Evaluation Of In-vivo Diuretic and In-vitro Litholytic Potential by Compound Ethanolic Bulb Extract of Allium cepa And Allium sativum In Experimental Model

Azhar Sulthana I., Suresh Kumar J., Mohan D., Naga Jyothi T., Sravani M., Smati K.,

doi:10.5281/zenodo.15045404

Research Paper | Open Access
Volume 03 | Issue 03 | Article Id IJPS/250303101

Evaluation Of In-vivo Diuretic and In-vitro Litholytic Potential by Compound Ethanolic Bulb Extract of Allium cepa And Allium sativum In Experimental Model
Azhar Sulthana I.* Suresh Kumar J. Mohan D. Naga Jyothi T. Sravani M. Smati K.
Narasaraopeta institute of Pharmaceutical Sciences, Narasaraopet.

Abstract

Urolithiasis, the formation of stones within the urinary system, is a prevalent and painful condition, characterized by recurrent episodes. It affects approximately 12% of the global population, with calcium stones accounting for approximately 80% of all urinary calculi. The present study investigates the potential litholytic and diuretic properties of compound ethanolic extract of Allium cepa (onion) and Allium sativum (garlic) as a natural remedy for urolithiasis. The bulbs of both plants were subjected to maceration to obtain the compound extract, and phytochemical screening identified presence of bioactive compounds, including carbohydrates, tannins, flavonoids, proteins, and phenols. The diuretic activity of the extracts was evaluated in comparison to furosemide (10 mg/kg), with doses of 10 mg/kg and 20 mg/kg used for testing. Litholytic activity was assessed against Cystone. In vitro experiments, including nucleation and aggregation tests, demonstrated that the extracts inhibited calcium oxalate (CaOx) crystal formation, with the highest inhibition at 1000 µg/mL. The compound ethanolic extract of Allium cepa and Allium sativum exhibited significant anti-urolithiatic activity, comparable to the standard drug Cystone. In In vivo, the extracts significantly increased urinary output and enhanced electrolyte excretion (Na+, K+, Cl-) in a dose-dependent manner, showing similar effects to furosemide. Analysis of urinary parameters indicated notable alterations in diuretic, saluretic, and natriuretic indices following treatment. This study suggests that the compound product of Allium cepa and Allium sativum possess promising anti-urolithiatic and diuretic effects, likely due to the presence of active compounds such as flavonoids, tannins, and phenols. These findings propose that these plants could serve as natural alternatives for the treatment of urolithiasis and the promotion of diuresis, warranting further clinical investigation to confirm their therapeutic efficacy.

Keywords

Allium cepa, Allium sativum, anti-urolithiatic, diuretic activity, phytochemical screening, nucleation, aggregation, furosemide.

Introduction

The increasing global prevalence of kidney-related disorders, such as nephrolithiasis (kidney stones) and fluid retention, has led to growing interest in their management. Common therapeutic strategies for addressing these conditions include diuretics, which stimulate fluid excretion, and litholytics, which aid in dissolving kidney stones. Traditional medicinal plants have long been recognized for their bioactive compounds, which exhibit significant pharmacological effects in treating such ailments. Among these plants, Allium cepa (onion) and Allium sativum (garlic) stand out due to their diverse bioactive constituents, such as sulfur-containing compounds, flavonoids, and phenolic acids, all of which contribute to their therapeutic effects. ^(1,4) Recent research has highlighted the diuretic properties of both Allium cepa and Allium sativum, with evidence showing their ability to increase renal excretion of sodium, chloride, and potassium. ⁽⁵⁾Additionally, both garlic and onion are known for their high content of sulfur compounds, particularly allicin in garlic, which are associated with antioxidant, anti-inflammatory, and antimicrobial properties. These characteristics suggest their potential as therapeutic agents for kidney-related conditions. ^(2,7) Nephrolithiasis, a condition marked by the formation of kidney stones, remains a significant health concern. Several studies on the litholytic properties of natural compounds have proposed that Allium sativum and Allium cepa may help in breaking down or dissolving kidney stones, potentially providing a non-invasive alternative to surgical treatments. ^(3,6) This study aims to assess the in-vivo diuretic and in-vitro litholytic effects of ethanolic extracts of Allium cepa and Allium sativum, utilizing both animal models and laboratory simulations. The outcomes of this research could provide scientific support for the use of these plants in treating fluid retention and kidney stones. ⁽⁷⁾

MATERIALS AND METHODS:

