ANTIUROLITHIATIC ACTIVITY OF LEAF EXTRACTS OF SYZYGIUM JAMBOS (L.) ALSTON AND ITS ZINC NANOPARTICLES: AN IN-VITRO AND IN-VIVO APPROACH

ANTIUROLITHIATIC ACTIVITY OF LEAF EXTRACTS OF SYZYGIUM JAMBOS (L.) ALSTON AND ITS ZINC NANOPARTICLES: AN IN-VITRO AND IN-VIVO APPROACH

Kangkan Deka ^*, Bibhuti Bhusan Kakoti and Moonjit Das

Department of Pharmaceutical Sciences, Dibrugarh University, Dibrugarh - 786004, Assam, India.

ABSTRACT: Urolithiasis is one of the painful urologic disorders that occurs in approximately 12% of the global population. Syzygium jambos (L.) Alston (Family: Myrtaceae), commonly known as Rose apple in English and Bogijamun in the Assamese, is an important medicinal plant found extensively in Assam and in the Indian continent. Scientific studies confirmed that the extracts of various parts possess anticancer activity, antioxidant, analgesic, antimicrobial activities, and anti-inflammatory properties. Phytochemical and Pharmacological evaluation of the Plant was carried out with special reference to antiurolithiatic activity. The in -vitro evaluation was done using growth inhibition study of struvite crystals, and in-vivo evaluation was done by Ethylene glycol induced urolithiatic model in rats. Our investigation showed that leaf extracts and ZnO-NPs prevented the growth of urinary stones. Further studies should be done to understand pharmacological action and its possible mechanism through elaborate preclinical experimentation and clinical trials in preventing urolithiasis in susceptible populations.

Urolithiasis, Nanoparticles, Biochemical, Histopathology

INTRODUCTION: Urolithiasis is the formation of stone in the urinary system, i.e., in the kidney, ureter, and urinary bladder or in the urethra. The term Urolithiasis is derived from ‘ouron’ meaning ‘urine’ and ‘lithos’, meaning ‘stone’ ¹. Urolithiasis, a common kidney disorder in all over the world with an estimated lifetime risk of about 2%-5% in Asia, 8%-15% in Europe and America, and around 20% in the Middle East ². A complex process of imbalance between promoters and inhibitors in the kidneys leads to stone formation and involves several physicochemical events commencing with crystal nucleation, aggregation, and end with retention within the urinary tract ³.

The cause of urolithiasis is still unknown but probably positive family history, overweight, obesity, or increased BMI. Some other causes include low urine volume <1500 ml/day, high dietary animal protein intake, increased urine excretion of calcium oxalate, uric acid, and cystine ⁴. Among the several types of kidney stones, the most common (about 80%) are calculi of calcium oxalate (CaOx) crystals existing in the form of CaOx Monohydrate (COM) and CaOx Dihydrate (COD).  Calcium containing stones, especially COM (Whewellite), COD (Weddellite), and basic calcium phosphate (Apatite) happens to be in the range of 75-90%, followed by magnesium ammonium phosphate (Struvite) with a range of 10-15%, uric acid stone (3-10%) and cystine stone (0.5-1%) ⁵.

Extracorporeal Shock Wave Lithotripsy (ESWL) and drug among every single utilized treatment drastically changed the urological practice and almost grow to be the standard strategy for getting rid of kidney stones; however, accompany demerits like the traumatic effects of shock waves, the persistence of residual stone fragments which may cause infection. In addition, ESWL can cause acute kidney damage, reduced renal function, hemorrhage, and high blood pressure. Thus, it is worth the effort to search for an alternative to these means by using medicinal plants or phytotherapy ⁶. The extensive literature of Ayurveda states that a number of plants are useful in urinary stone treatment; most plants still have to be explored for pharmacological treatments ⁷.

Researches on the several plants for example Launaea procumbens ⁸, Dolichos biflorus ⁹, Acorus calamus L. ⁷, Peperomia tetraphylla ¹⁰, Nigella sativa L. ¹¹, Peucedanum grande ¹², Bryophyllum pinnatum ¹³, Aerva lanata ¹⁴, Biophytum sensitivum ¹⁵, Musa paradisiaca ¹⁶, Bergenia ligulata ¹⁷, Ipomoea eriocarpa ¹⁸ have shown promising result in the treatment of urolithiasis.

