Antiurolithiatic Activity of Ethanolic Extract of Piper cubeba Dried Fruits: An In-vitro and In-vivo Study

Urolithiasis is frequently combatted in its various forms during urological hurdles and is a major health issue. Some common causes include insufficient urinary drainage, external bodies in urinary area, bacterial contagions, and excessive oxalate along with calcium diets, gout and intestinal dysfunction1. It comprises as a worldwide health issue with an incidence of up to 5 % in the overall public, but its prevalence is greater in certain geographic areas like 70 % in Gulf nations, 15 % in the US and Turkey, 11 % in India as well as 4-8 % in United Kingdom2,3.This condition is almost twofold as mutual in males as in females along with its occurrence is rises as per age in adults4.


INTRODUCTION
Urolithiasis is frequently combatted in its various forms during urological hurdles and is a major health issue. Some common causes include insufficient urinary drainage, external bodies in urinary area, bacterial contagions, and excessive oxalate along with calcium diets, gout and intestinal dysfunction 1 . It comprises as a worldwide health issue with an incidence of up to 5 % in the overall public, but its prevalence is greater in certain geographic areas like 70 % in Gulf nations, 15 % in the US and Turkey, 11 % in India as well as 4-8 % in United Kingdom 2,3 .This condition is almost twofold as mutual in males as in females along with its occurrence is rises as per age in adults 4 .
Though, no analyses have been documented consequently as on antiurolithiatic activity of Piper cubeba. Hence, the given study was made an attempt to found the scientific data for the antiurolithiatic activity of EEPC beside ethylene glycol produced urolithiasis in rats.

Collection of plant material
Dried fruits of plant material purchased as of Sirigiri Venkappa ayurvedic stores, Kurnool, Andhra Pradesh, India in July 2017, authenticated and conformed by Raw material herbarium and museum, NISCAIR, Delhi, India by a reference no.(NISCAIR/ RHMD/ Consult/ 2017/3091-40). "The dried fruits have been pulverized to get the coarse powder used for extraction.

Preparation of extract
The coarse powder of Piper cubeba fruits crammed in a thimble and was subjected to extraction by using Sohxlet apparatus, with ethanol. The extract was filtered, and the residue obtained at laboratory temperature after concentration in the bath of water was additionally evaporated as well as stored in desiccator till utilization.

Phytochemical screening
The ethanolic extract of Piper cubeba dried fruits (EEPC) sample was exposed to initial phytochemical screening for alkaloids, glycosides, tannins, phenols, steroids, flavonoids as well as terpenoids following standard procedures 8 .

HPTLC analysis of "ethanolic extract of Piper cubeba dried fruits
The ethanolic extract of Piper cubeba dried fruits (EEPC) was applied at a concentration of 2 µl using the CAMAG Automatic TLC sampler 4 applicator over pre-coated silica gel 60 F254 TLC plates (Merck) 0.2 mm thick, 5 cm × 20 cm, as a stationary phase. Plates have been industrialized through using "ethyl acetate: formic acid: acetic acid: water (100:11:11:26) v / v / v/ v, n-hexane: ethyl acetate (1:1) v / v, by the mobile phase" means for flavonoids 9 and terpenoids respectively. The plates were industrialized in CAMAG -glass chamber which was twin trough saturated with saturated pad for 20minutes, to a distance of 70 mm. Developed plates have been derivatized with Anisaldehyde sulphuric acid and Natural product reagent respectively for terpenoids and flavonoids using CAMAG derivatizer. The tracks are scanned densitometrically using Camag scanner at a wave length of 540nm for terpenoids and 366mm for flavonoids, and the finger print profiles are recorded for terpenoids and flavonoids. The data was analysed using Camag software, Vision CATS.

Evaluation of in-vitro antiurolithiatic activity
The Piper cubeba ethanolic extract on CaOx crystallization was governed through the extent of variations in turbidity owing to crystal nucleation along with aggregation. The turbidity of Calcium oxalate was recorded at 620 nm at 37 ° C and pH 6.8. The UV / Vis (Lab India) spectrophotometer was utilized to turbidity determination of the rise in calcium oxalate.

