INTRODUCTION:

The use of medicinal plants for the treatment of urinary tract calculi in Algeria, and especially in Africa, remains fairly anchored in popular practices where it constitutes the main curative basis¹. In this sense, we have set ourselves the objective of evaluating the litholytic and inhibitory effects of a plant that is fairly well known in the Algerian coast, called Fraxinus excelsior L, on the magnesium ammonium phosphate (PAM), commonly referred to as Struvite^2-4.

Due to its abundance, Fraxinus excelsior L is identified among the medicinal plants used not only for its diuretic ⁵, anti-inflammatory and antirheumatic⁶, antioxidant⁷, analgesic⁸ antipyretic⁹, hypoglycemic¹⁰, hypotensive¹¹ effects but also cited for its litholytic effect¹² in the local pharmacopoeia. However, no study highlights its effect on the lithiasis infection, which is often associated with the formation of struvite. This type of lithiasis is the consequence of persistent and recurrent infections of the urinary tract¹³. It is generally caused by ureolytic microorganisms^14-16, of which Proteus mirabilis is well known for its ability to produce active urease¹⁷. The latter converts urea into ammonia and carbon dioxide, which are respectively precursors for the formation of struvite and carbapatite^18-20. This process is accompanied by urinary alkalinization, which allows a stabilization of these components^21-23. In the situation of urolithiasis, infectious lithiasis is recurrent and difficult to treat due to the fact that the growth of struvite and/or carbonate apatite crystals take place around the microorganisms that provide protection and greatly improve the perpetuation of their urease activity²⁴. Accordingly, the preventative aspect to deal with the infectious lithiasis and its recurrence so as to ensure, to the fullest extent, the litholytic and inhibitory effects of certain plants on this type of lithiasis, remains indeed a genuine eventuality.

MATERIAL AND METHODS:

Plant material:

The leaves of the Fraxinus excelsior plant (Figure. 1b) were harvested in March 2018 from the region of Seraidi (Province of Annaba: Eastern Algeria). This plant was identified by Pr. R., Mecheri; Botanist at the Faculty of Medicine -Badji Mokhtar Annaba University.

Preparation of aqueous extract:

The aqueous extract was prepared by infusing 20 g of Fraxinus excelsior dried leaves in 200 ml of boiling distilled water for a period of 20 min. The filtrate was then evaporated in Rotary Evaporator (BUCHI, R215) at 40 ° C. Thus, the aqueous extract obtained was sealed in an airtight container and stored at 4°C until use. The yield is prepared according to the following equation ^{25, 26}:

R% = (m/m₀).100

R%: Percent yield

m: Mass of the dry residue remaining after evaporation in grams; and

m₀: Mass of the sample before drying in grams.

Figure 1. (a) Fraxinus excelsior Tree and (b) Fraxinus excelsior Leaves

Determination of Total Phenolic Content:

The total phenolic content of the extract was determined with Folin-Ciocalteu reagent (FCR) according to the method reported in Slinkard and Singleton ²⁷. This assay consists of preparing a first solution consisting essentially of 125 µl of aqueous extract, 500 µl of distilled water and 125 µl of FCR reagent. After 3 min, 1250 μl of 2% sodium carbonate (Na2CO3) and 1000 μl of distilled water were added, and the reaction mixture was stirred in the dark for 90 min. The absorbance was then measured by a spectrophotometer (DR2800, HACH LANGE) at a wavelength of 760 nm. The results are expressed in milligrams of gallic acid equivalent per gram of plant extract (mg GAE /g extract).

Determination of Total Flavonoid Content:

The total flavonoid content of the extract was determined according to the protocol of Türkğluet et al ²⁸. A volume of 125 μl of aqueous extract is diluted in 2550 μl of methanol (CH3OH), 100 μl of potassium acetate solution (CH3COOK) and 100 μl of aluminum nitrate (Al (NO3) 3) at 10%. After 40 min at room temperature, the absorbance was assessed at a wavelength of 415 nm and the total flavonoid concentration was expressed in milligrams of quercetin equivalent per gram of plant extract (mg QE /g extract).