Collection of Plant material: Fifty bulbs of plant sample (A.cepa and A.sativum) were purchased from a local market in Narasaraopet, Andhra Pradesh. The plant material was identified and authenticated by the Department of Botany and Microbiology, Acharya Nagarjuna University. The bulbs of the Allium cepa and Allium sativum were properly washed and shade dried for a period of two weeks, crushed in a mortar, and later pulverized into a fine powder using an electric blender.
Extraction of bioactive compounds from the plant material: 100g of the powdered plant materials (A.cepa and A.sativum) were poured into different beakers and 500 ml of ethanol were poured into each beaker respectively. The contents are stirred using a sterile glass rod and allowed to stand for 72 hours at room temperature (25⁰C ± 1). The contents were filtered through a filter paper (Whattman No. 1) and the filtrate concentrated and evaporated using water-bath at the temperature of +95⁰C. Extracts are then mixed and kept at 20⁰C prior use. ⁽⁸⁾
Animals: Male wistar rats weighing around 100g to 200g were selected. Animals were placed in cages made of polypropylene at random times with bedding made of rice husks. Animals were housed at a relative humidity of 30%–70% and at a temperature of 24°C ± 2°C. All of the animals were served a typical commercial pellet rat diet and were given unlimited access to water.
Assessment of Anti-urolithiatic activity of compound extract:

Nucleation Assay: Calcium chloride (5 mmol/L) and sodium oxalate (7.5 mmol/L) solutions were prepared in a Tris-HCl (0.05 mol/L) and sodium chloride (NaCl) (0.15 mol/L) buffer at pH 6.5. Compound ethanolic extracts of Allium cepa and Allium sativum were diluted to concentrations ranging like 100, 200, 400, 800, 1000 µg/mL in distilled water. One millilitre of each ethanolic extract concentration was mixed with 3 mL of calcium chloride (CaCl₂) solution, followed by the addition of 3 mL of sodium oxalate (Na₂C₂O₄) solution. The resulting mixtures were incubated for 30 minutes at 37ºC. The optical density (OD) of the mixtures was then measured at a wavelength of 620 nm. The percentage inhibition of nucleation by the extract was calculated using the formula provided and compared to the inhibition calculated for the standard Cystone.

% Inhibition = [1 – (OD test/OD control) × 100] ⁽⁹⁾

OD test = OD of the test (compound ethanolic extract) OD control = OD of the negative control

CaCl₂ + Na₂C₂O₄ → CaC₂O₄ + 2NaCl

Aggregation Assay: Sodium oxalate (Na₂C₂O₄) and calcium chloride (CaCl₂), each at a concentration of 50 mmol/L, were mixed and heated in a water bath for one hour, then incubated at 37°C for 12 hours. The resulting mixture was evaporated to yield CaOx crystals. One millilitre of a Tris-HCl (0.05 mol/L) and sodium chloride (NaCl) (0.15 mol/L) buffer at pH 6.5 was added to 1 mg of CaOx crystals. Three millilitres of the CaOx solution were added to each concentration (100, 200, 400, 800, 1000 µg/mL) of extract/standard (Cystone) and incubated at 37°C for 30 minutes. The absorbance of both the sample and standard was measured at 620 nm. The percentage inhibition of CaOx crystal aggregation was then calculated using the provided formula. % Inhibition =(1-OD(TEST)/OD (CONTROL)×100 ⁽¹⁰⁾

Preliminary Phytochemical Screening: The compound Ethanolic Bulb extract that is obtained from the Maceration process followed by evaporation for concentrated extract is subjected to various Phytochemical screenings to understand the types of phytochemicals present in the extract. The screening tests includes Carbohydrates [Molisch test, Benedict test, Fehling test], Tannins [Ferric chloride test], Flavonoids [Lead acetate test], Proteins [Million test, Biuret test], Phenols [Bontrager test]. ⁽¹¹⁾
Determination of Diuretic activity of compound extract in animal models:

Grouping of animals: We chose 24 healthy male wistar rats and divided them into 4 groups with 6 rats in each group. Group 1 served as control group and administered with normal saline (5 ml/kg). Group 2 served as positive control group and administered with Furosemide (10 mg/kg). Group 3 and 4 administered with 10mg/kg and 20mg/kg respectively.
Experimental Design: The diuretic effects of the compound ethanolic extract of A.cepa and A.sativum were evaluated in male wistar rats using the Lipschitz Test. Male wistar rats were divided into 4 groups, each consisting of six rats. Group I served as the normal control and was given the vehicle (0.9% in normal saline, 5 ml/kg body weight). Group II received Furosemide (10 mg/kg, p.o.) in the vehicle. Groups III, IV were treated with 10 mg/kg and 20 mg/kg, p.o. doses of the compound ethanolic extract of A.cepa and A.sativum in the vehicle. After treatment, all rats were hydrated with saline and placed in metabolic cages (2 rats per cage) that were designed to separate urine and faeces, and maintained at a temperature of 21°C±0.5°C. Urine volume was measured after a 2-hour collection period for 8 hours, during which the animals were not given food or water. Various parameters, such as total urine volume and concentrations of sodium, potassium, and chloride in the urine, were assessed. ⁽¹²⁾
Estimation of Urinary Electrolytes: Urine electrolytes (sodium, potassium and chloride) were determined by Ion-Selective Electrode [ISE] method.
Statistical analysis: Experimental results were expressed as mean + SEM (n=6). Statistical Analysis was performed with one-way-ANOVA followed by Dunnett’s t-test.

