Abstract

Piper guineense is an aromatic and food plant used in traditional medicine for its therapeutic properties. Among the pathologies against which this plant is used is arterial hypertension (HTA). In clinic, diuretics are used as first-line therapy alone or in combination with other antihypertensives to treat hypertension. Thus, the objective of this work is to evaluate the diuretic potential of aqueous, hydroethanolic and essential oil extracts of P. guineense bark in wistar rats. Diuretic activity parameters and pH were measured from fresh urine collected from rats placed in metabolic cages after six hours of observation. Aldosterone was quantified in plasma after centrifugation of blood collected by puncture of the retro orbital sinus of rats. The results obtained show that aqueous, hydroethanolic and essential oil extracts (75 and 150 mg/kg, b.w.) possess diuretic properties. They increase the elimination of sodium (P<0.001) and water (natriuretic and saludiuretic effects). At a dose of 150 mk/kg, the hydroethanolic extract basifies the urinary pH, inhibits the activity of carbonic anhydrase and decreases the plasma concentration of aldosterone (P<0.001) in rats. From these facts, the hydroethanolic extract would act both like Furosemide and Spironolactone (50 mg/kg). However, the aqueous extract and the essential oil have no effect on urinary pH, carbonic anhydrase activity and plasma aldosterone concentration in rats, but they increase sodium and water secretion. This shows that the aqueous extract and the essential oil would act according to the same mechanism as furosemide (20 mg/kg).

1. Introduction

The use of medicinal plants for treatment remains the main recourse for the vast majority of populations in developing countries ¹. According to the World Health Organization, these populations use plants to meet their primary health care needs, partly for their accessibility but above all for the limited financial means faced with conventional pharmaceutical products ². Among the plants used is Piper guineense (P. guineense) which is an aromatic and food plant of the Piperaceae family which has a wide use in traditional medicine and is therefore of great ethnopharmacological and food interest ³. It is a perennial climbing vine reaching 20 m in height. These fruits, leaves, barks and roots are used on the one hand for the preparation of herbal remedies for the treatment of infectious, inflammatory and cardiovascular diseases and on the other hand as an ingredient or spice to flavor dishes. In recent studies, P. guineense was found to be one of the most valuable spices with numerous human health benefits ^{4, 5, 6}. It is in this context that thiswork focused on the evaluation of the diuretic potential of aqueous, hydroethanolic extracts and of the essential oil of the bark of the vine of P. guineense in rats.

2. Material and Methods

Adult female wistar rats aged 20 ± 2 weeks and weighing between 180 and 200 g were used. All these animals were supplied by the animal facility of the Faculty of Science and Technology where they were kept under standard lighting conditions (12 hours of light, 12 hours of darkness) at an ambient temperature of 25 ± 2°C. These rats had free access to standard chow and tap water.

aqueous extract

The aqueous extract of the bark of P. guineense was prepared by 10 % maceration.The extract was obtained by mixing 100 g of the plant material in 1 L of distilled water. This mixture was macerated under magnetic stirring for 24 hours. The maceration obtained was then filtered on wattman N°1 paper and then concentrated to dryness using a rotary evaporator of the Buchi II type.

The extract was obtained by mixing 100 g of plant material in a 1 L hydroethanolic solution (50% v/v). This mixture was left to macerate for 72 hours. The maceration obtained was then filtered on wattman N^o1 paper and then concentrated using a Buchi II type rotary evaporator.The dry extract obtained was used to prepare the test solution.

500 g of fresh bark of P. guineense were extracted by hydrodistillation, using a Clevenger type apparatus for 5 h. The essential oil was extracted with diethyl ether and dried over anhydrous sodium sulfate ⁷.

The baseline diuresis of each rat was measured before the evaluation of the diuretic activity to ensure the proper functioning of the renal function of the rats subjected to the experiment. This was measured by administering distilled water to the rats at a rate of 50 mL/kg. The elimination time of the first drop of urine or latency time was noted and the urinary excretion measured after six hours. Rats excreting at least 40% of the administered volume were selected for testing ⁸. The diuretic activity was evaluated according to the method reported by ⁸. The rats were fasted on food 18 hours before the tests, with free access to tap water. Eleven (11) batches of five (5) rats each were constituted and treated per os as follows:

• batch 1, control negative with out over load hydrosaline:distilled water (5 mL/kg, bw)

• batch 2, positive control 1: hydrosaline overload of 0.9% NaCl (25 mL/kg, bw) + distilled water (5 mL/kg, bw);

• batch 3, positive control 2: hydrosaline overload of NaCl 0.9% (25 mL/kg, bw) +DMSO (5 mL/kg, bw)

• batch 4, reference control1: hydrosaline overload of NaCl 0.9% (25 mL/kg, bw) + Furosemide (20 mg/kg, bw);

• batch 5, reference control 2: hydrosaline overload of NaCl 0.9% (25 mL/kg, bw) + Spironolactone (50 mg/kg, bw);

• treated batches 6 and 7: hydrosaline overload of NaCl 0.9% (25 mL/kg, bw)+the aqueous extract of the barks of P. guineense at the respective doses of (75 and 150 mg/kg, bw);

• treated batches 8 and 9: hydrosaline overload of NaCl 0.9% (25 mL/kg, bw) + the hydroethanolic extract of the bark of P. guineense at the respective doses of ( 75 and 150 mg/kg, bw);

• treated batches 10 and 11: hydrosaline overload of 0.9% NaCl (25 mL/kg, bw) + essential oil from P. guineense bark diluted in DMSO at the respective doses of (75 and 150 mg/kg, bw).

