Assessment of potential antiurolithiatic property of Carissa carandas Linn. leaves on zinc disc insertion -incited urolithiasis in wistar male albino rats

The goal of the research was to assess the antiurolithiatic property of Carissa carandas Linn. leaf extract in rats. The rats were segregated into 6 groups of 6 rats each. Calcium oxalate urolithiasis was surgically incited by insertions of pre-weighed and sterile zinc disc in bladders of animals. This was also followed by supplementing 0.75% v/v ethane-1, 2-diol (Ethylene glycol) in quaf ing water ad libitum for 28 days. Upon postsurgical recovery period (3 days), Cystone (750 mg/kg) and three doses of EELCC (Ethanolic extract of leaves of Carissa carandas Linn.) namely 100, 200, and 400 mg/kg b.w., were given to zinc disc inserted animals for the duration of 28 days by oral route. Antiurolithiatic property was assessed by measuring the urinary volume, weight of the calculi, estimating the pH and analyzing the proportion of diverse biological markers in urine and serum specimens. An outstanding reduction in urine output and pH were noticed in zinc disc inserted rats, which were intercepted by the remedial extract. The extract also produced a signi icant enhancement in rate of glomerular iltration (GFR) and reduced the calculi deposition throughout the inserted zinc disc. The elevated levels of serum and urinary biochemical parameters like creatinine, urea, calcium, blood urea nitrogen (BUN), oxalate, and uric acid were also prevented by the extract. A signi icant (P< 0.01) potential antiurolithiatic property is observed at 400 mg/kg of EELCC.


INTRODUCTION
Urolithiasis is a typical urinary disorder that indicates calculi emanating in any place in the renal system, which are the kidneys' and bladder. It is in luencing almost 12% of the world population with a noticeable recurrence after an aciurgy dislodge (Lenin et al., 2001) which demands an emergency requisite for proxy remedy. Surgical insertion of foreign substance such as zinc disc in the animal's bladder, eventually causes acquisition of a calculus throughout the zinc disc. After insertion of a zinc disc, if ethane-1,2-diol (Ethylene glycol) is given orally to the rats with the crystals growing throughout the insert mostly constitute CaOx (Khan, 1997). Other than zinc discs, some of the other substances such as plastic discs, CaOx crystals, and non-absorbable surgical thread pieces can also be inserted in the bladder to incite the urolithiasis (Pawar and Vyawahare, 2016). The plant Carissa carandas (Family: Apocynaceae) is asserted to be bene icial for diverse indisposition; however, its anti-urolithiatic potential has not been validated scienti ically. Hence, the current research was planned to assess the potential antiurolithiatic property of Carissa carandas Linn. leaves on zinc disc insertion-incited urolithiasis in wistar male albino rats.

Chemicals (AR Grade)
Ethylene glycol (Ethane-1,2-diol) and all other chemicals, and miscellaneous biochemistry analyzing kits for assessment of serum and urinary biochemical parameters were acquired from Merck Life Science Pvt. Ltd., Nellore, India. Cystone (Himalaya Drug Company, Bangalore, India) was acquired from the Apollo Pharmacy, Nellore.  (Hati et al., 2014). The dried, crude concentrated extract (50 g, dark brown semi-solid, yield 12.5% w/w) was labeled as EELCC.

Phytochemical testing
The EELCC was treated to qualitative testing of the diverse phyto-constituents by grade procedures (Rajaram et al., 2013).

Ethical clearance (Before the inception of the research)
The protocol of research was endorsed by the IAEC (Institutional Animal Ethics Committee) with endorse no: IAEC/XIII/03/RIPER/2019 and following general rules of the CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) with reg. no: 878/PO/Re/S/05/CPCSEA), New Delhi, India.

Experimental animals
A total of 36 healthy (Seven to eight week-old) wistar male albino rats, weighing 200-250 g, were procured from Adita Biosys Private Limited, Bangalore (Reg. no: 1868/PO/Bt/S/16/CPCSEA) and permitted to get habituated for a week. The rats were housed in washed PP (Polypropylene) cages containing sterile paddy husk as bedding at room temperature (26 ± 2 o C), humidity (45-55%), light intensity (325 lux) with 12 h dark-12 h light sequence throughout the research duration and were bestowed with quality chow and RO water ad libitum.

Acute toxicity investigation
No separate toxicity examination for the curative agent was done since the ethanolic extract was found to be safe as per OECD (Organization for Economic Co-operation and Development) guidelines 423 (Shamim, 2014).

Selection of dose
As the boundary dose did not exhibit signs of toxicity, at the doses of 100, 200 and 400 mg/kg, p.o., which were 1/20 th , 1/10 th and 1/5 th of 2000 mg/kg respectively was taken up for the research. Cystone in the dose of 750 mg/kg was taken as standard drug.

