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In Vitro Studies on Synergistic Effects of Limonia Acidissima And Apple Cider Vinegar on AntiUrolithiatic Activity
Abstract
Traditionally Wood apple (Limonia acidissima) and Apple cider vinegar was found to be used to treat and prevent kidney stones. The present study is dedicated towards proving the same and providing scientific proof to these traditional ideas. Minerals like calcium, oxala tes, phosphates, uric acid etc. form insoluble aggregates through formation of monohydrates and di -hydrates. This process is initiated by nucleation and then aggregation of insoluble masses. Phytoconstituents like flavonoids, terpenoids, ascorbic acid, acetic acid, potassium, magnesium etc. contain inhibitory properties and hence are urolithiatic in nature. In vitro tests for nucleation and aggregation has been conducted for Wood apple, Apple cider vinegar and their (1:1) synergistic sample and absorbance at 620 nm was recorded to obtain a turbidity slope. The tests were conducted in presence of sodium acetate/ NaCl buffer of pH 5.7 to mimic the biological conditions. 1:10 dilution of commercially obtained Apple cider vinegar sample showed better % inhibition of nucleation and aggregation when compared to the inhibition shown by standard drug cystone. 10 mg/ mL methanolic extract of dried pulp of wood apple showed higher % inhibition when compared to cystone and Apple cider vinegar. Synergistic sample containing both wood apple and apple cider vinegar in equal proportion showed the highest % inhibition for nucleation and aggregation of calcium oxalate crystals. This proved the synergistic activity of wood apple and apple cider vinegar for urolithiasis. Further studies are being conducted check for aggregation and degradation activities of the mentioned test extracts.
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The global escalation in antibiotic resistance cases means alternative antimicrobials are essential. The aim of this study was to investigate the antimicrobial capacity of apple cider vinegar (ACV) against E. coli, S. aureus and C. albicans. The minimum dilution of ACV required for growth inhibition varied for each microbial species. For C. albicans, a 1/2 ACV had the strongest effect, S. aureus, a 1/25 dilution ACV was required, whereas for E-coli cultures, a 1/50 ACV dilution was required (p < 0.05). Monocyte co-culture with microbes alongside ACV resulted in dose dependent downregulation of inflammatory cytokines (TNFα, IL-6). Results are expressed as percentage decreases in cytokine secretion comparing ACV treated with non-ACV treated monocytes cultured with E-coli (TNFα, 99.2%; IL-6, 98%), S. aureus (TNFα, 90%; IL-6, 83%) and C. albicans (TNFα, 83.3%; IL-6, 90.1%) respectively. Proteomic analyses of microbes demonstrated that ACV impaired cell integrity, organelles and protein expression. ACV treatment resulted in an absence in expression of DNA starvation protein, citrate synthase, isocitrate and malate dehydrogenases in E-coli; chaperone protein DNak and ftsz in S. aureus and pyruvate kinase, 6-phosphogluconate dehydrogenase, fructose bisphosphate were among the enzymes absent in C.albican cultures. The results demonstrate ACV has multiple antimicrobial potential with clinical therapeutic implications.
The leaves of Chenopodium album Linn. are traditionally used for treatment of kidney diseases and urinary stones. The present work investigated the effect of aqueous extract of leaves of C. album (CAAE) on in-vitro crystallization of CaOx and brushite crystals. Crystallization was studied by using nucleation and aggregation assay of calcium oxalate (CaOx) crystals and growth assay of calcium oxalate monohydrate and brushite crystals. The effects of CAAE and cystone on slope of nucleation and aggregation as well as growth of calcium oxalate crystallization were evaluated spectrophotometrically. The densities of the formed crystals were compared under microscope. The effects of CAAE and citric acid on growth of brushite crystals were carried out by using single diffusion gel growth technique, and the parameters evaluated were length, morphology and average size of the deposited crystals. CAAE significantly inhibited the slope of nucleation and aggregation of CaOx crystallization, and decreased the crystal density. It also inhibited the growth and caused the dissolution of brushite crystals. The standard drug cystone or citric acid also exhibited similar effects. The study reveals that the leaves of C. album were found effective in the prevention of the experimentally induced urinary stones and substantiate the traditional claim. It is concluded that the leaves of C. album have beneficial inhibitory effect on in-vitro crystallization of CaOx and CHPD (brushite) crystals.
Purpose:
For most cases, urolithiasis is a condition where excessive oxalate is present in the urine. Many reports have documented free radical generation followed by hyperoxaluria as a consequence of which calcium oxalate (CaOx) deposition occurs in the kidney tissue. The present study is aimed to exam the antilithiatic potency of the aqueous extract (AE) of Terminalia arjuna (T. arjuna).
