In Vitro – In Vivo Evaluation of Antiurolithiatic activity of piperine from

Piper nigrum

 

Yenduri Suvarna¹*, S K. Abdul Rahaman²

¹Research Scholar, Department of Pharmaceutical Analysis, University College of Pharmaceutical Sciences, Acharya Nagarjuna University, Guntur-522510

²Department of Pharmaceutical Analysis, Nirmala College of Pharmacy, Atmakur, Mangalagiri, Guntur-522503, Andhra Pradesh, India.

 

ABSTRACT:

Background: A natural product is a organic compound or substance produced by a living organism that is, found in nature. Natural products show significant pharmacological or biological activity that can be of therapeutic benefit in treating diseases. Now a day’s Urolithiasis or Urinary calculi or Kidney stone formation becoming very prevalent in the world and it is reported that Urolithiasis is the 3^rd most prevalent disease among the kidney diseases. Therefore there is an immediate urge in searching for alternative treatment for urolithiasis. Fruits of Piper longum Linn were commonly called as Black pepper have a long history in Indian traditional medicine and in Ayurveda for the treatment of Gastrointestinal and Respiratory complications. 1-(5(1,2-benzodioxol-5-yl)-1-oxo-2,4-pentadieneyl) piperidine commonly known as piperine were reported to possess many pharmacological activities. Purpose: This study evaluated the effect of Piperine on anti-urolithiatic activity in invitro and invivo models. Method: The anti-urolithiatic activity of Piperine was evaluated by using invitro methods like titrimetric method and aggregation assay. In vivo studies were done using male wistar rats. Results: The results of this study proved that Piperine has a significant anti-urolithiatic activity in rats (in-vivo) as well as in in-vitro models. Two test doses of piperine (40,80 mg/kg P.O) are evaluated using urolithiasis induced rats in in-vivo, titrimetric and aggregation in in-vitro models and it showed significant inhibition of crystallization with a significance of p<0.01 and p<0.05 when compared with the standard drug cystone (750mg/kg P.O.) Different serum parameters such as calcium, urea, uric acid, creatinine and urine parameters such as calcium and oxalate are assessed to evaluate anti urolithiatic activity of piperine in in-vivo study. The results were presented as mean±SEM. Difference among data was statistically analysed using One-way ANOVA to determine the level of significance using Graph pad Prism Differences between the data were considered significant at P < 0.05 and P<0.01. Conclusion: In in-vitro method of evaluation, piperine (20mg/kg) demonstrated a significant anti-urolithiatic activity than piperine (10mg/kg) when compared with a standard drug Cystone and in in-vivo models, piperine (40mg/kg) demonstrated a significant anti- urolithiatic activity than piperine (80mg/kg) when compared with a standard drug Cystone.

 

 

1. INTRODUCTION:

Urolithiasis is nothing but stone formation in the urinary tract. Kidney stones are hard, solid particles that form in the urinary tract and may occur due to crystal nucleation, aggregation and growth of insoluble particles. In many cases, the stones are very small and can pass out of the body without any problem. However, if a stone blocks the flow of urine, excruciating pain may result and medical treatment may be needed. Calcium containing stones especially calcium mono hydrate, calcium oxalate dehydrate and basic calcium phosphate are the most commonly occurring ones to an extent of 75-90% followed by magnesium ammonium phosphate to an extent of 10-15% and uric acid upto 3-10% and cysteine 0.5-1%^[1]. In most of the cases the commonly occurring stones are calcium oxalate or magnesium ammonium phosphate type and urolithiasis is affecting approximately 12% of the population, with reference rate of 70-80% in male and 47-60% in female.

 

Extracorporeal shock wave lithotripsy (ESWL) and drug treatment revolutionized urological practice almost became the standard procedure for eliminating kidney stones. But shock waves had many deleterious effects, residual stone fragments persisted and infection could occur. Moreover, ESWL may cause acute renal injury, a decrease in renal function, hemorrhage and hypertension^[2]. Because of these harmful effects new compounds should be invented with the high efficiency and low toxicity. Piperine is the compound extracted from black pepper which is having many pharmacological activities; therefore it is worthwhile to choose the Piperine for its anti urolithiatic activity.

 

2. MATERIALS AND METHODS:

2.1 Isolation of Piperine:

The fruits of Piper nigrum were taken and were shade dried for 7-8 days. The fruits were powdered, 200gm of crude drug powder was taken and 200ml alcohol was added, shaken well till all the powder is thoroughly mixed with the solvent. This was refluxed for 45min and filtered. the obtained filtrate was treated with warm alcoholic potassium hydroxide and the beaker containing the solution was placed in ice bath and allowed for crystallization. Fine yellow needles of piperine was separated^[3].

