Anticancer activity of Bacopa monnieri through apoptosis induction and mitophagy-dependent NLRP3 inflammasome inhibition in oral squamous cell carcinoma.

BACKGROUND: Bacopa monnieri (BM) is traditionally used in human diseases for its antioxidant, anti-inflammatory and neuroprotective effects. However, its anticancer potential has been poorly understood. AIM: The aim of this study was to explore the detailed anticancer mechanism of BM against oral cancer and to identify the bioactive BM fraction for possible cancer therapeutics. RESULTS: We performed bioactivity-guided fractionation and identified that the aqueous fraction of the ethanolic extract of BM (BM-AF) had a potent anticancer potential in both in vitro and in vivo oral cancer models. BM-AF inhibited cell viability, colony formation, cell migration and induced apoptotic cell death in Cal33 and FaDu cells. BM-AF at low doses promoted mitophagy and BM-AF mediated mitophagy was PARKIN dependent. In addition, BM-AF inhibited arecoline induced reactive oxygen species production in Cal33 cells. Moreover, BM-AF supressed arecoline-induced NLR family pyrin domain containing 3 (NLRP3) inflammasome activation through mitophagy in Cal33 cells. The in vivo antitumor effect of BM-AF was further validated in C57BL/6J mice through a 4-nitroquinolin-1-oxide and arecoline-induced oral cancer model. The tumor incidence was significantly reduced in the BM-AF treated group. Further, data obtained from western blot and immunohistochemistry analysis showed increased expression of apoptotic markers and decreased expression of inflammasome markers in the tongue tissue obtained from BM-AF treated mice in comparison with the non-treated tumor bearing mice. CONCLUSION: In conclusion, BM-AF exhibited potent anticancer activity through apoptosis induction and mitophagy-dependent inhibition of NLRP3 inflammasome activation in both in vitro and in vivo oral cancer models. Moreover, we have investigated apoptosis and mitophagy-inducing compounds from this plant extract having anticancer activity against oral cancer cells.

Ocimum tenuiflorum extract (HOLIXERTM): Possible effects on hypothalamic-pituitary-adrenal (HPA) axis in modulating stress.

Ocimum tenuiflorum is a sacred medicinal plant bestowed with multiple health benefits. This plant is traditionally considered an adaptogen. Many scientific studies have indicated the anti-stress potential of Ocimum tenuiflorum but with higher doses. The present study investigated the effects of HolixerTM (a clinically studied standardized Ocimum tenuiflorum extract) on modulating stress using two in vivo models, namely the swim endurance study in mice and forced swim test in rats. In addition, we explored the mechanism of action of HolixerTM on the HPA axis using two in vitro cell-based assays to check for its inhibitory effect on cortisol release and CRF1 receptor antagonistic activity. Ocimum tenuiflorum extract enhanced the swimming time in mice, reduced the stress-induced increase in immobility time, and prevented the increase in corticosterone in rats subjected to the forced swim test. Further, Ocimum tenuiflorum extract inhibited cortisol release and exhibited a significant CRF1 receptor antagonist activity. Thus, Ocimum tenuiflorum extract was found effective in managing stress, and the effect could be due to the inhibition of cortisol release and the antagonistic effect on the CRF1 receptors.

A flavonoid rich standardized extract of Glycyrrhiza glabra protects intestinal epithelial barrier function and regulates the tight-junction proteins expression.

