In silico and In vitro Evaluation of Anti-urolithiatic Activity of Ethanolic Extract of Syzygium cumini Stem Bark

 

Sathish Babu. P¹, Gokula Krishnan², Anand Babu.K¹* Chitra. K¹

¹Faculty of Pharmacy, Sri Ramachandra University, Porur, Chennai-116, TN, India

²Deparment of Biomedical Sciences, Sri Ramachandra University, Porur, Chennai-116, TN, India

 

ABSTRACT:

Urolithiasis is a series of several physicochemical event which includes aggregation, nucleation, growth, super-saturation and retention within the kidneys and it is a complex process. The aim of the present study was to investigate the In silico and In vitro anti-urolithiatic activity of ethanolic extract of Stzygium cumini stem bark. The objective of the present study was to predict the phytoconstituents possibly present in the extract by literature survey, to systematically perform In silco molecular docking studies and to perform Invitro anti urolithiatic activities by Titrimetry, Spectrophotometry and nucleation assay. The protein structure and ligand structures were collected from online databases and the docking studies were performed using HEX8.0 software. The calcium oxalate stones were prepared using homogeneous precipitation technique and the assays were performed using cystone as standard and comparing the results with that of the extract. The Results obtained from the docking studies showed satisfying energy binding values. The results obtained from titrimetric analysis (based on the amount of CaO stones present) for control (0.00087), Standard (0.00526) and extract (0.00067) showed effective results. Similarly, the Spectrometric studies to analyze the amount of CaO present also showed to a peak in between the standard and the control. There were less crystals observed in the extract containing sample compared to that of control in Aggregation assay. Obtained results showed to prove that the Ethanolic extract of Syzygium cumini has a potential urolithiatic activity.

 

 

 

 

 

INTRODUCTION:

Kidney stone formation is the oldest and serious painful urologic disease with significant prevalence in the population due to change in lifestyle and dietary factors. Kidney stones that form in the urinary tract are a hard and solid particles. In many cases, the stones can pass out of the body without any problems and hence it is very small. However, excruciating pain may result, if a stone (even a small one) blocks the flow of urine, and prompt medical treatment may be needed.

 

Literature surveys have proved that plants generally have been used in the treatment urolithiasis. Various parts of the Syzygium cumini tree like seeds, skeels, stem, bark, fruits have shown to prove that it has effective anti urolithiatic activity from traditional usages. So Syzygium cumini extracts can be effectively used to investigate the urolithiatic activity.¹ 

 

Molecular Docking is used as a key tool in computer assisted drug design and structural molecular biology. The objective of ligand-protein docking is to prognosticate the predominant binding site of ligand with a protein of known three dimensional structure.² By literature survey, certain compounds which are possibly extracted through solvent ethanol have been identified and the structures of these compounds can be docked with the receptor involved in urolithiasis drug binding to investigate the drug binding affinity of the compounds present in the extract.³

 

Invitro studies like nucleation assay, spectrophotometric assay and aggregation assay are normally done to identify the anti urolithiatic activity of an extract.

 

The aim of the present study was to investigate the In silico and in vitro anti-urolithiatic activity of ethanolic extract of Stzygium cumini stem bark.      

 

MATERIALS AND METHODS:

In-silico docking studies:

 

Figure 1: Materials and method involved in In silico Docking Studies.

 

The natural compounds for performing the docking studies were chosen based on the literature survey. As the extract of the Syzygium cumini stem bark has been used for isolation of various compounds and the compounds isolated were listed along with their chemical structures and those were chosen for the docking studies.^4,5

 

In-vitro Anti- Urolithiatic activity:^6-12

Collection of Plant Material and Extraction:

In the month of December 2015, the Syzygium cumini bark was collected from Tiruneveli district of Tamil Nadu. The extraction process was done by cold maceration technique. The plant material was dried in shade and powdered coarsely. The powdered bark was then divided into two halves of 500 g each and placed into two containers and ethanol (Solvent) was added until it dissolved. The filtrate was then concentrated in a china dish and used.

