1. INTRODUCTION:

Because of its high frequency and likelihood of recurrence, urolithiasis has become a complex disease. Calcium oxalate (CaOX) accounts for up to 80% of the stones examined¹. Kidney stone formation is a complicated process that involves a number of physicochemical processes, including as supersaturation, nucleation, growth accumulation, and retention inside the renal tubules². Specific drugs, such as thiazide diuretics and alkali citrate, are used in urolithiasis treatment but less compelling is the clinical evidence for their effectiveness³. Endoscopic stone removal is the most common treatment for urolithiasis today, and procedures like extracorporeal shock wave lithotripsy (ESWL) have revolutionized urolithiasis treatment but do not eradicate the danger of new stone formation⁴.

Which cause side effects such as haemorrhage, hypertension, tubular necrosis, subsequent fibrosis of the kidneys and also increase stone recurrence? But⁵ they are very costly. Hence it is important to find an alternative to these methods by using medicinal plants or phytotherapy. Since ancient times, a significant number of herbs have been used in India to treat urinary stones⁶. Basil is well-known for its traditional therapeutic properties, and it is allowed in a number of countries⁷.

Ocimum basilicum L. has traditionally been used for analgesic and anti-inflammatory activity⁸, hypoglycemic and hepato-protective activity⁹, anti-hyperlipidemic and anti-ulcerative activity¹⁰, cardioprotective and stimulant activity¹¹, sedative, hypnotic, and anticonvulsant activity¹², memory retention and anti-stroke activity¹³, Antimicrobial Activity¹⁴, Antimycobacterial and Antiviral Activity¹⁵, Antioxidant Activity¹⁶, Effect on Testicular Toxicity and Fertility¹⁷. There has been recent activity reported. However, no scientific investigation on the antiurolithiatic properties of Ocimum basilicum L. seeds has been published to far. As a result, the goal of this study is to prove the scientific validity of the antiurolithiatic activity of seeds of Ocimum basilicum L. hydroalcoholic extract in ethylene glycol-induced urolithiasis.

2. MATERIALS AND METHODS:

2.1. Plant material and extraction:

The plant material (seeds of O. basilicum)was collected from nearby tribal area and Dr. A.S.Reddy, Sardar Patel University, Gujarat, India, taxonomically identified, authenticated and certified the plant materials and sample of plant material was deposited in the herbarium as a vouched specimen for future reference. In a Soxhlet extractor ^18-19, continuous hot extraction was carried out using hydroalcoholic solvent. Hydroalcoholic extract (HOB) was prepared and used for further animal experiments.

2.2. Animals and ethical consideration:

Adult Male Wistar rats weighing 180-225 g were used and kept at the animal house, under controlled standard condition. The study protocol was approved by the Institutional Animal Ethical Committee of Indukaka Ipcowala College of Pharmacy, Gujarat, India. (Protocol No. CPCSEA/IAEC/IICP/2019/01).

2.3.Antiurolithiatic Activity:

In EG model animals were divided into five groups of 6 animals in each. Group I (Normal Group) animals received regular food and drinking water throughout the study duration and remaining Group II, III, IV and V received calculi inducing agent EG (0.75% w/v) in drinking water for 28th days. Group III, IV and V rats were received HOB (250 mg/kg), (400 mg/kg), and Cystone (750 mg/kg) from 1^st to 28^thday respectively. On last day of experiment (29^thday) collect urine, serum and isolate kidneys for estimation of biochemical parameters andhistopathology.

2.4.Biochemical parameter estimation:

On the 29^thday, animals were kept in individual metabolic cages and 24 h urine samples were collected. Blood was collected under light aesthetic condition from retro orbital plexus. It centrifuged at 15000 RPM for 25 minutes separated the Serum. Rats were sacrificed by cervical decapitation. Each animal's abdomen was sliced open to separate both kidneys²⁰.Using widely available coral clinical device kits and an analytical approach, various biochemical parameters were determined. Urolithiasis inhibitors (magnesium), urolithiasis promoters (calcium, oxalate, uric acid, and inorganic phosphate)²¹, serum creatinine²², serum uric acid²³, blood urea nitrogen (BUN)²⁴, serum urea²⁵, and antioxidant parameters (MDA, GSH, SOD, and catalase)²⁶ are the parameters that are measured.