Dissolution of Struvite crystals:

The crystallization process was induced by the presence of Proteus mirabilis isolated from apatient with urolithiasis and then cultured on Tryptic Soy Broth (TSB) for 18-h at 37 ° C. This in-vitro crystallization process is similar to that which occurs during the in-vivo growth of struvite stones, as long as it is initiated by urease producing microorganisms.

The synthetic urine solution was prepared according to the recipe described by Agnieszka et al., ²⁹. This synthetic urine is an aqueous solution containing in g/L the following components: CaCl₂.2H₂O: 0.651, MgCl₂.6H₂O: 0.651, NaCl: 4.6, Na₂SO4: 2.3, Sodium Citrate: 0.65, Sodium Oxalate: 0.02, KH₂PO4: 2.8, KCl: 1.6, NH₄Cl: 1.0, Urea: 25.0, Creatine: 1.1 and TSB: 10.0. The pH was initially adjusted to 5.8 and the prepared urine was sterilized by filtration using a 0.22μm filter into sterile vials, and finally stored at 4 ° C for a 1-week before use. These mineral components correspond to the average concentration observed over a full normal 24-hour cycle. TSB had been added to stimulate bacterial growth without interfering with the precipitation process ³⁰. Crystallization occurs after addition of the suspension of P. mirabilis in 2ml of artificial urine at an appropriate concentration of 0.5 Mc Farland standard and an incubation at 37 ° C for 24 hours.

A volume of 62.5µl of aqueous extract was then added at various concentrations: 0.0625; 0.125; 0.25; 0.5 and 1 mg/ml. The suspension of bacteria in synthetic urine without additives was prepared as a control. After 60min of incubation, a microscopic examination technique was carried out to compare the results obtained in the absence and presence of the tested extract. The pH measurements of each sample were carried out at the start and the end of the experiment.

Inhibition of Struvite crystallization:

The chemical synthesis of struvite crystals in the urine was performed according to the protocol of Sadki and Atmani ³¹, which consists in mixing two solutions of respective composition:

· A: potassium dihydrogen phosphate (KH2PO4): 0.1M.

· B: magnesium chloride (MgCl2) 41g, ammonium chloride (NH4Cl) 50g; 20ml of ammonium hydroxide (NH4OH) were added and diluted to 50ml with double-distilled water, then diluted 10 times.

The mixture of two volumes of 1ml each of these solutions was induced by adding 62.5µl of the plant extract to the solution “A”. The aqueous extract was prepared at different concentrations: 0.0625; 0.125; 0.25; 0.5; and 1mg/ml, and the control solution was prepared by replacing this extract with an equal volume of distilled water. After 30 min of incubation at 37 ° C, the growth of crystallized had been then monitored using PLM in all mixtures.

Fourier Transform Infrared (FTIR) Spectroscopy:

The micrographic observations were followed by FTIR analytical technique. To help with this process, the residues of various concentrations were recovered by centrifugation at 4000rpm for 10min and then dried in the open air. Thus, each dried pellet was mixed with Potassium Bromide (KBr) at a rate of 0.5-1.0g per 100g of KBr in an agate mortar. The powder obtained was compressed to a pellet of 13 mm-diameter and 1mm-thickness. The technique was applied using a Bruker ALPHA II FTIR Spectrometer in the 4000-400 cm-1 wave number range with a database used for analyzing the spectra by identifying the noticeable vibrations ^{32, 33}.

RESULTS:

Extraction yield:

The mass extracted from F excelsior plant leaves recovered after evaporation is 3.33g, which corresponds to an extraction yield of 16.7%.

Total phenolic and flavonoid contents:

The total phenolic content based FCR reagent ranged between 60.92±0.94mg GAE/g of extract using the calibration curve (y = 0.0102x – 0.0307; R2 = 0.9981). Also, the result obtained for the concentration of The total flavonoid content expressed in mg QE / g of extract ranged between 19.32±0.75 based on the calibration curve (y = 0.0066x – 0.0058; R2 = 0.9985).

Litholytic effect of Fraxinus excelsior on struvite crystals:

The Figure (Figures. 2b, 2c) represents the formation of struvite crystals induced by biological synthesis (presence of P. mirabilis) after incubation at 37°C for 24 h. The results show struvite crystals with various morphologies of coffin-shaped and X-shaped struvite crystals³⁴. In addition, it can be seen that the profile of facets is intact, which indicates conditions favorable for germination and crystal growth without serious constraints.

