Potential Metabolite Nymphayol Isolated from Water Lily (Nymphaea stellata) Flower Inhibits MCF-7 Human Breast Cancer Cell Growth via Upregulation of Cdkn2a, pRb2, p53 and Downregulation of PCNA mRNA Expressions.

Controlled production of cyclin dependent kinases (CDK) and stabilization of tumor suppressor genes are the most important factors involved in preventing carcinogenesis. The present study aimed to explore the cyclin dependent apoptotic effect of nymphayol on breast cancer MCF-7 cells. In our previous study, we isolated the crystal from a chloroform extract of Nymphaea stellata flower petals and it was confirmed as nymphayol (17-(hexan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol) using x-ray diffraction (XRD), Fourier transform infrared (FTIR), and mass spectroscopy (MS) methods. The cytotoxic effect of nymphayol on MCF-7 cells were analyzed using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The cellular and nuclear damage was determined using propidium iodide (PI) and acridine orange/ethidium bromide (AO/ErBr) staining. Tumor suppressor and apoptosis related mRNA transcript levels were determined using real-time polymerase chain reaction (RT-PCR). Nymphayol potentially inhibits MCF-7 cell viability up to 78%, and the IC50 value was observed as 2.8 µM in 24 h and 1.4 µM in 48 h. Treatment with nymphayol significantly increased reactive oxygen species (ROS) level and the tunnel assay confirmed DNA damage. We found characteristically 76% apoptotic cells and 9% necrotic cells in PI and AO/ErBr staining after 48 h treatment with 2.8 µM of nymphayol. Gene expression analysis confirmed significantly (p ≤ 0.001) increased mRNA levels of cyclin dependent kinase inhibitor 2A (Cdkn2a), retinoblastoma protein 2 (pRb2), p53, nuclear factor erythroid 2-factor 2 (Nrf2), caspase-3, and decreased B-cell lymphoma 2 (Bcl-2), murine double minute 2 (mdm2), and proliferating cell nuclear antigen (PCNA) expression after 48 h. Nymphayol effectively inhibited breast cancer cell viability, and is associated with early expression of Cdkn2a, pRb2, and activation of p53 and caspases.

Nymphaea nouchali Burm. f. hydroalcoholic seed extract increases glucose consumption in 3T3-L1 adipocytes through activation of peroxisome proliferator-activated receptor gamma and insulin sensitization.

Nymphaea nouchali Burm. f. (Family - Nymphaeaceae) is a well-known medicinal plant used in the Indian ayurvedic system of medicine for treating diabetes. The seeds especially have been prescribed for diabetes. The hydroalcoholic extract of N. nouchali seeds has been demonstrated to possess anti-hyperglycemic effects in diabetic rats, but the functional mechanism remains unknown. The nuclear receptor, peroxisome proliferator-activated receptor gamma (PPARγ) is noted to play an important role in glucose and lipid homeostasis. This study was hence focused in evaluating the effect of the extract on PPARγ activation, adipocyte differentiation, and glucose consumption in 3T3-L1 cells. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), followed by adipogenesis assay using Oil Red O technique. Glucose consumption of preadipocytes and adipocytes in the presence of the extract was also determined. Real-time polymerase chain reaction was performed to identify the expression of genes involved in glucose consumption in the adipocytes. MTT assay confirmed the extract to be nontoxic, and Oil Red O staining confirmed enhanced adipocyte differentiation of 3T3-L1 cells in a dose-dependent manner. The extract also increased the expression of PPARγ target gene, which in turn enhanced the expression of GLUT-4. The data, therefore, suggests that N. nouchali seed extract promotes adipocyte differentiation and glucose consumption by inducing PPARγ activation, which in turn increases mRNA GLUT-4 expression and subsequently enhances insulin-responsiveness in insulin target tissues.

Nymphayol increases glucose-stimulated insulin secretion by RIN-5F cells and GLUT4-mediated insulin sensitization in type 2 diabetic rat liver.

