Cynodon dactylon, Emblica officinalis, Kalanchoe pinnata and Bambusa nutans is reported to have diuretic and antiurolithiatic potential against ethylene glycol and ammonium chloride, and glyoxalate induced urolithiasis along with in vitro calcium oxalate (CaOx) crystal growth inhibition property. The ethyl acetate fraction of these plants have a high content of antioxidative phytocompounds like polyphenols and flavonoids. This study aims at the exploration of antilithiatic potential of C. dactylon, E. officinalis, K. pinnata, and B. nutans ethyl acetate fraction on 3 % glycolic acid diet feeding induced nephrolithiasis in rats. On the 28th day of glycolic acid diet feeding, urine samples were collected to determine oxalate, calcium, phosphorus, sodium, and potassium concentration. On the 29th day, blood samples were collected after sacrifice to determine oxalate, calcium, phosphorus, and magnesium level and kidney tissue were observed for histopathological changes. Glycolic acid feeding resulted in a significant increase in serum oxalate, calcium, and phosphorus level with a decrease in magnesium. Urinary level of oxalate, calcium, and phosphorus was also increased with a decrease in sodium and potassium. C. dactylon, E. officinalis, K. pinnata, and B. nutans markedly ameliorates glycolic acid induced nephrolithiasis by reversing the abnormal serum and urinary levels of oxalate, calcium, and phosphorus. Ethyl acetate fraction of E. officinalis and K. pinnata showed decreased deposition of microcrystals in kidney tubules with a prominent reduction in glomerular and tubular damage. These observations indicate the prominent protective effect of E. officinalis and K. pinnata in the prevention of Change Italic form to Normal form.

•    Cynodon dactylon
•    Emblica officinalis
•    Kalanchoe pinnata
•    Bambusa nutans
•    Glycolic acid
•    Urolithiasis

Sohgaura AK, Bigoniya P, Shrivastava B (2020) Assessment of Potential Antilithiatic Activity of Cynodon dactylon, Emblica officinalis, Kalanchoe pinnata and Bambusa nutans on Dietary Glycolic Acid Induced Nephrolithiasis. J Pharmacol Clin Toxicol 8(1):1139.

Nephrolithiasis is a common life threatening renal disease people get affected all over the world. Many factors can promote this disorder like some drugs, life style, climate, food habit etc. This disease is also associated with other disorder as chronic renal disease, bone loss, coronary disease, hypertension, type 2 diabetes, and other metabolic syndrome [1]. Nephrolithiasis is a complex process resulted from successive physicochemical events including supersaturation, nucleation, growth, aggregation, and retention within renal tubules [2]. Hyperoxaluria is one of the major risk factors of idiopathic calcium oxalate (CaOx) nephrolithiasis in human. The primary hyperoxaluria condition of the kidney was first time described by Lepoutre in 1925 [3]. Oxalate is a natural by-product of metabolism which is harmlessly excreted through the urine in normal individuals. However, hyperoxaluria or increased urinary excretion of oxalate can be dangerous due to its propensity to crystallize at physiologic pH and form CaOx crystals [4]. Epidemiological data showed that the majority of kidney stones are composed mainly of CaOx [5]. Most individuals have urine crystallization inhibiting capacity that does not allow nephrolithiasis to occur, but this natural inhibition is in deficit in stone formers [6].

Many remedies have been tried and tested to treat nephrolithiasis. In most cases, the management of nephrolithiasis involves both surgical and therapeutic approaches, which is relatively costly, painful, and require expert hands with the availability of appropriate equipment. Despite tremendous advances in the field of medicine, there is genuinely no satisfactory drug available to treat renal calculi. Research in search of herbomineral preparations is still ongoing [7]. A large number of Indian medicinal plants are used in the treatment of nephrolithiasis, and they are reported to be active with no side effects [8]. The plants C. dactylon (Poaceae), E. officinalis (Euphorbiaceae), K. pinnata (Crassulaceae) and B. nutans (Graminae) have reported diuretic and antilithiatic properties against ethylene glycol and ammonium chloride, and glyoxalate induced urolithiasis in different animal models in our previous studies. We have reported rich presence of antioxidative phytocompounds like polyphenols and flavonoids in ethyl acetate fractions of B. nutans shoot, E. officinalis fruit, and K. pinnata leaf along with excellent in vitro CaOx crystal growth inhibition activity. We are exploring the whole spectrum of antiurolithiatic potential of the selected plants targeting the ethyl acetate fraction.