After the treatments, each rat of a batch was placed in a metabolic cage.

Next, for each batch,urine was collected andthe following parameters were noted: the latency time or delay (time of appearance of the first drop of urine after placing the animals in the metabolic cage), the volume of urine excreted per hour for six hours then at the end twenty-four hours. Urinary pH was measured from a sample of fresh urine from each rat using a digital pH meter ⁹. The collected urine was stored in the refrigerator (-32°C) for subsequent analyses. volumetric urinary excretion (VUE), diuretic action (DA) and diuretic index (DI) were calculated for all batches and diuretic activity was deduced using the scale of ¹⁰.

At the end of the experiment, all rats were sedated with diethyl ether. Blood samples were taken by puncture at the level of the retro orbital sinus. The blood samples were collected in the dry tubes, then centrifuged (TD4A-WS DEESK) at 3000 revolutions for 15 minutes and the serum made it possible to determine the concentration of aldosterone by radioimmunoassay using the commercial kit (Rat Aldosterone ELISA Kit, Bioassay Technology Laboratory, China, Cat. No. E051Ra) which was performed according to the manufacturer's protocol.

The urinary concentrations of sodium ions (Na⁺), potassium (K⁺), calcium (Ca²⁺), magnesium (Mg²⁺) and chlorides (Cl^-) were determined using commercial kits (Cypress diagnostics, Belgium) using a spectrophotometer brand flame (EasyLyte® PLUS) and a brand analyzer (BioMate 3S, Thermo scientific, USA). Using the data obtained from the analysis of urinary electrolytes, the natriutic index (IN) or Na⁺/K⁺ ratio, the saluiretic index (SI) and the index of inhibition of carbonic anhydrase (CAI) were calculated by the following formulas:

The Na⁺/K⁺ ratio (IN) > 1, indicates a satisfactory natriuretic index and if >2, it indicates a favorable urinary excretion without excessive loss of potassium (K⁺), but if > 10 it indicates a favorable urinary excretion of potassium (K⁺), effect thrifty. The IAC is between 0.8 and 1.00, excludes the activities of carbonic anhydrase inhibition, but if IAC<0.8, it is considered to have strong activity of carbonic anhydrase inhibition ^{11, 12}.

3. Results

Table 2 shows the effect of aqueous, hydroethanolic and essential oil extracts from the bark of P. guineense in wistar rats on the urinary excretion of rats. Like the molecules used, the aqueous (150 mg/kg) and hydroethanolic (150 mg/kg) extracts cause significant elimination (P < 0.001 and P < 0.01 respectively) of the urine in the first thirty minutes after administration. , and this delay is shorter in rats treated with Furosemide 20 mg/kg (P<0.001), Spironolactone (P<0.01).All the test doses used caused continuous urine production up to the twenty-fourth hour compared to the control group, with the maximum elimination peaks from the first hour for furosemide, the aqueous and hydroethanolic extracts, and at the second hour for the Spironolactone (50 mg/kg) and essential oils (75 and 150 mg/kg) batches. With the volumetric urinary excretions respectively (EUV: 2.31%) for the batches of rats treated with Furosemide (20 mg/kg), (EUV: 1.72%) for the batches of rats treated with Spironolactone (50 mg/kg), 1.38, 1.43 and 1.12% for batches of rats treated with respectively aqueous, hydroethanolic extracts and essential oil (75 mg/kg) and (EUV: 1.5, 2.05 and 1.20%) respectively aqueous, hydroethanolic extracts and essential oil (150 mg/kg) (Table 2). As indicated in Table II, the urine collected from the groups treated with Furosemide and those treated with Spironolatone showed a pH of 6.68±0.03 and 7.05 respectively. However, the batches of rats treated with the aqueous extracts, hydroethanolic and essential oil (75 and 150 mg/kg) showed pH values of 7.06±0.10, 7.32±0.11, 7.25±0.08, 8.05±0.22, 7.06±0.11 and 709±0.08 respectively.