Syzygium jambos (L.) Alston (Family: Myrtaceae), commonly known as Rose apple in English and Bogi-jamun in the Assamese, is an important medicinal plant found extensively in the Indian continent. Scientific studies confirmed that the plant extract had anticancer activity, extracts of the rose apple leaves have antioxidant, analgesic, and anti-inflammatory properties, and the bark extracts displayed antimicrobial activities ¹⁹.

In recent years, an increased interest in the utilization of metal nanoparticles is seen due to their diverse applications in various disciplines such as medicine, biology, physics, chemistry, and material sciences ²⁰. Zinc oxide nanoparticles (ZnO NPs), in particular, are the perfect candidate for biological applications as they are environment friendly, non-toxic, bio-safe, and biocompatible. Moreover, the United States- Food and Drug Administration (FDA) listed ZnO and the other four zinc compounds as generally recognized as safe (GRAS) material (FDA, 2015). The ZnO NPs have been used in sunscreens, paints, and coatings as they are transparent to visible light and extremely absorbent to UV and are also used as an ingredient in antibacterial creams, ointments, and lotions, self-cleaning glass, ceramics, and deodorants ^21-24.

The present study aims at evaluating the antiurolithiatic activity of leaf extracts of Syzygium jambos (SJ) and its Zinc oxide Nanoparticles (ZnO-NPs) both in-vitro as well as in-vivo. Single diffusion gel growth technique and Ethylene glycol-induced urolithiasis model were used to evaluate the in-vitro and in-vivo antiurolithiatic activity, respectively.

MATERIALS AND METHODS:

Chemicals and Reagents: Sodium metasilicate (SMS), Ammonium Dihydrogen Phosphate (ADP), and magnesium acetate (MA), Zinc acetate, Sodium Hydroxide, Ethylene glycol, ammonium chloride, Cystone, which were procured from Sisco Research Laboratories Pvt. Ltd., and Research Lab Fine Chem Industries. Cystone was procured from Himalayan Drug Company.

Plant: Leaves of the plant Syzygium jambos (L.) Alston was collected from Dibrugarh University campus, Assam, India, in the month of August 2017. The Plant was authenticated by Dr. L. R. Saikia, Professor, Department of Life Sciences, Dibrugarh University, and a Voucher Specimen No. DULSc488 on 23/3/2018 and were preserved for future reference. Leaves were washed with water to remove dust and then shade dried. The dried leaves were then pulverized using a mechanical grinder to a coarse powder and stored in an airtight container to avoid moisture.

Preparation of Extract: Coarsely powdered leaves were subjected to successive extraction using the Soxhlation method. Solvents used for the extraction were in increasing order of polarity, i.e., Petroleum ether (40-60°), Chloroform, Ethyl Acetate (EASJ), and Methanol (MESJ). Each extract was concentrated by distilling off the solvent in a rotary evaporator and evaporated to dryness in Petri dish on a water bath. The crude extracts were stored in a desiccator to avoid moisture.

Biosynthesis of Zinc Oxide Nanoparticles (ZnO-NPs):

Preparation of Aqueous Leaf Extract for the Synthesis of ZnO-NPs: 15 g of coarsely powdered leaves were taken in 300 ml of distilled water in a 500 ml beaker, and the mixture was boiled at 60° for 10 min. The aqueous leaf extract was allowed to cool to room temperature, filtered through Whatman no. 1 filter paper, and the filtrate was stored for further experimental use.

Synthesis of ZnO-NPs: To 180 ml of the above extract, 400 ml of 91 mM of zinc acetate solution (8 gm of zinc acetate was dissolved in 400 ml of distilled water) was added dropwise under continuous stirring. To the above mixture 1N NaOH was added dropwise. The reaction mixture became yellowish, and a cream-colored precipitate of zinc hydroxide was formed. The reaction mixture was left for 30 min for complete reduction to zinc hydroxide. Then the precipitate was collected by centrifugation at 10000 rpm for 10 min at 4°. The precipitate was dried at 30°._­­­ ²⁵

Characterization of Biosynthesized ZnO-NPs:

UV–Visible Spectroscopy: Optical density of the reaction medium was measured through UV–Vis spectroscopy of the after diluting small aliquots of reaction mixture ten times diluted with Milli-Q water and transferred to the cuvette and analysis was done using UV- 1800 SHIMADZU UV- Spectrophotometer (Kyoto, Japan), in the wavelength range of 200–600 nm. The measurement was done in order to monitor the reduction of Zinc ion.

