Studies on the effect of phyllanthus emblica extract on the growth of urinary type struvite crystals invitro
© Bidhu et al. 2015
Received: 26 November 2014
Accepted: 4 May 2015
Published: 8 August 2015
Struvite also called as infection stones occur in the urinary system of humans particularly the women community. The present work was aimed to grow struvite crystals in-vitro using single diffusion gel growth technique and to understand the effect of phyllanthus emblica on its growth.
Sodium metasilicate gel of density 1.03 g/cc was used as the medium for growing the struvite crystals; The phyllanthus emblica extract of 5 % M/V concentration was used to understand its effect on struvite crystal crystal nucleation and growth. The well grown crystal samples were characterized by powder XRD, FTIR, UV–vis, Microhardness, SEM and Thermal analysis.
The grown struvite crystals had dendritic morphology in both the control and phyllanthus emblica extract added systems. However, it was observed that the nucleation was controlled in the phyllanthus emblica added system when compared to the control system.
The result showed that phyllanthus emblica reduces nucleation of struvite crystals.
Struvite ( magnesium ammonium phosphate: MgNH4PO4. 6H2O) is a complex mineral known to assume a number of natural morphological forms including coffin, wedge, short prismatic or short tabular forms . Struvite, accounts for 10-20 % of all renal calculi , and is one the major component of urinary stones. These stones can grow rapidly forming “staghorn-calculi”, which is a more painful urological disorder  threatening human life particularly the women. Priestly and Dunn  reported that these stones have a significant chance of causing death in affected patients. They are thought to be associated with urinary tract infections caused by ‘urease’ secreting bacteria [5, 6]. More recently struvite stones have been shown to be a mixture of struvite and carbonate apatite  . Struvite staghorn calculi can form a real threat to the kidney and hence their presence is an indication for active treatment in most individuals .
Stones that led to kidney loss were frequently struvite stones or stones producing a large mass effect in the kidney . Hence it is vital to study the growth control of struvite crystals. In the present investigation, struvite crystals are grown in vitro using single diffusion gel technique and the growth of these crystals in the presence of phyllanthus emblica fruit extract is investigated.
Traditional system of treating ailments of urinary stone problem using herbal medicines contributes to good health and some of them have been experimentally evaluated [10–15]. Phyllanthus emblica is used in traditional system for the treatment of a broad spectrum of disorders. Phyllanthus emblica is commonly known as Amla and is widely distributed in most tropical and subtropical regions. The extracts or active components of phyllanthus emblica are known to possess several pharmacological effects mainly as antidiabetic, antioxidant, immune modulatory, chemoprotective, hypolipidemic and anti-HIV-1 [16, 17]. Keeping in view the importance of the fruit of phyllanthus emblica, the present work was aimed to study its influence on the growth of struvite crystals in vitro.
All the chemicals and acids used in the present study are of AR grade. The phyllanthus emblica was thoroughly washed in pipe water and then with distilled water, sliced and dried in shade and was powdered using a grinder. This crude extract was used to perform the experiment. The phyllanthus emblica used in the present study was obtained from the vellimalai hills of southern part of Tamil Nadu. This fruit is harvested twice in a year.
Phyllanthus emblica extract preparation
The powdered phyllanthus emblica was dissolved in aqueous solution in the ratio 1:5 by weight. This mixture was heated to a temperature of 50 °C and stirred for about 1 h using a magnetic stirrer as to obtain a concentrated extract. This solution was then filtered using whatmann filter paper to remove the undissolved component. The resulting solution was used as the extract. This extract was stored in a freezer to avoid microbial contamination.
Sodium metasilicate of known mass was dissolved in distilled water using magnetic stirrer for about 1 h. This solution was then filtered using whattmann filter paper to remove the micro impurities if any and stored in a clean jar for further use.
In the present work, gel of density 1.03 g/cc was used for growing the struvite crystals. An aqueous solution of 1.0 M concentration of ammonium dihydrogen phosphate was mixed so that the pH value 6.0 could be set for the mixture. Utmost care was taken while mixing the solution so that no air bubbles are formed. 25 ml of this solution was then transferred to the crystallization vessels namely test tubes of length 140 mm and diameter 25 mm respectively for proper gel setting. After 24 h of gel setting, 10 ml of magnesium acetate solution of 1.0 M concentration was gently poured over the set gel along the sides of the test tube without disturbing the gel surface. The experiment was carried out at room temperature. Within 5 to 6 h, small crystals of struvite appeared. The growth was completed within a period of 20 days. The grown crystal was collected on the 25th day. These crystals were carefully collected in a clean petri dish and then harvested by removing the gel using distilled water.
