Mechanism of rutin mediated inhibition of insulin amyloid formation and protection of Neuro-2a cells from fibril-induced apoptosis.

Many metabolic and neurodegenerative diseases are associated with protein misfolding and aggregation. Insulin a key hormone, under certain conditions aggregates and forms pathological amyloid fibrils. Several polyphenols have been studied extensively to elucidate their inhibitory effect on amyloid formation. In the present study, we used insulin as an amyloid model to test the mechanism and efficacy of rutin as an anti-amyloidogenic molecule. By using electron microscopy, dynamic light scattering and circular dichroism spectroscopy, we show that rutin inhibits the insulin aggregate and fibril formation. Further, rutin interacts with insulin directly and inhibits fibril formation in a dose-dependent manner as demonstrated by micro scale thermophoresis experiments. The molecular docking study predicted the potential binding pocket of rutin at the interface of chain A and chain B of insulin thereby preventing it from forming the aggregates. Since, rutin is a natural anti-oxidant, we studied its role in diminishing amyloid fibril induced cytotoxicity and apoptosis. Rutin, decreases the insulin amyloid fibrils-induced Neuro-2a cytotoxicity by reducing reactive oxygen species (ROS) levels which in turn downregulates Bax and upregulates Bcl-2 and pBad proteins. These findings suggest the potential action of rutin in preventing protein misfolding, cell death, and serves as a lead structure to design novel anti-amyloidosis compounds.

Combination treatment of podophyllotoxin and rutin promotes mouse Lgr5+ ve intestinal stem cells survival against lethal radiation injury through Wnt signaling.

It has been well established that radiation-induced gastrointestinal injury is manifested through loss of intestinal crypt stem cells and disruption of the mucosal layers, resulting in diarrhoea, weight loss, electrolyte imbalance, infection and mortality. Podophyllotoxin and rutin in combination (G-003M) has been reported to regulate endogenous cellular antioxidant defense systems and inflammatory response. However, the mechanism by which G-003M ameliorates radiation-induced intestinal stem cell (ISC) injury remains unclear. Here, we hypothesize the radioprotective potential of G-003M would amplify the intestinal crypt stem cells through upregulation of Wnt/ß-catenin signaling and accelerate the reconstitution of the irradiated intestine. Our results showed significant functional and structural intestine regeneration in irradiated animals following G-003M treatment which resulted in improved animal survival. Immunohistochemical examination revealed an enhancement in Lgr5+ ve crypt stem cells. Increased ß-catenin nuclear translocation resulted in upregulation of ß-catenin target genes that supported ISC renewal and expansion in G-003M-treated mice, as compared to IR-treated mice. However, G-003M could not rescue the Wnt knockdown cohorts (XAV939 treated) which exhibited greater incidence of intestinal apoptosis, DNA damage and crypt depopulation upon radiation exposure. These findings suggest the involvement of Wnt pathway during G-003M mediated amelioration of IR-induced ISC injury. G-003M also minimised acute inflammation by restricting the infiltration of immune cells into the intestinal venules. Furthermore, G-003M treated animals showed improved anti-tumor response compared to FDA approved Amifostine. Taken together, our findings suggest that G-003M may be used as a potential countermeasure for radiation injuries as well as an adjuvant during anti-cancer therapy.

Rutin from different parts of Capparis spinosa growing wild in Khuzestan/Iran.

A qualitative and quantitative analysis of rutin from leaves, fruits and flowers of Capparis spinosa growing wild in Khuzestan was achieved. After soxhelet extraction of fats in diethyl ether, rutin was extracted by maceration using 50% EtOH. The ethanol extracts of these parts were separated by preparative TLC on silica gel precoated plate with a mixture of butanol: acetic acid (4:1, by volume) as the developing solvent. The spots were visualized under ultraviolet light (254 nm). Rutin was qualified by comparison of its R(f) value with that of standard. UV/Vis spectrum of separated rutin was also compared with those of standards and showed characteristic wavelengths at 260 and 360 nm. Purified rutin was quantified by UV/Vis spectrophotometric determination at 360 nm. The calibration curve was linear in the range of 0.156-2.5 microg mL(-1) with detection limit of 0.0731 microg mL(-1). The purity of extracted rutin from leave, flower and fruit determined by high performance liquid chromatography were 90.41, 87.25 and 64.56%, respectively. The amounts of rutin in leaves, fruits and flowers were 61.09, 6.03 and 43.72 mg per 100 g of dried powder, respectively. By analyzing the spiked samples of leave, flower and fruit the recovery of the UV/Vis method was in the range of 102-107.6%.