BACKGROUND: Intestinal epithelial barrier dysfunction predisposes to many gastrointestinal, metabolic, and psychological disorders. A flavonoid rich extract of Glycyrrhiza glabra (FREG) has previously been reported to possess anti-inflammatory, antioxidant, and antiulcer properties. AIM: To investigate the effect of FREG (GutGard®) on restoring intestinal barrier function in tumor necrosis factor-alpha (TNF-α) stimulated human colonic adenocarcinoma cell monolayer (Caco-2) and 2,4,6-Trinitrobenzenesulfonic acid (TNBS) induced ulcerative colitis in rats. METHODS: In in vitro, human intestinal Caco-2 cell monolayers were treated with TNF-α in the presence or absence of FREG and the paracellular permeability to FITC-conjugated 4-kD dextran (FD4) was measured to evaluate protection against the barrier dysfunction. In in vivo, intestinal barrier dysfunction was induced in male albino Wistar rats via intrarectal instillation of TNBS. Subsequently, the rats were treated orally with either FREG at 6.25, 12.5, and 25 mg/kg body weight, or Mesacol (250 mg/kg) for 5 days. On day 5, intestinal epithelial permeability was assessed with FD4 leakage into the serum. Also, colonic inflammation, colon morphology, histology and macroscopic score, weight to length ratio were evaluated. The activity of myeloperoxidase (MPO), TNF- α, secretory IgA levels and tight junction proteins expression were evaluated in rat's colon. RESULTS: FREG protected the intestinal epithelial barrier integrity in human intestinal Caco-2 cells in vitro. FREG administration significantly improved the intestinal epithelial barrier function as evident from significant reduction in FD4 leakage. The colon morphology, histology score, macroscopic score, colon weight to length ratio also indicates beneficial effects of FREG on barrier function. In addition, FREG regulated the tight junction proteins, and markedly decreased TNF-α, MPO levels and significantly increased the secretory IgA levels in TNBS induced colitis rats. CONCLUSION: The study findings support the protective action of FREG on intestinal epithelial barrier integrity indicating its potential in protecting from implications of leaky gut.

Safety of NR-INF-02, an Extract of Curcuma Longa Containing Turmerosaccharides, in Healthy Volunteers: A Randomized, Open-label Clinical Trial.

CONTEXT: Turmeric (Curcuma longa) is a common medicinal plant used in traditional medicine that also has been scientifically validated for its antioxidant, anti-arthritic, anticancer, analgesic, and anti-inflammatory properties. Researchers have still not much explored the beneficial effects of the curcuminoid-free portion of turmeric. NR-INF-02 is a proprietary, patented aqueous extract of Curcuma longa comprising turmerosaccharides with a novel phytochemical composition. OBJECTIVE: The study intended to evaluate the safety and tolerability of NR-INF-02 in healthy adult volunteers at doses of 1000 and 2000 mg, administered for 84 days. DESIGN: The study employed a randomized, open label, two-arm, parallel-group design. SETTING: The trial was carried at 2 sites, the Meenakshi Multispecialty Hospital in Chennai, Tamil Nadu, India and the Vijaya Super Specialty Hospital in Nellore, Andhra Pradesh, India. PARTICIPANTS: Participants were healthy adult, male or female volunteers, aged 18-65 years with a body mass index of ≥18.5 kg/m2 and ≤ 24.9 kg/m2 and a body weight of at least 55 kg for men and 48 kg for women. INTERVENTION: Participants were randomly divided into 2 groups with 24 participants each for a total of 48 participants. They received either 1000 or 2000 mg of NR-INF-02 for 84 days. OUTCOME MEASURES: The incidence of adverse events and the changes from baseline in clinical laboratory parameters-including hematological, biochemical, and urinalysis parameters-were assessed at baseline, at day 42, and postintervention at day 84 as primary endpoints for safety. Secondary endpoints were the changes in vital signs and the difference in the results of an electrocardiogram (ECG) between baseline and days 42 and 84. RESULTS: The NR-INF-02 at doses of 1000 and 2000 mg demonstrated a 4.17% and 20.83% incidence of adverse events (AEs), respectively. The AEs were mild to moderate and were either probably or possibly related, but not definitively, related to treatment. A detailed examination of hematological, biochemical, and urological parameters and of ECG results and vital signs didn't indicate any untoward effects for any participant. CONCLUSION: The study found NR-INF-02 to be safe and tolerable at both tested doses for the given duration of the trial for healthy adult volunteers.

