Targeting the antiurolithiatic activity by entrapping the bioactive compounds of Asparagus racemosus in chitosan nanoparticles on ethylene glycol induced renal calculi in Wister albino rats

The objective of our research is to investigate the antiurolithiatic intervention of bioactive compounds of Asparagus racemosus loaded Chitosan nanoparticles (BACARNPs) on ethylene glycol engendered renal calculi in male Wister rats. The ef iciency of bioactive compounds of A. racemosus (BACAR) at 800 mg/kg p.o and BACARNPs at 800 mg equivalent weight of BACAR/kg p.owas validated in ethylene glycol 0.75% (v/v) and ammonium chloride 1% (w/v) mediated renal calculi in rats. Cystone (750 mg/kg, p.o.) has been used as a standard drug. Urinary variables comprise calcium, magnesium, oxalate, phosphate, uric acid, creatinine, urine pH, urine volume, and Creatinine clearance; Serum parameters include creatinine, blood urea nitrogen (BUN), calcium and uric acid; calcium and oxalate deposition in the kidney were assessed. In vivo antioxidant parameters include lipid peroxidation, superoxide dismutase, catalase, and glutathione were determined and histopathological studies were also examined. In both control groups, a substantial increase in urinary excretion of calcium, oxalate, and their intensi ication in the kidney; enhanced amounts of phosphate, uric acid, and reduced magnesium levels in urine; elevated serum creatinine, BUN, calcium and uric acid; Creatinine clearance was declined were observed and normalized in treated groups. In vivo antioxidant parameters and histopathological variations reinstated to conventional form. Chitosan serves as a ligand to renal epithelial cells leads to improved agglomerationof BACAR in kidney compared to BACAR administered solitarily results in increased antiurolithiatic activity.


INTRODUCTION
Renal calculi, the third customary disorders of the kidney system. This is a multifarious illness that enables the development of calculi in every portion of the urinary tract, includes the kidneys, ureter, bladder, and urethra. Generally males are more affected than females, predominantly between 20-50 years of age with the recurrence frequency of 70-80% in males and 40-50% in females (Ghosh et al., 2000).
The compilation of calculi involves multiple elemen-tary events from supersaturation of urine, crystallization of constituents, aggregation, and retaining in the urine. The majority of the calculi i.e., 80% of calculi are of calcium oxalate or in collaboration with the calcium phosphate and 15% of calculi are of infection or struvite calculi and 5-10% are of uric acid calculi (Vyas et al., 2011). Investigation have also evident that the renal cell damage serves as a crystal binding site and encourages the emergence and aggregation of CaOx crystal in the renal tubular structures.
Multiple treatment strategies are employed to monitor urinary calculi conditions depends on the calculi size, location, severity of obstruction, kidney function. Extracorporeal shock wave lithotripsy, ureteroscopy, and percutaneous nephrolithotomy have often used methods for the revocation of calculi based on size. These procedures are relatively painful, expensive, increased rate of retreatment and cause undesirable consequences, namely tubular necrosis, hypertension, hemorrhage, and associated renal ibrosis, culminating in renal cell damage, as well as recurrence of calculi.
Treatment with synthetic drugs is based on the calculi type associated with complications and recurrence of calculi (Bergsland et al., 2013). These concepts failed to treat the disorder at the root level responsible for recurrence of the calculi in the future. Therefore, cavity was developed to ill in the interventions of calculi illness, which should meet the demands such as in luencing the cause at the priliminary stage, limiting complications, lowering the incidence of recurrence, and cost effective.
In the present scenario in order to meet out the multiple objectives in calculi treatment, the priority is oriented on herbal remedies. There is a growing interest in the herbal products in the ield of health care because of having con idence and irmly documented in the minds because they are secure, inexpensive, multiple pharmacological activities, lack of risks, and readily accessible. In the case of urolithiasis, they focus on the prevention of early stage calculi initiation by retrieval of severely injured renal cells to normal, which were adversely affected by oxidation of reactive oxygen species (ROS) and behave as a crystal binding sites leading to formation of calculi.
The antioxidant property of bioactive compounds in the extract is effective in preventing the cell injury and diuretic effect hinders the urinary supersaturation which is the key determinant in calculi initiation. Such outstanding features of herbal medication mitigate both the development and recurrence of calculi.
Roots are the therapeutic component of the plant that is extensively used for anti-infective, antioxidant, anti-rheumatic, diuretic, hepatoprotective, antimicrobial, analgesic, antipyretic, anticancer, antidiabetic, and anti-in lammatory effects. Out of diverse phytochemical constituents the steroidal saponins are proved of comprising antioxidant, diuretic, and alkalinizing effects which are potential therapeutic agents in urolithiasis (Joshi et al., 2010).
Bioavailability and targeting of desired bioactive compounds in the extract to the respective site is a crucial move to enhance the pharmacological action to the maximum. Hence recently, nanotechnology has drawn the attention of researchers to augment the treating ef iciency by fabricating green nanoparticles entrapping bioactive compounds of extract essential for pharmacological action. In the current research to reinforce the antiurolithiatic therapy, BACAR was entrapped in Chitosan nanoparticles using low molecular weight Chitosan (40 KDa) which serves as a ligand to the megalin receptors on renal cells resulting in substancial accumulation in the kidney (Yuan et al., 2009).

