INTRODUCTION:

Free radicals, are highly reactive chemical species formed due to the presence of one or more unpaired electrons^1,10. These include reactive oxygen species (ROS) and reactive nitrogen species (RNS), which are generated during normal metabolic processes and under environmental stress conditions¹. Excessive production of these reactive species can damage cellular macromolecules such as DNA, proteins, and lipids, thereby disrupting normal cellular functions².

Oxidative stress arises when endogenous antioxidant defense mechanisms are insufficient to neutralize the excessive generation of free radicals^3,9,13. This imbalance has been implicated in the development of several pathological conditions, including diabetes, cardiovascular disorders, inflammatory diseases, and renal complications^4,9. Among renal disorders, urolithiasis is a recurrent condition characterized by the formation of calculi within the urinary tract, predominantly composed of calcium oxalate crystals⁵.

The prevalence of kidney stone disease has increased globally due to sedentary lifestyle, altered dietary patterns, and metabolic disturbances⁶. Although current therapeutic approaches such as lithotripsy and surgical removal are effective, they are often associated with high cost, adverse effects, and frequent recurrence⁶. This has led to increased interest in plant-based therapies as safer and cost-effective alternatives for urolithiasis management^7,12.

Several medicinal plants rich in flavonoids, phenolics, and saponins have demonstrated inhibitory effects on calcium oxalate nucleation, aggregation, and crystal growth^8,11. In vitro calcium oxalate crystallization models are widely used as preliminary screening tools for evaluating anti-urolithiatic activity^7,8.

Ziziphus nummularia (Burm. f.) Wight & Arn., a member of the family Rhamnaceae and commonly known as Jharberi, is distributed across India, Pakistan, and Iran¹⁴. The plant has been traditionally used for the treatment of metabolic and inflammatory disorders. Phytochemical studies have reported the presence of flavonoids, phenolics, and glycosides in its leaves and fruits, which may contribute to antioxidant and anti-urolithiatic activities^17,22.

Antioxidant assays such as DPPH and superoxide radical scavenging methods are widely accepted for evaluating free radical neutralization through different mechanisms^15,16,18. Therefore, the present study was designed to comparatively evaluate the anti-urolithiatic and antioxidant potential of hydroalcoholic leaf and fruit extracts of Ziziphus nummularia using validated in vitro models.

METHODS:

Collection of Plant Materials, Identification and Authentication

Leaves and fruits of Ziziphus nummularia were collected, authenticated, and extracted using hydroalcoholic solvents²².

Fig 1.1.A Shrub of Ziziphus nummularia with Ripe Fruits

DPPH Radical Scavenging Assay:

The antioxidant potential of the extracts was evaluated using the DPPH free radical scavenging method, which measures the ability of antioxidants to donate hydrogen atoms or electrons to neutralize stable radicals¹⁵. The reaction mixtures were incubated in the dark to prevent photo-oxidation, and absorbance was measured spectrophotometrically. Radical scavenging activity was calculated as percentage inhibition compared with the control^16,19.

Superoxide Radical Scavenging Assay:

Superoxide radical scavenging activity was assessed using a standard assay system based on the inhibition of superoxide anion generation. This method evaluates the capacity of extracts to neutralize biologically relevant superoxide radicals involved in oxidative stress-mediated cellular damage^16,20,21.

Fig. 1.2 The role of Oxidative Stress in Various Organs

In Vitro Anti-Urolithiatic Activity:

Anti-urolithiatic activity was assessed using calcium oxalate crystallization assays, including nucleation, aggregation, and dissolution studies. These assays were performed according to previously reported methods with minor modifications^5,6. The inhibitory effect of extracts was calculated by comparing the rate of calcium oxalate formation in the presence and absence of plant extracts.