Safety Evaluation of Standardized Extract of Curcuma longa (NR-INF-02): A 90-Day Subchronic Oral Toxicity Study in Rats.

NR-INF-02 is a standardized extract containing turmerosaccharides from Curcuma longa that has anti-inflammatory, analgesic, and chondroprotective potential. In view of its potential uses, NR-INF-02 was evaluated for its safety in Wistar rats at an oral dose of 250, 500, and 1000 mg/kg in a 90-day repeated dose subchronic toxicity study. NR-INF-02 administered at 250, 500, and 1000 mg/kg for 90 days did not show any mortality or clinical signs of toxicity. Body weight gain, food consumption, ocular and neurological examination, and hematological, blood biochemical, hormone, and urine analysis revealed no evidence of toxicity of NR-INF-02 treatment in rats. Absolute and relative organ weights were comparable to control rats. The study did not reveal any major treatment related gross pathological and histopathological alterations in the tissues or organs examined. Thus, based on study observations, the no-observed adverse effect level (NOAEL) was found to be 1000 mg/kg body weight in albino Wistar rats.

Is Andrographis paniculata extract and andrographolide anaphylactic?

Andrographis paniculata, "King of bitters" is a popularly known medicinal plant extensively used in many parts of the world for treatment of various diseases. Since recent past, anaphylactic/allergic type adverse events were reported upon A. paniculata usage, the study aimed to evaluate the anaphylactic and anaphylactoid potential of A. paniculata extract and andrographolide (a major phytoactive of A. paniculata). The anaphylactic potential was evaluated using active systemic anaphylaxis (ASA) assay in guinea pigs. Further, the release of allergic mediators was measured in immunoglobulin E (IgE) sensitized and non-IgE sensitized Rat Basophilic Leukemia (RBL-2H3) cell lines in-vitro. A. paniculata extract or andrographolide sensitized guinea pigs following the challenge antigen administration orally and intravenously did not demonstrate any clinical signs of anaphylaxis. IgE sensitized and non- IgE sensitized RBL-2H3 cells treated with A. paniculata extract did not induce release of allergic mediators. Whereas IgE sensitized and non- IgE sensitized RBL-2H3 cells treated with andrographolide demonstrated mild to moderate release of allergic mediators. A. paniculata extract has no anaphylactic and anaphylactoid potential in in-vivo and in-vitro studies. Whereas, andrographolide effects on allergic mediators in in-vitro studies needs to be scrutinized if they are of biologically important.

Effects of turmeric curcuminoids and metformin against central sensitivity to pain in mice.

The reported experimental study was conducted to compare the effects of repeated daily oral doses of curcuminoids (CLE) with metformin as potential antidepressants and analgesics. Effects of a single and ten daily oral doses of CLE (5, 20, 80 mg/kg/day) and of 50 mg/kg/day metformin (MET) were compared in mice hot plate test (HPT) for analgesics. On the 11th treatment day, all animals were subjected to foot shock stress triggered hyperthermia test, and on the 12th treatment day to tail suspension test (TST) for antidepressants. Immediately thereafter, their blood levels of glucose, insulin and cortisol were quantified. Dose dependent analgesic activity of CLE was observed in HPT, whereas the metformin dose tested suppressed only pain hypersensitivity in the test. But statistically significant effects of both of them were observed in TST, and both of them also afforded protections against body weight loss and slight elevation in core temperatures induced by daily handling and repeated testing. CLE or metformin had no significant effects in foot shock stress triggered transient hyperthermic responses or on blood glucose, insulin and cortisol levels. Reported results reveal that curcuminoids as well as metformin are stress response modifiers with antidepressants like activities, but only low dose curcuminoids possess centrally acting analgesics like activities. They suggest that the bio-assay system used in this study is well suited for identifying curcuminoids like plant metabolites with analgesic and anti-stress activities, and that low dose curcuminoids are more effective as analgesics than low dose metformin.