FTIR spectra of urinary pellet samples (resulting from centrifugation) showed that they are formed essentially of struvite, small amounts of calcium phosphate apatites and calcium oxalate, thus confirming the nature of the crystals observed (Fig. 2d).

Figure 2. Micrographs of struvite crystals induced by P. mirabilis after 24 h at 37 ° C. (a-c) in a control solution; (d) FTIR analysis showing PAM nature and their predominance.

Figure 3 illustrates the litholytic effect of the Aqueous Extract for 60min on struvite crystals that were already induced by P. mirabilis after 24 hours at 37 ° C. For low concentrations, Figures 3a-3b show that the struvite crystals remain intact (at 0.0625 mg/ml), or weakly degrade (at 0.125 mg/ml). Beyond 0.25 mg/ml, the degradation of struvite crystals is all the more accentuated with the increase in the concentration (Figures. 3c, 3e). As regards the high concentrations, the degradation of the struvite crystal structure is accompanied by the appearance of calcium phosphate granules (Figure. 3d, 3e). FTIR spectra of the residue recovered by ultracentrifugation (1 mg/ml of aqueous extract solution; Figure. 3-f) reveals that the amount of struvite has decreased considerably relative to that of calcium oxalate with their crystalline form that predominate; Carbapatite (CA) and Amorphous Calcium Phosphate (ACP). FTIR spectra also shows protein absorbance bands related to the presence of bacteria that induce struvite crystal formation.

Figure 3. Crystals induced by P. mirabilis observed in the presence of the aqueous extract of F excelsior at various concentrations: 0.0625 (a); 0.125 (b); 0.25 (c); 0.5 and 1 mg/ml (e) using PLM and FTIR analysis clearly highlighting the nature of the revealed components

Inhibitory effect of struvite crystallization:

In the absence of an inhibitor:

In terms of counting the crystals formed in the absence of an inhibitor, a high crystal density (size and number) was recorded (Figure. 4a). These crystals show characteristic struvite facets (Figure. 4A).

In the presence of an inhibitor:

For low concentrations of aqueous extract (0.065 to 0. 25 mg/ml), the crystal density remains quite high (Figures. 4b, 4c and 4d). This density decreases considerably at high concentrations (0.5 and 1mg/ml) (Figures. 4e, 4f). Morphologically, the crystals obtained appear in lamellae and lamellae aggregates, the edges of which are broken and/or curved (Fig. 4B, C, D, E and F). FTIR analysis revealed that these are struvite crystals (Figure. 5).

Figure 4: Evaluation of inhibitory action both; in the absence (a, A) and the presence of the aqueous extract of F excelsior at various concentrations: 0.0625 (b, B); 0.125 (c, C); 0.25 (d, D); 0.5 (e, E) and 1mg/ml (f, F) using PLM on chemically synthesized struvite crystals.

Figure 5: FTIR Spectra of chemically induced crystals in the presence a 0.0625 mg/ml of the aqueous extract of F excelsior concentration after 30 min of incubation at 37 ° C.

DISCUSSION:

Traditionally, several medicinal plants are used to prevent and treat urinary lithiasis^35,36. It is, however, difficult to determine the exact mechanism attributed to the bioactive constituents³⁷. In this regard, several authors have evaluated the in vitro effect of different plant extracts on the formation of struvite crystals^38,39,40. The present study was undertaken to evaluate in vitro the litholytic and inhibitory effects of the aqueous extract of Fraxinus excelsior on the struvite crystals. To achieve this, the crystallization of struvite was induced by two processes: biological (in the presence of P. mirabilis) and chemical syntheses. As for the biological approach, a 24-hour incubation makes it possible to visualize by polarization microscopy an abundant crystalline population composed mainly of struvite crystals, which appear not only in different facets but also in crystalline aggregates⁴¹. It’s worth note at this level that the crystalline structure of these crystals is intact and that their germination and growth took place without major constraints. The FTIR spectra of the recovered urine residue reveals struvite absorbance bands of calcium phosphate (Carbapatite), calcium oxalate and protein⁴². Besides that, the formation of other components is justified by the presence of calcium chloride dihydrate (CaCl₂, 2H₂O) and sodium oxalate (Na₂C₂O₄) that respectively induces the formation of carbonate apatite and of calcium oxalate in the composition of the synthetic urine. The presence of proteins in the analyzed urinary pellet is directly linked to that of bacteria.

