Malus domestica (Apple) is one of the most widely cultivated cash crops of Nepal. Jumla and Mustang are two major pocket areas for the production of apple. Flavonoids including quercetin and rutin are potent antioxidants present in apples. This study was designed to quantify and compare the presence of quercetin and rutin in different plant parts (peel, leaf, and bark) among various cultivars of Malus domestica from two pocket zones of Nepal. A new HPLC-UV method was developed and validated for the quantification of quercetin and rutin. Polyphenols, flavonoids, and carbohydrate contents were determined by colorimetric methods. 2,2′-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay was carried out to measure in vitro antioxidative activity. Acid hydrolysis of each extract was carried out by the standard method to measure aglycone quercetin content after hydrolysis of its glycosides. The total rutin content ranged from 3.69 ± 1.34 to 374.50 ± 2.35 mg/100g dry extract weight. Before the acidic hydrolysis, the total quercetin content ranged from 2.96 ± 0.13 to 171.05 ± 0.95 mg/100g dry extract weight whereas its amount increased highly after the hydrolysis and it ranged from 80.84 ± 19.65 to 7445.32 ± 29.25 mg/100g dry extract weight. Total polyphenol content ranged from 19.48 ± 0.23 to 123.48 ± 1.84 µg gallic acid equivalent/mg of dry extract weight. Similarly, flavonoid content ranged from 2.21 ± 0.72 µg to 755.54 ± 1.91 µg quercetin equivalent/mg of dry extract weight. Total carbohydrate content ranged from 144.15 ± 3.73 to 484.65 ± 2.63 µg glucose equivalent per 0.5 mg dry extract weight. All the extracts showed the various degrees of antioxidant activity in a dose-dependent manner. Among them, stem bark of the Jonathan Jumla showed potent antioxidant activity with IC50 value of 13.003 µg/mL. The present study provides the information about variation of the phytochemical content among the different cultivars, parts, and geographic locations. Furthermore, it revealed that bark of Malus domestica cultivars had high quercetin and rutin content with high antioxidant activity.
Hypoglycemia, a complication of insulin or sulfonylurea therapy in diabetic patients, leads to brain damage. Furthermore, glucose replenishment following hypoglycemic coma induces neuronal cell death. In this study, we investigated the molecular mechanism underlying glucose deficiency-induced cytotoxicity and the protective effect of d-β-hydroxybutyrate (D-BHB) using SH-SY5Y cells. The cytotoxic mechanism of metformin under glucose deficiency was also examined. Cell viability under 1 mM glucose (glucose deficiency) was significantly decreased which was accompanied by increased production of reactive oxygen species (ROS) and decreased phosphorylation of extracellular signal-regulated kinase (ERK) and glycogen synthase 3 (GSK3β). ROS inhibitor reversed the glucose deficiency-induced cytotoxicity and restored the reduced phosphorylation of ERK and GSK3β. While metformin did not alter cell viability in normal glucose media, it further increased cell death and ROS production under glucose deficiency. However, D-BHB reversed cytotoxicity, ROS production, and the decrease in phosphorylation of ERK and GSK3β induced by the glucose deficiency. ERK inhibitor reversed the D-BHB-induced increase in cell viability under glucose deficiency, whereas GSK3β inhibitor did not restore glucose deficiency-induced cytotoxicity. Finally, the protective effect of D-BHB against glucose deficiency was confirmed in primary neuronal cells. We demonstrate that glucose deficiency-induced cytotoxicity is mediated by ERK inhibition through ROS production, which is attenuated by D-BHB and intensified by metformin.
This study was aimed to determine the antibacterial activity of root bark, leaves, and pericarp extract of Diploknema butyracea and to evaluate the prospective antioxidant activity, total flavonoid, polyphenol, and carbohydrate content. The plant parts were collected and extracted by cold maceration, using hexane, ethyl acetate, methanol, and distilled water. Phytochemical screening of different samples of D. butyracea in different solvents revealed the presence of varied extent of alkaloid, saponin, terpenoid, anthraquinones, tannin, cardiac glycoside, flavonoid, carbohydrate, polyphenol, protein and amino acid, resin, and phytosterol. Our study showed that methanolic root bark extract exhibited the potent antimicrobial activity against Staphylococcus aureus, Staphylococcus epidermidis, and Klebsiella pneumonia with an average zone of inhibition of 17.33 mm, 14.33 mm, and 13.0 mm, respectively. Surprisingly, all of the extracts were insensitive to Escherichia coli. The lowest minimum bactericidal concentration (MBC), 4.6 mg/ml, was observed with the aqueous pericarp extract against S. epidermidis and the highest was of 50 mg/ml shown by ethyl acetate pericarp against K. pneumonia. Our results showed that both the polar and nonpolar components present in the different parts of D. butyracea exhibit prominent antibacterial activities against different bacterial strains. The in vitro 2,2′-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity showed that the methanol extract of root barks displayed the most potent antioxidant activity (IC50 : 6.1 µg/ml). The total polyphenol content of the plant part extracts was observed between 19.48 ± 0.23 and 123.48 ± 1.84 µg gallic acid equivalent/mg of dry extract weight. Likewise, flavonoid content ranged from 40.63 ± 1.28 µg to 889.72 ± 3.40 μg quercetin equivalent/mg of dry extract weight and total carbohydrate content ranged from 11.92 ± 0.60 µg to 174.72 ± 0.60 µg glucose equivalent per/mg dry extract weight. Overall, our study showed that the root bark, pericarp, and leaves extract of D. butyracea evinced prominent antibacterial properties against various pathogenic bacterial strains.
Neuroblastomas are the most common solid extracranial tumors in childhood. We investigated the anticancer effect of cearoin isolated from Dalbergia odorifera in human neuroblastoma SH-SY5Y cells. SH-SY5Y cells were treated with various doses of cearoin. The viability was measured by MTT assay. DCFDA fluorescence assay and Griess assay were used for the measurement of intracellular reactive oxygen species (ROS) and nitric oxide (NO), respectively. Western blot analysis was performed to clarify the molecular pathway involved. Cearoin induced cell death in a dose-dependent manner. Cearoin increased the phosporylation of ERK, the conversion of LC3B-I to LC3B-II, decrease in Bcl2 expression, the activation of caspase-3, and the cleavage of PARP, indicating the induction of autophagy and apoptosis. Furthermore, cearoin treatment increased the production of ROS and NO. Co-treatment with the antioxidant N-acetylcysteine completely abolished cearoin-mediated autophagy, ERK activation and apoptosis, suggesting the critical role of ROS in cearoin-induced anticancer effects. Moreover, co-treatment with ERK inhibitor PD98059 partially reversed cearoin-induced cell death, indicating the involvement of ERK in cearoin anticancer effects. These data reveal that cearoin induces autophagy, ERK activation and apoptosis in neuroblastoma SH-SY5Y cells, which is mediated primarily by ROS generation, suggesting its therapeutic application for the treatment of neuroblastomas.
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