Materials and Methods:
The antilithiatic activity of Terminalia arjuna was investigated in vitro nucleation, aggregation and growth of the CaOx crystals as well as the morphology of CaOx crystals using the inbuilt software ‘Image-Pro Plus 7.0’ of Olympus upright microscope (BX53). Antioxidant activity of AE of Terminalia arjuna bark was also determined in vitro.
Results:
Terminalia arjuna extract exhibited a concentration dependent inhibition of nucleation and aggregation of CaOx crystals. The AE of Terminalia arjuna bark also inhibited the growth of CaOx crystals. At the same time, the AE also modified the morphology of CaOx crystals from hexagonal to spherical shape with increasing concentrations of AE and reduced the dimensions such as area, perimeter, length and width of CaOx crystals in a dose dependent manner. Also, the Terminalia arjuna AE scavenged the DPPH (2, 2-diphenyl-1-picrylhydrazyl) radicals with an IC50 at 13.1µg/mL.
Conclusions:
The study suggests that Terminalia arjuna bark has the potential to scavenge DPPH radicals and inhibit CaOx crystallization in vitro. In the light of these studies, Terminalia arjuna can be regarded as a promising candidate from natural plant sources of antilithiatic and antioxidant activity with high value.
Limonia acidissima (known as Vila in Siddha medicine) belonging to the Family Rutaceae, is a well known Traditional herb used since time immemorial. From ancient days to recent civilization, human beings depend on nature for running their life smoothly. Siddha Traditional system of medicine consist large number of plants with various medicinal and pharmacological importances and hence represents the highness of bioactive molecules. The Authors of this paper decided to collect and compile the literary documents, recent Pharmacognostical aspects, and Experimental research works of Limonia acidissima. Plants remain a vital source of drugs and now a day’s much emphasis have been given to Nutraceuticals. Various parts of the plant possess astringent, cardiotonic, Carminative and refrigerant actions. This review will be helpful to create interest towards the less known and less documented Limonia acidissima and may be useful in developing new formulations with more therapeutic values.
Hedychium species is one of the important aromatic, ornamental medicinal plants of North east India. In the present study, total flavonoid content and antioxidant activity of aqueous rhizome extract of three Hedychium species namely H. rubrum, H. coronarium and H. spicatum were investigated. In antioxidant activity: free radical scavenging activity, reducing power and total antioxidant activity were determined and expressed in terms of standard used for the respective assay. Total flavonoid content of aqueous rhizome extract of H. rubrum, H. coronarium and H. spicatum in terms of quercetin equivalent (QE) was 3.22, 2.77 and 0.74 μg/100g of extract respectively. In DPPH assay H. rubrum (32.3%) showed the highest free radical scavenging activity followed by H. coronarium (21%) and lowest activity in H. Spicatum (5.76%). In reducing power method, both standard ascorbic acid and sample extracts were found to be increased in a dose dependent manner. Total antioxidant activity of aqueous rhizome extract of H. rubrum, H. coronarium and H. spicatum in terms of ascorbic acid equivalent (AAE) was 207.3, 157.5 and 102.6 μg/ml of extract. The present data indicates that aqueous rhizome extract of all the three Hedychium species are potential source of antioxidant which may be due to the presence of flavonoid in the extracts. Among the three Hedychium species, H. rubrum was found to have the highest total flavonoid content and antioxidant activity.
Rates of nucleation and aggregation of calcium oxalate crystals were derived from 20-min time course measurements of OD620 after mixing solutions containing CaCl2 and K2C2O4 at 37°C, pH 5.7, ionic strength (IS) 0.21, with constant stirring (500 rpm); final assay concentrations were 4.25 mM calcium and 0.5 mM oxalate, respectively. The maximum increase of OD620 with time, termed S
N, mainly reflects maximum rate of formation of new particles and thus crystal nucleation. After equilibrium has been reached, OD620 progressively decreases despite ionized calcium staying constant and no new particles being formed, due to crystal aggregation. Rate of aggregation, S
A, is derived from the maximum decrease in OD620 with time. S
N and S
A are not independent, as indicated by a positive correlation (r=0.844, P=0.0001). Among the modifiers studied, citrate at 0.5–2.5 mM lowered both S
N and S
A in a concentration-dependent manner (P<0.01 for all comparisons vs control). Chondroitin-6-sulfate at 6.25–25 mg/l moderately lowered S
N, whereas it strongly inhibited aggregation (P<0.01 vs control). At 6.8–20.4 mg/l, albumin did not affect nucleation, whereas it inhibited aggregation in a concentration-dependent manner (P<0.005 vs control for all comparisons).