The addition of the aqueous extract after this incubation allowed us to observe with a polarized light a consequent degradation of the crystalline structure of the struvite crystals at concentrations ≥ 0.5mg/ml. This indicates the significant litholytic effect of the aqueous extract of Fraxinus excelsioron struvite crystals for high concentrations. In this context, the present results are consistent with those obtained by Stefanus et al.,⁴³ and Chauhan et al., ⁴⁴ studies respectively on the leaves of Orthosiphon aristatus Bl. Miq and Boerhavia diffusa Linn, which indicate that both extracts cause notable defects with defragmentation of struvite crystals at high doses. The fragments thus obtained are easy to be removed via the urinary tract.

The FTIR analysis of the pellets recovered in the presence of the aqueous extract of Fraxinus excelsior shows a predominance of absorbance bands relating to carbonated amorphous phosphate calcium and carbapatite at the expense of those of PAM, which confirms the struvite dissolution.

Struvite is a pH-dependent crystalline species; it forms in alkaline environment (pH >7) and its treatment involves urinary acidification⁴⁵. After the measurement of pH, it was found that the formation of struvite crystals is accompanied by urinary alkalinization, which results from the urease activity of P. mirabilis⁴⁶.

After the addition of AE, and regardless of the independently measured concentrations, the pH remained rather high (pH = from 8.9 to 9.1), and therefore is favorable to struvite stabilization, proving that the dissolution observed can be attributed to the action of certain active constituents present in the extract; such as, polyphenols and flavonoids, which are known by their litholytic potential⁴⁷. The chemical synthesis of struvite was characterized by rapid kinetics in which the formation of struvite crystals was observed with both the absence and the presence of the aqueous extract and just after 30 min of incubation. This result had been revealed using PLM and FTIR spectra.

The in vitro crystallization of struvite shows that the potentialof struvite crystal growth inhibition is proportional to the increasing concentrations of the aqueous extract tested; the more the concentration of the aqueous extract of Fraxinus excelsior increases, the more the average crystal size and number decrease. Sadki and Atmani³¹ also reported an in vitro growth inhibition study of struvite crystals using the aqueous extract of E. multiflora. The results revealed a similar inhibitory effect in which a dose of mg/ml aqueous extract tested induces a reduction in the size and number of crystals (40%) compared to the crystals in the control tube. Further, the study results demonstrated a change in crystal morphology. Therefore, if the inhibition of struvite crystallization can be avoided, infectious lithiasis could also be avoided.

The inhibitory effect of struvite crystallization by the supplementation of the aqueous extract of Fraxinus excelsior was expressed both; morphologically and by the enumeration of the formed struvite crystals. Indeed, for all of the aqueous extract concentrations, the morphology of the observed struvite crystals is quite particular and is characterized by lamellae and lamellae aggregate structure with a set of curved edges and contours. In addition, a fairly small number of these crystals were recorded for of the aqueous extract concentrations 0.5 and 1mg/ml. At low concentration of the aqueous extract, the germination and growth of struvite crystals are in fact governed by a fairly good velocity of kinetics fast enough to outweigh the inhibitory effect of the extract.

The latter exhibits by a noticeable modification of the crystals morphology. At a high concentration, the inhibitory potential of the aqueous extract is relatively significant not only for the crystals morphology but above all for the consequent reduction in their number. The results of this study are in consistent with those reported by Kaleeswaran et al., ⁴⁸ and Reshma et al.,⁴⁹ who showed that the aqueous extracts of Pedalium murex Linn and Scoparia Dulcis exerted, in addition to the litholytic effect, Fraxinus excelsior an in vitro inhibitory effect on struvite crystallization. Thus, the phenolic compounds present in may be responsible for the inhibitory and/or litholytic activities on the crystallization of struvite¹⁷.

