antioxidant. In Thailand, Pluchea indica (L.) Less. tea (PIT) has been commercially available as a health-promoting drink. This study focused on free radical scavenging activities of PIT, and its ability to protect isolated human low-density lipoproteins (LDL) from oxidation by chemical agents. A preliminary study to investigate the antioxidant nature of PIT was undertaken. These included common antioxidant assays involving 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2-azinobis-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), hypochlorous acid (HOCl), and its potential to scavenge peroxynitrite. In separated experiments, isolated human LDL was challenged with either 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), copper (Cu2+), or 3-Morpholinosydnonimine hydrochloride (SIN-1) to induce LDL oxidation. PIT exhibited antioxidant activity in all test systems and performed significantly better than CST in both DPPH (P < 0.05; IC50PIT = 245.85 ± 15.83 and CST = 315.41 ± 24.18 µg/ml) and peroxynitrite scavenging assays. PIT at 75 µg/ml almost fully prevented the peroxynitrite over a 5 h period. Moreover, it displayed similar properties to CST during the antioxidation of isolated human LDL using AAPH, Cu2+, SIN-1, and hypochlorous acid scavenging assays. However, it revealed a significantly lower ABTS scavenging activity than CST (P < 0.05; IC50PIT = 30.47 ± 2.20 and CST = 21.59 ± 0.67 µg/ml). The main constituents of the PIT were identified using LC-MS/MS. It contained 4-O-caffeoylquinic acid (4-CQ), 5-O-caffeoylquinic acid (5-CQ), 3,4-O-dicaffeoylquinic acid (3,4-CQ), 3,5-O-dicaffeoylquinic acid (3,5-CQ), and 4,5-O-dicaffeoylquinic acid (4,5-CQ). In conclusion, caffeoyl derivatives in PIT could play an important role in potent antioxidant properties. So, it may be further developed to be antioxidant beverages for preventing atherosclerosis and cardiovascular diseases associated with oxidative stress.

An insight into anti-adipogenic properties of an Oroxylum indicum (L.) Kurz extract.

BACKGROUND: Oroxylum indicum fruit extract (OIE) has been reported to inhibit the development of adipocytes. However, the exact mechanism of its metabolic activity is not clearly defined. This study attempted to investigate whether OIE was involved in disrupting the cell cycle, glucose metabolism, and mitochondrial function in 3 T3-L1 cells. METHODS: The effect of the OIE on cell cycle progression was measured by flow cytometry along with observing the expression of the cycle regulator by immunoblotting. The effect of the OIE on glucose metabolism was investigated. The amount of glucose uptake (2-NBDG) influenced by insulin was determined as well as the protein tyrosine phosphorylation (PY20), and glucose transporter4 (GLUT4) expression was determined by immunoblotting assay. Mitochondria are also essential to metabolic processes. This study investigated mitochondrial activity using fluorescent lipophilic carbocyanine dye (JC-1) and mitochondria mass by MitoTracker Green (MTG) staining fluorescence dyes. Finally, cellular ATP concentration was measured using an ATP chemiluminescence assay. RESULTS: Treatment with OIE plus adipogenic stimulators for 24 h arrested cell cycle progression in the G2/M phase. Moreover, 200 µg/mL of OIE significantly diminished the expression of the insulin receptor (IR) and GLUT4 protein compared to the untreated-adipocytes (P < 0.05). The mitochondrial membrane potential (MMP) was significantly reduced (24 h) and increased (day 12) by OIE compared to untreated-adipocytes (P < 0.05). However, OIE maintained MMP and ATP at a similar level compared to the pre-adipocytes (day 12). Transmission electron microscope (TEM) results demonstrated that OIE could protect mitochondria deformation compared to the untreated-adipocytes. CONCLUSION: These results suggest that the inhibitory effect of the OIE on adipogenesis may potentially inhibit the cell cycle and phosphorylation of IR, leading to a decrease in glucose uptake to the cells. The OIE also slows down the mitochondrial activity of the early phase of cell differentiation, which can also inhibit the development of fat cells.

Pluchea indica (L.) Less. Tea Ameliorates Hyperglycemia, Dyslipidemia, and Obesity in High Fat Diet-Fed Mice.