Erectogenic and Aphrodisiac Property of Moringa oleifera: Involvement of Soluble Epoxide Hydrolase Enzyme.

Soluble epoxide hydrolase (sEH) inhibitors have been reported to improve penile erection; therefore, sEH could be useful for management of erectile dysfunction. Methanolic and aqueous extracts of 30 Indian medicinal plants were screened for their sEH inhibition potential. Fifteen extracts showed >50% inhibition when screened at 50 µg/mL in sEH inhibition assay. Methanolic extract of Moringa oleifera Lam. (Moringaceae) seeds (MEMO) was most potent with IC50 1.7 ± 0.1 µg/mL and was selected for in vitro studies on isolated rat corpus cavernosum smooth muscle and in vivo sexual behaviour studies on healthy and diabetic rats. Rats were divided into five groups, each containing six animals and treated orally with either water, vehicle (1% Tween-20), MEMO (45 and 90 mg/kg/day for 21 days), and standard drug, sildenafil (5 mg/kg/day for 7 days). An equal number of female rats were used, and the effect of MEMO and sildenafil was compared with that of vehicle. MEMO significantly relaxed isolated rat corpus cavernosum smooth muscle at 0.1-100 µg/mL in vitro and significantly increased (p < 0.05) sexual activity, intracavernous pressure/mean arterial pressure in normal and diabetic rats. The increase in erectile function of rats by MEMO could be because of its sEH inhibitory activity. Copyright © 2016 John Wiley & Sons, Ltd.

Anti-stress Activity of Ocimum sanctum: Possible Effects on Hypothalamic-Pituitary-Adrenal Axis.

The present study investigated anti-stress potential of Ocimum sanctum in chronic variable stress (CVS) paradigm. Further, the possible mechanism of anti-stress was explored in vitro using cell and cell-free assays. Rats were administered O. sanctum followed by CVS regimen for a period of 16 days. On days 4, 8, 12, and 16, body weight and immobility time in forced swim test were measured. In addition, the possible inhibitory effect of O. sanctum and ursolic acid on cortisol release and CRHR1 receptor activity were studied in cell-based assays, while inhibitory effects on 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) and catechol-O-methyltransferase (COMT) were studied in cell-free assays. CVS group demonstrated less body weight gain and higher immobility time than O. sanctum administered groups, while oral administration of O. sanctum significantly increased body weight gain and decreased the immobility time. Further, O. sanctum and its constituents inhibited cortisol release and exhibited a significant CRHR1 receptor antagonist activity. Also, they had specific inhibitory activity towards 11ß-HSD1 and COMT activity. Thus, O. sanctum was found to be effective in the management of stress effects, and anti-stress activity could be due to inhibition of cortisol release, blocking CRHR1 receptor, and inhibiting 11ß-HSD1 and COMT activities. Copyright © 2016 John Wiley & Sons, Ltd.

Anti-Inflammatory Activity of Polysaccharide Fraction of Curcuma longa Extract (NR-INF-02).

The aim of the study was to investigate the safety and anti-inflammatory effects of polysaccharide fraction (F1) of Curcuma longa extract (NR-INF-02) in classical rodent models of inflammation. F1 was evaluated for its acute oral toxicity and found to be safe upto 5000 mg/kg body weight in rats. The anti-inflammatory activity of F1 was evaluated in acute (carrageenan - induced paw edema; xylene - induced ear edema) and chronic (cotton pellet - induced granuloma) models of inflammation. The results of the study demonstrated that F1 significantly (p ≤ 0.05) inhibited carrageenan-induced paw edema at 1 h and 3 h at doses of 11.25, 22.5 and 45 mg/kg body weight in rats. Also, F1 at doses of 15.75, 31.5 and 63 mg/kg significantly inhibited the xylene induced ear edema in mice. In a chronic model, F1 at 11.25, 22.5 and 45 mg/kg doses produced significant reduction of wet and dry weights of cotton pellets in rats. Overall results indicated that F1 of NR-INF-02 significantly attenuated acute and chronic inflammation in rodent models. This study emphasizes on the importance of Curcuma longa polysaccharide's role in acute and chronic inflammation.