RESULTS:

Preliminary Phytochemical Screening: The percentage yield of the compound extract is found to be 94.8%. Qualitative phytoconstituent determination in the compound extract showed the presence of.

Table: 1 Preliminary Phytochemical Screening

S. No.	Phytoconstituents	Name Of Test	Results
	Carbohydrates	Molisch’s	Present
		Benedict’s	Present
		Fehling’s	Present
	Tannins	Ferric chloride	Present
	Flavonoids	Lead acetate	Present
	Proteins	Million’s	Present
	Proteins	Biuret’s	Present
	Phenols	Bontrager’s test	Present

b) Nucleation Assay: The compound ethanolic extract of A.cepa and A.sativum inhibited crystal formation as well as promoted crystal dissolution in a dose dependent manner and was comparable to the activity of Cystone. The highest percentage of nucleation inhibition was obtained at a concentration of 1000 µg/ml (83.72%). The Compound Ethanolic extract of A. cepa and A. sativum was found to possess significant anti-urolithiatic activity when compared to standard Cystone.

Fig:1 Photomicrographs of CaOx crystal density in different solutions; A. control, B. cystone, C. Compound Ethanolic extract of A. cepa and A. sativum 400, D. Ethanolic extract of A. cepa and A. sativum 800 and E. Compound Ethanolic extract of A. cepa and A. sativum 1000 (µg/ml) magnification 100x

Table: 2 Nucleation assay of Compound Ethanolic extract of A. Cepa and A. sativum

Values are expressed as Mean ± Sem (n=3), significant *P<0.5, **P<0.01, ***P<0.001 compared to standard (cystone)

S.NO.	Compound *Ethanolic extract of A. cepa and A. sativum* (µg/ml)**	% Inhibition
S.NO.		Compound Ethanolic extract of A. cepa and A. sativum	Cystone
	100	38.20 ± 0.63*	28.28 ± 0.29
	200	38.67 ± 0.58*	30.82 ± 0.29
	400	47.10 ± 1.12*	41.65 ± 0.28
	800	67.11 ± 1.89**	50.88 ± 0.86
	1000	82.96 ± 1.77***	75.85 ± 1.31

Aggregation Assay: The compound ethanolic extract of A.cepa and A.sativum inhibited crystal formation as well as promoted crystal dissolution in a dose dependent manner and was comparable to the activity of Cystone. The highest percentage of aggregation inhibition was obtained at a concentration of 1000 µg/ml (50.98%). The Compound Ethanolic extract of A.cepa and A.sativum was found to possess significant anti-urolithiatic activity when compared to standard Cystone.

Table: 3 Aggregation assay of Compound Ethanolic extract of A. Cepa and A. sativum Values are expressed as Mean ± Sem(n=3), significant *P<0.5, **P<0.01, ***P<0.001 compared to standard (cystone).

S.No.	Compound Ethanolic extract of A. cepa and A. sativum (µg/ml)	% Inhibition
S.No.		Compound Ethanolic extract of A. cepa and A. sativum	Cystone
	100	32.60 ± 0.55***	23.33 ± 0.24
	200	29.56 ± 0.14***	33.55 ± 0.76
	400	32.41 ± 0.56***	42.96 ± 0.20
	800	52.25 ± 0.62***	41.06 ± 0.99
	1000	50.47 ± 1.34***	45.60 ± 0.38

Fig: 2 Effect of Compound Ethanolic extract of A. cepa and A. sativum and Cystone on Nucleation, Aggregation

Diuretic Activity: The results obtained with an evaluation of the diuretic activity of Ethanolic extract of A. cepa and A. sativum are shown in Table 4,5. From the result, it can be observed that compound ethanolic extract of A. cepa and A. sativum has shown a significant diuretic activity by increasing urinary output and increased excretion of sodium, potassium, and chloride when compared to control. The effect of Ethanolic extract of A. cepa and A. sativum was found to be dose-dependent, i.e., among the two doses studied, a higher dose produced more effect. A comparison was made with the standard diuretic drug furosemide, the diuretic effect observed after treatment with Ethanolic extract of A. cepa and A. sativum was found to be significant in terms of urinary output, sodium, potassium, and chloride concentrations. Determination of urinary electrolyte concentration revealed that Ethanolic extract of A. cepa and A. sativum was effective in increasing urinary electrolyte concentrations for all the three ions tested (Na⁺, K⁺, Cl^-).

Table: 4 Effect of Compound ethanolic extract of A. cepa and A. sativum on excretion of electrolytes.