Nucleation assay
The effect "of the ethanolic extract of Piper cubeba" over the growth of calcium oxalate (CaOx) crystal was through nucleation assesses. Individually, calcium chloride(3mM) as well as sodium oxalate(0.5mM) explanations were filtered three times through the pore size of 0.22μm filter, from that 950μL of calcium chloride was took and to this added 100μL of extract at different concentrations (50μg -3200μg/mL). Then added 950μL of sodium oxalate solution for initiation of crystals. Then finally the solutions were attractively moved at 800 rpm with an inspiring bar. The temperature 37ºC was preserved. At 620nm the solution optical density has been monitored. The nucleation rate was determined through associating "the induction time (the delay ahead of the crystals exterior that have extended a critical size" then consequently turn out to be optically demonstrable) if the control with the extract occurrence of no need to addition of corm extracts 10 .

Aggregation assay
The ethanolic extract of Piper cubeba influence on calcium oxalate (CaOx) crystal aggregation was resolved through aggregation assay. 0.8mg/mL COM crystals were buffered with 0.05M Tris containing sodium chloride (0.15M) at pH 6.5. Those all were performed at the temperature 37°C in the presence and corm extract absence afterward the stirring apprehends. The aggregation rate was predictable as below mentioned formula, through associating the turbidity slope in the sample and through that turbidity in the controller 11 . Evaluation of in-vivo pharmacological studies:

Selection of animals
Healthy wistar albino rats whose weight was approximately 150-180 g are collected from animal house, CES college of pharmacy, Kurnool. In polypropylene cages the animals were kept, preserved at 27 ± 2 ° C with in a qualified "humidity of 65 ± 10%, through 12 h of light and dark cycles. Animals were fed with regular pellet diet manufactured through Nutrivet life sciences, Pune, India" and protocol have been accepted through the IAEC with protocol No.: IAEC/CESCOP/2017-10.

Acute toxicity studies
In accordance to OECD 423 regulations, for acute toxicity studies male albino mice (20 g body weight) have been used 12 . For oral administration of EEPC to the overnight fasted animals, EEPC has been suspended in 1% sodium CMC. To distinguish behavioural alterations, the animals were continuously monitored for 3 h along with each 30 min aimed at next 3 h as well as then up to 24 h. Animals were also experiential aimed at the next 14 days to identify humanity as well as social variations.
For the effective dosage non-median lethal dose's (LD 50 ) 1/10 th part is used 13 .

Ethylene glycol induced urolithiasis in wistar albino rats
Atmani et al 14 . described a method that was used for the antiurolithiatic activity evaluation of ethanolic extract of Piper cubeba in albino rats. Thirty-six rats were allocated in six groups, every group involving of six rats.

Serum analysis:"
After last dose of the EEPC treatment, blood was composed through retro-orbital plexus below slight anaesthetic conditions. By centrifugation Serum was unglued (Research Centrifuge, R-22, Remi India) at 10,000×g for 10 mins as well as examined aimed at creatinine, uric acid as well as blood urea nitrogen (BUN) through semi-auto analyser (Mispa Excel Chemistry analyser) with diagnostic kits of Excel Diagnostic Pvt. Ltd, Hyderabad.

Urine analysis
On the 28 th day individual animal was kept in separate metabolic cages; 24 hr of urine sample was collected. Provide the water for rats all through the collection of urine, urine sample was subjected aimed at estimation of Calcium (Calcium diagnostic Kit, Agappe Diagnostics Ltd, Kerala, India), Oxalate 15 and Phosphate 16 .

