Fluoride and Biological Mineralization II: Mechanism of Action of Fluoride to Influence the Collagen-Induced In Vitro Mineralization and Demineralization Reactions.

Fluoride had been shown to inhibit collagen-induced in vitro mineralization without affecting demineralization at its lower concentrations (> 1X10-5 and < 1X10-4 M) and stimulate mineralization in addition to inhibiting demineralization at its concentration > 1X10-4 M. The present studies were designed to investigate the mechanism by which fluoride acts to produce these concentration-dependent effects. The inhibition of mineralization occurring at the lower concentrations of fluoride was found to be due to the inactivation of the specific calcium binding sites of collagen involved in initiating the process of mineralization. Stimulation of mineralization obtained at the higher concentrations of fluoride was found to be due to the activation of the specific phosphate-binding sites of the collagen and the formation of a relatively less soluble and highly stable fluorapatite instead of hydroxyapatite. At its higher concentrations, fluoride was also found to inhibit demineralization by binding to the mineral phase associated with collagen. A model has been presented to explain the mechanisms whereby fluoride may act to produce the above observed effects.

Delving into the Antiurolithiatic Potential of Tribulus terrestris Extract Through -In Vivo Efficacy and Preclinical Safety Investigations in Wistar Rats.

Modern treatment interventions for kidney stones are wrought with side-effects, hence the need for alternative therapies such as plant-based medicines. We have previously documented through in vitro studies that statistically optimized aqueous extract of Tribulus terrestris (Zygophyllaceae family) possesses antiurolithic and antioxidant potential. This provides strong scientific foundation to conduct in vivo efficacy and preclinical safety studies to corroborate and lend further proof to its ability to prevent and cure kidney stones. The preventive and curative urolithiatic efficacy in experimentally induced nephrolithiatic Wistar rats, along with preclinical toxicity was evaluated following oral administration of statistically optimized aqueous extract of T. terrestris. Treatment showed augmented renal function, restoration of normal renal architecture and increase in body weight. Microscopic analysis of urine revealed excretion of small sized urinary crystals, demonstrating that treatment potentially modulated the morphology of renal stones. Tissue enzymatic estimation affirmed the antioxidant efficacy of treatment with reduced free radical generation. Significant upregulation of p38MAPK at both the gene and protein level was noted in hyperoxaluric group and interestingly treatment reversed it. Acute oral toxicity study established the Median Lethal Dose (LD50) to be greater than 2000 mg/kg body weight (b.wt.) No observed adverse effect level (NOAEL) by repeated oral toxicity for 28 days at 750 mg/kg b.wt. was noted. This study lends scientific evidence to the safe, preventive and curative potential of statistically optimized aqueous extract of T. terrestris at a dose of 750 mg/kg b.wt. and suggests that the extract shows promise as a therapeutic antiurolithic agent.

Response surface methodology based extraction of Tribulus terrestris leads to an upsurge of antilithiatic potential by inhibition of calcium oxalate crystallization processes.

Tribulus terrestris has significant antilithiatic efficacy established via both in vitro as well as in vivo studies and is used in numerous anti-urolithiatic herbal formulations viz. Cystone, Uriflow, Uritone and Neeri. However, to fully utilize its antilithiatic potential, the influence of different extraction parameters on antilithiatic ability of T. terrestris aqueous extract needs elucidation. Thus, the current study was undertaken using statistically optimized extraction conditions for aqueous extract preparation. Response surface methodology was employed to observe the influence of three variables i.e. temperature (°C), time (h) and solid: liquid ratio (S: L) on the extraction yield (%) and protein content (mg/g) of T. terrestris aqueous extract. RSM results revealed that the high S:L ratio, low temperature and reduced incubation time were optimal conditions for aqueous extraction. Under such extraction conditions the protein content reached the value of 26.6±1.22 mg/g and the obtained extraction yield was 27.32±1.62%. The assessment of antilithiatic activity of 4 selected extracts (AE1-4), revealed enhanced nucleation and aggregation inhibition of calcium oxalate crystals with AE1 and AE2, which in addition significantly altered the size and morphology of calcium oxalate monohydrate (COM) crystals compared to AE3 and AE4. In vitro cell culture based studies on renal epithelial cells (MDCK, NRK-52E and PK 15) proved that the AE1 showed higher cytoprotective potency by increasing cell viability as compared to the oxalate treated group. The free radical scavenging activity of aqueous extract lowered the reactive oxygen specie's induced damage and potentially reduced the signals of programmed cell death due to oxalate injury. In addition, modulation of the COM crystal morphology was enhanced by AE1 as compared to AE2. The FTIR and GC-MS analysis of AE1, showed the presence of biomolecules which could aid in the attenuation of lithiatic process. In the light of these results the utility of the RSM approach to fully optimize the antilithiatic potential of T. terrestris cannot be undermined.

