Diuretic Activity of Fruit Extract of Xanthium strumarium L in Albino Rats

M.A Halkai¹, Arun Patil^*¹, MD Akhil Ahmed¹, AK Beknal¹

¹Department of Pharmacognosy, HKES’s College of Pharmacy, Gulbarga Karnataka, India 585 105.

*Corresponding Author E-mail: asp_room@rediffmail.com

ABSTRACT:

Objective: The present study is an attempt to investigate the diuretic effect of fruit extracts, phytochemical screening, isolation and identification of phytoconstituents from petroleum ether, benzene, chloroform, alcohol and aqueous extracts of Xanthium strumarium.

Materials and methods: The animals were divided into four groups each. Group-I was treated only with normal saline solution and serve as control. Group-II was received furosemide at a dose of 5 mg/Kg, p.o. and it was considered as positive control group. Group-III & Group-IV received the petroleum ether extract (PEE) at doses of 250 and 500mg/Kg, p.o. respectively. After oral administration of PEE, the urinary output of each group was recorded at different time intervals from the graduated urine chamber at metabolic cage. The volume of urine collected was measured at the end of 5 h.

Results: We observed a potent diuretic and electrolyte activity in petroleum ether extract of Xanthium strumarium L fruits. The Diuretic activity was not observed in chloroform and alcoholic extracts.

KEYWORDS: Diuretic activity, Xanthium strumarium L fruits, Furosemide.

INTRODUCTION:

Ayurveda, the Indian traditional system of medicine, lays emphasis on promotion of health a concept of prevention of diseases and strengthening of both physical and mental health. It is recognized in Ayurveda that the diuretic system is involved in the etiology, pathophysiological mechanisms of various kidney diseases, heart, electrolyte balance and spleen inflammation. It held the doctrine that modulation of diuretic response would alleviate the diseases and the concept of Rasayana in Ayurveda. Ayurveda medicine thus constitutes a rich source of active substance for diuretic and electrolytes on herbal preparations. Among the wide range of drugs claimed to possess the rasayana effect^1.

Diuretics increase the rate of urine flow and sodium excretion and are used to adjust the volume and/or composition of body fluids in variety of clinical situation, including hypertension, heart failure, renal failure, nephritic syndrome and cirrhosis. A number of diuretics like mannitol, thiazides, furosemide, and ethacrinic acid are used in practice². Xanthium Strumarium L (Cocklebur) is a flowering plant belonging to the family Asteraceae, it is native to the America, Eastern Asia, South Africa and Australia. It is coarse, herbaceous annual herb growing about 50-120 cm tall, which invades agricultural lands³.

Xanthium Strumarium L compositae, is a common weed found in India⁴. The whole plant, specially root and fruit, is used as medicine. According to Ayurveda, Xanthium Strumarium L is anthelmentic, antipyretic, epilepsy, diuretic, cooling laxative, fattening, alexiteric and tonic, digestive and improves appetite, complexion, and memory. It cures leucoderma, poisonous bites of insects, salivation and fever. Seed yields semi-drying edible oil (30-35%) which resembles sun flower oil and used in bladder infection and herpes, can be used as manure where shell can be used as activated carbon⁵.

Cocklebur was cultivated as a leafy vegetable in China. Young floral tops and the two leaves below are boiled in water and eaten as a pot-herb in Assam. The herb as such is suspected to be poisonous, but the toxic substances are removed by washing and cooking⁶ In English, Xanthium is known as cocklebur or burweed. The genus Xanthium includes 25 species; all of American origin X. spinosum L and X. strumarium L are used medicinally in Europe, North America and Brazil; X. canadens Mill was used in North America and Brazil and X. strumarium L in China, India and Malaysia⁷. Two species of Xanthium, X. indicum and X. strumarium have been reported in India. It commonly grows in waste places and along river banks in warmer parts. It is commonly called chotagokhru due to the shape of its fruit which look likes the cow's toe. In many parts of India, it is known as adhasisi, as this weed is used for the treatment of common disease hemicrania."