Kidney homogenate analysis and histopathology
At the study period end, the rats were euthanized using a CO 2 chamber along with the abdomen was cut open to eliminate mutual kidneys as of every rat. The extracted kidneys remained washed off irrelevant tissue as well as rinsed in saline which is ice-cold physiological, utilized for histopathology as well as homogenate study. The left kidney of the individual rats was excellently chopped along with 20% of "homogenate was organised in the Tris-Hcl buffer (0.02 mol / l, pH 7.4)" and tested for Calcium, oxalate as well as phosphate. The right kidney have been immovable in formalin of 10% neutral buffered, and dealed with a sequence of graded alcohol as well as xylene and fixed in paraffin wax, partitioned at 5µm as well as marked with Hematoxylin and Eosin aimed at inspection beneath polarized light, With the help of Olympus Digital Camera, histopathological examination of slides was carried out under polarized light microscope (40X) and photographs were taken.

Statistical analysis
Every value is articulated "as mean ± SEM. The data was statistically studied through utilizing one-way ANOVA" surveyed through Dunnett's t assessment in GraphPad Prism 5.03 version software.

Preliminary phytochemical screening of ethanolic extract Piper cubeba dried fruits
The screening of Phytochemical discloses the phenolics presence, tannins, steroids, terpenoids and flavonoids in ethanolic extract of Piper cubeba dried fruits. "

HPTLC analysis of ethanolic extract of Piper cubeba dried fruits"
To obtain the reproducible results with high resolution different combinations of solvents with various ratios were tested. Satisfactory results were obtained "with n-hexane: ethyl acetate (1:1) v/v as well as ethyl acetate: water: formic acid: acetic acid (100:26:11:11) v/v/v/v" for terpenoids and flavonoids respectively. The "ethanolic extract of Piper cubeba dried fruits showed the presence of 7 terpenoids, 2 flavonoids with different Rf values and % of area when scanned at 540nm ( Figure  1) and 366nm ( Figure 2) respectively for terpenoids and flavonoids after derivatization (Table 1).

In-vitro antiurolithiatic activity
In the present study the different graded concentrations i.e. from 50µg/ ml to 3200 µg/ml of ethanolic extract of Piper cubeba dried fruits were utilized aimed at invitro antiurolithiatic activity evaluation. The reticence of crystal formation was directly proportional to the increase in the concentration of EEPC, with maximum activity was pragmatic at 3200 µg/ml in CaOx crystal nucleation and aggregation assays (Graphs 1 and 2).

Acute toxicity studies
The limit test was completed with a measure of EEPC (2000 mg / kg, b.w) given by oral path to a group of mice utilizing an oral feed needle (22 gauge). Upon treatment, mice were examined for 14 days and no changes in normal behavior were detected, as a result of which it was concluded that the EEPC was experimentally non-toxic in standard mice along with that the non-medium lethal dose of 1/20 th (100 "mg / kg b.w), 1/10th of the dose (200 mg / kg b.w), 1/5th of the dose (400 mg / kg b.w)" was considered as smaller, medium and high doses for further pharmacological studies.

Serum analysis
The current study treatment with ethylene glycol (0.75%, p.o) results in a substantial ( ### p<0.001) elevation in higher level of (43.36 ± 1.04, 6.93 ± 0.24, 6.741 ± 0.52) serum BUN, creatinine and uric acid respectively when comparison is made with normal group. These changes were restored significantly in the rats treated with standard drug cystone (750mg/kg, p.o) treated group animals acceptably lowers the BUN levels, creatinine along with uric acid respectively ( *** p<0.

Urine analysis
In wistar albino rats hyperoxaluria was generated through administration of Ethylene glycol (0.75%

Kidney histopathology
Histopathological examination was performed on the kidneys of rats treated with ethylene glycol (0.75 % v / v), which revealed the existence of the bonds "of calcium oxalate within the renal tubules" along with renal tubules dilation was observed along with interstitial tenderness (Figure 3). The amount of bonds of "calcium oxalate in the renal tubules along with the dilation of renal tubules in Group III ( Figure 3C) rats was substantially lower than Group II ( Figure 3B). EEPC treatment with varying doses "(100 mg / kg, BW, p.o, 200 mg / kg, BW, p.o, 400 mg / kg, BW, p.o)" i.e. category IV-VI ( Figure 3D-3F);Suggestively lowers the deposition of calcium oxalate crystal, dilation of renal tubules as well asinterstitial inflammation compared to Group-II ( Figure 3B).

