Evaluation of the Diuretic activity of the Methanolic Extract of Bougainvillea spectabilis

 

N.V.L Suvarchala Reddy V¹, Alekhya B², Ganga Raju M²

¹Senior Assistant Professor, Department of Pharmacology, GRCP, Bachupally, Hyderabad.

²Department of Pharmacology, Gokaraju Rangaraju College of pharmacy, Hyderabad

 

ABSTRACT:

Although Bougainvillea spectabilisis very popular and attractive ornamental plants in most areas with warm climates belonging to the family Nyctaginaceae. The plants belonging to this family possess diuretic activity. Therefore, the aim of the present study was to evaluate the diuretic activity of the methanolic extract of Bougainvilea spectabilis of aerial parts. Bougainvillea spectabiliswas orally administered to adult male albino rats at 200 and 400 mg/kg, and its diuretic activity was evaluated and compared to the reference drug furosemide (20 mg/kg, administered i.p.). Acute toxicity of the extract was assessed. Compared to control rats, there was significant higher urinary output, as well as sodium and potassium ion excretion, in animals administered the methanolic extract of Bougainvillea spectabilis. The diuretic activity of aerial parts of Bougainvillea spectabilis has been attributed to the presence of flavonoids. This could be responsible for the effect demonstrated herein, which was similar to that produced by the reference drug furosemide. The mechanism of action of furosemide is by inducing a loss of water through the inhibition of NaCl reabsorption. The results suggest that this receptor-mediated mechanism may account for the diuretic effect of Bougainvillea spectabilis as well.

 

 

 

INTRODUCTION:

Diuretics drugs increase the rate of urine flow and adjust the volume and composition of body fluids. Drug-induced diuresis is beneficial for the treatment of many maladies such as congestive heart failure (CHF), chronic renal failure, nephritis, cirrhosis, hypertension and pregnancy-induced toxemia.^1,2 However, many of the diuretics currently used in clinical practice have been associated with a number of adverse effects, including electrolyte imbalance, metabolic alterations, the onset of diabetes, activation of the renin-angiotensin and neuroendocrine systems, and impairment of sexual function.^2,3

 

Therefore, it is important to consider alternatives that have greater effectivenessand fewer side effects. Many of the herbs used in folk medicine have yet to be scientifically evaluated for their effectiveness and safety.⁴

 

Bougainvillea spectabilis leaves are used to cure variety of disorders like for diarrhea and to reduce stomach. It is used for cough and sore throat. Infusions of flowers used as treatment for low blood pressure. Some pharmacological studies report revealed that it has medicinal properties such as antidiabetic, antifertility potential, antibacterial, anti-inflammatory⁹, antiviral and larvicidal. The chemical characterization of some Nyctaginaceae family plant species, such as bougainvinonespeltogynoids, essential oils including methyl salicylate, terpinolene, α-(E)-ionone, pinitol, β-sitosterol, quercetin, and quercetin-3-O-rutinoside.⁷ Bougainvillea spectabilishas been employed as a diuretic in most areas with warm climate, but this use still lacks a scientific basis. The aim of the present study was to evaluate the diuretic activity of methanolic extract of Bougainvillea spectabilis.

 

MATERIALS AND METHODS:

Plant collection:

Specimens of Bougainvillea spectabiliswild were collected and authenticated in the month of December 2017, native of South America. (Voucher no. 286) is preserved in the herbarium of the biological research centre at the Osmania University in Hyderabad, and was identified by Professor & Head of Department of Botany of that institute.

 

Methanolic extraction of Bougainvillea spectabilis:

The air-dried aerial part of the plant (1kg) was extracted successively with methanol solution. Extractions in these organic solvents were all conducted by heating the solid plant residue in the appropriate solvent at reflux for 16 h. The crushed plant material is loaded into the thimble, which is placed inside the Soxhlet extractor. The side arm is lagged with glass wool. The solvent is heated using the isomantle and will begin to evaporate, moving through the apparatus to the condenser. The condensate then drips into the reservoir containing the thimble. Once the level of solvent reaches the siphon it pours back into the flask and the cycle begins again. The process should run for a total of 16 h, afterward, insoluble fatty materials were removed by filtration. The filtrate was evaporated under vacuum to give defatted extracts^{1, 2}. The obtained extract was stored in dry ampoules for the further study.

 

Experimental animals:

Adult male Albino rats (250 - 300 g) were housed in transparent polycarbonate cages four per cage. Animals were maintained in a room that had little noise, a controlled temperature (22-25 C), 8 to 10 air changes per minute and natural lighting. They were given food (a standard rodent diet of Purina lab chow) and water ad libitum, and underwent an adaptation period of three days. The experimental protocol was approved by the Institutional Animal Ethics Committee (approval no: 1175/PO/Re/S/08/CPCSEA, 28/04/2017).