Chemicals
Analytical grade chemicals (Sigma Aldrich, Merck India Ltd., and Hi-media) procured from Bros Scienti ics, Tirupati, India, were utilized in the current investigation. Cystone, (Himalaya Drug Company, Bangalore, India) was procured from the Apollo pharmacy, Tirupati.

Collection and Preparation of aqueous Extract of Asparagus racemosus
Roots of Asparagus racemosus were accessed from the Sri Srinivasa ayurvedic pharmacy, Tirupati. It was identi ied and authenticated by Dr. K. Madhava chetty, Assistant professor, Department of botany, Sri Venkateswara University, Tirupati. Voucher specimen (voucher No: 0698) were submitted to the research centre. The roots were dried and coarsely  grated. The 100 g of root powder macerated with 1L of distilled water for 48 h at room temperature. The extract was concentrated, and the earned semisolid mass of 13 g was preserved in an airtight container free from moisture, heat, and air and tagged as BACAR.

Preparation of Bioactive compounds of Asparagus racemosus loaded chitosan nanoparticles (BACARNPs)
The BACAR was entrapped in Chitosan nanoparticles by employing the ionic gelation method. The optimization of BACARNPs is accomplished with the use of Box Behnken Design (BBD). BACARNPs were dispensed at a dose equivalent to 800 mg of BACAR

Acute Toxicity Studies
Oral acute toxicity studies were accomplished in following the OECD guidelines 423.

Experimental Design
Rats were categorized into nine groups of six animals each (n = 6). In rats, CaOx calculi were instigated by administering the drinking water consisting of 0.75% (v/v) of ethylene glycol (EG) and 1 % (w/v) of ammonium chloride (AC) for 15 days. The treatment protocol was designed as follows, (Atmani et al., 2003) ,

Group II
Preventive control (EG + AC + vehicle from day 1 to 15)

Group VI
Curative control (EG + AC from day 1 to 15, vehicle from day 16 to 30)

Group IX
Curative BACARNPs (EG + AC from day 1 to 15, BACARNPs Equivalent of 800 mg/kg, orally from day 16 to 30)

