Evaluation of the diuretic activity of ethanolic and ...

Evaluation of the diuretic activity of ethanolic and aqueous extracts of

Tabernaemontana divaricata in rats

Short Title: Diuretic potential of Tabernaemontana divaricata plant extracts

Chanchal Raj1*, Shailesh Kewatkar2, A. Balasubramaniam3, Ashish Jain1, Prakash Turiya4,

Sandeep Dhull5, Nadeem Sayyed5

1 Department of Pharmacognosy, Shri D. D. Vispute College of Pharmacy and Research Center,

New Panvel, Navi Mumbai, Maharashtra, India 2 Departments of Pharmacognosy, Rajarshi Shahu College of Pharmacy, Buldana, Maharashtra,

India 3 Dean, School of Pharmacy, ITM University, Gwalior, Madhya Pradesh, India 4 Consultant Medical Writer, Indore, Madhya Pradesh, India 5 Department of Clinical Research, Meril Life Sciences Pvt. Ltd, Vapi, Gujarat, India

*Corresponding Author

Chanchal Navin Raj

Shri D. D. Vispute College of Pharmacy and Research Center,

New Panvel, Navi Mumbai 410206,

Maharashtra, India

Email- [email protected]

Mob-+91 9826668034

Conflicts of interest: There are no conflicts of interest

Journal of Seybold Report ISSN NO: 1533-9211

VOLUME 25 ISSUE 9 2020 Page: 4236

Abstract

Tabernaemontana divaricata an evergreen plant belonging to the family Apocynaceae exhibited

its application in the ailment called strangury as per traditional literature. Thus, considering the

above facts, the objective of the present study was decided to contribute something new via

evaluating the diuretic potential of this plant by administering ethanolic and aqueous extracts to

healthy rats. Male Wistar rats were administered with different doses of the ethanolic extract

(TDEE), and aqueous extracts (TDAE) of T. divaricata leaves each at 100, 200 and 400 mg/kg

p.o. in normal saline solution (25 ml/kg p.o.), also a normal saline solution (25 ml/kg oral

administration) used as control and Furosemide 10 mg/kg p.o. Administration of TDEE at 100,

200 and 400 mg/kg resulted in a significant increase in urine volume and electrolyte excretion,

although less than that found with furosemide. TDAE at a test dose level of 100, 200 mg/kg was

found to be insignificant when compared to the standard drug. Instead, at 400 mg/kg, TDAE

showed enhanced electrolyte excretion. TDEE at test doses are more effective in increasing

electrolyte excretion than TDAE in a dose-dependent manner. The results of the study confirmed

and supported the traditional use of T. divaricata as diuretics.

Keywords: Diuretics, Electrolytes excretion, Furosemide, Strangury, Tabernaemontana

divaricata



Introduction

Diuretics are the agents that potentiate the urine excretion from the body. The use of diuretics

becomes mandatory for specific diseases or disorders, where it becomes important to maintain

the water and electrolytes balance in the body [1]. Failing which may lead to severe

manifestations causing life-threatening problems. Congestive heart failure (CHF), situations of

fluid overload like acute and chronic renal failure, nephritis, cirrhosis, hypertension, and

pregnancy-induced toxemia [2-3] are some of the disorders where diuretics proved to be life-

saving therapy.

Among the various disorders, hypertension is much of concern as it has implicated in the main

etiology for cardiac and renal failure, ischemic heart disease and cerebrovascular disease. The

worldwide premature death caused by hypertension has reckoned at 7.1 million, and the burden

of disease at 4.5% (WHO, 2003). Many diuretic drugs in practice are Furosemide, thiazides,

Acetazolamide, Mannitol, and Ethacrynic acid [4].

Diuretics give relief from pulmonary congestion and peripheral edema. This in turn, decreases

the cardiac workload, plasma volume, and oxygen demand, which eventually lower blood

pressure [5]. Diuretics with different sites of action and mechanisms of action are prescribed

based on the type of therapy needed in various disorders. Carbonic anhydrase inhibitors e.g.,

Acetazolamide. Osmotic diuretics e.g., Mannitol and Urea. Mannitol and other osmotic diuretics

restrict the tubular reabsorption of water and electrolytes. Loop diuretics, high-ceiling diuretics,

or Inhibitors of Na+-K+-2Cl– symport, e.g., Furosemide, Bumetanide, Ethacrynic acid,

Torsemide.