Preparation of the semi-permeable membrane from eggs:

The glass rod was used to puncture the Apex of the eggs, in order to squeeze out the entire content. Empty eggs were washed thoroughly with distilled water and the eggs were placed in a beaker consisting 4ml concentrated HCl in 200ml distilled water. Decalcification of semi permeable membrane occurs, when the setup is kept for overnight. On the next day, semi permeable membranes from the egg shells were removed carefully, thoroughly washed with distilled water and for neutralization of acid traces it is placed in ammonia solution, and then rinsed it with distilled water. It was stored in the moistened condition in refrigerator at a pH of 7-7.4. The prepared membranes were tied onto a double sided open ended tube to pack the components for incubation.

 

Synthesis of calcium oxalate by homogenous precipitation:

To synthesis calcium oxalate by homogenous precipitation method 1.47gm of calcium chloride dihydrate was dissolved in 100ml distilled water and 1.34gm of sodium oxalate was dissolved in 100 ml of 2N H2SO4. Calcium oxalate is precipitated out with stirring when both were mixed equally in a beaker. Ammonia solution is used hence the resultant calcium oxalate was freed from traces of sulfuric acid; finally washed with distilled water and then dried at a temperature 60°C for 2hours.

 

Preparation of 0.02M KMnO₄ solution:

To prepare 0.02M KMnO₄ solution, 0.32gm of KMnO₄ was dissolved in 100ml of distilled water and boiled for 30minutes. After cooling excess of MnO₄ was removed by filtration.

 

Estimation of Calcium oxalate by Spectrophotometeric method:

Group I: 1ml of calcium oxalate (1mg/ml) added with 1ml of distilled water

Group II: 1ml of calcium oxalate (1mg/ml) added with 1ml of Cystone solution (400mg/ml)

Group III: 1ml of calcium oxalate (1mg/ml) added with 1ml of ethanolic extract of Syzygium cumini (20mg/ml)

 

The egg semi permeable membrane is packed together with all groups and tied with thread at one end and allowed suspended in a conical flask containing 150 ml 0.1 M Tris buffer each. The another end of thread is tied with the stick placed on the mouth of conical flask and aluminium foil is covered to it. All groups were plaxed in the incubator, pre heated to 37°C for 4 hours, incubated for for three days. From sutured semi permeable membrane, the entire content of each group was transferred into test tube individually. 4ml of 1N H₂SO₄ and 60-80 ml of 0.02M KMnO₄ were added and kept aside for 2 hours. After 2 hours, the colour change from dark pink to colourless was observed. This Change of colour intensity from dark pink to colourless was measured against 620nm spectrophotometrically.

 

Estimation of Calcium oxalate by Titrimetric Method:

1mg of the calcium oxalate and 10mg of the extract/compound/standard is weighed exactly and packed it together in semi evaluation. Setup was allowed to suspend in a conical flask containing 100ml 0.1 M TRIS buffer. One group served as negative control (contained only 1mg of calcium oxalate). Placed the conical flask of all groups in an incubator, preheated to 37ºC for 2 hours, for about 7-8 hours. The semi-permeable membrane contents were removed from each group into a test tube. 2 ml of 1 N sulphuric acid is added and titrated with 0.9494 N KMnO₄ till a light pink colour end point is obtained.1ml of 0.9494 N KMnO4 equivalent to 0.1898mg of 4 Calcium. In the beginning, to identify the quantity of calcium oxalate actually test substance(s) could dissolve, the amount of undissolved calcium oxalate is subtracted from the total quantity used in the experiment.

 

Estimation of Calcium oxalate by Nucleation assay (Turbidity method):

The Spectrophotometric assay was used to determine the inhibitory activity of the extracts on the nucleation of calcium oxalate crystals. 100µl of 50 mM sodium oxalate solutions and 100µl of 4 mM calcium chloride to 0.5ml of human normal urine, both prepared in a buffer containing 0.5ml of 0.05 mM Tris buffer and 0.5ml of 0.15mM NaCl solution at 37ºC and pH 6.5, adjusted to volume by adding 1.5ml of distilled water, hence crystallization is initiated.  By comparing the induction time of crystals (the time of appearance of crystals that reached a critical size and hence optically detectable) in the presence of the extract and that of the control with no extract, the rate of nucleation was determined. The optical density (OD) was recorded at 620nm, and the percentage inhibition calculated as (1-OD (experimental)/ OD (control))/100.

