In silico, in vitro, and in vivo acute and sub-acute toxicity profiling of whole plant methanol extract of Equisetum diffusum D. Don from the sub-Himalayan West Bengal, India, having ethnobotanical uses.

BACKGROUND: Equisetum diffusum D. Don commonly known as 'Himalayan horsetail', has been traditionally used in the treatment of back pain, bone fracture and dislocation, and arthritis by various tribal communities of India. Our previous study confirmed the anti-inflammatory efficacy of the plant through in silico, in vitro, and in vivo model studies. Therefore, the current research is focused on safety dose evaluation for the first-time of the whole-plant methanol extract (EDME) of E. diffusum through appropriate in silico, in vitro, and in vivo approaches. METHOD: The whole plant, along with its rhizomes, was collected, and the methanol extract was prepared. The in silico ADMET study was performed to predict the pharmacokinetics profile and toxicity of all the identified phyto-compounds of EDME previously screened by GC-MS study. In vitro cytotoxicity study of EDME was performed using two cell lines: kidney (HEK293) and liver (Huh7) cell lines. The in vivo toxicity study of EDME was validated by the acute toxicity (OECD 423, 2002) and sub-acute toxicity assays (OECD 407, 2008) in the Wistar Albino rat model. RESULTS: The in silico ADMET study of all 47 bioactives predicted good pharmacokinetic and low toxicity profiles. In vitro cytotoxicity showed higher IC50 values of EDME viz., 672 ± 15.7 µg/mL and 1698 ± 6.54 µg/mL for both kidney (HEK293) and liver (Huh7) cell lines, respectively, which were considered as low-toxic. Based on acute oral toxicity, the LD50 value of the extract was considered "non-toxic" up to a feeding range of 2000 mg/kg of body weight. The regular consumption of the extract for an extended period (28 days) was also qualified as safe based on the body and organ weight, hematological, biochemical, and histoarchitecture results in the sub-acute toxicity assay. CONCLUSION: The detailed in silico, in vitro, in vivo (acute and sub-acute oral toxicity) studies gave us a new insight to the safety dose evaluation of Equisetum diffusum, which may serve as a reliable documentation for undertaking the experimental validation of the ethnobotanical uses of the plant which would help in the field of drug development for the treatment of inflammation related complications.

Fruit waste: a current perspective for the sustainable production of pharmacological, nutraceutical, and bioactive resources.

Fruits are crucial components of a balanced diet and a good source of natural antioxidants, that have proven efficacy in various chronic illnesses. Various kinds of waste generated from fruit industries are considered a global concern. By utilizing this fruit waste, the international goal of "zero waste" can be achieved by sustainable utilization of these waste materials as a rich source of secondary metabolites. Moreover, to overcome this waste burden, research have focused on recovering the bioactive compounds from fruit industries and obtaining a new strategy to combat certain chronic diseases. The separation of high-value substances from fruit waste, including phytochemicals, dietary fibers, and polysaccharides which can then be used as functional ingredients for long-term health benefits. Several novel extraction technologies like ultrasound-assisted extraction (UAE), pressurized liquid extraction (PLE), and supercritical fluid extraction (SFE) could provide an alternative approach for successful extraction of the valuable bioactives from the fruit waste for their utilization as nutraceuticals, therapeutics, and value-added products. Most of these waste-derived secondary metabolites comprise polyphenols, which have been reported to have anti-inflammatory, insulin resistance-treating, cardiovascular disease-maintaining, probiotics-enhancing, or even anti-microbial and anti-viral capabilities. This review summarizes the current knowledge of fruit waste by-products in pharmacological, biological, and probiotic applications and highlights several methods for identifying efficacious bioactive compounds from fruit wastes.

Revealing Probiotic Potential of Enterococcus Strains Isolated From Traditionally Fermented Chhurpi and Healthy Human Gut.