 

Acute oral toxicity study:

Methanolic extract of Bougainvillea spectabilis aerial parts was tested on albino Swiss mice up to a dose of 2000mg/kg, bd. wt. The animal did not exhibit any signs of toxicity or mortality up to 2000 mg/kg, bd. wt. various morphological and behavioural characters were observed during the study. The other parameters like food and water consumption were also observed. All the animals were found to be safe even after 14 days of observation. Hence the extract was found to be safe up to 2000 mg/kg, bd.wt.¹².

Evaluation of diuretic activity:

Animals were maintained under standard condition of temperature and humidity and underwent for an adaptation period of three days. The animals were divided into four groups (n=6). Group 1, as the negative control, received normal saline solution; group 2 received the reference diuretic, furosemide (Lasix, SANOFIAVENTIS) at 20 mg/kg administered intraperitoneally^12,13; groups 3 and 4 received the methanolic extract of Bougainvillea spectabilis at 200 mg/kg p.o. and 400 mg/kg p.o. respectively, and the diuretic activity was carried out based on the method of Lipschitz .¹⁴

 

Immediately after administration by gavage using an 18 G intragastric cannula, the animals were placed in metabolic cages (4 per cage), especially designed to separate urine and faeces, and kept at a controlled temperature of 22-25 °C. At the end of 6 h, the volume of urine collected was measured. During this period, no food and water was available to the animals. During the one-week experimental period, the parameters measured were total urine volume, and concentration of Na⁺ and K⁺ in the urine. Na⁺, K⁺ concentrations were determined by ‘flame photometer’.

 

Statistical analysis:

Results are expressed as the mean ± SEM. Data was analyzed by one-way analysis of variance (ANOVA) followed by Dunnetts multiple comparison tests. A value of p <0.05 was considered statistically significant.

 

RESULTS AND DISCUSSION:

The preliminary phytochemical investigation of methanolic extract of Bougainvillea spectabilis aerial parts showed the presence of phenolic compounds, alkaloids, flavonoids, saponins, terpenoids, tannins, carbohydrates etc.

 

Table 1: Preliminary Phytochemical analysis of Methanolic extract of Bougainvillea spectabilis

Phytochemical Constituents	Results
Carbohydrates	++
Alkaloids	++
Saponins	++
Tannins	++
Flavanoids	++

 

Acute toxicity test:

The experimental extracts of Bougainvillea spectabilis were used in concentrations of 200 mg/kg and 400 mg/kg, with animals showing no signs of acute toxicity. No macroscopic alterations were noted in the viscera of the treated rats.

 

Effect on urine volume:

The animals were observed with no signs of dehydration at 6 h intervals. The reference diuretic (furosemide) significantly increased urine output compared to the control (p >0.001), with a diuretic index of 2.77. Administration of the test drug at 200 and 400 mg/kg also resulted in a significant increase in urine volume, although less than that found with the reference drug. The diuretic index for these two doses was 1.65 and 1.99, respectively, compared to 2.77 found for furosemide (Table 2).

 

Table 2-Effect of oral administration of the methanolic extract of Bougainvillea spectabilis of aerial parts on urinary volume

Group	Urine volume (mL/6 h)	Diuretic index (6h interval)
Control MEBS 200 mg/kg MEBS 400 mg/kg Furosemide	6.17±0.10 10.2±0.19** 12.3±0.45** 17.01±0.40*	--- 1.65 1.99 2.77

 

MEBS (Methanolic extract of Bougainvillea spectabilis). Values are expressed as the mean ± SEM; *p < 0.001 compared to the control group, **p < 0.001 compared to Furosemide group (ANOVA followed by Dunnett’s test).

 

Diuretic index=volume of test group / volume of control group.

 

Effect on urinary electrolyte excretion:

Methanolic extract of Bougainvillea spectabilis showed a significant increase in the excretion of sodium and potassiumwas found. Moreover, increase in the Na⁺/K⁺ ratio was also found. The increase in electrolyte excretions with the methanolic extract (at both doses) was less than that found with furosemide (Table 3).

 

Table 3–Effect of oral administration of the methanolic extract of Bougainvillea spectabilis of aerial parts on urinary electrolyte excretion

Treatment	Dose	Total Na+	Total K+	Na+/K+
Normal saline MEBS MEBS Furosemide	25 ml/kg 200 mg/kg 400 mg/kg 20 mg/kg	150.1±13.2 201.2±3.21** 232.4 ± 0.62* 322.3 ± 4.31*	403±4.34 105.4±9.1** 120.2±13.1** 136.1±8.22*	0.37 1.90 1.93 2.36

 

MEBS (Methanolic extract of Bougainvillea spectabilis). Values are expressed as the mean ± SEM; *p < 0.001 compared to the control group, **p < 0.001 compared to Furosemide group (ANOVA followed by Dunnett’s test).