To study the effect of phyllanthus emblica extract on the growth of struvite crystals, the following method was adopted. The prepared extract (4 ml) was separately added along with 10 ml of the supernatant solution namely 1.0 M of magnesium acetate and then transferred over the set gel in seven crystallization vessels in order to monitor its effect on the crystal growth in all the vessels. The above procedure was carried out simultaneously with the control system to compare the growth and morphology of struvite crystal grown in the control system and in the presence of phyllanthus emblica extract. The amount of extract solution added is 4 ml which is very little compared to gel ie, the mass ratio of extract to gel < < 1 and hence the addition of extract will not change the pH of the gel. Also the solution was added after setting of gel hence evaluating the pH of the gel may destroy the integrity of the gel.
The grown crystals were collected after 25 days. The particles present in the extract may adhere to the crystal surface and do not bind with magnesium, ammonium or phosphate. It was observed that nucleation was controlled and less number of struvite crystals were formed in comparison with the crystals formed in the control system. Thus an inhibiting effect was observed due to the addition of phyllanthus emblica extract.
To further investigate the inhibiting effect, the water soluble particles of phyllanthus emblica at 5 % W/V concentration was separately added to the supernatant solution in the test tubes right from the beginning of the growth of the crystals and compared with the growth of the struvite crystals in the control system (without phyllanthus emblica extract or its water soluble particles). The prepared phyllanthus emblica extract was precipitated using acetone. This precipitate was separated from the solution and warmed for 5 min to remove acetone if any present in the precipitate. This precipitate was added along with 10 ml of supernatant solution (magnesium acetate) of 1.0 M concentration and this combination was added gently over the set gels in the test tubes. The experiment was carried out at room temperature. The studies were carried out in seven crystallisation vessels for both control system and the phyllanthus emblica extract added system. The experiments were repeated thrice to ensure the repeatability.
The crystals grown in the control system and water soluble particles of emblica extract at 5 % W/V added system were subjected to various characterization techniques such as powder XRD, FTIR, UV–vis, Microhardness, SEM and Thermal analysis. Powder XRD analysis was carried out using XPERT-PRO diffractometer system using Cu Kα radiation of wavelength 1.5406 Ao. The data were analyzed using the related computer software. The sample was scanned in the range of 10° to 80° at a scan rate of 2°/min. The FTIR spectra was recorded at room temperature by means of Bruker, AphaT, instrument using KBr pellet technique in the wavenumber range 4000–550 cm−1. The UV–vis analysis was carried out using double beam UV–vis spectrophotometer in the range of 200–800 nm. Microhardness studies were carried out using a Vicker’s hardness tester (Leitz Wetzlar) fitted with a Vicker’s diamond pyramidal indenter and attached to an incident light microscope to understand the mechanical properties of the grown crystals. The microstructures of the grown crystal samples were observed using a scanning electron microscope (Tescan Vega 3). The thermal behaviour of the grown samples were measured from ambient to 700 °C at a constant heating rate of 10 °C/min in the nitrogen atmosphere.
Results and discussion
Powder XRD analysis
FTIR spectral analysis
UV- Visible analysis
Vickers Microhardness studies
where, P is the applied load (kg), d is the average diagonal length of the Vicker’s impression in mm after unloading.1.8544 is a constant of a geometrical factor for the diamond pyramid.
where k is an arbitrary constant and n is the Meyer’s index, also known as work hardening coefficient.
The invitro crystallization of struvite crystals were carried out successfully using the gel technique. The role of phyllanthus emblica extract on the struvite crystal formation was investigated and it was found to have an inhibiting effect on the growth of struvite. The grown crystals had dendritic morphology. The number of crystals obtained in the phyllanthus emblica extract added system was found to be less than the control system. Hence, phyllanthus emblica reduces nucleation of struvite crystals and thus act as an inhibitor for struvite crystal formation.
The grown crystals were confirmed by powder XRD and FTIR analysis. The UV–vis analysis showed that the energy gap decreases for the phyllanthus emblica extract added struvite crystals. The microhardness studies further confirmed that the addition of phyllanthus emblica softens the struvite crystals. The SEM images showed that the grown crystals had multilayered depositions. The TGA-DTA analysis confirmed that the thermal stability decreases for the struvite crystals grown in the presence of phyllanthus emblica extract.
The authors are thankful to St. Josephs College, Trichy for timely characterization of the samples.
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