Pluchea indica (L.) Less. (P. indica) tea has been used for a health-promoting drink, especially in Southeast Asia. The effect of P. indica tea (PIT) on amelioration of hyperglycemia; dyslipidemia that was total cholesterol (TC), LDL-cholesterol (LDL-C), HDL-cholesterol (HDL-C), and triglyceride (TG); and obesity in high fat diet-induced (HFD) mice was investigated. Oral glucose tolerance test (OGTT) displayed that PIT at 400 and 600 mg/kg orally ameliorated hyperglycemia with a dose-dependent manner compared to the untreated group. Moreover, PIT at these dosages exhibited significantly lower TC, LDL-C, TG, and perigonadal fat weight in HFD treated mice compared to HFD mice (P < 0.05) with a dose-dependent manner. In contrast, HDL-C was higher than in the HFD group, but not a significant difference (P > 0.05). The PIT chemical analysis results demonstrated that PIT contained total phenolic content (TPC), total flavonoid content (TFC), 4-O-caffeoylquinic acid (4-CQ), 5-O-caffeoylquinic acid (5-CQ), 3,4-O-dicaffeoylquinic acid (3,4-CQ), 3,5-O-dicaffeoylquinic acid (3,5-CQ), 4,5-O-dicaffeoylquinic acid (4,5-CQ), beta-caryophyllene, and gamma-gurjunene that may play an important role in inhibiting hyperlipidemia and hyperglycemia. Also, histological analysis expressed that the mean area and amount of perigonadal fat adipocytes of PIT treated groups were significantly lower and higher than the HFD group (P < 0.05), respectively. The toxicity test of PIT at 600 mg/kg/day in mice showed that serum creatinine, alanine transaminase (ALT), alkaline phosphatase (ALP), and complete blood count (CBC) levels of HFD and PIT treated groups were not significantly different compared to the normal control diet group (NCD) (P > 0.05). These results suggest that PIT does not become toxic to the kidney, liver, and blood. In conclusion, PIT has the potential to develop into healthy food supplement or medicine for the prevention and treatment of hyperglycemic, hyperlipidemic, and obese patients.

Intracellular ROS Scavenging and Anti-Inflammatory Activities of Oroxylum indicum Kurz (L.) Extract in LPS plus IFN-γ-Activated RAW264.7 Macrophages.

Oroxylum indicum (L.) Kurz has been used as plant-based food and herbal medicine in many Asian countries. The aim of the present study was to examine the antioxidant and anti-inflammatory activities of O. indicum extract (O. indicum) in RAW264.7 cells activated by LPS plus IFN-γ. The phytochemical compounds in O. indicum were identified by GC-MS and LC-MS/MS. Five flavonoids (luteolin, apigenin, baicalein, oroxylin A, and quercetin) and 27 volatile compounds were found in O. indicum. O. indicum presented antioxidant activities, including reducing ability by FRAP assay and free radical scavenging activity by DPPH assay. Moreover, O. indicum also suppressed LPS plus IFN-γ-activated reactive oxygen species generation in RAW264.7 macrophages. It possessed the potent anti-inflammatory action through suppressing nitric oxide (NO) and IL-6 secretion, possibly due to its ability to scavenge intracellular ROS. The synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectroscopy results showed the alteration of signal intensity and integrated areas relating to lipid and protein of the activated RAW264.7 macrophages compared to unactivated cells. This is the first report of an application of the SR-FTIR technique to evaluate biomolecular changes in activated RAW264.7 cells. Our results indicate that O. indicum may be used as a potential source of nutraceutical for the development of health food supplement or a novel anti-inflammatory herbal medicine.

Antiadipogenesis of Oroxylum indicum (L.) Kurz Extract via PPARγ2 in 3T3-L1 Adipocytes.

Oroxylum indicum is regarded as a traditional food with medicinal properties and is used widely throughout Asia. It has previously been demonstrated that O. indicum extract (OIE) was able to suppress the differentiation of 3T3-L1 preadipocytes to adipocytes. However, the mechanism underlying the antiadipogenesis of this plant has not been fully investigated. The present study aimed to explore the impact of OIE at 50 to 200 µg mL-1 on the molecular mechanism involved in the antiadipogenic activity in 3T3-L1 cells at day 0 of their differentiation to adipocytes. The morphology and biochemistry of the cells on day 12 were investigated and compared to the relevant controls. Adiponectin was measured using enzyme-linked immunosorbent assay (ELISA). The mRNA expression of peroxisome proliferator-activated receptor-gamma 2 (PPARγ2), sterol regulatory element-binding proteins 1c (SREBP-1c), fatty acid synthetase (FAS), glucose transporter (GLUT4), and leptin in adipocytes was determined by real-time PCR. The results demonstrated that the OIE at 200 µg mL-1 exhibited strongest suppression on intracellular lipid accumulation. The levels of adiponectin were dramatically increased in the untreated adipocytes, whereas significantly decreased in the 200 µg mL-1 OIE-treated adipocytes (P < 0.05). Expression of the mRNAs revealed that OIE-treated adipocytes at 200 µg mL-1 significantly inhibited the expression of PPARγ2 and SREBP-1c and lowered the level of expression of GLUT4, FAS, and leptin compared to the control (P < 0.05). These findings suggest that OIE inhibits adipocyte differentiation along with the downregulation of PPARγ2, SREBP-1c, and GLUT4, leading to the decrease in the expression of FAS and adipokine (leptin and adiponectin). Thus, OIE might be developed for hyperlipidemia and obesity prevention.