 

2.2 Experimental animals:

Six to seven week-old male  Wistar albino rats weighing 150-200g were obtained from the animal house of Bangladesh Council of Scientific and Industrial Research (BCSIR), Chittagong, Bangladesh. The animals were housed individually in stainless steel wire meshed plastic cages in a temperature (23±2⁰C) and humidity (55%-60%) controlled room with a 12 h light-dark cycle. The animals were supplied with standard rat pellet diet and drinking water and ethylene glycol during the entire period of the study^[4][5]. Animals were maintained and experiment was carried out according to the rules and regulations of ICH.

 

2.3 Chemicals and reagents:

All the chemicals used were of analytical grade. Ethylene glycol was obtained from Sigma Aldrich Chemicals Pvt. Limited, Bangalore, India. Cystone syrup manufactured by the Himalaya Drug Company, Bangalore, was purchased from the market of Bangalore. Kits used in this study for the determination of calcium, blood urea nitrogen (BUN), creatinine and phosphorus were purchased from Lab Care Diagnostics (India) Pvt. Ltd., Bangalore^[6].

 

2.4 Acute toxicity testing:

The acute oral toxicity study was carried out in 12 male rats as per the guidelines set by the Organization for Economic Cooperation and Development (OECD) number 425 (up and down procedure) and maximum tolerable dose (MTD) was determined^[7]. Administration of piperine up to a dose of 80mg/kg did not show any mortality and MTD was determined on the basis of observed toxicological sign and symptoms.1/2th of MTD i.e. 40mg/kg and 80mg/kg were selected.

 

3. Anti-Urolithiatic activity:

A) In-vitro procedure for anti-urolithiatic activity:

1) Titrimetric method

2) Aggregation assay

 

B) In-vivo method to evaluate anti-urolithiasis in rats:

3.1 In vitro method:

3.1.1 Preparation of Calcium oxalate crystals: 10ml 1M Calcium chloride, 10ml 1M Sodium oxalate, 10ml 2N Sulphuric acid were mixed in a beaker for formation of slurry. The solution was filtered to collect the precipitate. The precipitate was washed with ammonia to remove traces of sulphuric acid. The precipitate was dried in hot-air oven at 60^oC for 4hrs.The crystals obtained were incubated in TRIS BUFFER at a pH 7.4^[8] and were assayed by using aggregation assay and titrimetry method.

 

3.1.2. Titrimetric Method:

3.1.2.1 Preparation of semi-permeable membrane from farm eggs: The semi permeable membrane of eggs lies in between the outer calcified shell and the inner contents like albumin and yolk. Shell was removed chemically by placing the eggs in 1M HCl for an overnight which caused complete decalcification. They were further washed with distilled water and carefully with a sharp pointer a hole is made and the contents were emptied out completely. From the decalcified eggs, the egg membrane was washed thoroughly with distilled water and it was placed in ammonium solution^[9].

 

3.1.2.2 Procedure:

1mg of the calcium oxalate and 10mg of the extract/compound/standard were weighed accurately and packed it together in semi Permeable membrane. This was allowed to suspend in a conical flask containing 100ml 0.1M TRIS buffer. Conical flasks of all groups were placed in an incubator at 37˚C for about 7-8 hours. The contents of semi-permeable membrane were removed from each group into a test tube; 2ml of 1N sulphuric acid was added and titrated with 0.9494N potassium permanganate till a light pink colour end point was obtained. The amount of calcium oxalate dissolved by test substance under study was determined by subtracting undissolved calcium oxalate from the amount of calcium oxalate taken initially^[10].

 

1ml of 0.9494 N KMnO4 is equivalent to 0.1898mg of Calcium

 

3.1.3. Nucleation assay:

The rate of nucleation of the calcium oxalate crystals was determined by a spectrophotometric assay. The inhibition of nucleation rate (calcium oxalate crystallization) was studied in absence and presence of inhibitors. The calcium oxalate monohydrate (COM) crystals were prepared by adding 1ml of 0.025M calcium chloride and 2ml of 0.025M sodium oxalate to a buffer solution containing a mixture of 1ml of 0.05mM Tris buffer and 1ml of 0.15mM sodium chloride (pH 7.4). Absorbance at 620nm was recorded. Solutions were prepared similarly by adding Cystone/Piperine 10mg/kg/ Piperine 20mg/kg dose. The rate of nucleation was estimated after 10 min by comparing the absorbance in the presence of the extract against control with standard and control^{[11] [12]}.