The study aims at the screening of C. dactylon whole plant, E. officinalis fruit, K. pinnata leaf and B. nutan shoot ethyl acetate fraction rich in flavonoid and polyphenol for antinephrolithiasis potential against glycolic acid rich diet feeding on the rat. In-vivo experimental induction of nephrolithiasis in the rat is commonly done with calcium oxalate and other oxalate metabolic intermediates like glycolate and ethylene glycol [9-11]. Ethylene glycol (EG) administration is a well-known model of nephrocalcinosis. EG metabolizes into glycolate, glyoxylate, and oxalate leading to CaOx crystal development in both urine and kidneys [12]. Glycolic acid and ethylene glycol are precursors of oxalic acid. Oxalic acid is produced by dehydrogenation of glycolic acid. Glycolate is oxidized to oxalate by lactate dehydrogenase enzyme [13,14]. Glycolic acid is an in vivo precursor of oxalate biosynthesis [15]. It is worth to study antiurolithiatic potential of C. dactylon whole plant, E. officinalis fruit, K. pinnata leaf and B. nutan shoot ethyl acetate fraction against urolithiasis induced by glycolic acid rich diet feeding.

Collection and extraction of plant

The C. dactylon whole plant, E. officinalis fruit, K. pinnata leaf, and B. nutan shoot was collected in between October 2014 to April 2015 from Rewa, Raisen and Bhopal districts of M.P. Al the plants were authenticated by Dr. Zia Ul Hasan, Prof, and Head, Department of Botany, Saifia College of Science Bhopal, M.P., and voucher herbarium specimen (456/Bot/saifia/14) was maintained. Cleaned plant material was dried under the shade, pulverized to form a coarse powder and processed for hydromethanolic extraction. Ethyl acetate fraction was enriched from hydro-methanolic extract following the method as described previously [16].

Experimental animal

In-vivo study was performed with due permission from the Institutional Animal Ethical Committee. Laboratory breed adult Wistar male rats (100-150gm) were housed in polypropylene cages with paddy husk bedding maintained at 22 ± 2C and 12 hr light-dark cycle. The animals were fed with standard pellet diet and water ad libitum. All experimental procedures were conducted in accordance with the ethical guidelines of CPCSEA, New Delhi.

Study protocol

Based on the acute toxicity reported previously dose range selected for E. officinalis, K. pinnata and B. nutans is 25, 50 and 100 mg/kg, and 100, 300 and 500mg/kg p.o., for C. dactylon [17]. The 90 rats were divided into fifteen groups comprising of six animals in each group. All the animals were treated as per the protocol daily for 28 days to assess the effect of ethyl acetate fraction (EA) of C. dactylon, E. officinalis, K. pinnata, and B. nutan. Glycolic acid 3% (w/v) containing diet was used as nephrolithiasis inducing agent [10]. The animals were grouped as follows:

GROUP 1: Fed with the standard diet for 28 days (Vehicle control).

GROUP 2: Glycolic acid diet (Negative control).

GROUP 3: Glycolic acid diet + 750 mg/kg Cystone (Positive control).

GROUP 4: Glycolic acid diet + 100 mg/kg EA C. dactylon

GROUP 5: Glycolic acid diet + 300 mg/kg EA C. dactylon

GROUP 6: Glycolic acid diet + 500 mg/kg EA C. dactylon

GROUP 7: Glycolic acid diet + 25 mg/kg EA E. officinalis

GROUP 8: Glycolic acid diet + 50 mg/kg EA E. officinalis

GROUP 9: Glycolic acid diet + 100 mg/kg EA E. officinalis

GROUP 10: Glycolic acid diet + 25 mg/kg EA K. pinnata

GROUP 11: Glycolic acid diet + 50 mg/kg EA K. pinnata

GROUP 12: Glycolic acid diet + 100 mg/kg EA K. pinnata

GROUP 13: Glycolic acid diet + 25 mg/kg EA B. nutan

GROUP 14: Glycolic acid diet + 50 mg/kg EA B. nutan

GROUP 15: Glycolic acid diet + 100 mg/kg EA B. nutan

Vehicle control rats had fed with regular diet along with free access to drinking water ad libitum. Rats of groups 2 to 15 also had free access to the normal diet containing additional 3% glycolic acid (w/v) for 28 days in order to promote hyperoxaluria and CaOx crystal deposition in the kidney. Animals of group 4 to 15 were treated with plant extracts per orally daily for 28 days.