Several studies related to anti-crystallization compounds of plant origin; such as, polyphenols and flavonoids, have reliable results in treating struvite crystals. Prywer and Torzewska⁵⁰ reported the effect of the Polyphenolic Curcumin Compound against P. mirabilis during struvite crystallization in an artificial urine. In the presence of curcumin, the results revealed that the induction time increases, and the efficiency of struvite formation decreases, in which it does not affect crystals morphology in relation to the absence of curcumin. Torzewska and Rozalski¹⁷ reported an in vitro crystallization inhibition by various phenolic substances on crystal formation induced by P. mirabilis. The results show that nearly 50% of the tested substances prevented the formation of struvite crystals and successfully inhibited urease activity, which is the driving force for struvite crystallization. Dayana Jeyaleela G and her collaborators⁵¹ confirmed the inhibitory potential of flavonoids extracted from the leaves of Meliadubia on struvite crystals. The results showed that the flavonoids act on urease by inhibiting the latter through hydrogen bonds formation and van der Waals interactions. In addition to its direct effects on urease, the flavonoids inhibited crystallization, distorting the struvite crystal surfaces.

The chemical composition analysis of the aqueous extract of Fraxinus excelsior shows that it contains a significant amount of polyphenols and flavonoids, which insinuates the formation of crystals containing active substance complex; the complexes formed are usually more water soluble than the crystals itself. These in vitro assays show that the aqueous extract of Fraxinus excelsior is effective in dissolving and inhibiting the crystallization. The prevention against infection lithiasis is primarily based on inhibiting crystal growth and dissolving struvite crystals.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGEMENTS:

Authors would like to thank the team of Laboratory of Biophysics, Faculty of Medicine, Annaba (Algeria), Laboratory of Biochemistry and Applied Microbiology, Annaba University (Algeria).

The authors also thank Professor Rym MECHERI (Department of Pharmacy, Faculty of Medicine -Badji Mokhtar University, Annaba, Algeria) for the plant identification.

REFERENCES:

1. Khitri W, Lachgueur N, Tasfaout A, Lardjam A et Khalfa A. Plantes antilithiasiques utilisées en médecine traditionnelle dans la ville d’Oran, Algérie: Approche ethnobotanique et phytochimique. Revue d’ethnoécologie 2016; 9: 2267–2419. https://doi.org/10.4000/ethnoecologie.2511

2. Neeraja Kamakshi U, Ganga Rao B, Venkateswara Rao B. Comparison of In vitro Antiurolithiatic Activity of Aervalanata, Sphaeranthusindicus, Merremiaemarginata. Research J. Pharm. and Tech. 2017; 10(6): 1653-1656. doi: 10.5958/0974-360X.2017.00291.8

3. Amritan Kachru, Monika Bisht, Mamta Baunthiyal. In vitro evaluation of anti-neprolithiatic activity of leaves and seeds of Macrotylomauniflorum on dissolution or removal of kidney stones. Res. J. Pharmacognosy& Phytochem.2016; 8(1): 05-12. doi: 10.5958/0975-4385.2016.00002.9

4. Dileep Singh Baghel, Amit Mittal, Saurabh Singh, Anand Kumar Chaudhary, Amit Bhatia, Shruti Chopra. Formulation, Evaluation and Assessment of In Vitro Potential of Gokshur Ghan Tablet against Urolithiasis (Mutrakrichra). Research Journal of Pharmacy and Technology. 2021; 14(4):1945-2. doi: 10.52711/0974-360X.2021.00344

5. Tita I, Mogosanu GD, Tita MG. Ethnobotanical inventory of medicinal plants from the South West of Romania. Farmacia 2009; 57(2): 141–156.

6. Von Kruedener S, Schneider W, Elstner EF. “A combination of Populustremula, Solidgovirgaurea and Fraxinus excelsior as an anti-inflammatory and antirheumatic drug. A short review”. Arzneimittel-Forschung 1995; 45(2): 169–171. https://pubmed.ncbi.nlm.nih.gov/7710441/

7. Middleton P, Stewart F, Al-Qahtani S, Egan P, Orourke C, Abdulrahman A, Byres M, Middleton M, Kumarasamy Y, Shoeb M, Nahar L, Delazar A, Deysarker S. Antioxidant, antibacterial activities and general toxicity of Alnusglutinosa, Fraxinus excelsior and Papaver rhoeas. Iran. J Pharm Res 2005; (2): 81–86. https://dx.doi.org/10.22037/ijpr.2010.620