Loop diuretics inhibit the activity of the Na+-K+-2Cl– symport in the thick ascending limb of

Henle’s loop. Inhibitors of Na+-K+-2Cl–symport in the thick ascending limb are immensely

efficient due to an amalgamation of two factors: first, near about 25% of the filtered Na+ load is

generally reabsorbed by the thick ascending limb, and second lack of reabsorption capacity of

nephron segments past the thick ascending limb. Thiazide diuretics and related agents or

Inhibitors of Na+Cl- symport e.g., Chlorothiazide, Hydrochlorothiazide, Chlorthalidone,

Indapamide, and Metolazone. Thiazide and thiazide related diuretics generally act as an inhibitor

of Na+Cl- cotransporter protein. This action curbs on the reabsorption of Na+ and Cl- ions into

the distal convoluted tubule, making the tubular fluid hyperosmotic, resulting in diuresis.

Potassium-sparing diuretics: these comprise of inhibitors of renal epithelial Na+ channels e.g.,

Amiloride and Triamterene. Amiloride and Triamterene block the Na+ channels and stop Na+-K+

exchange. Antagonists of mineralocorticoid receptors, i.e., aldosterone antagonists, e.g.,

Spironolactone. It combines with the aldosterone-receptor in the collecting duct and thus

prevents the binding of aldosterone with its receptors, thus preventing Na+ reabsorption and K+

secretion in the collecting duct.

Carbonic anhydrase inhibitors, a loop diuretic, and thiazide diuretics increase the delivery of

Na+ to the late distal tubule and collecting duct, a situation that often is associated with increased

K+ and H+ excretion. Thus, there is a natriuresis, diuresis, and potassium accumulation [6-8].

Drug-induced diuresis is favorable for the treatment of many disorders, as mentioned above.

However, in current clinical practice, many of the diuretics have been associated with many

adverse effects, metabolic alterations, including electrolyte imbalance, activation of the renin-

angiotensin and neuroendocrine systems, impairment of sexual function, onset of diabetes, etc.

[3, 9]. Hence, it is important to consider alternatives that have greater effectiveness and fewer



side effects. Furthermore, many of the herbs used in folk medicine have yet to be scientifically

evaluated for their efficacy and safety [10]. Scores of investigators demonstrated that studies of

herbs used in traditional medicine as diuretics were in progressive elevation in the last two

decades [11] and might be a promising way for human pathology treatment. Naturally occurring

diuretics in coffee and tea like theobromine, caffeine, and theophylline may validate the

traditional use of the plant as a diuretic [12]. Some plants show diuretic effects comparable to the

loop diuretics e.g., Furosemide [13]. The Indian Ayurvedic system of medicine is rich in treating

renal problems. Abelmoschus esculentus (Bhindaka), Bacopa monnieri (Brahmi), Barbarea

vulgaris (Cress), Boerhavia diffusa (Punarnava), Emblica officinalis (Amla) etc. are some of the

examples [14].

Chahndani, Tabernaemontana divaricata (TD, Family: Apocynaceae) in traditional medicine is

used to treat various diseases like ulceration, epilepsy, abdominal tumors, eye infections,

inflammation, mania, oedema, leprosy, diarrhea. It has also been used as anthelmintic,

aphrodisiac, antihypertensive, emmenogouge, purgative, remedy against poisons and tonic to the

brain, liver, and spleen vermicide, odontalgia, biliousness, paralysis, arthralgia, and melalgia and

blood disorders. TD also exhibited its application in the ailment called strangury, i.e., painful

discharge of urine as per traditional literature [15].

In the present study, we tested the diuretic activity of TD by evaluating the total urine

volume and urinary electrolyte excretion against Furosemide (10 mg/kg, p.o.) in rats.



Materials and methods

Plant material

The leaves of TD were collected in January 2010 from the Bhopal, Madhya Pradesh, India. The

plant was authenticated and identified by Dr. D. V. Amla, Deputy Director, National Botanical

Research Institute, Lucknow, Uttar Pradesh, India, and a voucher specimen no.

Tit/NBRI/CIF/141/2009 was deposited.

Preparation of extract

The leaves were dried in the shade, stored at 25ºC, powdered and passed through sieve no.40.

The dried powdered leaves of TD (500g) were first defatted with Petroleum Ether (60- 80°C) and

later extracted with ethanol and distilled water separately by maceration for 5 days. After

completion of the extraction, the solvent was removed by distillation and concentrated in vaccuo

(40°C) to yield ethanolic and aqueous extract, respectively.

Preliminary phytochemical screening of TD

The preliminary phytochemical investigation was carried out with ethanolic and aqueous extracts

of leaves of TD for the qualitative identification of phytochemical constituents. Phytochemical

tests were carried out by standard methods [16-17].

