Biochemical and molecular anticancer approaches for Boerhaavia diffusa root extracts in oral cancer.

Background: Boerhaavia diffusa is a medicinal herb with anti-inflammatory, antiproliferative, anticancer, and immunomodulatory properties, found across India. Aim and Objectives: The present study is designed to investigate the therapeutic potential for B. diffusa root extracts in oral cancer cell line. Materials and Methods: The aqueous and methanolic extracts of B. diffusa were prepared using Soxhlet apparatus. In order to determine the phytochemical constituents of B. diffusa, the extracts were subjected to gas chromatography-mass spectrometry analysis. The antioxidant potential of B. diffusa extracts was assessed by 2,2-Diphenyl-picrylhydrazyl, ferric ion-reducing antioxidant power, catalase and peroxidase assays. The effective concentration of B. diffusa root on cell viability was analyzed by [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The ability of B. diffusa root extracts to modify the cell-cycle phases was performed by FACS analysis. The apoptotic inducing potential of B. diffusa in oral cancer cells was confirmed by acridine orange-ethidium bromide and 4',6-diamidino-2-phenylindole staining. The protein profile of apoptotic processes was validated by the Western blot analysis; docking studies were also performed. Results: We observed that antioxidant activity was higher in B. diffusa methanolic extract compared with aqueous extract. The results showed that the methanolic and aqueous extracts of B. diffusa exhibited significant cytotoxic effect with IC50 value of 36 µg/ml and 30 µg/ml, respectively. The apoptotic DNA fragmentation and the apoptotic inducing potential in KB oral cancer cell line were higher for the methanolic extract compared with the aqueous extract. These results were also confirmed by in-silico analysis. Conclusion: The results indicate that extracts obtained from the roots of B. diffusa inhibit the progression of oral cancer. These compounds of pharmacological importance can be either used alone or in combination with other drugs to treat oral cancer.

Renoprotective effect of tectorigenin glycosides isolated from Iris spuria L. (Zeal) against hyperoxaluria and hyperglycemia in NRK-49Fcells.

Oxidative stress has been identified as an underlying factor in the development of insulin resistance, ß-cell dysfunction, impaired glucose tolerance and type 2 diabetes mellitus and it also play major role in kidney stone formation. The present study is aimed to elucidate the in vitro nephroprotective activity of two isoflavonoid glycosides, tectorigenin 7-O-ß-D-glucosyl-(1→6)-ß-D-glucoside (1) and tectorigenin 7-O-ß-D-glucosyl-4'-O-ß-D-glucoside (2) isolated from the n-BuOH fraction of Iris spuria L. (Zeal) rhizome MeOH extract against oxalate and high glucose-induced oxidative stress in NRK-49F cells. The results revealed that compounds 1 and 2 significantly increased the antioxidant enzyme activities and decreased MDA levels in both oxalate and high glucose stress. Treatment with these phytochemicals effectively down-regulated expression of crystal modulator genes and pro-fibrotic genes in oxalate and high glucose-mediated stress respectively. This study indicates cytoprotective, antioxidant, anti-urolithic and anti-diabetic effects of compounds 1 and 2 against oxalate and high glucose stress.[Figure: see text].

Rutin ameliorates metabolic acidosis and fibrosis in alloxan induced diabetic nephropathy and cardiomyopathy in experimental rats.

Diabetic nephropathy and cardiomyopathy are two major causes of mortality among patients with diabetes mellitus (DM). Since current diabetic medications are associated with various side effects, the naturally occurring plant-derived compounds are in demand. Bioflavonoids originating from vegetables and medicinal plants have beneficial effects on diabetes by improving glycemic control, lipid metabolism, and anti-oxidant status. The present study is focused on the effect of rutin against alloxan induced diabetic nephropathy and cardiomyopathy. Male albino Wistar rats were divided into four groups, each of six rats. Group I control rats received 0.9% saline as a single dose intraperitoneally. Group II rats were induced diabetes with a single dose of alloxan monohydrate (150 mg/kg body weight in 0.9% saline) intraperitoneally. Group III rats received 0.28 M of NH4Cl in drinking water for 3 days for the experimental induction of metabolic acidosis. Group IV rats were injected with a single dose of alloxan monohydrate (150 mg/kg bodyweight) and administered rutin hydrate (100 mg/kg) for a period of 4 weeks by oral gavage. Administration of rutin prevented urinary ketone body formation and decreased serum creatinine and urea levels in alloxan induced diabetic rats. Rutin supplementation reduced the levels of serum triglycerides and cholesterol in diabetic rats. Gene expression profiling of metabolic acidosis related genes (AQP2, AQP3 and V2R) and also histopathological results demonstrated the protective effect of rutin against diabetic ketoacidodis and fibrosis. The results of the present study revealed rutin administration prevents the progression of diabetic nephropathy and cardiomyopathy through amelioration of fibrosis and metabolic acidosis.

Oxidative stress and endoplasmic stress in calcium oxalate stone disease: the chicken or the egg?