 

Figure 2: Preparation of materials for Titrimetric and spectrophotometric methods

 

Figure 3: Nucleation assay Preparations

RESULTS AND DISCUSSSIONS:

In-silico docking studies:

Binding Positions of various Chemical Compounds with Receptor (Figures: 4-9)

      

                           Figure-4: Compound-A                              Figure-5: Compound-B                           Figure-6: Compound-C

 

      Figure-7: Compound-D                              Figure-8: Compound-E                           Figure-9:Compound-F

 

Table-1: Results of Insilco Docking studies:

Name of the compound	Code of the compound	Total Energy	Vander wal’s Interactions (VDW)	Hydrogen Bonds (HBond)	Average Connecting Pairs (AverConPair)
Delphinidin 3-gentiobioside	Compound- A	-342.67	-185.777	-69.709	87.19
Kaempferol	Compound- B	-226.43	-146.669	-29.663	49.99
Mearnsetin	Compound- C	-245.51	-155.479	-34.221	55.81
Myricetin 3-acetylrhamnoside	Compound- D	-289.88	-178.391	-41.369	70.12
Oleanolic acid	Compound- E	-286.80	-108.583	-40.363	68.22
Quercetin	Compound- F	-223.93	-148.663	-26.997	48.27

 

 

In general, Before every docking run, HEX allows the given ligand and receptor residue to rotate onto the z-axis. Hence potent docking of the ligand on all sites for the receptor to possibly attach are well prognosticated and docked in HEX. The docking results are shown in Table 1 respectively. The docked poses of all the ligands are shown in the figures 4 to 9. All the compounds have showed NEGATIVE VALUES i.e., each and every compounds have greater affinity towards the receptor. Thus these compounds can be effectively used in regulating the human calcium sensing receptor and among all these compounds, Delphinidin 3-gentiobioside showed a very high energy binding value of -342.67.

 

In-vitro studies:

Spectrophotometric estimation of calcium oxalate:

The ethanolic extract of Syzygium cumini has greater capability to dissolve calcium oxalate as foremost element for stone forming in urinary tract. The percentage of Calcium oxalate dissolved indicates the capability of the extract to eliminate the kidney stones formed. The details are as shown in Table 2.

 

Table 2: Results of Spectrophotometric estimation of Calcium Oxalate

Group	Absorbance (nm)	% Calcium oxalate undissolved
Control	0.0004	---
Standard (Cystone)	0.3240	87.06%
Extract	0.4380	72.051%

 

Figure 10: Spectrophotometric estimation of Calcium oxalate

 

Titrimetric estimation of Calcium oxalate:

The study of the urinary chemistry with respect to the stone-forming minerals will provide a good indication of the risk of stone formation. The percentage of Calcium oxalate dissolved indicates the capability of the extract to eliminate the kidney stones formed. The details are as shown in Table 3.

 

Table 3: Results of Titrimetric estimation of Calcium Oxalate

 

Figure 11: Titrimetric Estimation of Calcium oxalate

 

Nucleation assay:

The calcium oxalate particles crystallization within the urinary tract is attributed by Urine supersaturation. This is nucleation process where stone forming salts begins unite into clusters with summation of new constituents. Cystone standard solution show stronger inhibition activity than the extract in the nucleation of calcium oxalate salts. The crystal growth observed in the Extract were less compared to that of Cystone.

 

 

CONCLUSION:

Kidney stone elimination plays a major influence in the treatment of urolithiasis. The results obtained from insilico docking studies showed an effective inference on utilization of the isolated compounds as treating aids for urolithiasis. Similarly, the in vitro studies have proven to show that the Ethanolic extract of Syzygium cumini Stem bark are very efficient in the treatment of Urolithiasis. Thus the study proves that the extract as well and the isolated compounds of Syzygium cumini can be effectively used to control as well as eliminate the kidney stone formation.

 

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Accepted on 27.01.2017           © RJPT All right reserved