2.5. Histopathology examination:

Kidney was fixed with 10% v/v neutral formalin solution and sent to histology examination.²⁷

2.6. Statistical Evaluation:

The results among the groups were analysed by one-way ANOVA followed by Dunnett's test using Graph pad Prism version 6. Results were considered significant when the value of p < 0.05.

3. RESULTS:

3.1. Physical andurinary parameters:

When compared to normal, only EG had a substantial (P <0.05) drop in body weight, PH, and urine volume. When compared to the similar results of the normal control group, an increase in kidney weight was detected. The animals given Cystone, HOB (250, 500 mg/kg) had substantial increases in body weight, PH, and urine volume, as well as a significant decrease (P<0.05) in kidney weight. HOB -500 had a response that was virtually identical to Cystone, the usual medication. [Table-1].

EG treatment increased uric acid, calcium, phosphorus, and oxalate levels in urine substantially (p<0.05), but creatinine clearance and magnesium levels were lower than in the normal control group [Table-1]. When compared to the lithiatic control group, HOB treatment (250, 500 mg/kg) significantly improved dose-dependent creatinine clearance and magnesium levels, as well as significant reductions in uric acid, phosphorus, calcium, and oxalate levels (P< 0.05 in all cases).

Table 1: Effect of hydro-alcoholic extract of Ocimumbasilicum L. on various physical and urine parameters in ethylene glycol induced urolithiasis

No	Parameters	GP I Normal Control	GP II EG Treated	GP III HOB (250mg/Kg)	GP IV HOB (500mg/Kg)	GP V Cystone (750 Mg/Kg)
1	Body weight (gm)	225.0 ± 0.66 1.438	204.0 ± 1.43*	220.2 ± 2.10**	221.3 ± 1.83**	223.2 ± 1.88**
2	Kidney weight (gm)	0.962 ±0.019	1.454 ± 0.07*	1.059 ± 0.03**	0.980 ± 0.007**	0.9863 ± 0.01**
3	PH	8.000 ± 0.02	6.097 ± 0 .10*	7.467 ± 0.16**	7.633 ± 0.06**	7.643 ± 0.08**
4	Urine volume(ml/24hrs)	8.850 ± 0.25	6.017 ± 0.26*	8.900 ± 0.14**	9.117 ± 0.26**	9.667 ± 0.14**
5	Calcium (mg/24 hr)	5.16 ± 0.37	11.82 ± 0.17*	7.83 ± 0.23* **	5.83 ± 0.14**	5.74 ± 0.22**
6	Cretinine(mg/24 hr)	1.19 ± 0.02	0.318 ± 0.02*	1.08 ± 0.03* **	1.12 ± 0.01**	1.15 ± 0.02**
7	Cretinine(mg/24 hr)	1.19 ± 0.02	0.318 ± 0.02*	1.08 ± 0.03* **	1.12 ± 0.01**	1.15 ± 0.02**
8	Magnesium(mg/24 hr)	2.49 ± 0.08	1.45 ± 0.20*	2.08 ± 0.02* **	2.15 ± 0.03**	2.26 ± 0.04**
9	Oxalate (mg/24hrs)	4.15 ± 0.17	7.45 ± 0.18*	5.15 ± 0.21* **	4.66 ± 0.16**	4.35 ± 0.21**
10	Phosphorus(mg/24 hr)	4.78 ± 0.11	8.45 ± 0.16*	5.68 ± 0.18* **	5.45 ± 0.35**	5.13 ± 0.16**

Mean ± S.E.M. Number of animals in each group = 6.*p<0.05compared to normal control group, ** p<0.05 when compared with EG treated group by one way ANOVA followed by Dunnet test. SEM: Standard error of mean. ANOVA: Analysis of variance.

Table 2: Effect of hydro-alcoholic extract of Ocimumbasilicum L. Linn. Seeds on various serum and anti-oxidant parameters in ethylene glycol induced urolithiasis.