MATERIALS AND METHODS:

Animals of either sex weighing 150-200/g were procured from central animal house, M.R Medical College, Gulbarga and were used for the study. Animals were kept for 1 week to acclimatize to laboratory conditions before starting the experiment; they were given free access to food and water ad libitum, expect during experimentation. The fruits of Xanthium straumarium L were obtained from local area of Gulbarga, Bidar, and Aurad, India and authenticated by Dr. Srinath Rao, Chairman of department of Botany, Gulbarga University, Gulbarga, Karnataka, India.

Preparation of Extract:

The fruits were collected in the end of June and first week of July. They were shade dried and powdered to a coarse fine grade. The method is based on the extraction of active constituents present in the drug, using various solvents ranging from non-polar to polar. The extraction was carried using Soxhlet apparatus. The powder was passed through sieve no. 16 and 22. 250/g powder was subjected to extraction using solvent i.e. petroleum ether was added slowly through Soxhlet apparatus. Solvent required was 800-900mL. Temperature was partially increased and maintained according to respective boiling temperature. The powder was carried for successive extraction by using benzene, chloroform and alcohol. Distilled water was used for maceration. The extracts were collected, filtered and solvents were evaporated using rotary flash evaporator. The extracts were concentrated and stored in refrigerator. Qualitative chemical test were performed to the above extracts of Xanthium strumarium L to identify the various phytoconstituents^8,9. The results of preliminary phytochemical investigation are shown in Table 1.

Table 1: Preliminary phytochemical investigation of X. strumarium L fruits

Phytochemical constituents	Petroleum ether extract	Benzene extract	Chloroform extract	Alcohol extract	Aqueous extract
Phenolic compounds and Tannins	+	-	-	+	-
Phytosterols	+	-	+	-	-
Carbohydrates	-	-	-	-	+
Saponins	-	-	-	-	-
Alkaloids	-	-	-	-	-
Flavonoids	-	-	-	-	-
Proteins and Amino acids	-	-	-	+	+

“+” = Present “-” = Absent

Diuretic Activity:

The method of Lipschitz et al¹⁰ was employed for the assessement of diuretic activity. Albino rats of either sex deprived of food and water 18 h prior to the experiment were divided into four groups. Group-I received only normal saline and served as control. Group-II received furosemide at a dose of 5 mg/Kg, p.o. and it was considered as positive control group. Group-III and Group-IV received the PEE, at doses of 250 and 500mg/Kg, p.o. respectively. After oral administration, each animal were placed in an individual metabolic cages specially designed to separate feces and urine at room temperature. The observed parameters were total urine volume for 5hours, Na⁺ and K⁺ excreted in urine. The concentration of electrolytes in urine is expressed in terms of mmol/L and the urine volume is expressed in mL/100g/5 h. Na⁺ and K⁺ concentrations were measured by flame photometer and Cl^-concentration was estimated by titration with silver nitrate solution (N/50) using three drops of 5% potassium chromate as an indicator.¹¹ The results obtained were compared with the control and analysed by Students‘t’ test.

Acute toxicity (LD₅₀) studies:

An attempt was made to identify LD₅₀ of pet ether aqueous extracts of Xanthium strumarium L fruits. Since no mortality was observed at 2000 mg/Kg. It was thought that 2000mg/Kg was the cut off dose. Therefore 1/10^th and 1/20^th dose (i.e. 200mg/Kg and 100mg/Kg) were selected for all further studies.

Statistical Analysis:

All values are expressed as mean±SEM. The values obtained for the above parameters in case of the extracts were compared with standard drug and control group by using one-way Analysis of Variance (ANOVA) followed by Dunnett’s test. P<0.05 was considered as significant.

RESULTS:

In the present study, petroleum ether, chloroform, and alcoholic extracts of Xanthium strumarium L fruits were subjected to preliminary chemical tests. Table 1 shows the presence of various chemical constituents. All extracts were subjected to pharmacological screening to evaluate acute toxicity studies and diuretic activity. All three extracts viz. petroleum ether, chloroform, and alcoholic extract of fruits of Xanthium strumarium L were screened for diuretic activity, and the extracts were administered orally at the dose of 200 mg/Kg b.w. Urine volume (ml), concentration of Na⁺, K⁺, and Na⁺/K⁺ratio electrolytes (mmol/L) in the urine were recorded. The ratios of the concentration of Na+/K+ and diuretic index at the end of 5 hours were calculated to assess the diuretic potential of the extracts of X. strumarium L (Table 2).