Estimation of urine biochemical parameters: All the animals were kept in individual cages, and urine samples of 24 hr were collected on 28th day after the last dosing with free access to water. Urine samples were analyzed for the level of oxalate, calcium, phosphates, sodium, and potassium with the help of Autoanalyser (Star 21 Plus, Rapid Diagnostics, India) using diagnostic kits (Span Diagnostics Ltd, India).

Estimation of serum biochemical parameters: The animals were sacrificed on 29th day under ether anesthesia followed by cardiac puncture. Blood was collected in a clean and dry test tube immediately after sacrifice and allowed to coagulate for 30 min in room temperature. Serum was separated by centrifugation at 3000 rpm for 10 min, and the supernatant was collected and used for estimation of oxalate, magnesium, calcium, and phosphates using diagnostic kits (Span Diagnostics Ltd., India) and Autoanalyser (Star 21 Plus, Rapid Diagnostics, India).

Histopathology of kidney: Hematoxylin-eosin stained kidney tissue sections were observed for histopathological changes and photographed using an optical microscope at 10× magnification [18].

Statistical analysis

The values in tables were expressed as Mean ± SEM. Statistical analysis was performed using one-way analysis of variance ANOVA followed by Dunnett multiple comparison tests. A P value of less than 0.05 was considered statistically significant.

Estimation of urine biochemical parameters

Nephrolithiasis induction by dietary glycolic acid is confirmed as it showed significant (P < 0.001) elevation of the urine biochemical parameters, i.e., oxalate, calcium, and Inorganic phosphates and decrease in sodium and potassium compared to the negative control group. The Ca/Ox ratio in the negative control group was found to be 0.372 compared to 0.362 of the vehicle control group. The ethyl acetate fraction of C. dactylon at 300 and 500 mg/kg dose showed significantly (P < 0.01 – 0.001) decreased urinary excretion of oxalate, calcium and inorganic phosphates with increased excretion of sodium and potassium. The Ca/Ox ratio is moderately increased as 0.507 and 0.487, respectively at 300 and 500 mg/kg dose. Urinary excretion of oxalate, calcium and inorganic phosphates was decreased significantly (P < 0.01 - 0.001) with an increase in sodium and potassium level by E. officinalis ethyl acetate fraction at 50 and 100 mg/kg dose. The Ca/Ox ratio is slightly increased as 0.415 and 0.427, respectively at 50 and 100 mg/kg dose.

K. pinnata leaf ethyl acetate fraction at 50 and 100 mg/kg dose significantly (P < 0.05 – 0.001) decreased urinary excretion of oxalate, calcium, and inorganic phosphates along with an increase in sodium and potassium level. Ca/Ox ratio are also slightly increased with a value of 0.442 and 0.462, respectively at 50 and 100mg/kg dose of K. pinnata ethyl acetate fraction. B. nutan ethyl acetate fraction at 50 and 100 mg/kg dose showed significant (P < 0.05-0.01) decrease in urinary excretion of oxalate, calcium, and inorganic phosphates with an increase in sodium and potassium excretion. B. nutan ethyl acetate fraction does not show a measurable difference in Ca/Ox ratio as compared to vehicle and negative control groups (Table 1).

Table 1: Urine biochemical parameter of glycolic acid induced nephrolithiatic rats treated with ethyl acetate fraction of C. dactylon, E. officinalis, K. pinnata and B. nutan.

Effect on serum biochemical parameters

The serum sample of glycolic acid induced nephrolithiatic negative control group animals showed significant (P < 0.01- 0.001) increase in oxalate, calcium, and phosphorus with a decrease in magnesium concentration as compared to the vehicle control group. C. dactylon ethyl acetate fraction at 300 and 500 mg/kg dose showed significant (P < 0.01–0.001) reduction in elevated levels of oxalate, calcium, and phosphorus, and also increased magnesium level compared to the negative control group. E. officinalis ethyl acetate fraction at 50 and 100 mg/ kg dose has significantly (P < 0.05 - 0.001) decreased oxalate, calcium, and phosphorus, and increased magnesium compared to the negative control group. Ethyl acetate fraction of K. pinnata at 50mg/kg dose has significantly (P < 0.05 - 0.01) decreased oxalate and calcium level of blood with an increase in magnesium level compared to the negative control group. At 100 mg/kg K. pinnata ethyl acetate fraction showed significant (P < 0.05 - 0.001) reversal of all four blood parameters. B. nutan ethyl acetate fraction at 50mg/kg dose has significantly (P < 0.05 - 0.01) decreased oxalate, calcium, and phosphorus, but with nonsignificant effect on magnesium level, whereas at 100 mg/kg dose B. nutan ethyl acetate fraction has normalized all the disturbed blood parameters (Table 2).