8. Flanagan J, Meyer M, Pasamar MA, Ibarra A, Roller M, Genoher NAi, Leiva S, Gomez-García F, Alcaraz M, Martínez-Carrasco A, Vicente V. Safety evaluation and nutritional composition of a Fraxinus excelsior seed extract, FraxiPure™. Food and Chemical Toxicology 2013; 53:10–17.https://doi.org/10.1016/j.fct.2012.11.030

9. Okpanyi SN, Schirpke-von PR, Dickson D. Anti-inflammatory, analgesic and antipyretic effect of various plant extracts and their combinations in an animal model. Arzneimittel-Forshung 1989; 39(6):698–703.https://pubmed.ncbi.nlm.nih.gov/2476136/

10. Eddouks M, Maghrani M. Phlorizin-like effect of Fraxinus excelsior in normal and diabetic rats. J Ethnopharmacol. 2004; 94(1):149–154.https://doi.org/10.1016/j.jep.2004.05.005

11. Eddouks M, Maghrani M, Zeggwagh NA, Haloui M, Michel J-B. Fraxinus excelsior L. evokes a hypotensive action in normal and spontaneously hypertensive rats. J Ethnopharmacol. 2005; 99(1):49–54.https://doi.org/10.1016/j.jep.2005.01.050

12. Salman A, Muhammad MH, Zafar AM. Antiurolithiatic plants: Multidimensional pharmacology. Journal of Pharmacognosy and Phytochemistry 2016; 5(2): 04–24.

13. R. Kalai, B. Sasirekha. Dietary Guidelines to Reduce the Risk of Renal Stones. Int. J. Nur. Edu. and Research. 2018; 6(1): 73-77. doi: 10.5958/2454-2660.2018.00018.2

14. Flannigan R, Choy WH, Chew B, Lange D. Renal struvite stones-pathogenesis, microbiology, and management strategies. Nat. Rev. Urol. 2014; 11(6):333–341. https://doi.org/10.1038/nrurol.2014.99

15. Manzoor MAP, Duwal SR, Mujeeburahiman M, Rekha P-D. Vitamin C inhibits crystallization of struvite from artificial urine in the presence of Pseudomonas aeruginosa. International Braz. J. Urol. 2018; 44(6):1234-1242. https://doi.org/10.1590/S1677-5538.IBJU.2017.0656

16. Chauhan CK, Joshi MJ, Vaidya ADB. Growth inhibition of Struvite crystals in the presence of herbal extract Commiphora wightii. Journal of Materials Science: Materials in Medicine 2009; 20(1): 85.https://doi.org/10.1007/s10856-008-3489-z

17. Torzewska A, Rozalski A. Inhibition of crystallization caused by Proteus mirabilis during the development of infectious urolithiasis by various phenolic substances. Microbiological research 2014; 169(7-8): 579-584.https://doi.org/10.1016/j.micres.2013.09.020

18. Vrunda Zalavadiya, Vipulshah, D.D. Santani. A Review on Urolithiasis and its Treatment using Plants. Research J. Pharmacognosy and Phytochemistry 2013; 5(1): 1-8.

19. Sujoy B, Aparna A. Potential clinical significance of urease enzyme. European Scientific Journal 2013; 9(21).

20. Kappaun K, Piovesan AR, Carlini CR, Ligabue-Braun R. Ureases: Historical aspects, catalytic, and non-catalytic properties. Journal of Advanced Research 2018; 13:3–17. https://doi.org/10.1016/j.jare.2018.05.010

21. Rieu P. Lithiases d’infection. Annales d’urologie 2005; 39:16–29. https://doi.org/10.1016/j.anuro.2005.01.001

22. Ramadevi S, Kaleeswaran B, Ilavenil S. Effect of traditionally used herb Pedalium murex L. and its active compound pedalitin on urease expression – For the management of kidney stone. Saudi Journal of Biological Sciences 2020; 27(3):833–839. https://doi.org/10.1016/j.sjbs.2020.01.014

23. K Geetha, R Manavalan, D Venkappaya. Effects and Causes of Lithiasis. Research J. Pharmacology and Pharmacodynamics. 2010; 2(4): 261-267