Animals

Male Wistar rats weighing 150- 200 g were provided by the animal house. Animals were made

available with the standard animal feed and water supply ad libitum before the experiments. The

animal studies were approved by the Institutional Animal Ethics Committee (Reg. no.

831/bc/04/CPCSEA), New Delhi, India. For each experimental study rats were starved for 18 h

with access to water only.



Drugs and chemicals

Furosemide (Lasix, SANOFIAVENTIS), potassium chromate, silver nitrate, etc. were used in the

present study.

Acute toxicity study

An acute toxicity study was carried out for the extracts of TD following the Organization of

economic co-operation and development (OECD) guidelines [18]. The extract was dissolved in

distilled water in a dose of 2 g/kg body weight and orally administered to overnight-fasted,

healthy rats (n = 6). The animals were observed continuously for 24 h for mortality.

Evaluation of diuretic activity

Animals were maintained under standard conditions of temperature and humidity and underwent

an adaptation period of three days. The animals were divided into eight groups (n= 6). Group I

served as the control and received normal saline solution (25 ml/kg oral administration); group II

received the reference diuretic, Furosemide 10 mg/kg p.o.; groups III, IV and V received the

ethanolic extract of TD (TDEE) at 100, 200 and 400 mg/kg p.o., respectively, in normal saline

solution (25 ml/kg p.o.) and groups VI, VII, and VIII received the aqueous extract of TD

(TDAE) at 100, 200 and 400 mg/kg p.o., respectively, also in normal saline solution (25 ml/kg

p.o.). The diuretic activity was assessed based on the method of Lipschitz et al. (1943) [19].

Immediately after the administration of all the doses; animals were placed individually in

metabolic cages that were specially designed to separate urine and feces and kept at a controlled

temperature of 20-25 0C. At the end of 12 h [20] the volume of urine was collected and



measured. The animals were deprived of food and water during the period of experimentation.

The parameters viz. body weight before and after the test period, total urine volume, and

concentration of Na+, K+, and Cl- were measured. The concentration of Na+ and K+ cations was

determined by a flame photometric method while that of Cl- was determined by titration with

silver nitrate solution (N/50) using three drops of 5% potassium chromate solution as an

indicator [21-22].

Statistical analysis

Results are expressed as the mean ± SEM. Data was analyzed by one-way analysis of variance

(ANOVA) followed by Dunnett’s multiple comparison tests. A value of P < 0.05 was considered

statistically significant.

Results

Preliminary phytochemical screening of TD

Phytochemical screening of TD revealed the presence of resins, alkaloids, tannins, amino acids,

proteins, flavonoids, saponins, phenols, steroids, glycosides and tri-terpenoids.

Effect on urine volume

The animals were observed for 12 h and showed no signs of dehydration. Group II animals that

were dosed with the reference diuretic (furosemide) significantly increased urine output as

compared to the control group (P < 0.001), with a diuretic index of 2.75 (Table 1).

Administration of the test drug i.e., TDEE at 100, 200 and 400 mg/kg, also resulted in a

significant increase in urine volume, although less than that found with the reference drug. In the

case of TDAE, at a test dose level of 100, 200 and 400 mg/kg the diuretic indices obtained were

1.88, 1.92 and 2.05, respectively, with a significant result at the test dose of 400 mg/kg, p.o. Thus



TDEE at test doses is more effective in enhancing the urine excretion as compared to TDAE in a

dose-dependent manner.

Effect on urinary electrolyte excretion

Immediately after the administration of all the doses, animals were placed individually in

metabolic cages. The volume of urine was collected and measured, and the concentration of

electrolytes was determined, which is mentioned in Table 2. It was found that TDEE showed a

significant increase in the excretion of sodium, potassium, and chloride ions in a dose-dependent

manner. The increase in electrolyte excretion with the ethanolic extract (at all doses) was less

than that found with furosemide. TDAE at a test dose level of 100, 200 were found to be

insignificant when compared to the standard drug, while at 400 mg/kg, it showed increased

electrolyte excretion. Thus TDEE at test doses is more effective in enhancing the electrolyte

excretion compared to TDAE and that in a dose-dependent manner.

Discussion

The diuretic effect of the orally administered ethanolic and aqueous extracts of TD has been

evaluated in normal rats, and compared with that produced by furosemide, a loop diuretic widely

used in clinical practice. The results obtained in the study are immensely helpful in establishing

the scientific evidence for the traditionally claimed diuretic efficiency of TD. An encyclopedia of

world medicinal plants has demonstrated the use of decoction of leaves of TD as an anti-

hypertensive and diuretic. Also, TD exhibited its application in the ailment called strangury, i.e.,

a painful discharge of urine as per traditional literature [23].