Nymphaea stellata (Willd.) has been used in traditional medicine for centuries to treat several illnesses, including diabetes. However, scientific evidence supporting its mechanism of action is lacking. Here, we showed that an N. stellata flower chloroform extract (NSFCExt) has significant plasma glucose lowering ability. Furthermore, an active compound was identified and purified by column chromatography, and the structure of this compound, nymphayol, was determined by X-ray crystallographic analysis. Nymphayol was tested for its effects on insulin secretion by RIN-5F cells cultured in low or high glucose medium; we found that nymphayol treatment improved glucose-stimulated insulin secretion in vitro. Additionally, insulin sensitization and glucose uptake were increased in L6 myotubes. Nymphayol was administered to type 2 diabetic male Wistar rats at several doses (5, 10 or 20 mg/kg/day) for 45 days. After nymphayol administration, the plasma glucose concentration was significantly (p⩽0.05) lower (60.33%) than in control diabetic rats, and the plasma insulin level increased in a dose-dependent manner. In addition, the cellular insulin response was analyzed in type 2 diabetic rats; oral administration of nymphayol increased IRS1 phosphorylation and GLUT4 protein expression in liver and muscle. Nymphayol significantly (p⩽0.05) restored the levels of HbA1c, hepatic glycogen and hepatic glucose-metabolizing enzyme (hexokinase, glucose-6-phosphate dehydrogenase, glucose-6-phosphatase, fructose-1, 6-bisphosphatase, glycogen synthase and glycogen phosphorylase) activity in diabetic rats. The administration of glibenclamide, a reference drug (600 µg/kg), also produced a significant (p⩽0.05) reduction in blood glucose in STZ-nicotinamide induced diabetic rats. The results suggest that nymphayol may be a useful therapy for diabetes because it stimulates insulin secretion and promotes glucose absorption.

Gastroprotective effect of nymphayol isolated from Nymphaea stellata (Willd.) flowers: contribution of antioxidant, anti-inflammatory and anti-apoptotic activities.

Gastric ulcer is an illness that affects a great number of people worldwide. The goal of the present research was to assess the anti-ulcerogenic activity of nymphayol (NYM), isolated from Nymphaea stellata, against an ethanol-induced ulcer model in rats. Administration of ethanol elevates the levels of the ulcer index (UI) along with causing tremendous increases in lipid peroxidation and myeloperoxidase (MPO) and significant decreases in gastric mucus, catalase (CAT), superoxide dismutase (SOD), glutathione (GSH), glutathione peroxidase (GPx), and prostaglandin E2 (PGE2). However, the NYM- (45 mg/kg) pretreated animals showed considerable increases in antioxidants, gastric mucus, and PGE2 level and significant decreases in UI, lipid peroxidation, and MPO level. Pro-inflammatory cytokines such as interleukin-6 (IL-6), interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ) were increased and the level of interleukin-10 (IL-10), an anti-inflammatory cytokine, was decreased in ethanol-induced ulcerated animals, and these inequalities were amended by NYM pretreatment. Pro-apoptotic markers including caspase-8, caspase-9, and caspase-3 were decreased and Bcl-2, an anti-apoptotic marker, was increased through NYM pretreatment, as compared with the ethanol-induced ulcer group. Pretreatment with indomethacin, SC560, rofecoxib, and Nω-Nitro-L-arginine methyl ester (L-NAME) considerably prevented the ulcer protective activity of NYM (45 mg/kg), indicating the involvement of cyclooxygenase (COX) and nitric oxide synthase (NOS) in NYM-mediated gastroprotection against ethanol-induced ulcer. These outcomes suggest that the gastroprotective effect of NYM might be mediated by adjustment of inflammatory mediators and apoptotic markers and increasing antioxidants.

Antinociceptive, Immunomodulatory and Antipyretic Activity of Nymphayol Isolated from Nymphaea stellata (Willd.) Flowers.