Table 2: Serum biochemical parameter of glycolic acid induced nephrolithiatic rats treated with ethyl acetate fraction of C. dactylon, E. officinalis, K. pinnata and B. nutan.

Histopathology of kidney

The tissue section of rat kidney treated with glycolic acid rich diet showed heavy deposition of CaOx microcrystals in tubules, moderate glomerular and tubular damage along with tubular dilation. Damage of the epithelial cell lining occurred with degeneration and thickening. Crystal deposition resulted in glomerular and peritubular congestion, blood vessel congestion, epithelial desquamation, and cellular inflammation.

Cystone moderately reduced CaOx microcrystals deposition, glomerular and tubular damage, epithelial cell damage, and inflammation. C. dactylon ethyl acetate fraction treated groups at 500 mg/kg dose showed an effective reversal of these symptoms and very less deposition of microcrystal in tubules with the presence of inflammatory damage. Ethyl acetate fraction of E. officinalis at 50 and 100mg/kg dose showed decreased deposition of microcrystals in kidney tubules with a noticeable reduction in glomerular and tubular damage. Tubular dilation and damage have also been reversed effectively by E. officinalis but with the presence of some inflammatory cell infiltration. The kidney tissue sections of K. pinnata ethyl acetate fraction treated group at 50, and 100mg/kg dose showed a moderate reduction in glomerular and epithelial damage and tubular dilation, though microcrystal deposition and inflammatory infiltration was not reversed effectively. The kidney tissue section of different doses of B. nutan ethyl acetate fraction treated groups showed a nominal reversal of glycolic acid induced histological damages with deposition of microcrystals in tubules, but a reduction in glomerular and tubular damage (Figure 1).

Figure 1 Kidney histopathology of glycolic acid induced nephrolithiatic rats treated with ethyl acetate fraction of C. dactylon, E. officinalis, K. pinnata, and B. nutan. Vehicle control (A); Glycolic acid diet (B); Cystone 750 mg/kg (C); EACD 100 mg/kg (D); EACD 300 mg/kg (E); EACD 500 mg/kg (F); EAEO 25 mg/ kg (G); EAEO 50 mg/kg (H); EAEO 100 mg/kg (I); EAKP 25 mg/kg (J); EAKP 50 mg/kg (K); EAKP 100 mg/kg (L); EABN 25 mg/kg (M); EABN 50 mg/kg (N); EABN 100 mg/kg (O). EACD = C. dactylon ethyl acetate fraction, EAEO = E. officinalis ethyl acetate fraction, EAKP = K. pinnata ethyl acetate fraction and EABN = B. nutan ethyl acetate fraction.

In the modern system of medicine treatment for urolithiasis is expensive and dissatisfactory as proper drug therapy is not available for clearing the kidney stones. Patients usually depend on alternative systems of medicine for getting relief of kidney stone [19]. Plant based phytotherapeutic agents are used for treating urolithiasis as they are efficacious and have lesser side effects and reduce the recurrence rate of renal stone [20]. Our study aims at exploration whole profile of antiurolithiatic potential of C. dactylon, E. officinalis, K. pinnata and B. nutan ethyl acetate fraction against all possible mechanisms of nephrolithiatic complications. The beneficial effects of C. dactylon, E. officinalis, K. pinnata and B. nutan ethyl acetate fraction against EG and ammonium chloride, and glyoxalate induced nephrolitisaia has already been reported along with in vitro CaOx crystal dissolution efficacy.