Spectrophotometric Estimation of Calcium Oxalate:

The experimental groups were prepared as follows:

Group I (Control): 1mL calcium oxalate (1mg/mL) + 1 mL distilled water

Group II (Standard): 1mL calcium oxalate (1mg/mL) + 1mL Cystone (400mg/mL)

Group III (Test): 1mL calcium oxalate (1mg/mL) + 1 mL hydroalcoholic leaf extract of Ziziphus nummularia (20mg/mL)

Group Iv (Test): 1mL calcium oxalate (1mg/mL) + 1 mL hydroalcoholic fruit extract of Ziziphus nummularia (20mg/mL)

Statistical Analysis:

Each test was carried out in duplicate and data was expressed as mean±SEM (standard error of the mean). For the statistical analysis of the data, group means were compared with one-way analysis of variance and post hoc analysis (SPSS 26.0 software). Dunnett’s post hoc test was applied to identify significance among groups; p<0.05 was statistically significant.

RESULTS:

The phytochemical screening of various extracts showed the presence of a Flavonoids, glycosides, tannins, Phenolic compounds and Saponins, whereas protein and amino acid are absent in the leaf extract of Z. nummularia. However, the Saponins are present in leaf extract and are absent in fruit extract of Z. nummularia.

Calcium Oxalate Dissolution:

Fruit extract:

87.34% inhibition (spectrophotometric), 14% undissolved (titrimetric)

Leaf extract:

72.05% inhibition, 16% undissolved

Standard (Cystone):

87.06% inhibition, 10% undissolved

Antioxidant Activity:

DPPH scavenging:

Leaves (9.7–75.86%), Fruit (lower)

Superoxide scavenging:

Leaves showed higher activity than fruit

Table1.1 DPPH radical scavenging activity of ethanolic extract of leaves and fruits of Z. nummularia

S. No.	Conc.(µg/ml) of standard extract	% Radical scavenging activity of standard ascorbic acid Ascorbic Acid	*% Radical scavenging activity Ziziphus nummularia* leaf extract**	*% Radical scavenging activity of Ziziphus nummularia* fruit extract**
1	0	0±0.00	0±0.00	0 ± 0
2	25	38.73±1.10	9.70±0.38	5.23 ± 0.49
3	50	87.99±1.17	20.42±0.70	9.28 ± 0.12
4	75	94.03±0.26	33.33±0.63	15.61 ± 0.63
5	100	95.76±0.23	45.48±0.76	26.16 ± 0.27
6	125	96.32±0.09	53.16±0.63	33.50 ± 0.09
7	150	96.91±0.13	63.20±0.24	42.95 ± 0.24
8	175	97.89±0.12	67.51±0.38	55.44 ± 0.52
9	200	98.64±0.27	75.86±0.27	60.67 ± 0.27

Values are mean±SE of each triplet test.

Table 1.3 % superoxide radical scavenging activity of leaves and fruits extract of Z. nummularia comparison with standard gallic acid

S. No.	Conc.(µg/ml)	% Radical scavenging activity of gallic acid	*% Radical scavenging activity Ziziphus nummularia* leaf extract**	*% Radical scavenging activity of Ziziphus nummularia* fruit extract**
1	0	0±0.00	0±0.00	0±0
2	25	36.45±1.10	10.30±0.28	6.23±0.01
3	50	56.52±1.17	24.31 ±0.18	8.22±0.10
4	75	89.63±0.52	33.73±0.01	24.61±0.03
5	100	96.21±0.23	47.14 ±0.02	26.89±0.02
6	125	97.45±0.09	53.19±0.01	32.50±0.07
7	150	97.74±0.14	65.22 ±0.01	35.99±0.01
8	175	98.89±0.12	67.54±0.37	50.14± 0.02
9	200	99.64±0.27	73.38 ±0.36	58.03±0.35

Table 1.4: IC₅₀ value of Superoxide radical scavenging activity

S. No.	Sample Name	IC₅₀ Value (µg/ml)
1	Gallic acid	25.624
2	ZNLS (Ziziphus nummularia Leaves Soxhlet)	125.253
3	ZNFS (Ziziphus nummularia Fruits Soxhlet)	178.12

Table: 1.5 Results of Spectrophotometric estimation of Calcium Oxalate

Group	Absorbance (nm)	% Calcium oxalate undissolved
Control	0.0004	---
Standard (Cystone)	0.3240	87.06%
Extract (leaf) Extract (fruit)	0.2280 0.2830	70.37% 87.34%