 

Na⁺ / K⁺ ratio = Total Na⁺/ K⁺

 

There are few reports on the diuretic activity of the Nyctaginaceae species. The diuretic effect of the orallyadministered methanolic extract of Bougainvillea spectabilis was evaluated in normal adult male Albino rats and compared with that produced by furosemide, a loop diuretic widely used in clinical practice. Diuresis has two components: an increase in urine volume (water secretion) and a net loss of solutes (i.e., electrolytes) in the urine. These processes may result from suppression of renal tubular reabsorption of water and electrolytes into the blood stream. Administration of the Bougainvillea spectabilis extract showed a significant increase in urine output and electrolyte excretion (p < 0.001) in a dose dependent manner (Tables 1 and 2), indicating the possibility of intrinsic and causal action, possibly receptor-mediated.

 

Some herbs induce diuresis by stimulating the thirst centre in the hypothalamus and thereby enhancing fluid intake^16,17. Some plants elicit diuresis due to their high salt content.¹⁸Such nonspecific mechanisms are unlikely to be involved in the effect of the test compound, in spite of the high Na⁺ level in urine, because the extract of Bougainvillea spectabilis did not alter the osmolality or specific gravity of urine. Thus, the diuretic effect is not related to an osmotic mechanism. Furthermore, osmotic diuretics are inactive when administered orally, and for this reason are usually administrated intravenously¹⁸.The diuretic effect of Bougainvillea spectabilis is also unlikely to be due to an impairment of the action of an antidiuretic hormone, because such impairment causes polyuria with low osmolality.

 

The reference drug furosemide showed a marked increase in urine volume and in urinary excretion of Na⁺ and K⁺, with a similar pattern as that found with the methanolic extract of Bougainvillea spectabilis (Tables 1 and 2), suggesting a similar mechanism of action in both cases. Furosemide, like other loop diuretics, acts by inhibiting NKCC2, the luminal Na⁺ and K⁺2Cl symporter in the thick ascending limb of the Henley loop. It also abolishes the corticomedullary osmotic gradient and blocks negative as well as positive free water clearance^{19, 20}. By inhibiting the transporter, the loop diuretics reduce the reabsorption of NaCl in the kidney and also diminish the lumen-positive potential that derives from K⁺ recycling. This electrical potential normally drives divalent cation reabsorption in the loop. Thus, by reducing this loop potential, diuretics induce an increase in Mg²⁺ and Ca²⁺²¹.

 

Some flavonoids have shown a diuretic effect. Several isoflavonoids, including genistein and daidzein, have been reported to cause inhibition of the Na⁺-K⁺- 2Cl^-cotransporter, as well as an increase in natriuresis and kaluresis²².Moreover, the flavonoid crisine has been shown to induce a significant increase in urine flow, glomerular filtration and Na⁺ and K⁺ excretion. Recently, it wasreported that seven methoxyflavonoids actively bound to adenosine receptor A1, provoking antagonism and therefore dieresis and sodium excretion²³.

 

In the present study, in reference to the elimination of Na⁺ and K⁺ the extract of Bougainvillea spectabilis showed a greater natriuretic than kaluretic effect. The Na⁺/K⁺ ratio can define the nature of the diuretic mechanism. The Na⁺/K⁺ ratio for furosemide is approximately 1, meaning that it eliminates the two electrolytes equally. On the other hand, with acids this ratio is less than one (with a greater excretion of K⁺ than Na⁺), and with spironolactone it is greater than one (with a lower excretion of K⁺ than Na⁺)²⁴.

 

There is an association between urine volume and Na⁺ concentration in the urine. This is logical, considering that the action mechanism of a great number of diuretics on the market is by decreasing the reabsorption of this ion, which induces osmosis of water out of the organism²⁴. The isolation and chemical characterization of the compounds present in different endemic species of the Bougainvillea genuswas found and showed the presence of flavonoids. Hence, flavonoids are probably responsible for the diuretic effect of MEBS.

 

CONCLUSION:

The present study demonstrates the diuretic activity of the methanolic extract of Bougainvillea spectabilis, which increased urinary volume and electrolyte (sodium and potassium) excretion. The diuretic pattern of the methanolic extract was similar to that of the reference drug (furosemide), suggesting a similar mechanism of action. Further study of Bougainvillea spectabilis is necessary in order to isolate the compounds present in this species, as well as identify which compounds are responsible for the diuretic effect shown by the methanolic extract. Additionally, it is necessary to determine the mechanism or mechanisms of action involved in the diuretic effect.

 

ACKNOWLEDGEMENTS:

The authors are grateful to the Principal and the Management of the Gokaraju Rangaraju College of Pharmacy, for the constant support and encouragement during the course of the work.

 

CONFLICT OF INTEREST:

The authors have no conflict of interest

 

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Received on 08.10.2018         Modified on 30.10.2018

Accepted on 18.11.2018  ©A&V Publications All right reserved