DISCUSSION
In urolithiasis, CaOx urolithiasis is the utmost predominant kind of every urinary diseases related to the stone. Important magnitudes concerned in its pathological biomineralization comprise crystal nucleation, accumulation and evolution. Present analysis was aimed to discourse these crucial events concerned in CaOx stone development as a source to take a look at the effectiveness of P. cubeba as an antiurolithiatic agent. In-vitro conditions, nucleation is an essential in the "pathogenesis of CaOx urolithiasis. Nucleation fundamentally represents a thermodynamically pushed occasion of phase alteration wherein dissolved substances in a supersaturated solution" impulsively crystallize [17][18][19] Similar phase change and formation of CaOx crystals was witnessed while carrying out nucleation assay. The reticence of crystal formation was directly proportional to the increase in the concentration of EEPC, with maximum activity was pragmatic at 3200 µg/ml in CaOx crystal nucleation.
This implies the anticrystallization actions of EEPC from CaOx crystallization. A particular probable mechanism of anticrystallization actions of EEPC might be the proficiency of it's to be complicated with totally free calcium as well as oxalate ions, therefore obstructing the development of CaOx complexes, as has also been suggested for Sarghassum wightti 19 .
Aggregation is an important factor "of crystal retention as huge crystal agglomerates are actually the people which make renal tubular obstruction" therefore encouraging stone formation. EEPC showed considerable inhibitory impact on CaOx crystal accumulation.
In vivo conditions, oxalate is usually excreted as unaltered in urine. Nevertheless, hyperoxaluria facilitates the development of calcium oxalate renal calculi 20 , because urinary oxalic acid appears to complex with calcium and form insoluble CaOx crystals in the kidney 21 .
Consequently, conditions which promote the absorption of oxalate from the production of food (or) endogenous oxalate may cause the formation of CaOx stone 22 .
It has been confirmed that ingested ethylene glycol is converted to oxalic acid by the presence of liver enzyme glycolate oxidase 23 , thus facilitating the accumulation of CaOx in the kidneys.
In the current analysis rats fed by means of ethylene glycol (0.75%v/v) resulted substantial rise in serum stages of BUN, creatinine and uric acid as well as promotes excessive excretion of urinary levels of calcium, oxalate and phosphate, indicates calcium oxalate stones formation in kidneys. However, treatment with EEPC, significantly lowers the raised levels of BUN, creatinine along with uric acid along with urinary levels of calcium, oxalate and phosphate in a dose reliant on style.
Microscopic investigation of the sectional rat kidney treated with "ethylene glycol shows the presence of calcium oxalate bonds, dilatation of renal tubules accompanied through interstitial inflammation. However, cotreatment through EEPC reduces the number of calcium oxalate bonds, dilatation of renal tubules as well as prevents the damage to renal tubules in dose dependent manner.
The phytoconstituent found in the citation can be accountable for the movement, aimed at instance flavonoids, tannins, phenolic, steroids and terpenoids. In previous literature, flavonoids 24 and terpenoids 25 performs a significant part in the antiurolithiatic activity. Attempt also made to standardise the extract by performing finger printing of flavonoids and terpenoids by HPTLC.

CONCLUSION
The findings of this assessment provide strong evidence that the EEPC avoids the calcium oxalate crystals development in in vitro. Oral administration of EEPC to ethylene glycol mediated urolithiasis results in a dose-dependent decrease in raised serum levels of BUN, Creatinine as well as uric acid and urinary calcium, oxalate and phosphate. Therefore, EEPC expressed considerable antiurolithiatic activity against urolithiasis caused by ethylene glycol in rats.