 

3.2 In vivo method:

Ethylene glycol model was used to induce urolithiasis for 28 days^[13]. Thirty animals were randomly divided into five groups as group I, II, III, IV and V containing six in each. Group I served as a vehicle-treated control and maintained on regular rat food and drinking water ethylene glycol. All the remaining groups received calculi inducing treatment for 28days which comprised of 0.75% v/v ethylene glycol in drinking water ethylene glycol. Group II which received ethylene glycol only, served as model control for 28days. Group III was administered CYSTONE 750mg/kg body weight/day (The Himalaya Drug Company, India) served as standard. Groups IV and V served as a treatment groups which received Piperine at doses of 40 and 80 mg/kg body weight, respectively. Extract and standard drugs were suspended in distilled water and given once daily by oral route using the gastric tube.

 

3.2.1 Collection and analysis of urine:

All animals were kept in individual metallic cages and 24 hours urine samples were collected on 28^th day of calculi induction treatment. The volume, pH and crystalluria were determined^[14]. Urine was acidified with a drop of concentrated HCl and stored at 20ᵒC for determination of calcium, magnesium, phosphate, uric acid and glycosaminoglycans (GAGs) using commercially available kits (Span Diagnostics Ltd., India; Biocolor Ltd., UK). The oxalate and citrate were estimated by the method described previously by Hodgkinson, 1970; Rajagopal, 1984.

 

3.2.2 Collection and analysis of serum:

After the experimental period, blood was collected from retro-orbital under light ether anesthesia and animals were sacrificed by high dose of anesthetics. Serum was separated by centrifugation at 10,000g for 10min and analysed for the calcium, creatinine, uric acid, urea and blood urea nitrogen (BUN) using commercially available diagnostics while oxalate was measured by the method of Hodgkinson, 1970.

 

3.2.3 Kidney histopathology and homogenate analysis:

The abdomen was incised and opened, and both kidneys were removed from each animal. Isolated kidneys were cleaned off extraneous tissue, weighed and rinsed with ice-cold normal saline. The left kidney was fixed with 10%v/v neutral formalin and after harvesting, sliced horizontally and sent to histology services (Samarth Pathology Laboratory, Surat) for Haematoxylin and Eosin staining^[15] Calcium oxalate crystal depositions were counted by light microscope using polarized filter and given a score as follows: o1¼0, o10¼1, o30¼2, o50¼3, o75¼4 and 475¼5. Same histology slides were subjected to microscopic examination for the presence of glomerular congestion, tubular casts, peritubular congestion, epithelial adhesion, blood vessel congestion, interstitial edema and inflammatory cells, and given the scores accordingly. The right kidney was finely chopped and 20% homogenate prepared in Tris –HCl buffer (pH7.4). Total kidney homogenate was used for assaying tissue calcium and oxalate, malondialdehyde (MDA) (Ohkawa etal.,1979), reduced glutathione (GSH) (Beutler etal.,1963), and catalase (Hugo, 1984).

 

3.2.4 Statistical analysis:

The results were presented as mean ± SEM. Difference among data was statistically analysed using One-way ANOVA followed by the Turkey post test to determine the level of significance using Graph pad Prism. Differences between the data were considered significant at P <0.05.

 

4. RESULTS:

The present study was designed to evaluate anti-urolithiatic activity of Piperine in both in-vitro and in-vivo models

 

 

 

4.1 In vitro method:

4.1.1 Titrimetric method:

In titrimetric method, calcium oxalate crystals treated with piperine 10mg/kg and 20mg/kg, cystone (standard), control were evaluated for the amount of calcium oxalate dissoluted by titration (Table-1). It was found that calcium oxalate treated with piperine 10mg/kg dose has shown maximum dissolution of calcium oxalate when compared with control, standard and piperine 20mg/kg dose (Fig-1).

4.1.2 Aggregation assay:

In aggregation assay method, anti-urolithiatic activity of piperine was studied in terms of inhibition of calcium oxalate formation by comparing and measuring turbidity spectrophotometrically. The amount of calcium oxalate present in the solution in presence of cystone is less which indicates more anti-urolithiatic activity (Table-1). Piperine of dose 10 mg/kg has shown the approximate result as that of standard i.e. cystone (Fig-1).