X-Ray Diffraction (XRD): XRD analysis was carried out for the washed and dried sample of ZnO-NPs using Ultima IV (Rigaku, Japan) at the wavelength of 1.5406 Å. XRD was performed in the 2h range of 20–80 degrees at 40 kV and 40 mA with a divergence slit of 10 mm in 2h/h continuous scanning mode. The particle size of the formed nanoparticle was calculated by using the Debye Scherrer formula.

D = kλ/β cosθ......(Equation a)

Where D is the mean size of crystallites (nm), K is crystallite shape factor a good approximation is 0.9, λ is the X-ray wavelength, β is the Full Width at Half the Maximum (FWHM) in radians of the X-ray diffraction peak, and θ is the Braggs' angle (deg.).

Evaluation of in-vitro Antiurolithiatic Activity: Struvite crystals were grown by single diffusion gel growth technique, and the growth inhibition study of the Struvite crystals was carried out in the presence of the different concentration of EASJ and MESJ and ZnO-NPs ²⁶.

Crystal Growth: SMS solution having a specific gravity of 1.03 and an aqueous solution of 0.5M ADP were mixed until a pH of 7.2 was attained. About 20 ml of the gel solution was then transferred into tubes (test tubes and glassware were autoclaved at 120° for 15 min) with a length of 140mm and a diameter of 25 mm. After gelation, 10 ml of the supernatant solution of 1.0M magnesium acetate (control solution) and 1.0M magnesium acetate prepared with 0.5-2% concentrations of the leaf extracts and ZnO-NPs were gently poured on the gel. Composition and pH of the supernatant solutions are shown in Table 1.

TABLE 1: COMPOSITION OF THE SUPERNATANT SOLUTION

After pouring the supernatant solution, the test tubes were capped with air-tight stoppers. In order to avoid microbial contamination, the entire procedure was carried out in an aseptic condition at room temperature in laminar airflow.

It was anticipated for the following reaction to occur between the two reactants in the gel:

NH₄H₂PO₄•2H₂O + (CH₃COO)2Mg•4H₂O → NH₄MgPO₄•6H₂O + 2CH₃COOH

The apparent lengths of the growing/dissolving crystals in each of the test tubes were measured at regular intervals with the help of a traveling microscope of least count 0.001 cm. The mean length of the crystals, as well as single-factor analysis of variance (ANOVA), were calculated. The percentage inhibitions of the samples were also calculated for each day of observation. The total mass of struvite crystals in each test tube was measured after the removal of crystals, and the yield per test tube of the crystals was obtained.

Characterization Techniques: Fourier transforms infrared (FTIR) spectrum was recorded and interpreted using BRUKER ALPHA-T FT-IR Spectrometer (Billerica, US).

Statistical Analysis: Data are expressed as mean ± standard deviation (SD) from three separate observations. Statistical analysis of the difference between groups was evaluated by one-way ANOVA followed by Dunnett’s multiple comparison test using GraphPad Prism 5 software. Level of significance P was considered at <0.0001.

Evaluation of in-vivo Antiurolithiatic Activity: Based on the data obtained from the in vitro studies, the in-vivo antiurolithiatic study was carried out using methanolic leaf extract (MESJ) and Zinc Oxide Nanoparticles (ZnO-NPs) of aqueous leaf extract of Syzygium jambos (L.) Alston in male wistar rats. The ethylene glycol-induced urolithiasis model was used for the study.¹⁸ Necessary permission in regards to the animals was obtained prior to the study from the IAEC of Dibrugarh University (Approval no. IAEC/DU 167 dtd. 12.06.2018).