In human nephrolithiasis, most stones contain calcium and are located within urinary cavities as monohydrate calcium oxalate, dihydrate calcium oxalate and/or calcium phosphates in various proportion. Oxalate urinary calculi can be experimentally induced in male rats by single large glycolic acid oral dosing [21], or by diet feeding for 4 to 6 weeks [10,15,22,23].Administration of glycolic acid diet for four weeks leads to hyperoxaluria and CaOx tubular crystal deposition not only within cortex and medulla but also in pelvic cavities [22]. Glycolic acid administration increases the urinary calcium level. It has been stated that hypercalciuria favors precipitation of CaOx from urine [24]. Glycolic acid diet provokes hyperoxaluria, which is due to the fast conversion of glycolic acid to oxalate by the oxalate synthesizing enzyme, glycolic acid oxidase and lactate dehydrogenase in the liver.

Glycolic acid is known to increase the incidence of oxalate lithiasis, and our results are in concord with these reports [25].

Cystone has reduced serum, and urinary oxalate levels may be due to its inhibitory action on oxalate synthesizing liver enzyme indicating that cystone can play an essential role in the prevention of disorders associated with kidney stone formation [26]. Ethyl acetate fractions of E. officinalis, K. pinnata, C. dactylon, and B. nutan have significantly reversed elevated serum and urinary calcium, oxalate and phosphorous levels. E. officinalis, K. pinnata, and C. dactylon has moderately increased Ca/Ox ratio of urine. Glycolic acid rich diet feeding showed heavy deposition of CaOx microcrystals in tubules along with tubular dilation and tubular damage. E. officinalis showed decreased deposition of microcrystals in kidney tubules with a prominent reduction in glomerular and tubular damage whereas K. pinnata and C. dactylon showed a moderate reduction in glomerular and epithelial damage and tubular dilation, though microcrystal deposition and inflammatory infiltration was not reversed effectively. Ethyl acetate fraction of E. officinalis and K. pinnata showed most potential antilithiatic effect against glycolic acid induced urolithiasis.

Phytocompounds exert antilithogenic effect by altering the ionic composition of urine, decreasing the calcium ion concentration or increasing magnesium and citrate excretion [27]. The plants with additional diuretic activity also have good lithotriptic activity [4]. Herbal medicine contains groups of phytocompounds that are capable of minimizing tissue injury enabled with multiple mechanisms of protective action [28]. Among the four plants, K. pinnata ethyl acetate fraction had the highest content of total flavonoid and polyphenol whereas B. nutans had the lowest flavonoid content through its polyphenol content was rich [16]. Ethyl acetate fractions of K. pinnata, C. dactylon and E. officinalis showed potential diuretic activity [17]. Ethyl acetate fraction of K. pinnata and E. officinalis was most effective against ethylene glycol induced nephrolithiasis on rats [29], whereas K. pinnata and B. nutan were highly effective on mice against glyoxylate nephrolithiasis [30].

Acquired and genetic kidney disorders are associated with excessive oxalate excretion, in particular with primary hyperoxaluria types I [31]. Oxalate is a useless metabolic end product, formed as a byproduct of the metabolism of glyoxylate and ascorbate, excreted in the urine where it constitutes a hazard because of the insolubility as the calcium salt. Glycolic acid (or hydroxyacetic acid) is the smallest α-hydroxy acid. Determination of oxalate, as well as glycolic acid, is essential for the diagnosis of primary hyperoxaluria type I (glycolic aciduria), caused by the low or absent activity of the liver-specific peroxisomal alanine:glyoxylate aminotransferase [32]. Hyperoxaluria induction with glyoxylate and glycolic acid treatment produced an increase in glycolic acid oxidase and lactate dehydrogenase enzyme level [26,33,34]. High content of glycolic acid can modify normal tubular epithelium into a crystal-binding epithelium [35]. The observed antiurolithiasis activity of E. officinalis and K. pinnata ethyl acetate fraction can be correlated to the concerted effect of flavonoids and polyphenolic compounds. Amla (E. officinalis) containing tannins, flavonoids, phenolic compounds, saponins, terpenoids, especially chebulinic acid, chebulagic acid, emblicanin, gallic acid, ellagic acid, and quercetin is highly valued in traditional Indian medicine [36]. Bogucka-Kocka et al., reported rich quantity of ferulic and caffeic acid in K. pinnata leaf [37]. All these phenolic and flavonoid constituents are well known for antioxidant and anti-inflammatory activity. Ethyl acetate fraction of all the four plants had shown a highly significant decrease in lactate dehydrogenase level of kidney tissue of mice treated with glyoxalate [30]. The results signify that E. officinalis and K. pinnata have modulating effect on down regulation of oxalate synthesis in rat kidney followed by glycolic acid dietary intake may be via antioxidant and antiinflammatory activity along with inhibition of oxalate synthesizing enzymes.

