Evaluation of the Diuretic and Electrolyte Excretion Effects of Syzygium Cumini Fruit Pulp Extract in Experimental Rats

Diuretics are drugs that increase the amount of urine produced by improving the excretion of water and electrolytes, mainly by preventing the renal tubules from reabsorbing sodium. They are frequently used in the treatment of diseases such as hypertension, congestive heart failure, renal problems, and edema and are necessary for preserving fluid and electrolyte balance (1,2). Looking for safer and more effective alternatives is necessary because, even though their therapeutic significance, currently available diuretics are commonly linked to side effects such as electrolyte imbalance, metabolic disorders, and renal problems (2). Throughout many decades, medicinal plants have been an important source of therapeutic agents, providing a variety of bioactive compounds with possible pharmacological benefits (3). Among these, Syzygium cumini, also referred to as jamun, is a historically significant medicinal plant that is utilized in many medical systems to treat gastrointestinal disorders, diabetes, and inflammation (4,5). The fruit pulp is particularly rich in phytoconstituents such as flavonoids, phenolics, and tannins, which are known to exhibit diverse biological activities (6,7). Although Syzygium cumini has been extensively studied for its antidiabetic and antioxidant properties, limited scientific evidence is available regarding its potential diuretic activity. Considering the presence of bioactive phytochemicals that may influence renal function and electrolyte balance, it is hypothesized that the fruit pulp extract may exhibit significant diuretic effects (8,9). Therefore, in order to assess the diuretic activity of methanolic extract of Syzygium cumini fruit pulp (MESCFP) in experimental models and investigate its potential mechanism of action by measuring urine output, electrolyte excretion (Na⁺, K⁺, Cl⁻), and associated diuretic indices, the current study was conducted.

MATERIALS & METHODS

Collection of plant material

Syzygium cumini fresh fruits were collected at Kalaburagi, Karnataka, India (15.32°N, 75.83°E). Dr. Pratibha G. Sangapurkar, a professor and chairman of the Department of P.G. Studies and Research in Botany at Gulbarga University in Kalaburagi, verified and taxonomically identified the plant material. For future reference, a voucher specimen (HGUK 217) was placed in the scientific herbarium.

Preparation of Fruit Pulp

The collected fruits were thoroughly washed with distilled water to remove adhering impurities. The pulp was carefully separated from the seeds, rewashed, and air-dried at room temperature. The dried pulp was then coarsely powdered and stored in an airtight container until further use.

Preparation of plant extract

The powdered material was initially defatted using hexane to remove lipophilic constituents. After filtration, the hexane fraction was discarded, and the defatted residue was subjected to extraction. Cold percolation was used for extraction with methanol as a solvent. To produce an effective extraction, the defatted powder was soaked in methanol at a 1:5 (w/v) ratio for 72 hours while shaking periodically. The mixture was then filtered through Whatman's filter paper, and the filtrate was concentrated under reduced pressure with a rotary evaporator to get the methanolic extract of Syzygium cumini fruit pulp (MESCFP) (10).

Phytochemistry Analysis

A preliminary qualitative phytochemical screening of the methanolic extract of Syzygium cumini fruit pulp (MESCFP) was performed using established methods to detect essential phytoconstituents such as alkaloids, flavonoids, tannins, saponins, glycosides, and anthraquinones (11).

Animals & Ethics

Wistar albino rats weighing between 150 and 200 grams were maintained under standard laboratory conditions with unlimited access to food and water. All experimental protocols followed CPCSEA guidelines and were approved by the Institutional Animal Ethics Committee (HKES/MTRIPS/IAEC/164/2024-25) (12).

Acute Oral Toxicity

Acute oral toxicity of the methanolic extract of Syzygium cumini was assessed according to OECD guideline 425 (13). Animals were observed for mortality and clinical signs of toxicity for 14 days following oral administration of the extract.

Diuretic Activity

Experimental Animals and Housing

Wistar albino rats were divided into groups of six each (n = 6). To get comfortable, the animals were housed individually in metabolic cages for 24 hours before the experiment. The cages had wire mesh bottoms and funnel systems for collecting quantitative pee, as well as stainless-steel sieves for separating feces. Animals were fasted for 18 hours before the experiment and given free access to water. Before administering the test and usual medicines, the urine bladder was gently emptied by exerting light pressure to the pelvic region. All animals were given normal saline orally to ensure a consistent water and electrolyte load.