Animals: The study was carried out with adult male Wistar rats weighing 90-140 gm. Animals were acclimatized to experimental conditions in cases and kept under standard environmental conditions (22 ± 3°; 12/12 h light/dark cycle). Water and grains were provided to all rats.

Ethylene Glycol-Induced Urolithiasis: All the 25 rats were divided in 5 separate groups with 5 animals in each group. Group I was taken as the control regimen, and regular food and drinking water were provided. Group II–V were given stone inducing treatment till 28th day, comprising of 1% ethylene glycol (w/v) with 1% ammonium chloride (w/v) for 4 days, followed by 1% ethylene glycol alone in the water. Group II (Disease control) received only stone‑inducing treatment till the 28^th day. Group III served as the standard control group and received antiurolithiatic drug, Cystone (500 mg/kg), from the 15^th day till the 28^th day. Group IV and V served as a curative regimen. Group IV received MESJ (250 mg/kg b.w.) from 1^st day till 28^th day, and Group V received ZnO-NPs (50 mg/kg b.w.) from 15^th day till 28^th day.

Measurement of Body Weight and Water Intake: Bodyweight (%) and water intake (24 h) were found out at the end of the 28^th day for each group.

Biochemical Analysis:

Collection and Analysis of Urine: Urine samples (24 h) have been accumulated with the aid of keeping the rats in metabolic cages. During the urine collection period, animals were provided with free access to drinking water. Urine samples were investigated for phosphorus, calcium, and magnesium.

Serum Analysis: Subsequent to sacrificing of the animals, blood was collected from the rats of each group, and serum was isolated by centrifugation at 6000 rpm for 15 min and investigated for phosphorus, calcium, urea, and creatinine.

Kidney Histopathology: Kidneys were isolated from animals of all 5 groups and were kept in 10% formalin solution to prevent the tissues from decaying. Sections were cut with 5 µm thickness followed by its mounting on slides subsequent to staining with hematoxylin and eosin. The slides were examined under a light microscope to observe kidney architecture and CaOx deposits.

Statistical Analysis: Results were expressed in terms of mean ± standard error mean. Differences among data were determined using a one-way ANOVA test followed by Dunnett’s multiple comparison test (GraphPad Software, Inc., version 5, CA, USA.), and P<0.05 was considered statistically significant.

RESULTS:

Characterization of ZnO-NPs:

UV-vis Spectroscopy: The formation and stability of the synthesized ZnO-NPs were checked by means of observing the UV–vis absorption spectra. Fig. 1 represents the UV-visible spectra of freshly prepared ZnO-NPs. Peak obtained at 355 nm clearly demonstrates the presence of ZnO-NPs in the reaction mixture. This result correlates with the already reported results, in which the absorption peak was found at 360 nm ²⁷.

FIG. 1: UV–VIS ABSORPTION SPECTRA OF BIOSYNTHESIZED ZnO NPs

X-ray Diffraction: The XRD pattern of the ZnO-NPs Fig. 2 matches with the standard Joint Committee on Powder Diffraction Standards (JCPDS) ZnO pattern (96-230-0451). Six sharp peaks are observed at 2θ values of31.88, 34.56, 36.41, 46.64, 56.6, and 62.9, which are indexed as (100), (002), (101), (012), (110), and (013) bands of hexagonal structures of ZnO-NPs. Using Debye Scherrer formula (Equation a), the average crystallite size of ZnO-NPs was found to be about 20 nm.

The above-obtained data matches that of the data generated by approaches carried out by Kumar et al. 2017,²⁰ Bala et al., 2015, ²⁷ Geetha et al. 2016,²⁸ which thus indicates that the applied procedure for the green synthesis of ZnO-NPs was successful in accomplishing the desired nanoparticle synthesis.

FIG. 2: XRD PATTERN SPECTRA OF BIOSYNTHESIZED ZnO NPs

In-vitro Antiurolithiatic Activity:

Crystal Growth and Morphologies: Struvite crystals with two morphological shapes, namely needle type, X-shaped dendritic type, were formed in the gel. Fig. 3 shows the struvite crystals in the gel medium with and without leaf extract solutions. Fig. 4 shows the different types of the morphology of the gel-grown struvite crystals.