Crystal modulators play a significant role in the formation of calcium oxalate stone disease. When renal cells are subjected to oxalate stress, the loss in cell integrity leads to exposure of multiple proteins that assist and/or inhibit crystal attachment and retention. Contact between oxalate and calcium oxalate with urothelium proves fatal to cells as a result of reactive oxygen species generation and onset of oxidative stress. Hence, as a therapeutic strategy it was hypothesised that supplementation of antioxidants would suffice. On the contrary to popular belief, the detection of oxalate induced endoplasmic reticulum mediated apoptosis proved the ineffectiveness of antioxidant therapy alone. Thus, the inadequacy of antioxidant supplementation in oxalate stress invoked the presence of an alternative pathway for the induction of kidney fibrosis in hyperoxaluric rats. In addition to settling this query, the link between oxidative stress and ER stress is not well understood, especially in urolithiasis.

Chromosomal integration of heterologous oxalate decarboxylase in Lactobacillus plantarum WCFS1 using mobile genetic element Ll.LtrB.

Lactobacillus plantarum WCFS1 (L. plantarum WCFS1) is commonly used as a potential cell factory because of its 'generally recognized as safe' status. The plasmid instability and the presence of antibiotic selection marker complicate the application of genetically modified L. plantarum in human clinical trials. In the present study, we aimed to integrate oxalate decarboxylase (oxdC) gene of Bacillus subtilis origin by targeted chromosomal mutation in L. plantarum using mobile genetic element Ll.LtrB as a therapeutic tool against calcium oxalate stone disease. oxdC expression cassette was constructed and integrated into a targeted gene, thymidylate synthase (thyA) in the L. plantarum genome. The dependence on external thymidine for growth and survival was established by live dead population assay and SEM (scanning electron microscopy) analysis. The western blotting assay showed the secretion of 44 kDa OxdC protein in the culture supernatant of L. plantarum ∆thyA:OxdC. The biologically contained recombinant strain significantly reduced the oxalate concentration by 53% and exhibited a loss of viability when introduced to environmental samples. Biologically contained L. plantarum secreting OxdC constructed using group II intron has the ability to degrade oxalate present in the extracellular environment and could be used as a therapeutic tool for the calcium oxalate stone disease.

Combination of ramipril and rutin alleviate alloxan induced diabetic nephropathy targeting multiple stress pathways in vivo.

Diabetic nephropathy (DN) is considered as one of the major microvascular complications of diabetes mellitus (DM) which leads to end stage renal disease (ESRD). Even though existing therapeutic options are effective in decreasing albuminuria, drugs targeting the preservation of GFR and prevention of ESRD may provide better strategy for the treatment. Since metabolic disorders are multifactorial, poly-herbal medications, and drug-herbal combination are in demand. Therefore, the present work is focused on the combinatorial renoprotective effect of rutin and ramipril on alloxan induced DN in experimental rats. Male Wistar rats were divided into five groups, group I-control, group II-diabetic rats, group III-diabetic rats treated with ramipril, group IV-diabetic rats treated with rutin, group V-diabetic rats treated with ramipril and rutin for a period of six weeks. Results revealed administration of alloxan induced hyperglycemia and alteration in antioxidant profile. However, combination of a bioflavonoid with an Angiotensin converting enzyme (ACE) inhibitor administration restored the antioxidant status in experimental DN rats. Over-expression of ACE, TGF-ß1 and decreased podocin expression in diabetic rats was significantly reversed in rats administered with both ramipril and rutin. In addition to attentuating oxidative stress and fibrosis, combinatorial therapy significantly down-regulated endoplasmic reticulum stress markers GRP78 and CHOP. Notably, combination of both ramipril and rutin in low doses reduced the side effects than the administration of monotherapy alone. Histopathological results revealed that combinatorial therapy was associated with a reduction in tubulointerstitial injury. The current study contributes the understanding of the multifactorial nature of DN and implies combinatorial treatment of ACE inhibitor with an antioxidant will be a promising therapeutic strategy for DN by their mechanism of action targeting various pathophysiological changes and stress pathways.

Designer probiotic Lactobacillus plantarum expressing oxalate decarboxylase developed using group II intron degrades intestinal oxalate in hyperoxaluric rats.

Increased intestinal absorption of oxalate causes hyperoxaluria, a major risk factor for kidney stone disease. Intestinal colonization of recombinant probiotic bacteria expressing oxalate-degrading gene (OxdC) is an effective therapeutic option for recurrent calcium oxalate (CaOx) stone disease. Therefore, we aimed to develop food-grade probiotic L. plantarum secreting OxdC using lactococcal group II intron, Ll.LtrB and evaluate its oxalate degradation ability in vivo. Male Wistar albino rats were divided into four groups. The rats of group I received normal rat chow and drinking water. Groups II, III and IV rats received 5% potassium oxalate containing diet for 28 days. Groups III and IV rats received L. plantarum and food-grade recombinant L. plantarum respectively from 15 to 28 days. Biochemical parameters and crystalluria were analysed in 24 h urine samples. At the end of experimental period, rats were sacrificed; intestine and kidneys were dissected out for colonization studies and histopathological analysis. Herein, we found that the administration of recombinant probiotics significantly reduced the urinary oxalate, calcium, urea, and creatinine levels in rats of group IV compared to group II. Furthermore, colonization studies indicated that recombinant probiotics have gastrointestinal transit and intestinal colonization ability similar to that of wild-type bacteria. In addition, gene expression studies revealed down-regulation of OPN and KIM-1 among group IV rats. Histopathological analysis showed less evidence of nephrocalcinosis in group IV rats. In conclusion, the study demonstrates that food-grade L. plantarum secreting OxdC is capable of degrading intestinal oxalate and thereby prevent CaOx stone formation in experimental rats.