The Effects of Cordyceps sinensis (Berk.) Sacc. and Gymnema inodorum (Lour.) Decne. Extracts on Adipogenesis and Lipase Activity In Vitro.

This study aimed to investigate the effects of Cordyceps sinensis extract (CSE) and Gymnema inodorum extract (GIE), used alone and combined, on antiadipogenesis in 3T3-L1 cells. Oil Red O staining was used to examine the effects of these extracts on inhibition of intracellular lipid accumulation in 3T3-L1 adipocytes and on lipid droplet morphology. Fourier transform-infrared (FTIR) microspectroscopy was used to examine biomolecular changes in 3T3-L1 adipocytes. The pancreatic lipase assay was used to evaluate the inhibitory effects of CSE and GIE on pancreatic lipase activity. Taken together, the results indicated that CSE, GIE, and their combination suppressed lipid accumulation. The FTIR microspectroscopy results indicated that CSE, GIE, and their combination had inhibitory effects on lipid accumulation in the adipocytes. Compared with the untreated adipocytes, the signal intensity and integrated areas of glycogen and other carbohydrates, the acyl chain of phospholipids, and the lipid/protein ratios of the CSE, GIE, alone, and combined treated adipocytes were significantly lower (p < 0.05). Combination treatment resulted in a synergistic effect on lipid accumulation reduction in the adipocytes. Principal component analysis of the biomolecular changes revealed six distinct clusters in the FTIR spectra of the sample cells. The pancreatic lipase assay results indicated that CSE and GIE inhibited the pancreatic lipase activity in a dose-dependent manner (mean ± standard error of the mean IC50 values, 2312.44 ± 176.55 µg mL-1 and 982.24 ± 44.40 µg mL-1, resp.). Our findings indicated that FTIR microspectroscopy has potential application for evaluation of the effectiveness of medicinal plants and for the development of infrared biochemical obesity markers useful for treating patients with obesity. These results suggested that use of CSE and GIE alone and in combination may be efficacious as a complementary therapy for hyperlipidemia and obesity management. However, clinical trials in animals and humans must first be completed.

Nitro-Carba test, a novel and simple chromogenic phenotypic method for rapid screening of carbapenemase-producing Enterobacteriaceae.

OBJECTIVES: In this study, a rapid and simple chromogenic method for screening of carbapenemase-producing Enterobacteriaceae (CPE), namely the Nitro-Carba test (NCT), was developed. METHODS: The NCT was validated using a total of 31 carbapenemase-producing isolates [9 Klebsiella pneumoniae carbapenemase (KPC), 11 metallo-ß-lactamase (MBL) and 11 OXA-48] and 56 non-carbapenemase-producing isolates. The assay relies on the hydrolysis of nitrocefin by carbapenemases in the presence of carbapenem antibiotics. Carbapenemases were extracted with lysis buffer prior to addition to wells with and without imipenem (IPM), meropenem (MEM) and ertapenem (ETP). Following addition of nitrocefin, a change in colour from yellow to red, indicating carbapenemase production, was observed within 20min. The susceptibility profiles of each bacterial isolate were also investigated. RESULTS: The NCT detected all 31 CPE within a timeframe of only 10s to 12min. All carbapenemase-producers hydrolysed nitrocefin in all wells. No colour change in wells with carbapenems was observed in non-carbapenemase-producers. The sensitivity for all three carbapenems was 100%, whilst the specificity of IPM, MEM and ETP was 64.3%, 91.1% and 100%, respectively. IPM, MEM and ETP had minimum inhibitory concentrations (MICs) against all carbapenemase-producing strains ranging from 0.5µg/mL to ≥256µg/mL, 0.25µg/mL to ≥256µg/mL and 1µg/mL to ≥256µg/mL, respectively. OXA-48-producing isolates showed lower MICs compared with MBL- and KPC-producing isolates. CONCLUSION: This assay is a promising method for detecting CPE rapidly. The NCT is a simple and reliable method capable of detecting CPE even in carbapenem-susceptible strains.

The Effect of Pluchea indica (L.) Less. Tea on Adipogenesis in 3T3-L1 Adipocytes and Lipase Activity.