This study substantiates the excellent antiurolithiasis potential of E. officinalis and K. pinnata against urolithiasis induced by glycolic acid feeding for 28 days. K. pinnata and E. officinalis ethyl acetate fractions exerted crystal growth inhibition properties along with the noteworthy reversal of urine and serum abnormal parameters. The observed potent antiurolithiatic activity of K. pinnata and E. officinalis ethyl acetate fraction with rich presence of flavonoid and polyphenol may be due to antioxidant and bioprotective mechanisms on the renal epithelium.

1. Sohgaura A, Bigoniya P. A review on epidemiology and etiology of renal stone. Am J Drug Discov Dev. 2017; 2: 54-62.

2. Khan SR. Animal models of kidney stone formation: an analysis, World J Urol. 1997; 15: 236-243.

3. Lepoutre C. Calculs multiples chez un enfant. Infiltration du parenchyme renal par de cristaux. J Urol (Paris). 1925; 20: 424.

4. Pareta SK, Patra KC, Mazumder PM, Sasmal D. Establishing the Principle of Herbal Therapy for Antiurolithiatic Activity: A Review. J Toxicol Pharmacol. 2011; 6: 321-332.

5. Daudon M, Bader CA, Jungers P. Urinary calculi: review of classification methods and correlations with etiology. Scanning Microsc. 1993; 7: 1081-1106.

6. Tiselius HG, Hallin A, Lindbäck B. Crystallization properties in stone forming and normal subject’s urine diluted using a standardized procedure to match the composition of urine in the distal part of the distal tubule and the middle part of the collecting duct. Urol Res. 2001; 29: 75-82.

7. Anderson EE, Rundles RW, Silberman HR, Metz EN. Allopurinol control of hyperuricosuria: A new concept in the prevention of uric acid stones. J Urol. 1967; 97: 344-347.

8. Nadkarni KM. Indian Materia Medica. Bombay, India: Popular book Depot; 1976.

9. Bharathi K, Prasad KVSRG, Sailaja B. Protective effect of Tridax procumbens L. on calcium oxalate uolithiasis and oxidative stress. Pharmanest Inter J Adv Pharm Sci. 2011; 2: 9-14.

10. Sandhyarani G, Swathi K, Gangarapu K. Effect of methanolic leaf extract of Cochlosperum vitifolium on glycolic acid induced urolithiasis in rats. Acta Biomed Scient. 2014; 1: 32-35.

11. Khan NI, Shinge JS, Naikwade NS. Antilithiatic effect of Helianthus Annuus Linn. leaf extract in ethylene glycol and ammonium chloride induced nephrolithiasis. Inter J Pharm Pharmaceut Sci. 2010; 2: 180- 184.

12. Mcmartin K. Are calcium oxalate crystals involved in the mechanism of acute renal failure in ethylene glycol poisoning. Clin Toxicol (Phila). 2009; 47: 859-869.

13. Jiang J, Johnson LC, Knight J, Callahan MF, Riedel TJ, Holmes RP, et al. Metabolism of [13C5] hydroxyproline in vitro and in vivo: implications for primary hyperoxaluria. Am J Physiol-Gastrointestinal Liver Physiol. 2012; 302: G637-G643.

14. Knight J, Jiang J, Assimos DG, Holmes RP. Hydroxyproline ingestion and urinary oxalate and glycolate excretion. Kidney Inter. 2006; 70: 1929-1934.

15. Khan ZA, Assiri AM, Al-Afghani HM, Maghrabi TM. Inhibition of oxalate nephrolithiasis with Ammi visnaga (AI-Khillah). Int Urol Nephrol. 2001; 33: 605-608.

16. Sohgaura AK, Bigoniya P, Shrivastava B. In-vitro antilithiatic potential of Kalanchoe pinnata, Emblica officinalis, Bambusa nutan and Cynodon dactylon. J Pharm Bioallied Sci. 2018a; 10: 83-89.

17. Sohgaura A, Bigoniya P, Shrivastava B. Diuretic potential of Cynodon dactylon, Emblica officinalis, Kalanchoe pinnata and Bambusa nutans. J Pharmacog Phytochem. 2018b; 7: 2895-2900.