The aim of this study was to measure the effects of normal (nTHP) and abnormal stone former Tamm-Horsfall protein (SF-THP) on calcium oxalate (CaOx) nucleation and aggregation as well as on crystal morphology, in presence or absence of citrate.
Nucleation and aggregation of CaOx crystals from a supersaturated, stirred solution (200 mM NaCl, 10 mM Na-acetate, pH 5.70, 5 mM Ca and 0.5 mM Ox) were studied by spectrophotometric time-course measurements of OD at 620 nm (OD(620)). Measured parameters were induction time t(I) (time to induce formation of detectable particles), S(N), (slope of increase of OD(620), mainly due to crystal nucleation), and S(A), (slope of decrease of OD(620) after equilibrium has been reached, due to crystal aggregation). Effects of citrate, nTHP and SF-THP on these parameters were measured, and scanning electron microscopy (SEM) was performed.
At 1.5, 2.5 and 3.5 mM, citrate increased t(I) and inhibited crystal nucleation (by 78-87%) as well as aggregation (by 63-70%), and smaller CaOx crystals (length/width ratio 1.7+/-0.1) than under standard conditions (length/width 3.9+/-0.5) were visible (P<0.001). Normal THP at 30 and 40 mg/l inhibited crystal nucleation and, more strongly, aggregation (inhibition 76-81%). SEM revealed a decrease in length/width ratio to 2.6+/-0.4 (P=0.051 vs standard conditions) and less aggregation than without nTHP. At all concentrations tested, SF-THP reduced t(I) (P=0.0001 vs standard conditions) and promoted aggregation (inhibition -48 to -33%); crystals were elongated with a length/width ratio of 4.3+/-0.6 (P<0. 05 vs nTHP). When simultaneously present with nTHP, citrate enhanced the inhibitory effects of nTHP, producing the smallest (length/width 1.5+/-0.1) and least aggregated crystals. Finally, 3.5 mM citrate turned promotory SF-THP into a crystallization inhibitor with abundant small and clustered, but not aggregated crystals.
Citrate appears to be the main determinant of CaOx crystallization rates and crystal morphology in the presence of nTHP as well as SF-THP. Its effects appear to predominate over those of THP, since even promotory SF-THP is turned into a crystallization inhibitor in the presence of citrate. This re-emphasizes at a morphological level what has been concluded from functional as well from clinical studies, namely that citrate is needed in urine at equimolar concentrations to calcium in order to prevent the formation of large crystal aggregates in presence of abnormal THP.
Previous studies on healthy people show that vinegar delays gastric emptying and lowers postprandial blood glucose and insulin levels. The aim of this study was to investigate the effect of apple cider vinegar on delayed gastric emptying rate on diabetes mellitus patients.
Ten patients with type 1 diabetes and diabetic gastroparesis, including one patient who had undergone vagotomy, were included and completed the investigator blinded crossover trial. The gastric emptying rate (GER) was measured using standardized real-time ultrasonography. The GER was calculated as the percentage change in the antral cross-sectional area 15 and 90 minutes after ingestion of 300 g rice pudding and 200 ml water (GER1), or 300 g rice pudding and 200 ml water with 30 ml apple cider vinegar (GER2). The subjects drank 200 ml water daily before breakfast one week before the measurement of GER1. The same subjects drank 200 ml water with 30 ml vinegar daily before breakfast for two weeks before the measurement of GER2.
The median values of GER1 and GER2 were 27% and 17%, respectively. The effect of vinegar on the rate of gastric emptying was statistically significant (p < 0.05).
This study shows that vinegar affects insulin-dependent diabetes mellitus patients with diabetic gastroparesis by reducing the gastric emptying rate even further, and this might be a disadvantage regarding to their glycaemic control.
ISRCTN33841495.
Objective: The present study was carried out to evaluate the invitro anti-urolithiasis activity of leaves and flower infusions of Convolvulus arvensis. Materials and methods: The inhibition of in-vitro calcium-oxalate crystal (a major component of most urinary stones) formation by various extracts was investigated by different methods. Synthetic urine supersaturated with calcium oxide was prepared and urolithiasis was investigated by inhibition assay, aggregation assay, and sedimentary crystal formation. Crystal formation in synthetic urine was studied at different time intervals using leaf and flower infusions at different concentrations 10, 25, 50, 75,100 mg/ml each respectively. Results: Among the two extracts when compared to control group, the inhibitory potency of leaf extract was found to be more significant (P<0.05), than the flower extract.