S. No	Treatment	Na⁺(mmol/L)	K⁺(mmol/L)	Cl^-(mmol/L)
1	Control	108.9 ± 1.33	45.0 ± 0.55	149.46 ± 1.83
2	Furosemide	115.8 ± 1.42	86.45 ± 1.06	351.69 ± 4.31
3	Compound extract(10mg/kg)	126.53 ± 1.55	80.37 ± 0.98	241.49 ± 2.96

Table: 5 Effect of Compound Ethanolic extract of A. cepa and A. sativum on Urine volume, Natriuretic index, and Saluretic index.

(Values are expressed as Mean ± Sem(n=6), significance (ns) compared to control.)

Sl. no	Treatment	Urine volume	Diuretic index	Natriuretic index	Saluretic index	Lipschitz value
1	Control	0.86 ± 0.011^ns	---	1.33	1	0.56
2	Furosemide (10mg/kg)	1.53 ± 0.019^ns	1.77	1.06	1.80	1
3	Compound extract(10mg/kg)	1.82 ± 0.022^ns	2.11	1.16	1.42	1.18
4	Compound extract(20mg/kg)	1.96 ± 0.024^ns	2.27	1.34	2.11	1.28

Fig: 3 Effect of Compound Ethanolic extract of A.cepa and A.sativum on Urinary Sodium, Potassium and Chloride (mmol/l) Ions Concentration.

DISCUSSIONS:

Medicinal plants have become a significant source for any disease due to the increased side effects of modern medicine. The present study is on Allium cepa and Allium sativum are used traditionally and has various medicinal uses. They have shown Antioxidant Activity, Antidiabetic Activity, Neuroprotective Activity, Antiplatelet Activity, Anti-inflammatory Activity, Anti-depressant Activity, Analgesic, Emetic, Anti-tumorous, Diuretic and Litholytic activity, Antiparasitic Activity, Antimicrobial activity.⁽¹³⁾ Diuretics are drugs that increase urine production in the kidneys. They are also used in the treatment of high blood pressure, urolithiasis, congestive heart failure, and pulmonary oedema. But the drawback of modern medicine is side effects which include electrolyte imbalance, metabolic alterations, headaches, dizziness, and dehydration.⁽¹⁴⁾ Lithiasis, one of the causes of acute and chronic renal failure. Generally, stones are hard and crystalline minerals. Normal urine contains many inhibitors for calculi formation. Kidney calculi are formed as a result of an imbalance between the inhibitors and promoters.⁽¹⁵⁾ The present study is to evaluate the in-vitro litholytic and in-vivo diuretic activity of Compound Ethanolic extract of A.cepa and A.sativum. To find out litholytic activity the nucleation assay, aggregation assay, were carried out. For diuretic activity Lipschitz method is adopted. Nucleation is an event where the dissolved substance of the supersaturated solution immediately crystallizes. Similar phase change and formation of CaOx crystals was observed while performing the nucleation assay. Significant inhibition in the nucleation of CaOx crystals was observed in the presence of Compound Ethanolic extract of A.cepa and A.sativum. The Ethanolic extract of A.cepa and A.sativum shown more activity than cystone. The possible mechanism for anti-crystallization may be Compound Ethanolic extract of A. cepa and A. sativum would have the ability to complex with free calcium and oxalate ions, thus preventing the formation of CaOx complexes.⁽¹⁶⁾ Aggregation is the process where numerous crystals in the solution come together and adhere to form as a large crystal. The formed large crystals agglomerates and cause renal tubular obstruction there by promoting stone formation. Most of the previous papers stated that the test drug or extract inhibited the crystallization by favouring the formation of calcium oxalate dihydrate (COD) crystals instead of calcium oxalate monohydrate (COM). The decrease in turbidity of Compound Ethanolic extract of A. cepa and A. sativum observed in the process of aggregation may be due to the possibility of more amounts of COD crystals in the solution. This indicates that Compound Ethanolic extract of A. cepa and A. sativum has inhibitory influence on aggregation.⁽¹⁷⁾ In the present study, we have evaluated diuretic properties of Compound ethanolic bulb extract of Allium cepa and Allium sativum. The animals were fasted overnight and administered Furosemide and test drug (10mg/kg and 20mg/kg). The urine was collected at 2-hour, 4 hour and 8 hour and urine output and electrolyte excretion were observed. The excretion of Na⁺, K⁺, Cl^-perceived with Compound Ethanolic extract of A. cepa and A. sativum and furosemide is may be due to inhibition Na⁺ /K⁺/Cl^-symport at distal loop of nephron. The Compound Ethanolic extract of A. cepa and A. sativum shown dose dependent activity. The activity may be due to active principles such as carbohydrates, flavonoids, proteins, tannins, and phenols in Compound Ethanolic extract of A. cepa and A. sativum.

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