Sr. No	Parameters	GP I Normal Control	GP II EG Treated	GP III HOB (250mg/Kg)	GP IV HOB (500mg/Kg)	GP V Cystone (750 Mg/Kg)
1	Urea(mg/dl)	20.46 ± 0.64	33.24 ± 0.11*	22.28 ± 0.31* **	21.59 ± 0.31**	18.99 ± 0.41**
2	Uric acid (mg/dl)	7.08 ± 0.038	10.30 ± 0.15*	7.71 ± 0.16**	7.48 ± 0.33**	6.72 ± 0.21**
3	Creatinine (mg/dl)	0.67 ± 0.00	1.54 ± 0.13*	0.86 ± 0.03**	0.76 ± 0.04**	0.76 ± 0.03**
4	Calcium (mg/dl)	7.19 ± 0.36	13.19 ± 0.30*	10.16 ± 0.12* **	8.00 ± 0.31**	7.44 ± 0.13**
5	Magnesium (mg/dl)	3.87 ± 0.06	1.48 ± 0.06*	3.67 ± 0.15**	3.62 ± 0.11**	3.79 ± 0.05**
6	Phosphorus (mg/dl)	3.36 ± 0.11	6.94 ± 0.02*	4.88 ± 0.32* **	3.95 ± 0.15**	3.67 ± 0.16**
7	MDA(nmol/mg protein)	0.053 ± 0.01	0.379 ± 0.04*	0.14 ± 0.02**	0.13 ± 0.01**	0.074 ± 0.00**
8	GSH(micro mol/mg protein)	0.056 ± 0.00	0.023 ± 0.00*	0.053 ± 0.00**	0.060 ± 0.01**	0.055 ± 0.00**
9	CAT(micro mol/ mg protein)	4.15 ± 0.34	1.27 ± 0.35*	4.74 ± 0.64**	4.36 ± 0.48**	4.63 ± 0.47**
10	SOD(U/mg protein)	39.04 ± 3.67	14.29 ± 1.21*	25.56 ± 2.21**	32.56 ± 2.10**	41.25 ± 41.25**

3.3. Histopathological examination:

In the normal group, the kidney microscopy section revealed normal architecture (Figure 1(a)), whereas in the disease control group, the kidney microscopy section revealed the presence of calcium oxalate crystals, serious damage to the glomeruli, medulla, interstitial spaces, tubules, mononuclear cell infiltration (Figure 1(b)), and major damage to the animal kidney section (figure 1(c )and 1(d)) Section crystal deposition was not found in the Cystone-treated population, and renal damage was almost completely restored (Figure 1) (e).

Figure. 1a) Normal Control

A) Normal Glomerular structure

B) Normal renal tubules

(Figure. 1b) Ethylene Control

A) Marked interstitial congestion

B) Oxalate stone

C and D) tubular damage and degeneration

(Figure.1c) Ethylene Glycol 250 Mg/Kg

A) Glomerular atrophy

B) Tubular dilation

(Figure.1d) Ethylene Glycol 500 Mg/Kg

A) Regenerated to normal Glomerular structure

(Figure. 1e) Cystone 750 Mg/Kg

A) Normal Glomerular structure.

1a:Microscopy of normal control group

1b:Microscopy of ethylene glycol control group.

1c:Microscopy of ethylene glycol + HOB (250mg/kg) group.

1d:Microscopy of ethylene glycol + HOB (500mg/kg) group.

1e:Microscopy of Cystonegroup.

Figure 1: Examination of Histopathology of rat kidney stained with hematoxylin and eosin

4. DISCUSSION:

Because the high structural degree and matrix composition of stones generated in the rats' and human' kidneys are similar, rat models of urolithiasis are useful experimental tools for studying the disease's pathogenesis. Male albino Wistar rats were used in this investigation to investigate for urolithiasis induction because their urinary system mimics that of humans²⁸, and previous research has shown that the amount of stone deposition in male rats is substantially lower than in female rats²⁹. The lower body weight of the lithiatic control group implies EG toxicity, resulting in oxalate build-up in intracellular regions, causing metabolic disturbance and cell damage^30, Intense discomfort can lead to a reduction in food consumption, which can lead to a reduction in body weight. In the illness control group, we also discovered a drop in body weight. However, the usual treatment of Cystone (750mg/kg; p.o.), low dose of HOB (250mg/kg; p.o.), and high dose of HOB (500mg/kg; p.o.) demonstrated good diuretic efficacy and hence clearance of calculi, preventing stone formation and associated pain and weight loss.