Struvite or magnesium ammonium phosphate hexahydrate (MAP) is one of the components of urinary stone. Struvite stones are commonly found in women. It forms in human beings as a result of urinary tract infection with urea splitting organisms. These stones can grow rapidly forming “staghorn-calculi”, which is a painful urological disorder. Therefore, it is of prime importance to study the growth and inhibition of struvite crystals. The growth inhibition effect of struvite crystals in sodium metasilicate (SMS) gel in the presence of sodium fluoride has been carried out. Crystals obtained have been analysed by powder and single crystal XRD, SEM–EDX, FTIR and TG–DTA. The results show that the presence of fluoride significantly affects struvite crystal growth and the characteristics of the crystallites produced. The mechanical property of the grown crystals has been investigated by Vickers microhardness testing. Work hardening coefficient was found to be >1·6 for both pure and doped samples which suggests that the crystal belongs to the family of soft material. Presence of sodium fluoride further softened the crystal.
Ethnopharmacological relevance:
Bergenia ligulata is widely used plant in South Asia, mainly India and Pakistan, as a traditional medicine for treatment of urolithiasis.
Aim of the study:
To rationalize the Bergenia ligulata use in kidney stones and to explain the underlying mechanisms.
Materials and methods:
The crude aqueous-methanolic extract of Bergenia ligulata rhizome (BLR) was studied using in vitro and in vivo methods.
Results:
BLR inhibited calcium oxalate (CaC(2)O(4)) crystal aggregation as well as crystal formation in the metastable solutions and exhibited antioxidant effect against 1,1-diphenyl-2-picrylhydrazyl free radical and lipid peroxidation in the in vitro. BLR caused diuresis in rats accompanied by a saluretic effect. In an animal model of urolithiasis, developed in male Wistar rats by adding 0.75% ethylene glycol (EG) in drinking water, BLR (5-10 mg/kg) prevented CaC(2)O(4) crystal deposition in the renal tubules. The lithogenic treatment caused polyuria, weight loss, impairment of renal function and oxidative stress, manifested as increased malondialdehyde and protein carbonyl contents, depleted reduced glutathione and decreased antioxidant enzyme activities of the kidneys, which were prevented by BLR. Unlike the untreated animals, EG intake did not cause excessive hyperoxaluria and hypocalciuria in BLR treated groups and there was a significant increase in the urinary Mg(2+), instead of a slight decrease.
Conclusions:
These data indicate the antiurolithic activity in Bergenia ligulata mediated possibly through CaC(2)O(4) crystal inhibition, diuretic, hypermagneseuric and antioxidant effects and this study rationalizes its medicinal use in urolithiasis.
In view of increasing resistance to existing antimicrobial agents, herbal drugs are being looked as very importance sourcefor discovery of new agents for treating various ailments related to bacterial infections. Cassia tora, Calendula officinalis andMomordica charantia are well known plants in Asia including India which posses wide range of pharmacological activities. Thesedrugs have been used in India as folk remedy in the form of decoctions and infusions to treat bacterial infections and also claimed tobe an effective against variety of skin conditions like psoriasis, acne, wounds etc. The present investigation was carried out to studythe unexplored area of these drugs towards their antibacterial activity with respect to their traditional use as antipsoriatic agents. Theherbs were subjected to successive extraction using different solvents and the extracts were subjected to antibacterial evaluationagainst both gram positive and gram negative organisms by cup plate technique. Among the various extracts, aqueous extracts werefound to be more effective against all the bacteria. Staphyllococcus aureus was more susceptible to the aqueous extracts among thetested organisms.
The understanding of the formation of urinary stones centers around three main mechanisms: the urinary concentration of stone-forming ions, the role of promoters, and the role of inhibitors of crystal formation and crystal aggregation. With respect to the promoting activity, lately emphasis has shifted from the role of the organic matrix to that of one salt inducing by epitaxy the precipitation of another salt. Among the inhibitors, it has become necessary to distinguish between those affecting crystal formation and those affecting crystal aggregation. For measuring the inhibitory activity, the various techniques and their relevance have been reviewed. It has been found that the main inhibitors for calcium phosphate and calcium oxalate precipitation are citrate, pyrophosphate, and perhaps magnesium. Those for calcium phosphate and calcium oxalate aggregation are glycosaminoglycans, pyrophosphate, and citrate. Among the synthetic inhibitors, the diphosphonates are the most powerful for both processes. The role and the therapeutic implications of these various concepts have been discussed.