Experimental design

The animals were separated into five groups:

• Group I (Control) administered 0.1% sodium carboxymethylcellulose (Na-CMC), p.o.
• Group II (Urea) administered urea (1 g/kg, p.o).
• Group III (Standard) administered furosemide (10 mg/kg p.o.).
• Group IV (MESCFP Low Dose) received 150 mg/kg of methanolic extract of Syzygium cumini fruit pulp (p.o.).
• Group V (MESCFP High Dose) administered MESCFP (300 mg/kg, p.o).

Urine samples were collected at 5 and 24 hours after medication administration. Urine volume was evaluated to assess sodium (Na⁺), potassium (K⁺), and chloride (Cl⁻) contents. Sodium and potassium levels were determined using a flame photometer. Lipschitz et al. (14) suggested a method for determining diuretic activity.

Evaluation of Diuretic Activity

The following parameters have been calculated:

• Urinary excretion (%) = (Total urine output / Total liquid provided) x 100 (15)
• Diuretic action is calculated as test group urine excretion divided by control group urine excretion (15).
• Diuretic activity is calculated by dividing the diuretic action of the test medication by that of urea or furosemide. (15)

Natriuretic activity was determined as the ratio of Na⁺ and K⁺.Saluretic activity was calculated as the sum of Na⁺ and Cl⁻ excretions. The inhibition of carbonic anhydrase was measured using the ratio Cl⁻/(Na⁺ + K⁺) (16).All treatment groups were given their diuretic and electrolyte excretion indices measured.

Diuretic Activity with Prostaglandin Involvement

Animals were divided into six groups (n = 6). Prior to treatment, all animals received normal saline (0.9% NaCl, 25 mL/kg, p.o.).

Experimental Design

• Group I (Control) received 0.1% Na-CMC, p.o.
• Group II (Lornoxicam): Lornoxicam (3 mg/kg, PO)
• Group III (MESCFP): 300 mg/kg, p.o.
• Group IV (MESCFP + Lornoxicam): MESCFP (300 mg/kg) + lornoxicam (3 mg/kg) given orally.
• Group V (Standard) administered furosemide (10 mg/kg, p.o.).
• Group VI (Furosemide + Lornoxicam): Furosemide (10 mg/kg) and lornoxicam (3 mg/kg), p.o.

The urine was collected for 15 hours, and the total Urine volume was recorded. Urinary electrolyte concentrations (Na⁺, K⁺, and Cl⁻) were determined and interpreted as mmol/L (17).

Statistical analysis

All experimental data were presented as mean ± SEM. One-way ANOVA was employed for statistical analysis, followed by Dunnett's multiple comparison test. A p-value of <0.05 was considered to be statistically significant (18).

RESULTS

Percentage Yield

The methanolic extract of Syzygium cumini fruit pulp (MESCFP) has resulted in a percentage yield of 10.40% w/w.

Preliminary phytochemical investigations

Preliminary qualitative phytochemical examination of Syzygium cumini fruit pulp (MESCFP) revealed the presence of alkaloids, flavonoids, saponins, tannins, glycosides, and anthraquinones (Table 1).

Table 1: Chemical constituents present in methanolic extract of Syzygium cumini fruits pulp

Diuretic Activity

Effect on Urine Volume, Urinary Excretion, Diuretic Action, and Diuretic Activity

Effects on urine volume, excretion, diuretic effect, and activity.

Table 2 shows how the methanolic extract of Syzygium cumini fruit pulp (MESCFP) affects urine volume, urinary excretion, diuretic action, and diuretic activity.