Aldose reductase and protein tyrosine phosphatase 1B inhibitory active compounds from Syzygium cumini seeds.

CONTEXT: Syzygium cumini (L.) Skeels (Myrtaceae), commonly known as jamun, is an Indian plant, traditionally well known for its medicinal properties including antidiabetic activity. OBJECTIVE: To isolate the antidiabetic compounds from Syzygium cumini seeds and evaluate their activity using aldose reductase (AR) and protein-tyrosine phosphatase 1B (PTP1B) inhibition assays. MATERIALS AND METHODS: The dried seeds were extracted with methanol and partitioned with ethyl acetate, butanol, and water. The extracts were screened for antidiabetic activity at a concentration of 100 µg/mL using in vitro AR and PTP 1B inhibition assays. RESULTS AND DISCUSSION: The highly enriched fractions obtained from broad ethyl acetate fraction yielded maslinic acid (1), 5-(hydroxymethyl) furfural (2), gallic acid (3), valoneic acid dilactone (4), rubuphenol (5), and ellagic acid (6). Structures were elucidated by (1)H-NMR and (13)C-NMR. The initial ethyl acetate fraction showed AR inhibitory activity with the IC50 value of 2.50 µg/mL and PTP1B enzyme inhibition with the IC50 value of 26.36 µg/mL. Compounds 3, 4, 5, and 6 were found to inhibit AR with IC50 values of 0.77, 0.075, 0.165, and 0.12 µg/mL while the compounds 4, 5, and 6 inhibited PTP1B with IC50 values of 9.37, 28.14, and 25.96 µg/mL, respectively. CONCLUSION: The results of this study demonstrate that the isolated constituents show promising in vitro antidiabetic activity and, therefore, can be candidates for in vivo biological screening using relevant models to ascertain their antidiabetic activity.

Hepatoprotective and antioxidant activity of standardized herbal extracts.

BACKGROUND: Phyllanthus emblica, Camellia sinensis, Mangifera indica, Punica granatum, and Acacia catechu have been shown to possess widespread pharmacological application against multitude of diseases namely cancer, diabetes, liver disorders, and oxidative stress. OBJECTIVE: We evaluated the hepatoprotective activity of the standardized herbal extracts against tert-butyl hydroperoxide (t-BH) induced toxicity and their mechanism of hepatoprotective action in human hepatocarcinoma cells (HepG2 cell line). MATERIALS AND METHODS: The hepatoprotective activity was studied by observing the effect of these herbal extracts on t-BH induced reduction in cell viability of HepG2 cells. In addition, the reducing power of the extracts and their ability to scavenge free radicals were evaluated using two antioxidant assay systems: cell free [oxygen radical absorbance capacity (ORAC), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid)] (ABTS)] and cell based [cellular antioxidant activity (CAA)]. RESULTS AND DISCUSSION: The results obtained showed that these extracts possess significant hepatoprotective activity. This may indicate that the plant extracts contain compounds, which can remove toxic metabolites following t-BH induced toxicity. The extracts exhibited significant antioxidant property as evident by the Trolox values and effective scavenging of DPPH and ABTS radicals. The extracts also demonstrated inhibition of AAPH-induced fluorescence in HepG2 cells. These results indicate the ability of the plant extracts to protect the liver cells from chemical-induced damage, which might be correlated to their radical scavenging potential. CONCLUSION: This study demonstrates that these extracts have potential hepatoprotective activity which is mainly attributed to the antioxidant potential, which might occur by reduction of lipid peroxidation and cellular damage.