FIG. 3: STRUVITE CRYSTALS GROWN IN THE GEL MEDIUM: (A) NO INHIBITOR (CONTROL); (B) 0.5%; (C) 1%; (D) 2%

FIG. 4: DIFFERENT TYPES OF MORPHOLOGY OF THE GEL-GROWN STRUVITE CRYSTALS. (A) NEEDLE, (B) X-SHAPED DENDRITIC

FTIR: The FTIR analysis of the struvite crystals carried out between 400 and 4,000 cm^-1 was in agreement with the reported spectra. Fig. 5 represents the spectra of gel-grown struvite crystals. The experimental and reported data of the vibrational bands are summarized below. There are four regions in the FTIR that occurred due to the various vibrations of water of crystallization, NH₄⁺ units, tetrahedral PO₄³⁻ units and metal-oxygen bonds. The absorption bands shown because of water of crystallization closely matches with the previously established peaks in several inorganic hydrated compounds ^{26, 31-32}. Tetrahedral NH₄⁺ has four vibrational modes, and all the four appeared in the FTIR spectra because of its general position in the struvite crystals ^{26, 32, 33}. The vibrational modes of tetrahedral PO₄³⁻ anions also matched the reported values ^{34, 35}.

Observed FTIR (cm^-1) frequencies: 2919.20 (N-H stretch, NH₄⁺ ion), 2854.35 (C-H stretch, CH₃), 2391.82 (H-O-H stretch, Water cluster), 1625.01 (H-O-H bend, Water cluster), 1457.57, 1375.32 (C-H bend, CH₃), 1259.64 (C-O stretch, Carboxylic acid), 976.65 (PO₄^3- symmetric stretch), 800.68 (C-C stretch).

Reference FTIR (cm^-1) frequencies: 2000-3000 (N-H stretch, NH₄⁺ ion), 2850-3000 (C-H stretch, CH₃), 2060-2460 (H-O-H stretch, Water cluster), 1590-1650 (H-O-H bend, Water cluster), 1375-1460 (C-H bend, CH3), 1000-1300 (C-O stretch, Carboxylic acid), 930-995 (PO₄^3- symmetric stretch), 800-1000 (C-C stretch).

FIG. 5: FOURIER TRANSFORM INFRARED SPECTRUM OF THE GEL-GROWN STRUVITE CRYSTALS

Growth-Inhibition Study: It was observed that as the concentration of the EASJ, MESJ, and ZnO-NPs increased, the average size of the struvite crystals in the hydrogel medium decreased, as shown in Table 2. The average length of the struvite crystals without inhibitor was 1.243 ± 0.092 cm, but was reduced to 0.866 ± 0.060, 0.668 ± 0.050 and 0.581 ± 0.048 cm at 0.5, 1 and 2% of the EASJ respectively and for MESJ it was 0.890 ± 0.096, 0.703 ± 0.077 and 0.654 ± 0.088 cm for 0.5,1 and 2% respectively. Similarly for ZnO-NPs reduction in crystal growth was 0.833 ± 0.101, 0.711 ± 0.094 and 0.606 ± 0.106 cm for 0.5, 1.0 and 2.0% respectively. From the ANOVA single factor analysis, the differences in average length were highly significant at P<0.0001.

TABLE 2: GROWTH OF STRUVITE CRYSTALS IN THE GEL FOR DIFFERENT CONCENTRATIONS OF EASJ, MESJ AND ZNO-NPs

Note: Level of significance: ***P< 0.0001 (one-way analysis of variance). ^▲ Each value represents mean ± SD (n = 3).

Percentage inhibition of the struvite crystals by the EASJ, MESJ, and ZnO-NPs was studied from the second day onward, and the results are summarized in Table 3. In the case of EASJ the maximum inhibition of 19.63% was observed with 2% extract after the fifth day, while the minimum 16.28% inhibition was observed at 0.5% extract after the fifth day; in case of MESJ the maximum inhibition of 30.56% was observed with 2% extract after the fifth day, while the minimum 24.68% inhibition was observed at 0.5% extract after the fifth day. Similarly, in the case of ZnO-NPs the maximum inhibition of 35.27% was observed with 2% extract after the fifth day, while the minimum 25.60% inhibition was observed at 0.5% extract after the fifth day. It was concluded that the percentage inhibition increased at higher concentrations of EASJ, MESJ, and ZnO-NPs.

