Evaluation of Antiurolithiatic activity of Moringa leaves by

UV Spectroscopic Method

 

Kabita Banik¹, S. Akshitha², O. V. Poojitha², Ch. Thulasi², V. Vineetha²

¹Assistant Professor, Bharat Institute of Technology, Mangalpally, Hyderabad, Telangana, India.

²B Pharmacy, Research Scholar, Bharat Institute of Technology, Hyderabad.

 

ABSTRACT:

The effect of M.olifera leaf against Calcium oxalate was tested by uv Spectrscopy method. calcium oxalate crystals were confirmed by UV analysis. Then the sample was treated against leaf extract and again UV analysis was done to confirm presence or absence of crystals. Two type of assay the done to confirming the property Aggression assay and nucleation assay. calcium Oxalate, Phosphate, uric acid and proteins are important markers as well as citrate and magnesium are inhibitors which were tested for the confirmation of the stone. LDH and oxalate are the enzymes of liver and kidneys are having an important role in Urolithiasis were also estimated. After studding the absorbance and turbidity of calcium oxalate solution we concluded the results, studies showed significant data to confirm the efficacy of M.olifera leaf having anti-urolithic activity. The results of present study suggest usefulness of M.olifera in the treatment of kidney stone disease.

 

 

 

INTRODUCTION:

Urolithiasis is the process of forming stones in the kidney, bladder, and/or urethra (Urinary tract). There are basic two aspects in the pathogenesis of kidney stone. Increased urinary excretion of stone forming constituent elements like calcium, phosphorus, uric acid, oxalate and cysteine and physico-chemical change that influence stone formation like pH of urine, stone matrix and protective substance in urine (Abdulkarim S, Long K, Lai O, Muhammad S, Ghazali H. Some physio-chemical properties of Moringa oleifera). Kidney stones are a common cause of blood in the urine and pain in the abdomen, flank, or groin. Kidney stones occur in 1 in 20 people at some time in their life.

 

Management of stone disease depends on the size and location of the stones. Stones larger than 5mm or stones that fail to pass through should be treated by some interventional procedures such as extracorporeal shock wave lithotripsy (ESWL), ureteroscopy (URS), or percutaneous nephrolithotomy (PNL). Although there are a few recent reports of beneficial effects of medical treatments in enhancing clearance of stones in the distal ureters, In this regard, many plants have been traditionally used to treat kidney stones and have been shown to be effective. In India, in the Ayurvedic system of medicine, ‘Pashanabheda’ group plants, claimed to be useful in the treatment of urinary stones. ‘Pashanabheda’ is the Sanskrit term used for a group of plants with diuretic and antiurolithiatic activities (Pashana = stone; Bheda = break). A majority of urinary stones are composed of phosphates, oxalates, cystine, and uric acid. Almost 80% of these calculi are composed of calcium oxalate (CaOx) (Summary of WHO Guidelines for the assessment of herbal medicines, Herbal Gram, 22 (1993), pp. 13-28.). Medical treatment and extracorporeal shock wave lithotripsy are extensively used for the removal of calculi. However, continuous exposure to shock waves may cause acute kidney injury, infection and decrease in kidney function, and an increase in stone relapse. Drug treatment has shown some feasibility, but not without side effects. Several pharmacological and clinical studies on traditional medicinal plants used in the treatment of kidney stones have publicized their therapeutic potential in various in vitro as well as in vivo models. The present study has been designed to study their n vitro antiurolithiatic property of the leaf extract of M.olifera by UV Spectroscopic method.

 

MATERIALS AND METHOD:

Plant material and extraction:

The fresh plant parts of Moringa olifera were collected from Telengana state. Petroleum ether, Chloroform, Ethanol, use as extracting solvent.

 

Apparatus Use:

Soxhlet apparatus , Rotary vacuum-evaporator (Yamato RE300, Japan), UV Spectrophotometer A1800 Simadzu

 

METHOD:

Preparation of Extracts:

The leaves of M.olifera were collected from Warangal district of Telengana, during the month of January, 2019. Leaves of M.olifera were shade dried and powdered to get coarse granules which were stored in an air tight container in the dark. The 25g of the powder was subjected to continuous extraction in Soxhlet apparatus using absolute ethanol for 7 h. The extract was filtered and evaporated under reduced pressure to give a viscous mass. The concentrated crude extracts were stored at 4°C in a refrigerator and used for further study.

 

Nucleation assay:

We chose the classical model for the study of oxalate crys-tallization because of its simplicity and satisfactory repro-ducibility. This model includes the study of crystallization without inhibitor and with it, in order to assess the inhibiting capacity of any chemical species used. Solution of calcium chloride and sodium oxalate were prepared at the final con-centrations of 5mmol/L and 7.5mmol/L respectively in a buffer containing Tris 0.05mol/L and NaCl 0.15mol/L at pH6.5. 950mL of calcium chloride solution mixed with 100 mL of herb extracts at the different concentrations (100 mg/mle1000 mg/ml). Crystallization was started by adding 950 mL of sodium oxalate solution. The temperature was maintained at 37°C. The OD of the solution was monitored at 620 nm. The rate of nucleation was estimated by comparing the in duction time in the presence of the extract with that of control.