The pH of the urine is a major factor in the formation of kidney stones. Low urinary pH reduces the solubility of calcium oxalate stones in urine, which promotes stone formation. The illness group's urinary pH was much lower in this study, implying that calcium oxalate solubility is limited and that urine is super-saturated with oxalates and calcium ions beginning with stone formation. The pH of urine in rats treated with Cystone and HOB was found to be considerably higher, suggesting that HOB treatment increased calcium oxalate stone solubility and decreased the number of ions in urine.

As seen in disease control groups, ethylene glycol consumption causes an increase in promoters such calcium, oxalate, uric acid, and phosphorus and a decrease in inhibitors like magnesium. However, treatment with a conventional medicine and low and high dose test samples considerably reduced promoters and inhibitors in various biological samples. Stones impede urine outflow and cause severe oxalate-induced Nephrotoxicity, resulting in waste nitrogenous in urine and reduced creatinine clearance³¹. Both standard and test therapies, on the other hand, considerably reduced the development of stone and hence reduced kidney damage, as seen by the slowed reduction in urea and creatinine clearance. As a result, administration of a hydroalcoholic extract of ocimum basilicum seeds reduced renal function decline.

When cells in the kidney are exposed to oxalate and/or CaOx crystals, they produce reactive oxygen species (ROS), which causes oxidative stress, which leads to injury and inflammation. Stone formation appears to be influenced by renal damage and inflammation. Due to down-regulated expression of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase, and glucose-6 phosphate dehydrogenase) as well as radical scavengers (vitamin E, ascorbic acid, and reduced glutathione), oxidative stress develops³².

Lipid peroxidation causes oxidative stress, which leads to renal cell injury and inflammation. The retention of calcium oxalate crystals and the formation of stones in renal tubules is facilitated by the loss of membrane integrity. CaOx kidney stone patients have been shown to have malondialdehyde (MDA) in their urine, indicating ROS in their kidneys, according to recent research. MDA excretion in the urine is thought to be a sign of renal epithelial cell injury³³.

Treatment with anti-oxidants and free radical scavengers has been shown to attenuate CaOx crystal-induced kidney damage in recent investigations (hyperoxaluria induced oxidative stress and urolithiasis.) Renal dysfunction and kidney stones can cause oxidative stress, which can be mitigated by the kidney's increased antioxidant enzymes. However, in the disease control group, failure to adjust resulted in cellular damage, as demonstrated by histopathology and a significant increase in lipid peroxidation and a drop in catalase levels.

The administration of both the standard and test medications resulted in a considerable reduction in lipid peroxidation and, as a result, a significant reduction in catalase levels in the kidney, implying efficacy in avoiding oxidative stress-induced damage. As a result, we may conclude that ocimum basilicum seeds are effective in preventing crystal aggregation, which may be due to its diuretic activity's ability to change the levels of promoters and inhibitors.

5. CONCLUSION:

Quantitative analyses of Ocimum basilicum L. extracts reveal the presence of antioxidant Phytochemicals such as flavonoids and polyphenols, which could have resulted in antilithiatic activity by dissolving the stone and acting as an antioxidant. As a result, more research is needed to determine the exact Phytochemicals contents of Ocimum basilicum L. and their mode of action in the prevention of urolithiasis.

6. AUTHORS' CONTRIBUTIONS:

Patel Ravindrakumar K: Principal researcher who did all sample practise and evaluations as well as writing the manuscript. Dr. Sandip Patel and Dr. Niraj Vyas: Guided, supervised, and assisted with statistical interpretation and paper evaluation.

ACKNOWLEDGEMENTS:

The authors are grateful to Indukaka Ipcowala College of Pharmacy, New Vallabh Vidyanagar, for giving this. Dr. Ghodasara, Department of Pathology, Anand Veterinary College, Anand, and Dr. Ghodasara, Department of Pathology, Anand Veterinary College, Anand, for providing histopathological studies services.

CONFLICT OF INTEREST:

The authors do not have any potential conflicts of interest.

Financial Assistance and Sponsorship:

This research got no specific funding from any funding agencies.

REFERENCES:

1. Daudon M. Bader CA. Jungers P. Urinary calculi: review of classification methods and correlations with etiology. Scanning Microsc. 1993 Sep;7(3):1081-104; discussion 1104-6.