A total of 47 plant extracts representing 132 genera and 172 species of plants distributed over 59 families were collected from various parts of Nigeria. The plant extracts were screened for the presence of alkaloids, saponins, tannins, phlobatannins and anthraquinones. The number of positive tests obtained was 176 (32.18%) for alkaloids, 242 (44.24%) for saponins, and 435 (79.52) for tannins. A few were positive for phlobatannins and anthraquinones.
Current studies have provided evidence that exposure of renal epithelial cells to oxalate and calcium oxalate crystals induces lipid peroxidation and injures the cells. Since oxidant/antioxidant balance is likely to play a critical role, we determined the effect of antioxidant scavengers on production of free radicals and injury to LLC-PK1 and MDCK cells from exposure to oxalate (Ox) or Ox + calcium oxalate monohydrate (COM) crystals.
LLC-PK1 and MDCK cells were grown in monolayers and exposed to 1.0 mmol. Ox or 1.0 mmol. Ox + 500 microg. /ml. COM crystals for 120 or 240 minutes. We measured the release of lactate dehydrogenase (LDH) as a marker for cell injury and malondialdehyde (MDA) as a marker of lipid peroxidation. Superoxide and hydroxyl radicals were measured in the presence or absence of 400 U/ml. catalase, or superoxide dismutase (SOD).
Exposure of LLC-PK1 cells to Ox resulted in a significant increase in MDA and release of LDH, which was further elevated when COM crystals were added. MDCK cells responded similarly to both challenges, but showed significantly less impact when compared with LLC-PK1 cells. Both treatments were associated with significant increase in the generation of hydroxyl and superoxide radicals by both cell types. In both cell lines, the addition of catalase or SOD significantly reduced the increase of MDA and release of LDH.
Results of the present study indicate that both Ox and COM crystals are injurious to renal epithelial cells and the injury is associated with generation of free radicals. Cells of proximal tubular origin are more susceptible than those of distal tubules and collecting ducts. Free radical scavengers, catalase and SOD provide significant protection.
Membrane injury facilitated the fixation of calcium oxalate crystals and subsequent growth into kidney stones. Oxalate-induced membrane injury was mediated by lipid peroxidation reaction through the generation of oxygen free radicals. In urolithic rat kidney or oxalate exposed cultured cells, both superoxide anion and hydroxyl radicals were generated in excess, causing cellular injury. In hyperoxaluric rat kidney, both superoxide and H2O2-generating enzymes such as glycolic acid oxidase (GAO) and xanthine oxidase (XO) were increased, and hydroxyl radical and transition metal ions, iron, and copper were accumulated. The lipid peroxidation products, thiobarbituric acid-reactive substances (TBARS), hydroperoxides, and diene conjugates were excessively released in tissues of urolithic rats and in plasma of rats as well as stone patients. The accumulation of these products was concomitant with the decrease in the antioxidant enzymes, superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glucose-6 phosphate dehydrogenase (G6PD) as well as radical scavengers, vitamin E, ascorbic acid, reduced glutathione (GSH), and protein thiol. All the above parameters were decreased in urolithic condition, irrespective of the agents used for the induction of urolithiasis. Oxalate binding activity and calcium oxalate crystal deposition were markedly pronounced, along with decreased adenosine triphosphatase (ATPase) activity. Lipid peroxidation positively correlated with cellular oxalate, oxalate binding, gamma-glutamyl carboxylase, and calcium level and negatively correlated with GSH, vitamin E. ascorbic acid, and total protein thiol. Antioxidant therapy to urolithic rats with vitamin E, glutathione monoester, methionine, lipoic acid, or fish oil normalised the cellular antioxidant system, enzymes and scavengers, and interrupted membrane lipid and protein peroxidation reaction, ATPase inactivation, and its associated calcium accumulation. Antioxidant therapy prevented calcium oxalate precipitation in the rat kidney and reduced oxalate excretion in stone patients. Similarly, calcium oxalate crystal deposition in vitro to urothelium was prevented by free radical scavengers such as phytic acid and mannitol by protecting the membrane from free radical-mediated damage. All these observations were suggestive of the active involvement of free radical-mediated lipid peroxidation-induced membrane damage in the pathogenesis of calcium oxalate crystal deposition and retention.
- A Sofowora
A. Sofowora, Phytochemical Screening of Medicinal
Plants and Traditional Medicine in Africa, Spectrum
Books Ltd, Ibadan, Nigeria, 1993.
Lab Report-Determination of Concentration Of Acetic Acid In Vinegar
- Eisya Azhari
Eisya azhari, Lab Report-Determination of
Concentration Of Acetic Acid In Vinegar, pp. 1-23.