 

Fig 1: Invitro antiurolithiatic activity by Titrimetry and Aggregation assay

 

Table 1: Evaluation of Piperine for anti-urolithiatic activity (Calcium oxalate concentration mg/ml) using Aggregation and Titrimetric methods

 

 

Fig 2: Estimation of serum creatinine, calcium, urea and uric acid levels in In vivo antiurolithiatic activity by serum analysis

Table 2: Effect of Piperine on various urinary and serum parameters in ethylene glycol induced urolithiasis

**P < 0.01 when compared to Ethylene glycol control  *P < 0.1 when compared to Ethylene glycol control

^#P < 0.1 when compared to Normal control

 

Fig 3: Estimation of urine calcium and Oxalate levels in In vivo antiurolithiatic activity by urine analysis

 

4.2 In vivo method:

In Vivo studies for anti-urolithiatic activity were done on male wistar rats of five groups and treated with water, ethylene glycol, cystone 750mg/kg, piperine 40mg/kg and 80mg/kg (Table-2). Urine and serum samples were collected from In vivo models after 28 days of calculi induction treatment and samples were analysed.

 

4.2.1 Serum analysis:

Serum analysis was done for the estimation of creatinine, urea, calcium and uric acid. It was found that piperine 40mg/kg has shown low levels of urea, uric acid and calcium but piperine 80mg/kg has shown low creatinine levels (Fig-2).

 

4.2.2 Urine analysis:

Urine samples were collected and evaluated for the amount of calcium and oxalate. It was found that among all the five groups, the in vivo models treated with piperine 40mg/kg has shown low calcium and oxalate content in urine which indicates significant anti-urolithiatic activity of piperine of dose 40mg/kg (Fig-3).

 

Fig 4e: Histopathology in a) Normal control b) Ethylene glycol control c) Cystone 750 mg/kg animal d) Piperine 40 mg/kg treated animal e) Piperene-80 mg/kg treated animal

4.2.3 Kidney Histopathology:

Kidney histopathological studies revealed that no calcium oxalate deposits were found in the kidney of positive control group. A significant deposition of calcium oxalate crystals was observed in negative control group and group treated with piperine 80mg/kg. Some occasional deposition of calcium oxalate crystals and dilation of tubules were observed in the kidney of standard group. On the other hand, very low deposition of calcium oxalate and dilation of tubules were observed in group treated with piperine 40mg/kg which indicates significant anti-urolithiatic activity (Fig-4a, Fig-4b, Fig-4c, Fig-4d, and Fig-4e).

 

5. DISCUSSION:

In the present study, anti-urolithiatic activity piperine isolated from fruits of Piper nigrum was evaluated by both invitro and in vivo methods. In In vitro methods, anti-urolithiatic activity of piperine was studied by performing titrimetric method and aggregation assay. The result of invitro studies revealed that piperine of dose 10 mg/kg has shown significant anti-urolithiatic activity when compared with standard (cystone) and piperine 20 mg/kg. The In vivo studies were done on male wistar rats by performing urine analysis, serum analysis and kidney histopathological studies. The results revealed that piperine 40mg/kg has shown significant anti-urolithiatic activity when compared with standard and piperine 80mg/kg. Piperine of doses 40 mg/kg and 80mg/kg have shown significant inhibition of crystallization with a significance of p<0.01 and p<0.05 when compared with the standard drug cystone (750mg/kg P.O.)

 

6. CONCLUSION:

From the present study, it was concluded that piperine isolated from Piper nigrum has shown significant anti-urolithiatic activity when compared with the standard (cystone). Invitro studies revealed that piperine 10 mg/kg has shown marked dissolution of calcium oxalate in titrimetric method and low concentration of calcium oxalate in aggregation assay method. In vivo studies revealed that piperine 40mg/kg has shown significant anti-urolithiatic activity in urine analysis, serum analysis and kidney histopathological studies. On the whole, piperine was found to show significant anti-urolithiatic activity than standard cystone.

 

7. ACKNOWLEDGEMENTS:

The authors would like to thank Sister P. Alphonsa (Secretary and Chairman of Nirmala College of Pharmacy, Atmakur) for her support throughout the study.

 

8. CONFLICT OF INTEREST:

The authors declare no conflict of interest for this work.

9. ABBREVIATIONS:

ESWL: Extracorporeal Shock Wave Lithotripsy

ICH: International Conference on Harmonisation

MTD: Maximum Tolerable dose

MDA: Malondialdehyde

GSH: Reduced Glutathione

ANOVA: Analysis of Variance

SEM: Standard Error of the Mean

BUN: Blood Urea Nitrogen

OECD: Organization for Economic Cooperation and Development

COM: Calcium Oxalate Monohydrate

GAGs: Glycosaminoglycans

 

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Received on 06.06.2019           Modified on 01.08.2019

Accepted on 29.08.2019         © RJPT All right reserved