Rottlerin, a polyphenolic compound from the fruits of Mallotus phillipensis (Lam.) Müll.Arg., impedes oxalate/calcium oxalate induced pathways of oxidative stress in male wistar rats.

BACKGROUND: Oxalate and/or calcium oxalate, is known to induce free radical production, subsequently leading to renal epithelial injury. Oxidative stress and mitochondrial dysfunction have emerged as new targets for managing oxalate induced renal injury. HYPOTHESIS: Plant products and antioxidants have gained tremendous attention in the prevention of lithiatic disease. Rottlerin, a polyphenolic compound from the fruits of Mallotus phillipensis (Lam.) Müll.Arg., has shown free radical scavenging, antioxidant activity and has been reported to interfere in signaling pathways leading to inflammation and apoptosis. In this study, the potential role of rottlerin, in rats exposed to hyperoxaluric environment was explored. METHODS: Hyperoxaluria was induced by administering 0.4% ethylene glycol and 1% ammonium chloride in drinking water to male wistar rats for 9 days. Rottlerin was administered intraperitoneally at 1mg/kg/day along with the hyperoxaluric agent. Prophylactic efficacy of rottlerin to diminish hyperoxaluria induced renal dysfunctionality and crystal load was examined along with its effect on free radicals generating pathways in hyperoxaluric rats. RESULTS: 0.4% ethylene glycol and 1% ammonium chloride led to induction of hyperoxaluria, oxiadtive stress and mitochondrial damage in rats. Rottlerin treatment reduced NADPH oxidase activity, prevented mitochondrial dysfunction and maintained antioxidant environment. It also refurbished renal functioning, tissue integrity and diminished urinary crystal load in hyperoxaluric rats treated with rottlerin. CONCLUSIONS: Thus, the present investigation suggests that rottlerin evidently reduced hyperoxaluric consequences and the probable mechanism of action of this drug could be attributed to its ability to quench free radicals by itself and interrupting signaling pathways involved in pathogenesis of stone formation.

The restrained expression of NF-kB in renal tissue ameliorates folic acid induced acute kidney injury in mice.

The Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) represent family of structurally-related eukaryotic transcription factors which regulate diverse array of cellular processes including immunological responses, inflammation, apoptosis, growth & development. Increased expression of NF-kB has often been seen in many diverse diseases, suggesting the importance of genomic deregulation to disease pathophysiology. In the present study we focused on acute kidney injury (AKI), which remains one of the major risk factor showing a high rate of mortality and morbidity. The pathology associated with it, however, remains incompletely known though inflammation has been reported to be one of the major risk factor in the disease pathophysiology. The role of NF-kB thus seemed pertinent. In the present study we show that high dose of folic acid (FA) induced acute kidney injury (AKI) characterized by elevation in levels of blood urea nitrogen & serum creatinine together with extensive tubular necrosis, loss of brush border and marked reduction in mitochondria. One of the salient observations of this study was a coupled increase in the expression of renal, relA, NF-kB2, and p53 genes and proteins during folic acid induced AKI (FA AKI). Treatment of mice with NF-kB inhibitor, pyrrolidine dithio-carbamate ammonium (PDTC) lowered the expression of these transcription factors and ameliorated the aberrant renal function by decreasing serum creatinine levels. In conclusion, our results suggested that NF-kB plays a pivotal role in maintaining renal function that also involved regulating p53 levels during FA AKI.

The most potent antilithiatic agent ameliorating renal dysfunction and oxidative stress from Bergenia ligulata rhizome.