In the present study, we aimed to analyze the antinociceptive, immunomodulatory and antipyretic activities of nymphayol were investigated in wistar rats and mice. Antinociceptive effect was evaluated by acetic acid induced writhing, formalin induced paw licking and hot-plate tests. Immunomodulatory activity was assessed by neutrophil adhesion test, humoral response to sheep red blood cells, delayed-type hypersensitivity, phagocytic activity and cyclophosphamide induced myelosuppression. Antipyretic activity was evaluated by yeast induced hyperthermia in rats. Nymphayol produced signifi cant (p<0.05) antinociceptive activity in acetic acid induced writhing response and late phase of the formalin induced paw licking response. Pre-treatment with nymphayol (50 mg/kg, oral) evoked a signifi cant increase in neutrophil adhesion to nylon fi bres. The augmentation of humoral immune response to sheep red blood cells by nymphayol (50 mg/kg) was evidenced by increase in antibody titres in rats. Oral administration of nymphayol (50 mg/kg) to rats potentiated the delayed-type hypersensitivity reaction induced by sheep red blood cells. Treatment with nymphayol showed a signifi cant (p<0.05) reduction in pyrexia in rats. The results suggest that nymphayol possesses potent anti-nociceptive, immunomodulatory and antipyretic activities.

Phytochemical analysis and in vitro antioxidant acitivity of hydroalcoholic seed extract of Nymphaea nouchali Burm. f

Objective: To evaluate the phytochemical constituents and the antioxidant activity of hydroalcoholic extract of Nymphaea nouchali seed locally prescribed as a diet for diabetes mellitus.Methods:the plant was assessed against 1,1 diphenyl-2-picryl hydrazyl (DPPH), nitric oxide and lipid peroxidation using standard protocols. Total phenolics, flavonoids and tannins were also determined.Results:Phytochemical analysis revealed the presence of phenols, flavones, tannins, protein, The antioxidant and free radical scavenging activity of hydroalcoholic extract of reducing sugars, glycosides, saponins, alkaloids and steroids. The activities of plant extract against DPPH, nitric oxide and lipid peroxidation was concentration dependent with IC50 value of 42.82, 23.58 and 54.65 μg/mL respectively. The total antioxidant capacity was high with 577.73 mg vitamin E/g of the extract and showed a moderately high vitamin C content of 197.22 mg/g. The total tannin content of hydroalcoholic seed extract was high (195.84 GE/g), followed by phenolics (179.56 GE/g) and flavonoids (23.55 QE/g).Conclusion:Our findings provide evidence that the crude extract of Nymphaea nouchali is a potential source of natural antioxidants and this justifies its use in folkloric medicine.

Antinociceptive, Immunomodulatory and Antipyretic Activity of Nymphayol Isolated from Nymphaea stellata (Willd.) Flowers

In the present study, we aimed to analyze the antinociceptive, immunomodulatory and antipyretic activities of nymphayol were investigated in wistar rats and mice. Antinociceptive effect was evaluated by acetic acid induced writhing, formalin induced paw licking and hot-plate tests. Immunomodulatory activity was assessed by neutrophil adhesion test, humoral response to sheep red blood cells, delayed-type hypersensitivity, phagocytic activity and cyclophosphamide induced myelosuppression. Antipyretic activity was evaluated by yeast induced hyperthermia in rats. Nymphayol produced significant (p<0.05) antinociceptive activity in acetic acid induced writhing response and late phase of the formalin induced paw licking response. Pre-treatment with nymphayol (50 mg/kg, oral) evoked a significant increase in neutrophil adhesion to nylon fibres. The augmentation of humoral immune response to sheep red blood cells by nymphayol (50 mg/kg) was evidenced by increase in antibody titres in rats. Oral administration of nymphayol (50 mg/kg) to rats potentiated the delayed-type hypersensitivity reaction induced by sheep red blood cells. Treatment with nymphayol showed a significant (p<0.05) reduction in pyrexia in rats. The results suggest that nymphayol possesses potent anti-nociceptive, immunomodulatory and antipyretic activities.