 

The growth of crystals was expected due to the following reaction:

 

 

Aggregation assay:

In this method CaOx monohydrate (COM) crystals were prepared by mixing calcium chloride and sodium oxalate at 50 mmol/L. Both solutions were equilibrated to 60° C in a water bath for 1h and then cooled to 37° C overnight. The crystals were harvested by centrifugation and then evaporated at 37 C. CaOXcrystals were used at a final concentration of 0.8 mg/ml, buffered with Tris 0.05 mol/L and NaCl 0.15 mol/L at pH 6.5. Experiments were conducted at 37 C in the absence or presence of the plant extract after stopping the stirring. The percentage aggregation inhibition rate (Ir) was then calculated by comparing the turbidity in the presence of the extract with that obtained in the control using following formula:

 

 

RESULTS AND DISCURSION:

Determination of absorption maxima:

Take the plant leaf extracts and diluted by 100µg/ml, 300µg/ml, 600µg/ml. Scanned the 3 sample by UV and the absorption maxima was found at 560nm for the plant leaf extract.

 

Turbidity calculates by Nucleation assay:

The formula of determine the turbidity as per lamber beer method:

 

Turbidity=(2.3*absorbence)/L

 

Table I: Table for Nucleation assay

Concentration of Herb Extract	Absorption at 560nm	Turbidity
100ug/ml	0.989	2.2747
300ug/ml	0.852	1.9596
500ug/ml	0.741	1.7043
700ug/ml	0.621	1.4283
1000ug/ml	0.412	0.9476

 

Figure 1: Effect of different concentration of leaf extract in Nucleation assay

 

Aggregation assay:

Calcium oxalate crystal at concentration of 0.8mg/ml was taken as control. In the control sample no herb extract was present.

 

Table II: CaOx crystallization without the addition of extract (control)

Concentration of Herb Extract	Absorption at 560nm	Turbidity
(Control)	0.945	2.1735

 

Determination of Rate of Inhibition (IR):

Table III: Effect of concentration M.Olifera extract on CaOx crystallization inhibition rate.

Concentration of Herb Extract	Absorption	Turbidity	IR
100ug/ml	0.651	1.4973	31.11111
500ug/ml	0.432	0.9936	54.28571
700ug/ml	0.356	0.8188	62.32804
1000ug/ml	0.258	0.5934	72.69841

 

Figure 2 : Effect of different concentration of leaf extract by Aggregation assay

 

The % inhibition of turbidity (aggregation) in the presence of herb extracts was lower than in the control, showing that crystals were less aggregated. The inhibited aggregation associated with the extract increased with concentration. by the addition of 0.01M sodium oxalate solutions. The effect of extract (100μg/ml - 1000μg/ml) was studied by the measurement of turbidity in presence or absence of extract at specific nm of a spectrophotometer. The herb extract of M.Olifera promoted the nucleation of calcium oxalate crystals, increasing their number but decreasing their size. It also promoted the formation of calcium oxalate dehydrates crystal, despite the presence of calcium oxalate monohydrate particles.

 

The main findings of the present study were that extracts from plants inhibited the crystallization of CaOx in solution, there were less and smaller particles with increasing concentrations of extract as shown in graph. Table: 2 showed maximum abs of crystals as it was without plant extracts while Fig. 2 showed comparatively less absorption. The increasing concentration of plant extracts (100, 500, 700, 1000 μg/ml) had inhibited the CaOx crystal growth.

 

Herbal extracts contain substances that inhibit the growth of CaOx crystals. This property of plants may be important in preventing kidney stone formation. CaOx crystals induced by urinary macromolecules was less tightly bound to epithelial cell surfaces, which are then excreted with urine. The extract may also contain substances that inhibit CaOx crystal aggregation, the agglomeration of particles is a critical step in urinary stone formation, as larger crystals are less likely to pass spontaneously in the urinary tract. If the extract keeps CaOx particles dispersed in solution they are more easily eliminated.

 

SUMMARY:

The effect of Moringa Olifera leaf extracts on CaOx crystallization was determined by the time course measurement of turbidity changes due to the crystal nucleation and aggregation. Study the Effect of different concentrations of leaf extracts of M.Olifera on CaOx crystallization was done. The precipitation of calcium oxalate at 37°C and pH 6.8 has been studied by the measurement of turbidity at 560nm. A spectrophotometer UV/Vis (Shimadzu) was employed to measure the turbidity of the formation of calcium oxalate. Both of the assays show positive results as per the objective of study. In aggregation assay the Inhibition rate was found 31.11 for 100µg/ml, 54.29 for 500µg/ml, and 62.33 for 700µg/ml and 72.70 for 1000µg/ml. So from this study we shown the Inhibition rate of crystal was increase according to the concentration of herb extract. And as per the Nucleation assay turbidity of the plant extract found 2.27 for 100µg/ml, 1.96 for 300µg/ml, 1.70 for 500µg/ml, 1.43 for 700µg/ml, 0.93 for 1000µg/ml. So from this study we shown the turbidity of CaOX solution was decrease along with the increasing concentration of herb extract.

 

CONCLUSION:

In aggregation assay the Inhibition rate of crystal was increase according to the concentration of herb extract. And the Nucleation assay from this study we shown the turbidity of CaOX solution was decrease along with the increasing concentration of herb extract. So we can conclude The leaf extract of M.Olifera have an inhibitory effect on CaOx crystallization which is beneficial in the treatment of Urolithiasis.

 

ACKNOWLEDGMENT:

We are grateful to Bharat Institute of Technology, Telangana for their support to this research.

 

CONFLICTS OF INTEREST:

There are no conflicts of interest.

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Accepted on 14.05.2020      ©Asian Pharma Press All Right Reserved