Biochemical examination in urine and serum
Urine and blood samples were extracted from all the groups of animals after termination of respective prescribed treatment schedules. Rats were hydrated orally with 6 ml of drinking water, placed in discrete metabolic cages and collected urine for 24 hours. The volume of urine was measured, and pH was explored. Urine was centrifuged at 2,500 rpm at 30 ± 2 • C for 5 min, and the supernatant was estimated for calcium, magnesium, phosphate, uric acid, using ERBA diagnostic kits. Oxalate was quanti ied using the method of Hodgkinson andWilliams, 1972 (Hodgkinson andWilliams, 1972). Blood samples from the retro-orbital venous plexus were retrived and serum was isolated by centrifuging at 1500 rpm for 15 min and used to validate creatinine, blood urea nitrogen (BUN), calcium, and uric acid using ERBA diagnostic kits. Semi Autoanalyser (Mispa excel, version: 1.4) was used for estimation of parameters. Creatinine clearance was considered as a renal function test and was computed using the formula (Cockcroft and Gault, 1976).

Kidney samples for In vivo antioxidant and histopathological studies
After the inished collection of urine and blood samples, rats were sacri iced by cervical decapitation; kidneys were attentively isolated and perfused with ice-cold saline. From each animal one kidney was meant for the determination of calcium and oxalate, in vivo antioxidant investigation includes lipid peroxidation represented as malondialdehyde (MDA) levels (Niehaus and Samuelsson, 1968), superoxide dismutase (SOD) (Misra and Fridovich, 1972), catalase (CAT) (Aebi and Catalase, 1984), and reduced glutathione (GSH) (Jollow et al., 1974). Another kidney was positioned in a 10% buffered formalin solution (pH 7.4) and polished with paraf in wax. The sections were processed employing a microtome and pigmented with hematoxylin and eosin and then inspected under a light microscope for renal impairment.

Statistical Analysis
The data acquired were analyzed by one-way ANOVA accompanied by Dunnettes multiple comparison test using Graph Pad Prism (version 5.0) p<0.05 was considered to be statistically signi icant. The values represented as mean ± standard error of the mean (SEM).
BACAR has been found to be safe in the acute oral toxicity studies, and has caused no mortality of up to 32000 mg/kg. The previous study manifests that BACAR exhibit antiurolithiatic behaviour at a dose of 800 mg/kg (Jagannath et al., 2015). Therefore, 800 mg/kg of BACAR were adopted for the present research.

Urinary Biochemical Parameters
On the ingestion of EG and AC in both control groups (II and VI) animals a signi icant escalation of calcium, oxalate, phosphate, and uric acid levels and decline magnesium levels was reported when compared to the normal group (I). These results conirmed the formation of CaOx calculi trigerred by EG and AC. An appreciable declined in calcium, oxalate, phosphate, uric acid levels, and improved magnesium levels were evidenced on treatment with cystone, BACAR, and BACARNPs, in the preventive (III, IV, and V) and curative (VII, VIII, and IX) treated groups demonstrated the preventive and curative effect on CaOx calculi (Karadi et al., 2006;Grases et al., 1989), (Table 1).
Standard urinary pH ranged approximately 6.0 to 7.0 in normal group (I) animals. Once CaOx calculi was installated, the pH in both control groups (II and VI) decreased dramatically to 5.0-6.0 relative to normal. The urine pH was signi icantly switched back normal for cystone, BACAR and BACARNPs treatment in both treated regimens owing to the alkalinizing effect of BACAR (King, 1967).
A signi icantly decreased output of urine is discovered in both control groups (II and VI) is observed due to obstruction in urinary low by the CaOx calculi as well as impairment of renal function unit responsible for the production of urine. Cystone, BACAR & BACARNPs therapy results in a substantial improvement in urinary ability in both treated group animals with the effect of preventing and curing of renal cell injury and the diuretic impact of BACAR (Table 2).
A signi icant decline in creatinine clearance was detected in the control groups (II and VI) and improved in the groups treated with cystone, BACAR & BACARNPs in both treated regimens (Table 2).
It con irmed the increased effectiveness of BACAR loading in chitosan nanoparticles (BACARNPs) over urine biochemical studies, urine pH and urine volume in comparison with BACAR alone and standard cystone.