Table 1.6: Results of Titrimetric estimation of Calcium Oxalate

Group	Undissloved Calcium oxalate
Control	62%
Standard (Cystone)	12%
Sample (leaf) Sample (fruit)	16% 14%

Fig: 1.5 Comparison of the inhibitory effect of leaf and fruit extracts of Ziziphus nummularia on calcium oxalate crystallization using spectrophotometric

DISCUSSION:

Previous Phytochemical investigations, including our earlier work, have reported the presence of flavonoids and phenolic compounds in the leaves and fruits of Ziziphus nummularia¹⁷.

The present investigation demonstrates that hydroalcoholic extracts of Ziziphus nummularia possess significant anti-urolithiatic and antioxidant activities. The fruit extract showed strong inhibition of calcium oxalate crystallization, comparable to the standard drug Cystone. This activity may be attributed to the presence of phenolic compounds capable of chelating calcium ions and interfering with crystal growth processes^26,27.

In contrast, the leaf extract exhibited higher antioxidant activity in both DPPH and superoxide radical scavenging assays. Enhanced antioxidant potential may help reduce oxidative stress-induced renal damage, thereby indirectly contributing to the prevention of stone formation^19,25. Comparable trends observed in both spectrophotometric and titrimetric estimations further validate the reliability of the experimental findings²⁴.

Overall, the comparative evaluation of leaf and fruit extracts highlights their distinct pharmacological potential and supports the traditional use of Ziziphus nummularia in urolithiasis management^19-23.

CONCLUSION:

Hydroalcoholic extracts of Ziziphus nummularia leaves and fruits significantly inhibit calcium oxalate crystallization and exhibit notable antioxidant activity. The fruit extract demonstrated anti-urolithiatic activity comparable to the standard drug Cystone, while the leaf extract showed superior antioxidant potential. These findings support the traditional use of Ziziphus nummularia in kidney stone management and suggest the need for further in vivo and phytochemical investigations.

CONFLICT OF INTEREST:

The author declares that there is no conflict of interest.

AUTHOR CONTRIBUTIONS (Credit):

Pooja Bagdi: Conceptualization, Methodology, Investigation, Data analysis, Pooja Bagdi, and Gaurav Kumar: Writing – original draft, Review & editing.

ACKNOWLEDGMENTS:

I sincerely thank the Principal of B.N. College of Pharmacy, Dr. Yuvraj Singh Sarangdevot, Dr. S.S. Sisodia (Head of Department, Pharmacology), and Dr. Jai Singh Vagela for providing laboratory facilities, equipment, and continuous support. I also express gratitude to Trinity Educational Institute and my colleague Dr. Gaurav Kumar (Prof. Lords University) for their valuable assistance during manuscript preparation.

REFERENCE:

1. Mandal M, Sarkar M, Khan A, Biswas M, Masi A, Rakwal R. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) in plants: maintenance of structural individuality and functional blend. Adv Redox Res. 2022; 5:100039.

2. Young IS, Woodside JV. Antioxidants in health and disease. J Clin Pathol. 2001; 54:176–186.

3. Abdollahi M, Ranjbar A, Shadnia S, Nikfar S, Rezaiee A. Pesticides and oxidative stress: a review. Med Sci Monit. 2004; 10(6): RA141–RA147.

4. Baynes JW, Thorpe SR. Role of oxidative stress in diabetic complications: a new perspective on an old paradigm. Diabetes. 1999; 48(1): 1–9.

5. Khan SR, Pearle MS, Robertson WG. Kidney stones. Nat Rev Dis Primers. 2016; 2:16008.

6. Butterweck V, Khan SR. Herbal medicines in the management of urolithiasis: alternative or complementary? Planta Med. 2009; 75(10): 1095–1103.

7. Saha S, Verma RJ. Inhibition of calcium oxalate crystallization in vitro by plant extracts. Urol Res. 2013; 41:203–210.