Oral administration of oxalate-enriched spinach extract as an improved methodology for the induction of dietary hyperoxaluric nephrocalcinosis in experimental rats.

Experimental induction of hyperoxaluria by ethylene glycol (EG) administration is disapproved as it causes metabolic acidosis while the oral administration of chemically synthesized potassium oxalate (KOx) diet does not mimic our natural system. Since existing models comprise limitations, this study is aimed to develop an improved model for the induction of dietary hyperoxaluria, and nephrocalcinosis in experimental rats by administration of naturally available oxalate rich diet. Male albino Wistar rats were divided into five groups. Group I, control; group II rats received 0.75% EG, group III rats fed with 5% KOx diet and group IV and V rats were administered with spinach extract of 250 and 500 mg soluble oxalate/day respectively, for 28 d. Urine and serum biochemistry were analyzed. After the experimental period, rats were sacrificed, liver and kidney tissue homogenates were used for antioxidant and lipid peroxidation assay. Relative change in expression of kidney injury molecule-1 (KIM-1) and crystal modulators genes in kidney tissues were evaluated. Tissue damage was assessed by histology studies of liver and kidney. Experimental group rats developed hyperoxaluria and crystalluria. Urine parameters, serum biochemistry, antioxidant profile, lipid peroxidation levels and gene expression analysis of experimental group II and III rats reflected acute kidney damage compared to group V rats. Histopathology results showed moderate hyperplasia in liver and severe interstitial inflammation in kidneys of group II and III than group V rats. Ingestion of naturally available oxalate enriched spinach extract successfully induced dietary hyperoxaluria and nephrocalcinosis in rats with minimal kidney damage.

Antioxidant activity of phenolic compounds from extracts of Eucalyptus globulus and Melaleuca styphelioides and their protective role on D-glucose-induced hyperglycemic stress and oxalate stress in NRK-49Fcells.

Phytochemicals serve as potential therapeutic agents for the prevention and treatment of diseases. In this study, we elucidate the renoprotective activity of compounds isolated from Eucalyptus globulus and Melaleuca styphelioides extracts in glucose- and oxalate-challenged NRK-49F cell model. The antioxidant potential of isolated compounds was evaluated based on their effect on antioxidant enzyme activities and lipid peroxidation levels. The results demonstrated that exposure of NRK-49F cells to glucose and oxalate stress augmented cell damage and attenuated antioxidant enzyme activities. The phytochemicals 2,2,8-trimethyl-6-formyl-chrom-3-ene-7-O-ß-D-glucopyranoside, Cornusiin B and tellimagrandin I treatment restored antioxidant enzyme activity, significantly lowered lipid peroxidation levels and effectively protected cells from glucose and oxalate stress equivalent to the known antioxidant, N-acetyl cysteine. Pterocarinin A significantly reversed cellular damage owing to glucose stress. In conclusion, the compounds isolated from E. globulus and M. styphelioides showed potential cytoprotective and anti-oxidative property against glucose- and oxalate-induced oxidative stress in NRK-49F cells.

Expression of heterologous oxalate decarboxylase in HEK293 cells confers protection against oxalate induced oxidative stress as a therapeutic approach for calcium oxalate stone disease.

Oxalates stimulate alterations in renal epithelial cells and thereby induce calcium oxalate (CaOx) stone formation. Bacillus subtilis YvrK gene encodes for oxalate decarboxylase (OxdC) which degrades oxalate to formate and CO2. The present work is aimed to clone the oxdC gene in a mammalian expression vector pcDNA and transfect into Human Embryonic Kidney 293 (HEK293) cells and evaluate the oxdC expression, cell survival rate and oxalate degrading efficiency. The results indicate cell survival rate of HEK293/pcDNAOXDC cells pre-incubated with oxalate was enhanced by 28%. HEK293/pcDNAOXDC cells expressing OxdC treated with oxalate, significantly restored antioxidant activity, mitochondrial membrane potential and intracellular reactive oxygen species (ROS) generation compared with HEK293/pcDNA. Apoptotic marker caspase 3 downregulation illustrates HEK293/pcDNAOXDC cells were able to survive under oxalate-mediated oxidative stress. The findings suggest HEK293 cells expressing oxdC capable of degrading oxalate protect cells from oxidative damage and thus serve as a therapeutic option for prevention of CaOx stone disease. [Formula: see text].