ETHNOPHARMACOLOGICAL RELEVANCE: The rhizome of Bergenia ligulata is referred by the Ayurvedic system for the treatment of kidney stone since decades and a few, in vitro and in vivo studies also support it. To identify the main phytochemical constituent(s) responsible for antilithiatic activity of its rhizome. MATERIALS AND METHODS: In order to identify the most potent antilithiatic metabolite, the crude extract of rhizome was fractionated using in vitro Calcium oxalate (CaOx) crystal growth inhibitory activity guided fractionation followed by its characterization via LC-MS, FTIR and NMR. Further, the antioxidant potential of purified molecule was assessed using in vitro assays (FRAP and H2O2 scavenging). In vivo activity of the metabolite was evaluated in hyperoxaluric rats given 0.4% ethylene glycol (EG) and 1.0% ammonium chloride (NH4Cl) for 9 days. RESULTS: Activity guided fractionation led to the isolation of most potent antilithiatic metabolite from the rhizome of Bergenia ligulata and spectroscopic analysis revealed it as bergenin. Bergenin showed reducing ability and H2O2 scavenging activity comparable with commercially available anitioxidant, α-tocopherol. At a dose of 10mg/kg body weight of the treated rat, it protected against deleterious effects of lithogenic treatment including weight loss, impaired renal function and oxidative stress, manifested as increased malondialdehyde, reduced redox ratio and decreased antioxidant enzyme activities in the kidneys of hyperoxaluric rats. The creatinine clearance and kidney damage were more improved by bergenin as compared to crude extract of rhizome. CONCLUSIONS: Since, bergenin maintained oxidant/antioxidant balance in hyperoxaluric rats, thus mechanistic insight of its antilithiatic activity was attributed to the antioxidant capability of bergenin. The results of the present study provide significant evidence that bergenin is an active component present in the rhizome of Bergenia ligulata for managing CaOx calculi.

A novel antilithiatic protein from Tribulus terrestris having cytoprotective potency.

Adhesion of calcium oxalate (CaOx) crystals to kidney cells is a key event in kidney stones associated with marked hyperoxaluria. As the propensity of stone recurrence and persistent side effects are not altered by surgical techniques available, phytotherapeutic agents could be useful as an adjuvant therapy. The present study is aimed at examining the antilithiatic potency of the protein biomolecules of Tribulus terrestris, a plant which is a common constituent of herbal marketed preparations to treat urolithiasis. Various biochemical methods with mass spectrometry were used to purify and characterize the purified protein. The protective potency of the protein was tested on the oxalate induced injury on renal epithelial cell lines (NRK 52E). An antilithiatic protein having molecular weight of ~ 60kDa was purified. This purified protein showed similarities with Carotenoid cleavage dioxygenase 7 (CCD7) of Arabidopsis thaliana after matching peptide mass fingerprints in MASCOT search engine. An EF hand domain was identified in CCD7 by SCAN PROSITE. Presence of an EF hand domain, a characteristic feature of calcium binding proteins and a role in the synthesis of retinol which is transported by retinol binding protein, a protein found in kidney stone matrix; of CCD7 support the role of TTP as an antilithiatic protein. The protective potency of TTP on NRK 52E was quite comparable to the aqueous extract of cystone. Our findings suggest that this purified protein biomolecule from Tribulus terrestris could open new vista in medical management of urolithiasis.

Preventive and curative effects of Achyranthes aspera Linn. extract in experimentally induced nephrolithiasis.

The present study was undertaken to evaluate the efficacy of Achyranthes aspera in preventing and reducing the growth of calcium oxalate stones in ethylene glycol induced nephrolithiatic model. Hyperoxaluria was induced in rats using ethylene glycol (EG, 0.4%) and ammonium chloride (1%) for 15 days and was then replaced with EG (0.4%) only. Upon administration of cystone (750 mg/kg body wt.), aqueous extract of A. aspera (500 and 1000 mg/kg body wt.), levels of renal injury markers (lactate dehydrogenase and alkaline phosphatase) were normalized with a decrease in serum urea and serum creatinine. Concurrent treatment reduced changes in the architecture of renal tissue and also decreased the size of crystals thereby helping in quick expulsion of the crystals. The present results indicated that Achyranthes aspera had an ability to maintain renal functioning and reduced renal injury.