In this study, the two lactic acid bacterial strains Enterococcus durans and Enterococcus lactis previously isolated from soft chhurpi, a traditionally fermented milk product prepared by the indigenous community of Sikkim Himalayas and healthy human gut were used. In this study, we attempted to investigate the probiotic attributes, safety, and health beneficial role, and hypercholesterolemia of Enterococcus durans and Enterococcus lactis. Both probiotic potential strains showed good hypocholesterolemic activity in vitro along with tolerance to acid pH (2 and 2.5), tolerance to three bile salts, oxbile, cholic acid, and taurocholic acid (0.5 and 1%), presence of BSH enzyme and its activity, and cell surface adherence. On assessing for safety, both LAB strains were sensitive to antibiotics and exhibited no hemolytic activity. The probiotic strains were tested in vivo in the Sprague-Dawley rats which were divided into five experimental groups: Normal Control (ND), probiotic strain Enterococcus durans HS03 (BSH-negative) and high-cholesterol diet (HCD1), probiotic strain Enterococcus lactis YY1 (BSH-positive) and high-cholesterol diet (HCD2), and a combination of both strains and high-cholesterol diet (HCD3) and Negative Control (HCD). The probiotic-treated groups HCD1, HCD2, and HCD3 showed a decrease in serum cholesterol levels up to 22.55, 6.67, and 31.06%; the TG and VLDL concentrations were 25.39, 26.3, and 33.21%; reduction in LDL-cholesterol was 33.66, 28.50, and 35.87%; and increase of HDL was 38.32, 47.9, and 41.92%. Similarly, the effects of total cholesterol and TG in the liver, kidney and liver histopathology, liver and body lipid index, and oxidative stress in rat liver were also studied. The fecal lactobacilli were more in the samples of the probiotic-treated groups and their fecal coliform and E. coli counts decreased relatively as compared to the control groups in 0, 7, 14, and 21 days. This is the first report on the probiotic potential of Enterococcus durans HS03 and Enterococcus lactis YY1 strains that gives a new insight into the cholesterol-lowering and probiotic product development with wide health attributes.

Validating potent anti-inflammatory and anti-rheumatoid properties of Drynaria quercifolia rhizome methanolic extract through in vitro, in vivo, in silico and GC-MS-based profiling.

BACKGROUND: The fronds of Drynaria quercifolia have traditionally been used in rheumatic pain management. The goal of the present study was to validate the potent anti-inflammatory and anti-rheumatoid properties of the methanolic-extract of its rhizome using in vitro, in vivo and in silico strategies. METHODS: The plant was collected and the methanolic extract was prepared from its rhizome. Protein denaturation test, hypotonicity and heat-induced haemolysis assays were performed in vitro. The in vivo anti-rheumatoid potential was assessed in Freund's complete adjuvant (FCA)-induced Wistar rat model through inflammatory paw-edema, haematological, biochemical, radiological and histopathological measurements. Moreover, metabolites of methanolic extract were screened by gas chromatography-mass spectrometry (GC-MS) and 3D molecular structures of active components were utilized for in silico docking study using AutoDock. RESULTS: In vitro results evinced a significant (p < 0.05) anti-inflammatory activity of the rhizome methanolic extract in a dose-linear response. Further, Drynaria quercifolia rhizome methanolic extract (DME) significantly ameliorated rheumatoid arthritis as indicated by the inhibition of arthritic paw-edema (in millimeter) in the rat rheumatoid arthritis models in both the low (57.71 ± 0.99, p < 0.01) and high dose groups (54.45 ± 1.30, p < 0.001) when compared to arthritic control. Treatment with DME also normalized the haematological (RBC, WBC, platelet counts and hemoglobin contents) and biochemical parameters (total protein, albumin, creatinine and ceruloplasmin) significantly (p < 0.05), which were further supported by histopathological and radiological analyses. Furthermore, GC-MS analysis of DME demonstrated the presence of 47 phytochemical compounds. Compounds like Squalene, Gamma Tocopherol, n-Hexadecanoic acid showed potent inhibition of cyclooxygenase-2 (COX-2), tumor necrosis factor (TNF-α), and interleukin (IL-6) in the docking analysis. CONCLUSION: Results from in vivo and in vitro studies indicated that DME possesses a potent anti-inflammatory and anti-arthritic activity. In silico studies delineated the emergent potent inhibitory effects of several bio-active components on the target inflammatory markers (COX-2, TNF-α and IL-6).