24. Daudon M, Jungers, P, Traxer O. Lithiase urinaire. Lavoisier, Médécine Sciences, Paris, 2012: 672p.

25. Falleh H, Ksoui R, Chaieb K, Karray-Bouraoui N, Trabelsi N, Boulaaba M, Abdelly C. Phenolic composition of Cynaracardunculus L. organs, and their biological activities. Compt Rend Biol2008; 331(5): 372-379.https://doi.org/10.1016/j.crvi.2008.02.008

26. Spandana. K, Shivani. M, Himabindhu. J, Ramanjaneyulu. K. Evaluation of In Vitro Antiurolithiatic Activity of Vignaradiata. Research J. Pharm. and Tech 2018; 11(12): 5455-5457. doi: 10.5958/0974-360X.2018.00994.0

27. Slinkard K, Singleton VL. Total phenol analyses: automation and comparison with manual methods. American Journal of Enology and Viticulture 1977; 28(1): 49-55.

28. Türkğluet A, Duru ME, Mercan N, Kivrak I, Gezer K. Antioxidant and antimicrobial activities of Laetiporussulphureus (Bull.) Murrill. a Food Chemistry 2007; 101(1):267–273. https://doi.org/10.1016/j.foodchem.2006.01.025

29. Torzewska A, Stączek P, Różalski A. Crystallization of urine mineral components may depend on the chemical nature of Proteus endotoxin polysaccharides. Journal of Medical Microbiology 2003; 52(6):471–477.https://doi.org/10.1099/jmm.0.05161-0

30. Prywer J, Ewa MB, Marcin O. Struvite crystal growth inhibition by trisodium citrate and the formation of chemical complexes in growth solution. Journal of Crystal Growth 2015; 418 :92–101.https://doi.org/10.1016/j.jcrysgro.2015.02.027

31. Sadki C, Atmani F. Évaluation de l’effet antilithiasique, oxalo-calcique et phospho ammoniaco-magnésien d’extrait aqueux d’Erica multifloraL. Progrès en urologie 2017; 27(16) :1058–1067.https://doi.org/10.1016/j.purol.2017.09.011

32. Daudon M, Protat MF, Réveillaud RJ. Analyse des calculs par spectrophotométrie infrarouge Avantages et limites de la méthode. Ann BiolClin1978; 36:475–489.

33. Swapnil R. Lahamage, Avinash B. Darekar, Ravindra B. Saudagar. Pharmaceutical Co-Crystallization. Asian J. Res. Pharm. Sci. 6(1): Jan.-Mar., 2016; Page 51-58. doi: 10.5958/2231-5659.2016.00008.4

34. Li H, Yao Q Z, Wang YY Li Y-L, Zhou G-T. Biomimetic synthesis of struvite with biogenic morphology and implication for pathological biomineralization. Scientific reports 2015; 5(1): 1-8.https://doi.org/10.1038/srep07718

35. El Habbani R, Lahrichi A, Sqalli Houssaini T, Kachkoul R, Mohim M, Chouhani BA and Chaqroune A. In vitro mass reduction of calcium oxalate urinary calculi by some medicinal plants. African Journal of Urology 2021; 27:28. https://doi.org/10.1186/s12301-021-00132-2

36. ChandakaMadhu, L. Harish, K. Narsimha Reddy, S. Sagar, D. Soumya, G. Pavan Kumar. Antiurolithic Activity of Aqueous Extract on Roots and Seeds of Plectranthustomentora on Ethylene Glycol Induced kidney Stones in Male Albino Rats. Asian J. Res. Pharm. Sci. 2(4): Oct.-Dec. 2012; Page 129-133.