Table 2: Effect of MESCFP on Urine Volume, Urinary Excretion, Diuretic Action and Diuretic Activity

MESCFP induced a dose-dependent increase in urine output in normal rats. MESCFP at 150 mg/kg significantly increased urine volume (0.900 ± 0.089 mL at 5 h and 1.567 ± 0.114 mL at 24 h) compared to the control group (0.621 ± 0.007 mL and 1.073 ± 0.013 mL, respectively, p < 0.01).  The respective urine excretion rates were 31.30% and 84.57% at 5 and 24 hours, respectively. The diuretic action was 1.75 and 2.72, while the diuretic activity was 0.94 and 2.82 at 5 and 24 hours, respectively. At a higher dose (300 mg/kg), MESCFP significantly increased urine production (1.893 ± 0.061 mL at 5 h and 3.513 ± 0.026 mL at 24 h) compared to the control (p < 0.001). Urinary excretion rates were 49.05% and 91.40%, respectively. The diuretic action was 3.04 (5 h) and 3.27 (24 h), with diuretic activity of 1.65 and 3.39, respectively. The standard drug, Furosemide (10 mg/kg), produced the highest urine volume (2.298 ± 0.010 mL at 5 h and 3.672 ± 0.012 mL at 24 h), which was extremely significant (p < 0.0001) compared to control. The urinary excretion values were 57.76% and 101.8%, confirming its potent diuretic effect. Urine output was also significantly higher in urea-treated animals (1.143 ± 0.009 mL at 5 hours and 1.983 ± 0.140 mL at 24 hours) than in the control group (p < 0.0001). Overall, MESCFP revealed strong and dosage-dependent diuretic efficacy; at 24 hours, the effects of the higher dose (300 mg/kg) were similar to those of furosemide.

Effect on Urinary Electrolyte Excretion

Table 3 indicates that the methanolic extract of Syzygium cumini fruit pulp (MESCFP) affects the excretion of electrolytes in the urine.

Table 3: Electrolyte’s excretion(mmol/L), of methanolic extract of Syzygium cumini fruit pulp in normal rats at 5 and 24th hour by oral administration.

Urinary sodium (Na⁺), potassium (K⁺), and chloride (Cl⁻) excretion levels were relatively low in the control group at both 5 and 24 hours.

Urea (1 g/kg) resulted in a significant improvement in electrolyte excretion. A marked elevation in K⁺ excretion was observed (79.00 ± 0.856 mmol/L at 5 h and 202.7 ± 0.714 mmol/L at 24 h), which was highly significant (p < 0.0001). Similarly, Cl⁻ excretion (134.3 ± 0.954 and 121.7 ± 2.334 mmol/L) and Na⁺ excretion (109.8 ± 1.249 mmol/L at 24 h) were significantly increased (p < 0.0001), indicating its osmotic diuretic effect. The standard drug, Furosemide (10 mg/kg), significantly increased Na⁺ excretion (218.3 ± 8.800 and 142.0 ± 2.206 mmol/L at 5 h and 24 h, respectively; p < 0.0001). It also produced a moderate but significant increase in K⁺ excretion (59.50 ± 2.604 mmol/L, p < 0.01 at 5 h; 102.8 ± 0.945 mmol/L, p < 0.0001 at 24 h) and Cl⁻ excretion (p < 0.0001), consistent with its loop diuretic action. MESCFP at 150 mg/kg (MESCFP I) showed a significant increase in Na⁺ excretion (171.0 ± 8.903 and 123 ± 1.265 mmol/L; p < 0.001) and Cl⁻ excretion (97.17 ± 0.909 mmol/L, p < 0.01 at 5 h; 96.17 ± 0.654 mmol/L, p < 0.001 at 24 h). A moderate increase in K⁺ excretion was also observed (54.00 ± 1.862 mmol/L, p < 0.01 at 5 h; 86.50 ± 0.839 mmol/L, p < 0.001 at 24 h). At the higher dose (300 mg/kg), MESCFP (MESCFP II) produced a more pronounced increase in Na⁺ excretion (194.4 ± 4.129 and 131.5 ± 1.025 mmol/L; p < 0.0001) and Cl⁻ excretion (100.8 ± 1.376 mmol/L, p < 0.001 at 5 h; 112 ± 1.406 mmol/L, p < 0.0001 at 24 h). K⁺ excretion was also significantly increased (55.50 ± 1.204 mmol/L, p < 0.001 at 5 h; 95.67 ± 0.892 mmol/L, p < 0.0001 at 24 h). Overall, MESCFP significantly enhanced urinary electrolyte excretion in a dose-dependent manner, particularly for Na⁺ and Cl⁻ ions. The higher dose (300 mg/kg) exhibited effects comparable to Furosemide, indicating prominent natriuretic and chloruretic activity.