Obesity and hyperlipidemia are a major problem in the world. Pluchea indica (L.) Less. tea (PIT) is a beverage that has various indications. This study focused on the effect of the PIT on inhibiting adipogenesis of 3T3-L1 cells and pancreatic lipase enzyme activity. The viability of 3T3-L1 cells was not significantly decreased after exposure to 200 to 1000 µg mL-1 PIT compared to controls (p > 0.05). The PIT at 750 to 1000 µg mL-1 exhibited a significantly reduced lipid accumulation compared to the control (p < 0.05). The inhibitory effects of the PIT at 250 to 1000 µg mL-1 on lipase activity were significantly increased compared to control (p < 0.05). The FTIR results showed that the integrated areas of lipids, proteins, nucleic acids, glycogen, and carbohydrates of the PIT-treated 3T3-L1 adipocytes were significantly lower than the untreated 3T3-L1 adipocytes (p < 0.05). These findings may indicate that the PIT is not only capable of inhibiting lipids and carbohydrate accumulation in adipocytes but also has a potential to inhibit pancreatic lipase activity. So, the PIT may be further developed to the novel lipid-lowering herbal supplement for the management of overweight or obesity.

Oroxylum indicum (L.) Kurz extract inhibits adipogenesis and lipase activity in vitro.

BACKGROUND: Oroxylum indicum (L.) Kurz (O. indicum) is found in Thailand. It has been used for the treatment of obesity. This study aimed to investigate the effects of an O. indicum extract (OIE) on the adipogenic and biomolecular change in 3T3-L1 adipocytes. METHODS: Initial studies examined the chemical components of OIE. The cell line 3T3-L1 was used to establish potential toxic effects of OIE during the differentiation of pre-adipocytes to adipocytes. The inhibitory effect of OIE on lipid accumulation in 3T3-L1 cells was investigated. Moreover, the impact of OIE on pancreatic lipase activity was determined. In further experiments, Fourier Transform Infrared (FTIR) was used to monitor and discriminate biomolecular changes caused by the potential anti-adipogenic effect of OIE on 3T3-L1 cells. RESULTS: Chemical screening methods indicated that OIE was composed of flavonoids, alkaloids, steroids, glycosides, and tannins. The percentage viability of 3T3-L1 cells was not significantly decreased after exposure to either 200 or 150 µg/mL of OIE for 2 and 10 days, respectively compared to control cells. The OIE exhibited a dose-dependent reduction of lipid accumulation compared to the control (p < 0.05). The extract also demonstrated a dose-dependent inhibitory effect upon lipase activity compared to the control. The inhibitory effect of the OIE on lipid accumulation in 3T3-L1 cells was also confirmed using FTIR microspectroscopy. The signal intensity and the integrated areas relating to lipids, lipid esters, nucleic acids, glycogen and carbohydrates of the OIE-treated 3T3-L1 adipocytes were significantly lower than the non-treated 3T3-L1 adipocytes (p < 0.05). Principal component analysis (PCA) indicated four distinct clusters for the FTIR spectra of 3T3-L1 adipocytes based on biomolecular changes (lipids, proteins, nucleic acids, and carbohydrates). This observation was confirmed using Unsupervised hierarchical cluster analysis (UHCA). CONCLUSIONS: These novel findings provide evidence that the OIE derived from the fruit pods of the plant is capable of inhibiting lipid and carbohydrate accumulation in adipocytes and also has the potential to inhibit an enzyme associated with fat absorption. The initial observations indicate that OIE may have important properties which in the future may be exploited for the management of the overweight or obese.

The Synergy and Mode of Action of Cyperus rotundus L. Extract Plus Ampicillin against Ampicillin-Resistant Staphylococcus aureus.

Cyperus rotundus L. has been used for pharmaceutical applications including antibacterial infections. Nevertheless, there is still no data regarding the mode of actions. This study aimed to determine the antibacterial activity and mode of actions of Cyperus rotundus extract (CRE) against ampicillin-resistant Staphylococcus aureus (ARSA) which poses a serious problem for hospitalized patients. The majority of chemical compounds of CRE were flavonoids and alkaloids. The minimum inhibitory concentrations (MICs) for ampicillin and CRE against all ARSA strains were 64 µg/ml and 0.5 mg/ml, respectively. Checkerboard assay revealed synergistic activity in the combination of ampicillin and CRE at the lowest fractional inhibitory concentration index (FICI) at 0.27. The killing curve assay had confirmed the synergistic and bactericidal activity of the combination against ARSA. Electron microscopic results showed that these ARSA cells treated with this combination caused peptidoglycan and cytoplasmic membrane (CM) damage and average cell areas significantly smaller than control. Also, this combination caused an increase in CM permeability of ARSA. CRE revealed the inhibitory activity against ß-lactamase. It is normally known that some drugs are derived from flavonoids or alkaloids. So, this CRE proposes the potential to develop a novel adjunct phytopharmaceutical to ampicillin for the remedy of ARSA.