Diuresis is associated with an increase in urine volume i.e., water excretion and a net loss of

electrolytes in the urine. The acute treatment of rats with TDEE and TDAE exhibited a

significant diuretic activity in a dose-dependent manner. Urine volume and cation excretion were



measured and found to be increasing with increased dose. Administration of TDEE at 100, 200,

and 400 mg/kg resulted in a significant increase in urine volume, although less than that found

with furosemide. Similar is the case with TDEE regarding electrolyte excretion. TDAE at a test

dose level of 100 and 200 mg/kg was found to be insignificant when compared to the standard

drug. Instead, at 400 mg/kg, TDAE showed enhanced electrolyte excretion. Thus TDEE at test

doses is more effective in increasing electrolyte excretion than TDAE in a dose-dependent

manner. Furosemide is well known for its potential saluretic and diuretic effects and from the

study, it was observed that TD extracts were acting similarly as furosemide. Furosemide is

categorized under loop diuretics or high ceiling diuretics, acts by inhibiting NKCC2, the luminal

Na-K- 2Cl symport in the thick ascending limb of the loop of Henle. It also annihilates the

cortico-medullary osmotic gradient and obstructs the negative as well as positive free water

clearance [24-25]. By inhibiting the transporter, the loop diuretic declines the reabsorption of

NaCl in the kidney and also diminishes the lumen-positive potential that derives from K

recycling. This electrical potential normally propels divalent cation reabsorption in the loop.

Thus, by reducing this loop potential, diuretics induce an increase in Mg and Ca [26].

Conclusion

Active principles of plants such as flavonoids, saponins, and organic acids are found to be

responsible for the diuretic effect. These natural products might be acting synergistically or

individually. The preliminary phytochemical analysis of TD revealed the presence of these

constituents. The present study with reference to the excretion of Na+, K+, and Cl-, showed a

more significant natriuretic effect than kaluretic one. The results of the study confirmed and

supported the traditional use of TD as diuretics.



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Table Legends:

Table 1: Effect of T. divaricata extracts and Furosemide on urine volume

Table2: Effect of T. divaricata extracts and Furosemide on electrolyte excretion

Table 1: Effect of T. divaricata extracts and Furosemide on urine volume

Treatment Dose (mg/kg) Urine volume

(ml/100 g/12 h)

Diuretic index

Control - 5.3 ± 0.6 -

Furosemide 10 14.6 ± 2.6*** 2.75

TDEE 100 11.1 ± 0.3* 2.09

TDEE 200 11.9 ± 0.7* 2.24

TDEE 400 13.4 ± 1.2** 2.52

TDAE 100 10.0 ± 1.0ns 1.88

TDAE 200 10.2 ± 1.0ns 1.92

TDAE 400 10.9 ± 0.7 * 2.05



TDEE: Ethanolic extract of T. divaricata; TDAE: Aqueous extract of T. divaricata

Values are expressed as the mean ± SEM; *P < 0.001 compared to the control group, **P < 0.01

compared to Furosemide group (ANOVA followed by Dunnett’s test).

Diuretic Index = Volume of test group/Volume of control group.

Table2: Effect of T. divaricata extracts and Furosemide on electrolyte excretion

Treatment Dose

(mg/kg)

Total Na+

(µmoles/kg)

Total K+

(µmoles/kg)

Total Cl-

(µmoles/kg)

Control - 1680 ± 1.0 665.8 ± 4.0 637.0 ± 52.4

Furosemide 10 3654 ± 1.6*** 1429 ± 5.9 *** 1836 ± 61.1***

TDEE 100 2275± 1.0* 859.5 ± 1.7 * 1174 ± 85.7**

TDEE 200 2365± 1.1** 929.5 ± 2.8 ** 1334 ± 97.9***

TDEE 400 2726 ± 1.1*** 1176 ± 4.8 *** 1678 ± 52.5***

TDAE 100 1870± 1.0ns 834.5 ± 2.6 * 932.3 ± 56.7*

TDAE 200 1980± 1.0ns 856.5 ± 4.7 * 941.5 ± 88.8*

TDAE 400 2203 ± 1.9 * 863.5 ± 2.4 * 1007 ± 77.9**

TDEE: Ethanolic extract of T. divaricata; TDAE: Aqueous extract of T. divaricata.

Values are expressed as the mean ± SEM.*P < 0.001 compared to the control group, **P < 0.01,

***P < 0.001, compared to Furosemide group (ANOVA followed by Dunnett’s test).



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