TABLE 3: PERCENTAGE INHIBITION OF THE STRUVITE CRYSTALS IN DIFFERENT CONCENTRATION OF EASJ, MESJ AND ZnO-NPs

After the growth study, struvite crystals were removed from the gel, and the total mass of the crystals for each concentration was measured. The results are summarized in Table 4. It was observed that the total mass of the crystals decreased with an increase in the concentration of the leaf extract.

TABLE 4: TOTAL MASS OF THE GROWN STRUVITE CRYSTALS IN DIFFERENT CONCENTRATIONS OF EASJ, MESJ AND ZnO-NPs

^▲Each value represents mean ± SD (n = 3)

In-vivo Antiurolithiatic Activity:

Measurement of Body Weight and Water Intake: Bodyweight and water intake that were recorded before the beginning of treatment were practically the same for all the groups. Table 5 represents the parameters recorded after the treatment. Loss in body weight of the animals was observed in the stone‑induced group (P<0.001 vs. Group I), whereas the other group animals showed a significant gain in their body weights after the experiment. There occurred no such noteworthy change in the water intake among the groups except in the stone‑induced group, which showed significantly high as compared to the control group (P < 0.001 vs. Group I).

Biochemical Analysis:

Urine Analysis: Table 5 shows the urine concentration of phosphorus, calcium, and magnesium present in Group I–V. Administration of 1% ethylene glycol in drinking water to the male Wistar rats caused a significant (P< 0.001 vs. Group I) increase of phosphorus and calcium concentration and a decrease in the magnesium concentration in urine of the stone‑induced group (Group II). However, treatment with MESJ (250mg/kg b.w.) and ZnO-NPs caused notable (P<0.001 vs. Group II) diminution in the phosphorus and calcium excretion and increased the magnesium excretion in urine in both the groups (Group IV and V, respectively) and were comparable to the standard group (Group III, cystone‑treated).

Serum Analysis: Renal function was assessed by estimating serum phosphorus, calcium, urea, and creatinine in Group I–V Table 5. The concentration of phosphorus, calcium, urea, and creatinine in the serum was significantly (P< 0.001 vs. Group I) increased in the stone‑induced group, indicating renal harm/damage. However, treatment with MESJ (250mg/kg b.w.) and ZnO-NPs significantly (P< 0.001 vs. Group II) reduced the concentrations of phosphorus, calcium, urea, and creatinine in the serum in both the groups to a close to a normal level and were comparable to the standard group.

Kidney Histopathology: Kidney histopathological examination revealed no CaOx crystal deposit or other abnormalities in the kidney of the control group (Group I), as shown in Fig. 6a. On the other hand, many CaOx crystal deposits in the renal tubules and congestion and dilation of the parenchymal blood vessels were seen in the renal tissue of the stone‑induced group (Group II) as shown in Fig. 6b. In the standard group (Group III), the kidney showed normal architecture with dilation of tubules in the corticomedullary junction, minima interstitial inflammation, and occasional renal tubules showed CaOx crystal deposits Fig. 6c. In MESJ (Group IV) and ZnO-NPs (Group V) groups, the kidney showed normal architecture, less tissue damage, and occasional renal tubules showed CaOx crystal deposit Fig. 6d and 6e.

TABLE 5: EFFECT OF SYZYGIUM JAMBOS ON PARAMETERS IN UROLITHIATIC MALE WISTAR RATS

Values for urine parameters are measured in 24 h urine sample. All values are stated as mean ± SD (n=5). ^aComparisons are made with Group I, ^bComparisons are made with Group II,*P<0.001 ^▲ns ^∎P<0.01^⧗P < 0.05, SD= Standard Deviation

FIG. 6: KIDNEY HISTOPATHOLOGY (A) GROUP I (CONTROL) (B) GROUP II (STONE INDUCED) (C) GROUP III (STANDARD) (D) GROUP IV (MESJ) (E) GROUP V (ZnO NP). ^*White arrow is to indicate stone