2. Laroubi A. Touhami M. Farouk L. Zrara I. Aboufatima R. Benharref A. Chait A. Prophylaxis effect of Trigonellafoenumgraecum L. seeds on renal stone formation in rats. Phytother Res. 2007 Oct;21(10):921-5. (DOI: 10.1002/ptr.2190).

3. Hess B. Pathophysiology. diagnosis and conservative therapy in calcium kidney calculi]. TherUmsch. 2003 Feb;60(2):79-87. German. (DOI: 10.1024/0040-5930.60.2.79).

4. Bashir S. Gilani AH. Antiurolithic effect of Bergenialigulata rhizome: an explanation of the underlying mechanisms. J Ethnopharmacol. 2009 Feb 25;122(1):106-16. (DOI: 10.1016/j.jep.2008.12.004).

5. Begun FP. Knoll CE. Gottlieb M. Lawson RK. Chronic effects of focused electrohydraulic shock waves on renal function and hypertension. J Urol. 1991 Mar;145(3):635-9. (DOI: 10.1016/s0022-5347(17)38410-0).

6. Bahuguna Y. Rawat MS. Juyal V. Gupta V. Antilithiatic effect of flowers of Jasminum auriculatum Vahl. International Journal of Green Pharmacy. 2009; 3(2):155–158. ((DOI: https://doi.org/10.22377/ij)

7. Lawrence BM. A review of the world production of essential oil. Perfumes and Flavors. 1985; 1-10.

8. El-Beshbishy H. Bahashwan S. Hypoglycemic effect of basil (Ocimumbasilicum) aqueous extract is mediated through inhibition of α-glucosidase and α-amylase activities: an in vitro study. ToxicolInd Health. 2012 Feb;28(1):42-50. (DOI: 10.1177/0748233711403193).

9. Jang MH. Piao XL. Kim JM. Kwon SW. Park JH. Inhibition of cholinesterase and amyloid-beta aggregation by resveratrol oligomers from Vitisamurensis. Phytother Res. 2008 Apr;22(4):544-9. (DOI: 10.1002/ptr.2406).

10. Fathiazad F. Matlobi A. Khorrami A. Hamedeyazdan S. Soraya H. Hammami M. Maleki-Dizaji N. Garjani A. Phytochemical screening and evaluation of cardioprotective activity of ethanolic extract of Ocimumbasilicum L. (basil) against isoproterenol induced myocardial infarction in rats. Daru. 2012 Dec 5;20(1):87. (DOI: 10.1186/2008-2231-20-87).

11. Satoh T. Sugawara Y. Effects on humans elicited by inhaling the fragrance of essential oils: sensory test. multi-channel thermometric study and forehead surface potential wave measurement on basil and peppermint. Anal Sci. 2003 Jan;19(1):139-46. (DOI: 10.2116/analsci.19.139).

12. Sarahroodi S. Esmaeili S. Mikaili P. Hemmati Z. Saberi Y. The effects of green Ocimumbasilicumhydroalcoholic extract on retention and retrieval of memory in mice. AncSci Life. 2012 Apr;31(4):185-9. (DOI: 10.4103/0257-7941.107354).

13. Opalchenova G. Obreshkova D. Comparative studies on the activity of basil--an essential oil from Ocimumbasilicum L.--against multidrug resistant clinical isolates of the genera Staphylococcus. Enterococcus and Pseudomonas by using different test methods. J Microbiol Methods. 2003 Jul;54(1):105-10. (DOI: 10.1016/s0167-7012(03)00012-5).

14. Siddiqui BS. Bhatti HA. Begum S. Perwaiz S. Evaluation of the antimycobacterium activity of the constituents from Ocimumbasilicum against Mycobacterium tuberculosis. J Ethnopharmacol. 2012 Oct 31;144(1):220-2. (DOI: 10.1016/j.jep.2012.08.003).

15. Rasul A. Akhtar N. Formulation and in vivo evaluation for anti-aging effects of an emulsion containing basil extract using non- invasive biophysical techniques. Daru. 2011;19(5):344-50.