Extract suspension preparation
Precisely weighed quantity of plant extract was dissolved in Sodium carboxymethyl cellulose (NaCMC) to prepare required suspension before administration (Two gram of plant extract was dissolved in 14 mL of 0.5% (w/v) NaCMC; 14 mL = 2000 mg of extract; 1 mL = 143 mg; 1 unit = 3.6 mg).

Zinc disc insertion incited urolithiasis in wistar male rats (Preventive regimen)
Urinary CaOx calculi were incited by surgical insertions of pre-weighed and sterile zinc disc in the bladders of animals. This was also followed by supplementing 0.75% v/v ethane-1,2-diol (Ethylene glycol) in quaf ing water ad libitum for 28 days (Revathy et al., 2016). The treatment schedule was planned as follows,

Body weight
The body weight of each rat was measured during the experimental period, once before and after the treatment.

Urine collection and examination
For this purpose the rats were retained on fasting for   24 h, on 29 th day of the research, rats were placed in separate diuresis cages, hydration with 5 mL (Every 6 h) of RO water and urine of 24 h was collected. Urinary volume was noted, and urinary pH was determined (Kumar et al., 2016). Urine was investigated for Creatinine, Calcium, Urea, Oxalate, Uric acid, and Blood urea nitrogen (BUN) content.

Urinary volume
On 29 th day of the experiment, rats were accommodated in individual diuresis cages for 24 hours and complete volume of urine was calculated by using the graduated cylinder and described in millilitres (Mariappan et al., 2016).

Urinary pH
The pH of animal's urine was analysed on 29 th day of the experiment using the pH meter (Rabie and Abdel-Halim, 2005).

Microscopic studies
On 29 th day of the experiment, urine microscopy of all the animals were done. Microscopic examination should be performed on centrifuged sample (Kaur et al., 2009).

Serum collection and examination
On the 29 th day of research, 1 mL of blood was drained from retro-orbital by anaesthetic conditions (Chinnala et al., 2013). Serum was segregated by centrifugation process at 15000 rpm for 20 minutes and investigated for Creatinine, Calcium, Oxalate, Blood urea nitrogen (BUN), Urea, and Uric acid content.

Radiographical examination
Radiography must be utilised to easily notice the  augmentation of deposition throughout the inserted zinc disc in the bladder. Radiographical investigation was done prior to immolating the rats to prove the development of calculi by digital X-ray instrument (Vargas et al., 1999).

Kidney and bladder weight
After urine and blood collection, all rats were immolated by the euthanasia practice of cervical dislocation; the stomach was cut unravel to carefully excise the couple kidneys and bladder, then weighed.

Weight of bladder calculi
After urine collection period, the weight of bladder calculi reconciled by immolating the rats by cervical dislocation. The bladders were enacted and zinc disc along with the cohered crystals were dislodged and packed in individual polyethylene bags.

Statistical study
All the data were speci ied as mean ± SEM of 6 rats (n=6). Data investigation was carried out by using software of GraphPad Prism (Version 8.0). The data were investigated by using one-way ANOVA proceeded by Dunnett's test (Multiple comparison) by using GraphPad Instat (Version 3.0) and P<0.05 value is contemplated as statistical signi icance.

RESULTS AND DISCUSSION
Phytochemical testing revealed the presence of tannins, lavonoids, saponins, triterpenes, phytosterols and phenols in the ethanolic extract of Carissa carandas L. leaves. In the oral acute toxicity investigation, EELCC was found to be safe as it did not cause any mortality or lethality or toxic reactions up to 2000 mg/kg. The surgical implantation of zinc disc along with 0.75% v/v ethane-1,2-diol (Ethylene glycol) in quaf ing water, induced urolithiasis caused a significant (P< 0.01) day to day (Fan et al., 1999) reduction in weight of body (g), decrease in 24 h urinary volume (mL) and pH (Khan et al., 1982) in lithiatic control when distinguished from normal control. The above changes were signi icance (P< 0.01) prevented in the EELCC at the doses of 100, 200, and 400 mg/kg of preventive treated groups in a dose wise mode when distinguished from lithiatic control (Table 1). Furthermore, the EELCC at the dose of 400 mg/kg displayed a greater signi icant (P< 0.01) in preventing the body weight reduction, increase in urinary volume and preventing the shift of pH from alkaline to acidic than Cystone (750 mg/kg).