DISCUSSION: For the evaluation of the in-vitro antiurolithiatic activity, struvite crystals were grown by using a single gel diffusion technique, and the growth inhibition of the struvite crystals in the presence of EASJ, MESJ, and ZnO NP were studied. It was observed that as the concentration of the EASJ, MESJ, and ZnO-NPs increased, the average size of the struvite crystals in the hydrogel medium decreased. From the growth inhibition of struvite crystals by EASJ, MESJ, and ZnO NP, two-stage inhibition hypothesis can be recommended. They are expected to inhibit the growing struvite crystals in two different stages: (i) by forming stable complexes with Mg²⁺ ion and therefore clogging the availability of Mg²⁺ ions for the growth of struvite crystals and (ii) by adsorption on the growing crystalline surface. A large amount of adsorption of an organic compound may induce desorption of complex formed by Mg²⁺ ions, thereby weakening the bonds of crystals resulting in fragmentation.

For the evaluation of in vivo antiurolithiatic activity, male Wistar rats were chosen to induce urolithiasis with 1% ethylene glycol (w/v) with 1% ammonium chloride (w/v). Likewise, to that of the previously reported studies, the present experiment also showed significant elevation in the concentration of phosphorus (hyperphosphaturia) and calcium (hypercalciuria) and lowered concentration of magnesium (hypomagnesemia) in the urine of the stone‑induced group Table 5 ^{36, 37}. The increased calcium may have occurred due to excessive tubular damage in the kidney prompting excretion of intracellular calcium via urine. Hypercalciuria leads to elevated phosphorus leakage. Elevated urinary phosphorus excretion along with oxalate stress appears to offer suitable surroundings for stone formation with the aid of forming calcium phosphate crystals, which epitaxially causes CaOx deposition ³⁶. Magnesium is considered as a potent inhibitor of CaOx crystals because it causes a decrease in supersaturation ^{18, 36}.

In the stone‑induced group, there was a substantial increase in phosphorus levels compared to the control group. The magnesium level was essentially diminished in the stone‑induced group as compared to the control group, due to supersaturation and metabolic acidosis. However, during the administration of MESJ and ZnO-NPs, the calcium and phosphorus level diminished to a near‑normal level, and magnesium excretion was restored in Group IV and V compared to the stone‑induced group, which demonstrates that MESJ and ZnO-NPs are effective in inhibiting hypercalciuria, hyperphosphaturia, and hypo-magnesemia.

In urolithiasis, the stones in the urinary tract obstruct urine outflow bringing about the reduced glomerular filtration rate. This leads to the deposition of waste products in the blood, specifically nitrogenous substances, for example, urea and creatinine ^{6, 7}. Accordingly, in the present test, serum concentrations of phosphorus, calcium, urea, and creatinine were determined. As shown in Table 5, the phosphorus, calcium, urea, and creatinine levels in the stone‑induced group were higher than the control group. However, treatment with MESJ and ZnO-NPs significantly reduced the phosphorus, calcium, urea, and creatinine levels. Our results are compatible with the previous findings. In the stone‑induced group, the high level of these parameters was because of the CaOx stone formation in the urinary tract, which led to the deposition of waste products in the blood. However, treatments with MESJ and ZnO-NPs inhibited the stone formation and brought down these parameters.

CONCLUSION: The present investigation showed that MESJ and ZnO-NPs Syzygium jambos prevented the growth of urinary stones. Further studies should be done to understand pharmacological action and its possible mechanism through elaborate preclinical experimentation and clinical trials in preventing urolithiasis in the susceptible population.

ACKNOWLEDGEMENT: Authors deeply acknowledge the facilities provided by Dibrugarh University in terms of library and experimental facilities.

CONFLICTS OF INTEREST: The authors declare no conflict of interest.

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    How to cite this article:

    Deka K, Kakoti BB and Das M: Antiurolithiatic activity of leaf extracts of Syzygium jambos (L.) alston and its zinc nanoparticles: an in-vitro and in-vivo approach. Int J Pharm Sci & Res 2021; 12(1): 336-46. doi: 10.13040/IJPSR.0975-8232.12(1).336-46.

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