16. Ch M. Naz S. Sharif A. Akram M. Saeed M. Biological and Pharmacological Properties of the Sweet Basil (Ocimum basilicum). Br J Pharm Res2015; 7(5):330–339. ((DOI:10.9734/BJPR/2015/16505)

17. Harborne JB. Phytochemicals Methods; A Guide to Modern techniques of plant analysis. 3rd Edition. Springer International. 1978; 7: 85.

18. Mukherjee PK. Quality Control of Herbal Drugs. 1st Edition. Business Horizone. Pharmaceutical Publishers. 2002; 380-422.

19. Karadi RV. Gadge NB. Alagawadi KR. Savadi RV. Effect of Moringaoleifera Lam. root-wood on ethylene glycol induced urolithiasis in rats. J Ethnopharmacol. 2006 Apr 21;105(1-2):306-11. (DOI: 10.1016/j.jep.2005.11.004).

20. Hodgkinsons A: Oxalic Acid in Biology and Medicine. London: Academic Press; 1977. P. 1–325.

21. Bandara T. Rokeya B. Khan S. Ali L. Ekanayake S. Jansz ER. Balasubramanium K. Effects of Gymnema lactiferum leaves on glycemic and lipidemic status in type 2 diabetes subjects. Bangladesh Journal of Pharmacology. 2009; 4(2):92–95. ((DOI: https://(DOI.org/10.3329/bjp.v4i2.1195)

22. CHANEY AL. MARBACH EP. Modified reagents for determination of urea and ammonia. Clin Chem. 1962 Apr;8:130-2.

23. FAWCETT JK. SCOTT JE. A rapid and precise method for the determination of urea. J ClinPathol. 1960 Mar;13(2):156-9. (DOI: 10.1136/jcp.13.2.156).

24. Jena AK. Vasisht K. Sharma N. Kaur R. Dhingra MS. Karan M. Amelioration of testosterone induced benign prostatic hyperplasia by Prunus species. J Ethnopharmacol. 2016 Aug 22;190:33-45. (DOI: 10.1016/j.jep.2016.05.052).

25. Gong P. Chen F. Liu X. Gong X. Wang J. Ma Y. Protective effect of caffeic acid phenethyl ester against cadmium-induced renal damage in mice. J Toxicol Sci. 2012;37(2):415-25. (DOI: 10.2131/jts.37.415).

26. Patel VB. Acharya N. Effect of Macrotylomauniflorum in ethylene glycol induced urolithiasis in rats. Heliyon. 2020 Jun 26;6(6):e04253. (DOI: 10.1016/j.heliyon.2020).

27. Karadi RV. Gadge NB. Alagawadi KR. Savadi RV. Effect of Moringaoleifera Lam. root-wood on ethylene glycol induced urolithiasis in rats. J Ethnopharmacol. 2006 Apr 21;105(1-2):306-11. (DOI: 10.1016/j.jep.2005.11.004).

28. Bouanani S. Henchiri C. Migianu-Griffoni E. Aouf N. Lecouvey M. Pharmacological and toxicological effects of Paronychia argentea in experimental calcium oxalate nephrolithiasis in rats. J Ethnopharmacol. 2010 May 4;129(1):38-45. (DOI: 10.1016/j.jep.2010.01.056).

29. Brent J. Current management of ethylene glycol poisoning. Drugs. 2001;61(7):979-88. (DOI: 10.2165/00003495-200161070-00006).

30. Selvam R. Calcium oxalate stone disease: role of lipid peroxidation and antioxidants. Urol Res. 2002 Mar;30(1):35-47. (DOI: 10.1007/s00240-001-0228-z).

31. Khan SR. Hyperoxaluria-induced oxidative stress and antioxidants for renal protection. Urol Res. 2005 Nov;33(5):349-57. (DOI: 10.1007/s00240-005-0492-4).

32. Selvam R. Kalaiselvi P. Govindaraj A. BalaMurugan V. Sathish Kumar AS. Effect of A. lanata leaf extract and Vediuppuchunnam on the urinary risk factors of calcium oxalate urolithiasis during experimental hyperoxaluria. Pharmacol Res. 2001 Jan;43(1):89-93. (DOI: 10.1006/phrs.2000.0745).

Received on 28.10.2021 Modified on 16.04.2022

Research J. Pharm. and Tech 2023; 16(7):3367-3372.

DOI: 10.52711/0974-360X.2023.00557