In vivo efficacy of Trachyspermum ammi anticalcifying protein in urolithiatic rat model.

ETHNOPHARMACOLOGICAL RELEVANCE: Many medicinal plants have been employed during ages to treat urinary stones though the rationale behind their use is not well established. Recently, we have successfully purified an anticalcifying protein from the seeds of Trachyspermum ammi (L.) Sprague ex Turril (Umbelliferae) using oxalate depletion assay and deciphered its inhibitory activity against calcium oxalate crystal growth. AIM: In this report, the antilithiatic activity of Trachyspermum ammi anticalcifying protein (TAP) was studied in urolithiatic rat model. METHODOLOGY: Urolithiasis was induced by exposure of 0.4% ethylene glycol (EG) and 1.0% ammonium chloride (NH(4)Cl) for 9 days. The efficacy of TAP was studied in another group given same dose of EG and NH(4)Cl in addition to 2mg/kg body weight of TAP. Further, we evaluated ability of TAP to inhibit the attachment of calcium oxalate (CaO(x)) crystal in kidney tissue and studied the consequences of CaO(x) adhesion on renal functioning and tissue integrity. RESULTS: The antilithiatic potential of TAP was confirmed by its ability to maintain renal functioning, reduce renal injury and decrease crystal excretion in urine and retention in renal tissues. CONCLUSIONS: Thus, the present investigation suggests the potential of TAP in preventing calcium oxalate deposition and forms the basis for the development of antilithiatic drug interventions against urolithiasis.

A novel calcium oxalate crystal growth inhibitory protein from the seeds of Dolichos biflorus (L.).

Recurrence and persistent side effects of present day treatment for urolithiasis restrict their use, so an alternate, using phytotherapy is being sought. Dolichos biflorus seeds, which are used as dietary food in India, possess antilithiatic properties. In the present study, a novel dimeric antilithiatic protein (98 kDa) from its seeds was purified based on its ability to inhibit calcium oxalate crystallization in vitro. Amino acid analysis of Dolichos biflorus antilithiatic protein showed abundant acidic amino acids. The mascot search engine presented sequence similarity with a calcium binding protein, calnexin of Pisum sativum from the m/z data obtained by MALDI TOF mass spectrometer. Above results demonstrate the anticalcifying/antilithiatic nature of a novel protein from the seeds of Dolichos biflorus and thus open new vistas for using plant proteins as therapeutic agents to treat urolithiasis.

Purification and characterization of an anticalcifying protein from the seeds of Trachyspermum ammi (L.).

Till date various plants extract have been studied to reduce the incidence of urolithiasis but the identification of naturally occurring calcium oxalate (CaOx) inhibitory biomolecules from plants was hampered in past by limitation in identification method. The present study is aimed at examining the efficacy of Trachyspermum ammi on CaOx crystallization in vitro and further by combining conventional biochemical methods with recent advances in mass spectrometry, a novel calcium oxalate (CaOx) crystal growth inhibitor was purified from the seeds of Trachyspermum ammi. An anticalcifying protein from the seeds of Trachyspermum ammi was purified by three step purification scheme; ammonium sulphate fractionation, anion exchange chromatography and molecular sieve chromatography based on its ability to inhibit calcium oxalate crystallization in vitro. An anticalcifying protein having molecular weight 107 kDa and isolectric point 6.2 was isolated. Amino acid analysis of Trachyspermum ammi anticalcifying protein (TAP) showed abundant presence of acidic amino acids (Asp and Glu). Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry of TAP showed similarities with an unnamed protein product of Vitis vinifera (CAO23876) after matching peptide mass fingerprints in MASCOT search engine. Two EF hand domains were identified in unnamed protein product of Vitis vinifera (CAO23876) by SMART normal module. Due to a significant similarity of TAP with unnamed protein product of Vitis vinifera, presence of two EF hand domains in TAP was anticipated, signifying its calcium binding properties which is a feature of most kidney stone inhibitory proteins.