Electrolyte Excretion Index and Diuretic Index

Table 4 indicates the electrolyte excretion indices (Na⁺, K⁺, and Cl⁻) and diuretic index of the methanolic extract of Syzygium cumini fruit pulp (MESCFP) at 5 and 24 hours.

Table 4: Effect of methanolic extract of Syzygium cumini fruit pulp on diuretic index and Electrolytic excretion index at 5 and 24^th hour of urine collection

The sodium excretion index was elevated in MESCFP-treated groups, with MESCFP I (1.73) and MESCFP II (1.56) at 5 h exceeding that of urea (1.20), suggesting a significant natriuretic effect. At 24 h, MESCFP II (1.29) and MESCFP I (1.21) continued to enhance sodium excretion, although to a lesser extent than furosemide (1.40).

Potassium excretion was highest in the urea-treated group (2.82 at 24 h), followed by furosemide (1.43), indicating pronounced kaliuresis. In contrast, MESCFP I (1.20) and MESCFP II (1.33) showed comparatively lower potassium excretion, suggesting a potassium-sparing effect.

Chloride excretion index was greatest with urea at both 5 h (1.47) and 24 h (1.38). MESCFP-treated groups demonstrated moderate increases (1.06–1.27), indicating balanced chloride excretion relative to standard treatments. Overall, MESCFP, particularly at 300 mg/kg, exhibited significant diuretic and natriuretic activity with relatively lower potassium loss, supporting its potential as a potassium-sparing diuretic.

Saluretic, Natriuretic, and Carbonic Anhydrase Activity

The combined electrolyte excretion indices of the methanolic extract of Syzygium cumini fruit pulp (MESCFP) are presented in (Table 5).

Table 5: Saluretic, Natriuretic, and CAI activity of methanolic extract of Syzygium cumini fruits pulp in normal rats at 5 and 24^th hr by oral administration

At 5 h, Furosemide (10 mg/kg) produced the highest saluretic effect (Na⁺ + Cl⁻: 317.6 mmol/L), followed closely by MESCFP II (300 mg/kg; 307.4 mmol/L), indicating a pronounced saluretic response. MESCFP I and urea exhibited comparatively lower values (236.4 and 254.4 mmol/L, respectively). At 24 h, MESCFP II (239.8 mmol/L) maintained substantial saluretic activity, comparable to furosemide (257.7 mmol/L), suggesting a sustained effect.

The natriuretic index (Na⁺/K⁺ ratio) at 5 h was highest in the control group (3.04), followed by MESCFP I (2.95), whereas urea and furosemide showed lower ratios (2.68 and 2.47, respectively), indicative of increased potassium loss with standard treatments. At 24 h, MESCFP I (1.345) and MESCFP II (1.288) maintained relatively higher Na⁺/K⁺ ratios compared to urea (1.072), supporting a potassium-sparing effect.

The carbonic anhydrase inhibition index (Cl⁻/(Na⁺ + K⁺)) was lowest in furosemide (0.506 at 5 h; 0.490 at 24 h) and MESCFP II (0.501 at 5 h; 0.483 at 24 h) compared to control (0.567 at 5 h; 0.470 at 24 h). This reduction suggests that MESCFP, particularly at higher dose, may exert mild carbonic anhydrase inhibitory activity, contributing to its diuretic effect. Overall, MESCFP demonstrated significant saluretic and natriuretic activity with relatively lower potassium loss and evidence of mild carbonic anhydrase inhibition, indicating a balanced and potentially safer diuretic profile.

Assessment of Diuretic Activity with Prostaglandin Involvement

Table 6 shows how the methanolic extract of Syzygium cumini fruit pulp (MESCFP) affects urine volume and electrolyte excretion both with and without Lornoxicam.

Table 6. Single-dose administration, with collection of urine for 15th hr after oral treatment with MESCFP, and study of involvement of prostaglandins in diuretic activity.