Consequence of EELCC on kidney, bladder and calculi weight
The surgical implantation of zinc disc along with 0.75% v/v ethane-1,2-diol (Ethylene glycol) in quaf ing water, induced urolithiasis caused a significant (P< 0.01) day to day, increase in (Eldin et al., 2008;Saha and Verma, 2015) weight of both (left and right) kidneys (g), weight of bladder (mg), and enormous (Perez-Hernadez et al., 2018) amount of stone (mg) formation hence increase content in the bladder in a lithiatic control when distinguished from normal control. The above changes were signi icantly (P< 0.01) prevented in the EELCC at the doses of 100, 200, and 400 mg/kg of preventive treated groups in a dose wise mode when distinguished from lithiatic control (Table 2 and Figure 3). Furthermore, the EELCC at the dose of 400 mg/kg displayed a greater signi icance (P< 0.01) in the prevention of increase in weight of kidneys and bladders, and decreased the building of accumulates throughout the zinc disc, hence decrease content in the bladder than Cystone (750 mg/kg).

Consequence of EELCC on serum and urinary biochemical parameters
The surgical implantation of zinc disc along with 0.75% v/v ethane-1,2-diol (Ethylene glycol) in quaf ing water, incited urolithiasis produced a signi icant (P< 0.01) upraise of diverse serum and urinary markers namely Creatinine, Oxalate, Uric acid, Calcium, Urea, and Blood urea nitrogen (BUN) in lithiatic control when distinguished from normal control (Dinnimath et al., 2017). The treatment groups of EELCC at the doses of 100, 200, and 400 mg/kg signi icance (P< 0.01) reverted the alterations of serum and urinary markers in a dose wise mode when distinguished from lithiatic control (Tables 3 and 4). The preventive treatment groups of the EELCC at the dose of 400 mg/kg dis-played a greater signi icant (P< 0.01) in reverting the alterations of serum and urinary markers and restored them to near normal value than Cystone (750 mg/kg).

Consequence of EELCC on microscopic studies of 24 h urine
Urine microscopy analysis of 24 h urine revealed the number of CaOx crystals, which is greater in the urine of lithiatic control as distinguished from normal control (Ragini and Padala, 2014). The treatment groups of EELCC at the doses of 100, 200, and 400 mg/kg showed signi icant (P< 0.01) reduction in number of CaOx crystals in a dose wise mode when distinguished from lithiatic control (Figure 2). Furthermore, the EELCC in a dose of 400 mg/kg showed no presence of calcium oxalate crystals when compared with Cystone (750 mg/kg).

Consequence of EELCC on stone formation by radiographs
The digital radiographs of animal's divulged appearance of CaOx crystals accumulates throughout the inserted zinc disc within 28 days, thereafter insertion in the lithiatic control (Vyas and Argal, 2013). The treatment groups of EELCC at the doses of 100, 200, and 400 mg/kg signi icantly (P< 0.01) decreased the formation of such deposits around the implanted disc when compared with lithiatic control group in a dose wise mode (Figure 1). Furthermore, the EELCC in a dose of 400 mg/kg signi icantly (P< 0.01) reduced the formation of CaOx crystal deposits around the implanted disc than Cystone (750 mg/kg). These indings were proved in actual calculi weights of the bladder ( Table 2).

Consequence of EELCC on microscopic examination of bladder tissue
Histopathology of normal control rats by section study of the bladder microscopy showed normal morphology. There were marked histological changes such as structural modi ication in the epithelial cells, tissue damage, in lammation and hyperplasia in lithiatic rats (Malipeddi and Das, 2016). However, bladder section of rats treated with EELCC at the doses of 100, 200, and 400 mg/kg showed improvement of the above noticed histological changes when distinguished from lithiatic control (Figure 4). Whereas, the EELCC treated rats (Preventive: 400 mg/kg) apparently showed a retained normal morphology and healing tissue, regenerative changes and healthy epithelial cells as similar to normal control than Cystone (750 mg/kg).

CONCLUSIONS
The feasible mechanisms underlying in this property is communicated jointly by antioxidant, diuretic, free-radical scavenging, and antiin lammatory properties of Carissa carandas. These indings can be applied to modern system of the medicine by incorporating it into a dosage form which will make it more acceptable for patients. This research projects the therapeutic potential of the ethanolic extract of leaves of Carissa carandas Linn. that may be developed as an alternative polyherbal anti-urolithiatic drug like Cystone.

ACKNOWLEDGEMENT
The irst author wishes to express his deepest gratitude to the Management and Dr. M. Gobinath, M. Pharm., Ph.D., Principal, Ratnam Institute of Pharmacy, Nellore, Andhra Pradesh, India, for bestowing all the necessary laboratory needs of the research and their constant support. The irst author is also grateful to Ms. N. Deepika, Asst. Professor, Dept. of Pharmacology, Joginpally B.R. Pharmacy College, Hyderabad, Telangana, India, for her valuable help and support.

Financial support and sponsorship
Nil.

Ethical approval
Endorsed by the IAEC (Institutional Animal Ethics Committee).