8. Patel PK, Patel MA, Saralai MG. In vitro screening of antiurolithiatic activity of medicinal plants. J Pharmacogn Phytochem. 2017; 6(4):137–142.

9. Soofiniya Y. Hypolipidemic and hypoglycemic effects of aerial part of Cynara scolymus in streptozotocin-induced diabetic rats. J Med Plants Res. 2011; 5(13): 2717–2723.

10. Valko M, Leibfritz D, Moncol J, Cronin MT, Mazur M, Telser J. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol. 2007; 39(1): 44–84.

11. Aslam M, Jafri MA, Javed K, Surendra S. Plant drug with hypoglycemic activity. Glimpses Plant Res. 1998; 12: 271–299.

12. Mirhoseini M, Baradaran A, Rafieian-Kopaei M. Medicinal plants, diabetes mellitus, and urgent needs. J Herb Med Pharmacol. 2013; 2(2): 11–17.

13. Munck A, Guyre PM, Holbrook NJ. Physiological functions of glucocorticoids in stress and their relation to pharmacological actions. Endocr Rev. 1984; 5(1): 25–44.

14. Katewa SS, Chaudhary BL, Jain A. Folk herbal medicines from tribal area of Rajasthan, India. J Ethnopharmacol. 2004; 92(1): 41–46.

15. Blois MS. Antioxidant determinations by the use of a stable free radical. Nature. 1958; 181(4617): 1199–1201.

16. Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem. 1971; 44(1): 276–287.

17. Bagdi P, Rathore V, Kumar G. Phytochemical investigation of extracts of Ziziphus nummularia (Burm. f.) Wight & Arn. leaves and fruits. Pharm Chem J. 2016; 3(4): 220–226.

18. Kumar S, Singh R. Antioxidant activity of medicinal plants: a comparative study. J Pharmacogn. 2020; 12(3): 45–52.

19. Sharma P, Rao M. Anti-urolithiatic activity of plant extracts in experimental models. Phytomedicine. 2019; 58: 152876.

20. Joshi V, Patel R. Calcium oxalate crystallization inhibition by herbal extracts. J Ethnopharmacol. 2021; 276: 113986.

21. Singh A, Kumar N. Phytochemical evaluation of Ziziphus nummularia leaves and fruits. Int J Pharmacogn. 2020; 7(4): 102–110.

22. Verma D, Gupta S. Anti-urolithiatic potential of traditional medicinal plants: a review. J Herb Med. 2021; 28: 100455.

23. Li X, Zhang Y, Liu Z, Kumar S, Singh R, Wang H. In vitro anti-urolithiatic activity of fruit extracts: a mechanistic study. Phytother Res. 2022; 36(7): 3170–3182.

24. Chen Y, Li J, Patel A, Singh R, Sharma P, Gupta N. Comparative antioxidant activity of leaf vs fruit extracts in medicinal plants. J Funct Foods. 2023; 102: 105483.

25. Ahmad T, Khan M. Calcium oxalate nucleation inhibition by polyphenols: an in vitro study. Biomed Pharmacother. 2021; 137: 111343.

26. Wang L, Li Q, Zhao Y, Chen H, Wu J, Sun X, Zhang Y. Role of flavonoids in kidney stone prevention: an in vitro approach. Molecules. 2020; 25(21): 5034.

27. Agarwal A, Tandon S, Singla SK, Tandon C. Role of polyphenols in the prevention of calcium oxalate crystallization: an in vitro evaluation. Urol Res. 2013; 41(3): 193–199.

Received on 29.01.2026 Revised on 03.03.2026

Accepted on 28.03.2026 Published on 08.07.2026

Available online from July 13, 2026

Res. J. Pharmacognosy and Phytochem. 2026; 18(3):228-232.

DOI: 10.52711/0975-4385.2026.00032

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

S. No.	Sample Name	IC₅₀ Value ꭒg/ml
1	Ascorbic acid (AA)	27.94
2	Ziziphus nummularia Leaves Soxhlet (ZNLS)	123.33
3	Ziziphus nummularia Fruits Soxhlet (ZNFS)	170.07