37. N. V. Khokhlenkova, M. V. Buryak, O. V. Povrozina, T. V. Kamina. Principles of the Urolithiasis Phytotherapy. Research J. Pharm. and Tech 2019; 12(9):4559-4564. doi: 10.5958/0974-360X.2019.00784.4

38. Zhu H, Sun X, Lu J, Wang M, Fang Y and Ge W. The effect of plum juice on the prevention of struvite calculus formation in vitro BJU Intrnationa 2012; 110: E362-E367. https://doi.org/10.1111/j.1464-410X.2012.11090.x

39. Chauhan CK, Joshi MJ. In vitro crystallization, characterization and growth inhibition study of urinary type struvite crystals. Journal of Crystal Growth 2013; 362: 330–337. https://doi.org/10.1016/j.jcrysgro.2011.11.008

40. Beghalia M, Belouatek A, Ghalem S, and Allali H. Inhibition and Dissolution Crystals of Magnesium Ammonium Phosphate by Acacia Radiana (Bark) and (Sheet) in Vitro Study Int'l Journal of Advances in Agricultural & Environmental Engg 2014; 1: 1523-1531.http://dx.doi.org/10.15242/IJAAEE.C514072

41. Prywer J, Torzewska A. “Bacterially induced struvite growth from synthetic urine: experimental and theoretical characterization of crystal morphology”. Crystal Growth and Design 2009; 9(8): 3538–3543. https://doi.org/10.1021/cg900281g

42. Jungers P, Daudon M, Le Duc A. Lithiase urinaire. Flammarion Médecine-Sciences 1989; 590 p.

43. Muryanto S, Sutanti S, Kasmiyatun M Inhibition of struvite crystal growth in the presence of herbal extract orthosiphonaristatus BL. MIQ. In MATEC Web of Conferences 2015; Vol. 58, p. 01013. EDP Sciences.https://doi.org/10.1051/matecconf/20165801013

44. Chauhan CK, Joshi MJ, Vaidya ADB. Growth Inhibition of Struvite Crystals in the Presence of Herbal Extract Boerhaavia diffusa Linn. American Journal of Infectious Diseases 2009; 5 (3): 170–179.https://doi.org/10.3844/ajidsp.2009.170.179

45. Scott MD, Andrew JR, Damien MB. Gastric patch pyeloplasty: development of an animal model to produce upper tract urinary acidification for treating struvite urinary calculi, J. Urol 2001; 166 (2): 684–687. https://doi.org/10.1016/S0022-5347(05)66043-0

46. Prywer J, Torzewska A. Effect of Curcumin Against Proteus mirabilis During Crystallization of Struvite from Artificial Urine. Evidence-Based Complementary and Alternative Medicine 2012; Article ID 862794, 7 pages.https://doi.org/10.1155/2012/862794

47. In vitro Antiurolithiatic activity of the Leaves and Flowers extracts of Paronychia argentea, a plant used in Traditional medicine in Algeria. Omar Mechraoui, Ali Imessaoudene, Mohamed Y. Maiz, Hicham Banouh, Lotfi Mouni, Abdelkrim Rebiai, Mohamed L. Belfar4, Noureddine Elboughdiri, Djamel Ghernaout, Bachir Ben Seghir. Asian J. Research Chem. 14(6): November – December 2021 DOI: 10.52711/0974-4150.2021.00069

48. Kaleeswaran B, Ramadevi S, Murugesan R, Srigopalram S, Suman T, and Balasubramanian T. Evaluation of anti-urolithiatic potential of ethyl acetate extract of Pedalium murex L. on struvite crystal (kidney stone). Journal of Traditional and Complementary Medicine 2019; 9(1):24–37.https://doi.org/10.1016/j.jtcme.2017.08.003

49. Reshma R, Mahima V, Badrinathan S, Himaja M, Sabina EP, ArunaiNambi Raj N. In vitro and In vivo stady on the effect of ScopariaDulcis in inhibiting the growth of urinary crystals. International Journal of Phytomedcinie 2014; 6: 617–624.

50. Prywer J, Torzewska A Effect of curcumin against proteus mirabilis during crystallization struvite from artificial urine. J. Evid. Based Complementary Altern. Med 2012; 1–7.https://doi.org/10.1155/2012/862794

51. Dayana Jeyaleela G, Rosaline VimalaJ, Senthil R, Anandagopu P and ManjulaK. Isolation, Characterization, Molecular Docking and in vitro Studies of Inhibitory Effect on the Growth of Struvite Crystal Derived from Meliadubia Leaf Extract. Asian Journal of Chemistry 2019; 31(11): 2628-2634.https://doi.org/10.14233/ajchem.2019.22217

Received on 16.07.2022 Modified on 18.08.2022

Asian J. Research Chem. 2022; 15(6):459-465.

DOI: 10.52711/0974-4150.2022.00080