Serum Biochemical Parameters
A consequential increased levels of creatinine, BUN, calcium and uric acid were reported when EG and AC were administered in both control groups (II and VI) relative to the normal group (I). This was witnessed by a reduction in glomerular iltration rate in reaction to calculi blockade in bowman's capsule which contributes to an accumulation of nitrogenous waste in the blood. Treatment with cystone, BACAR and BACARNPs in both treated regimens substantially inverted the concentrations of creatinine, BUN, calcium, and uric acid levels to usual in addition to enhanced glomerular iltration ef iciency due to the BACAR antioxidant activity. The antioxidant activity safeguards the renal cell damage due to reactive oxygen species (ROS) supposed to act as a calculi established site (Rathod et al., 2012), [ Table 3].
BACARNPs establish evidence of improving the serum parameters in comparison to BACAR administered alone and cystone due to increased deposition of BACAR in the kidneys.

Wet kidney weight and deposition of Calcium and Oxalate in the Kidney
A signi icant increase in the retention of calcium and oxalate levels in the kidney after consuming drinking water comprising EG and AC for 15 days, in both control groups (II and VI) relative to the normal group (I). A remarkable drop in calcium and oxalate amounts was witnessed in both treated group animals administered with cystone, BACAR, and BACARNPs (Patel et al., 2012). In response to that signi icant increased wet kidney weight in control groups (II and VI) and signi icant decreased in treated groups was noticed (Table 4).
Among the treated groups the BACARNPs extremely effective in declining the calcium and oxalate dumping due to the enhanced accumulation of BACAR in the kidneys with antioxidant and hypocalciuric effects.

In Vivo Antioxidant Parameters
In both control groups (II and VI) an extraordinary rise in MDA levels through an enhanced lipid peroxidation process was reported when EG and AC administered relative to the normal group (I). This is further evidenced by earlier studies that exposure to oxalate and CaOx crystals results in renal cell injury by intracellular ROS generation triggered by membrane lipid peroxidation (Namburu et al., 2017). Cystone, BACAR, and BACARNPs treatment effectively lowered MDA levels by obstructing the lipid peroxidation by antioxidant activity in both treated groups. A consequential depletion in the levels of enzymatic antioxidants SOD and CAT and non-enzymatic antioxidant GSH levels were noticed in both control groups (II and VI) when compared to the normal group (I). Such decreased levels weaken antioxidant defense against ROS, which may have also facilitated the aggregation and retention of oxalate and eventual dumping of CaOx calculi. The antioxidant activity of the cystone, BACAR, and BACARNPs in both treated regimens improved these enzyme levels to normal (Khan, 2014;Selvam, 2002) , (Table 5).
A signi icant re inement of In vivo antioxidant parameters in BACARNPs was noticed among the treated regimens by increased deposition of BACAR due to appropriate renal targetting.

Histopathological Studies
Histological examination revealed glomerular atrophy, dilation of renal tubules, and necrotic changes in control groups (II and VI). These astonishing histological changes might be attributed to oxalate induced lipid peroxidation. Our results stated that treatment with cystone, BACAR & BACARNPs has both defensive and preventive effects on the intratubular crystal deposition in both treated groups leading to a substantial reduction in the renal tissue damage index ( Figure 1). In igure A represents to histopathology of normal, B and F represents the preventive and curative control respectively, C, D, and E, and G, H, and I represents the preventive and curative cystone, BACAR, and BACARNPs respectively.
The BACARNPs treated group of animals recovered predominantly from histological changes over the rest of the treated groups.

CONCLUSIONS
In the present study, the indings, specially demonstrated the effectiveness of nanotechnology in enhancing the antiurolithiatic ef icacy of BACAR loaded in Chitosan nanoparticles by targeting the kidneys. As a result of ampli ied deposition of BACAR in the kidneys, the antiurolithiatic activity of BACARNPs has been shown to be predominantly effective over both BACAR alone and as well as standard cystone.