Table 2: Diuretic effect of Petroleum ether (40-60^oC) extract of X. strtumarium L (fruits) in healthy albino rats.

Group	Treatment	Mean urine volume(ml)	Electrolyte Na+	Concentration (mmol/l) K+	Na+/K+ ratio	Diuretic
I	Normal saline (5ml/Kg,p.o)	3.99±0.08	68.3 ±0.11	489.11 ±0.28	12.12	__
II	Furosemide (5mg/ Kg, p.o)	9.95±0.09**	173.36±0.27**	876.8 ±2.94**	19.77	2.13
III	PEE (250mg/ Kg, p.o)	4.65 ±0.06*	61.866 ±0.16*	346.17 ±1.10	9.22	0.71
IV	PEE (500mg/Kg, p.o)	6.68±0.09**	92.28±0.19**	451.85±0.09**	11.45	0.93

Data are presented as mean±SEM; n=6, ns=non-significant, * P<0.05 compared to control, **P<0.01 compared to control, *** P<0.001 compared to control (ANOVA followed by Dunnett’s test).‘ T ’ stands for urine collected for extracts, ‘C’ stands for urine collected for control and ‘S’ stands for urine collected for standard drugs

In the control group, the volume of urine for 5hours was found to be 3.99±0.08 and in standard group it was found to be 9.95±0.09**. In petroleum ether extract groups, the volume of urine for 5 hours was found to be (250mg/Kg, p.o) 4.65 ±0.06*, (500mg/Kg, p.o) 6.68±0.09** respectively. In the control group, the excretion of sodium for 5 hours was found to be 68.3±0.11mmol/l and in the standard group it was found to be 173.36±0.27**mmol/L. In the petroleum ether extracts groups, the excretion of sodium for 5 hours was found to be 61.866±0.16*(250mg/Kg, p.o), 92.28±0.19** (500mg/Kg, p.o) mmol/L, respectively. In the control group, the excretion of potassium for 5 h was found to be 489.11±0.28 mmol/L, and in the standard group, it was found to be 876.8±2.94**mmol/L. In the petroleum ether, extracts groups the excretion of potassium for 5 h was found to be 346.17 ±1.10 and 451.85±0.09**mmol/L. The ratio of the concentration of sodium ions to that of the potassium ions in the control group was found to be 12.12 and in the standard group, it was found to be 19.77. In the petroleum ether extract was found to be 09.22 and 11.45, respectively.

DISCUSSION:

The diuretic activities of the extracts were significant when as compared to control. The graded doses of the petroleum ether extract in normal saline showed a significant increase in Diuresis, Natriuresis, Kaliuresis, Glomerular Filtration Rate (GFR). The petroelum ether extract cause increase urine elimination and increase in Na+ and K+ excretion as compared to normal saline. Xanthium Strumarium L. extract significantly increased the GFR may be due to

a) A detergent like interaction with structural components of glomerular membranes.

b) A decrease in renal perfusion pressure, attributable to decrease in the resistance of the afferent arteriole or an increase in the resistance of the efferent arteriole or

c) The direct effect on the arteriole wall affecting glomerular blood flow.

The preliminary phytochemical analysis of these extracts reveal the presence of carbohydrates, phytosterols, tannins, protein and amino acids, Inorganic elemental analysis was also carried out by testing the ash of the fruits. The analysis has shown the presence of Ca⁺², Fe⁺² and P⁺³(calcium, iron and phosphorus) elements in the fruits.

ACKNOWLEDGEMENT:

The authors are thankful to authorities of HKE society and HKES’s College of Pharmacy, Gulbarga, Karnataka, India for providing the necessary facilities.

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Received on 17.02.2016 Modified on 27.02.2016

Res. J. Pharmacology & P’dynamics. 8(1): Jan.-Mar., 2016; Page 13-16

DOI: 10.5958/2321-5836.2016.00004.5