18. Dodoala S, Diviti R, Koganti B, Prasad KVSRG. Effect of ethanolic extract of Phyla nodiflora (Linn.) greene against calculi producing diet induced urolithiasis. Ind J Nat Prod Resour. 2010; 1: 314-321.

19. Galib BS, Kar ACB. Lithotriptic activity of Swadamstradi Kwatha Seadamstradi Chan Vati-an experimental evaluation. J Drug Res Ayurvedic Sci. 2006; 27: 8-18.

20. Kumaran MGS, Prasad SR, Mitra SK. Evaluation of efficacy and safety of cystone syrup in lower ureteric calculus. Ind J Cli Practice. 2007; 18: 31-36.

21. Al-Ghamdi SS, Al-Ghamdi AA, Shammah AA. Inhibition of calcium oxalate nephrotoxicity with Cymbopogon schoenanthus (Al-Ethkher). Drug Metab Lett. 2007; 1: 241-244.

22. Ogawa Y, Yamaguchi K, Morozumi M. Effects of magnesium salts in preventing experimental oxalate urolithiasis in rats. J Urol. 1990; 144: 385-389.

23. Shah JG, Patel BG, Patel SB, Patel R. Effect of Hordeum vulgare Linn. seeds on glycolic acid induced urolithiasis in rats. Pharmacog Commun. 2012; 2: 34-39.

24. Lemann JJ, Worcestor EM, Gray RW. Hypercalciuria and stones. Am J Kidney Disease. 1991; 27: 386-391.

25. Fossati P, Tarenghi G, Musitelli C. Enzymatic colorimetric method for the determination of inorganic phosphorus in serum and urine. J Clin Chem Clin Biochem. 1988; 26: 399-404.

26. Mitra SK, Gopumadhavan S, Venkataranganna MV, Sundaram R. Effect of cystone, a herbal formulation, on glycolic acid-induced urolithiasis in rats. Phytother Res. 1998; 12: 372-374.

27. Arafat OM, Tham SY, Sadikun A, Zhari I, Haughton PJ, Asmawi MZ. Studies on diuretic and hypouricemic effects of Orthosiphon stamineus methanol extracts in rats. J Ethnopharmacol. 2008; 118: 354-360.

28. Barry H. Antioxidant effects: A basis for drug selection. Drugs. 1991; 42: 569-605.

29. Sohgaura AK, Bigoniya P, Shrivastava B. Ameliorative effect of Kalanchoe pinnata, Emblica officinalis, Bambusa nutans and Cynodon dactylon on ethylene glycol and ammonium chloride induced nephrolithiasis. Pharmacologyonline. 2019; 1: 408-428.

30. Sohgaura AK, Bigoniya P, Shrivastava B. Antilithiatic effect of C. dactylon, E. officinalis, K. pinnata and B. nutans ethyl acetate fraction on glyoxylate induced nephrolithiasis Tropical J Pharmaceutical Res. 2019; (under communication).

31. Hibbard E, Williams MD, Lloyd H, Smith JMD. Disorders of oxalate metabolism. Am J Med. 1968; 45: 715-735.

32. Blau N. Oxalate, Glycolate, Glycerate, Sulfate, and Citrate. Blau N, Duran M, Gibson K, editors. Laboratory Guide to the Methods in Biochemical Genetics. Berlin, Heidelberg: Springer; 2008.

33. Selvam R, Bijikurien T. Methionine feeding prevents kidney stone deposition by restoration of free radical mediated changes in experimental rat urolithiasis. J Nutr Biochem. 1991; 2: 644-651.

34. Khan SR, Glenton PA. Experimental induction of calcium oxalate nephrolithiasis in mice. J Urol. 2010; 184: 1189-1196.

35. Verhulst A. Crystalluric and tubular epithelial parameters during the onset of intratubular nephrocalcinosis: illustration of the ‘fixed particle’ theory in vivo. Nephrol Dialysis Transplant. 2009; 24: 3659- 3668.

36. Hasan MR, Islam MN, Islam MR. A review on phytochemistry, pharmacological activities and traditional uses of Emblica officinalis. Inter Current Pharmaceutical J. 2016; 5: 14-22.

37. Bogucka-Kocka A, Zidorn C, Kasprzycka M, Szymczak G, Szewczyk K. Phenolic acid content, antioxidant and cytotoxic activities of four Kalanchoë species. Saudi J Biol Sci. 2018; 25: 622-630.