Table 3 shows the effect of aqueous, hydroethanolic and essential oil extracts of the bark of P. guineense on the urinary excretion of electrolytes in rats. Compared to the negative control batch (5 mL/kg), the animals having received (NaCl 0.9% +5 mL/kg) show a significant increase in the urinary elimination of sodium (P<0.01), potassium (P<0 .01) and chlorine (P<0.05). However, the urinary concentrations of calcium (P>0.05) and magnesium (P>0.05) remain unchanged. On the other hand, compared to the positive control (NaCl 0.9% + 5 mL/kg), the batches of animals having received furosemide (20 mg/kg), Spironolatone (50 mg/kg), the aqueous and hydroethanolic extracts (75 and 150 mg /kg) showed a significant increase in urinary excretion of sodium, potassium and chloride respectively (P<0.001), (P<0.001), (P<0.01) and (P<0.001). Concerning the urinary excretion of calcium and magnesium, only furosemide (20 mg/kg) and the hydroethanolic extract (150 mg/kg) showed a significant increase respectively (P<0.01) and (P<0. 05). In addition, the essential oil (75 and 150 mg/kg) did not cause any significant change (P>0.05) in the urinary concentrations of sodium, potassium, chlorine, calcium and magnesium compared to the positive control (NaCl 0.9% + DMSO 5ml/kg).

Table 4 shows that in the rats treated with the various products, the diuretic action is greater than one and this action is more pronounced in the rats which received respectively Furosemide 20 mg/kg (2.75), the hydroethanolic extract 75 and 150 mg/kg (1.62 and 2.10) then Spironolactone 50 mg/kg. The natriuretic index is higher in rats treated respectively with Furosemide 20 mg/kg (2.83), the hydroethanolic extract 75 and 150 mg/kg (2.21 and 2.83) then Spironolactone 50 mg/kg (2.32). Apart from the reference molecules used which have diuretic indexes equal to 1, the aqueous, hydroethanolic extracts and essential oil have diuretic indexes of less than one. Furthermore, the indices of carbonic anhydrase inhibition in rats treated with Furosemide 20 mg/kg, Spironolactone (50 mg/kg), the aqueous extract (75 and 150 mg/kg), the oil essential (75 and 150 mg/kg) are greater than 1 as in the controls. Only the rats treated with the hydroethanolic extract at doses of 75 and 150 mg/kg show indexes of inhibition of carbonic anhydrase between 0.8 and 1.

Figure 1 shows that the administration of 0.9% NaCl + distilled water (5 mL/kg) does not modify the plasma concentration of circulating aldosterone (P>0.05) compared to control rats having received distilled water (5 mL/kg). Similarly, the administration of the aqueous extract at doses of 75 and 150 mg/kg does not influence the plasma concentration of aldosterone. However, oral administration of the extract and hydroethanolic at doses of 75 and 150 mg/kg significantly reduced (P<0.05) the aldosterolemia of rats like Spironolactone administered at a dose of 50 mg/kg (P<0.05). On the other hand, since furosemide administered at a dose of 20 mg/kg, the aqueous extract 75 mg/kg and the essential oil administered at doses of 75 and 150 mg/kg do not significantly modify (P>0.01) the aldosterolemia of the rats compared respectively with the 0.9 % NaCl control + distilled water (5 mL/kg) and the 0.9% NaCl control + DMSO. (5mL/kg).

4. Discussion

The aim of the present study was to evaluate the diuretic potential of aqueous, hydroethanolic and essential oil extracts of P. guineense bark in wistar rats. The aqueous and hydroethanol extracts (150 mg/kg) cause, like furosemide (20 mg/kg), the elimination of the first drop of urine earlier compared to control rats (NaCl 0.9 % + distilled water 5 mL /kg). On the other hand, the essential oil (75 and 150 mg/kg) causes, as in the control rats (NaCl 0.9 % + DMSO 5 mL/kg), the first drops of urine half an hour after the administration of the products. These extracts therefore reduce the urine elimination time. This result suggests that the aqueous and hydroethanol extracts have a fleeting, rapid diuretic effect in the rat, like furosemide, which would act by blocking the tubular reabsorption of water at the branch level. ascending from the nephron ¹³. Like furosemide (20 mg/kg), the aqueous and hydroethanolic extracts (75 and 150 mg/kg) cause maximum elimination of urine from the first hour. However, the essential oil (75 and 150 mg/kg) causes maximum elimination at the second hour like spironolactone (50 mg/kg). In addition, all the test products used cause the elimination of urine during the first six hours and continues until the twenty-fourth hour like furosemide. Previous work has shown that furosemide at doses between 10 and 20 mg/kg exerts its diuretic effect during the first five hours ¹⁴. These results suggest that these extracts act like furosemide. Also, in rats treated with aqueous and hydroethanolic extracts at a dose of 150 mg/kg, there is a volumetric urinary excretion greater than 150 % as in rats having received furosemide and spirinolactone. These results suggest that these extracts at 150 mg/kg have, like furosemide, an important diuretic activity, according to the scale of Kau et al ¹⁰. In fact, under the same experimental conditions, significant diuretic activity of furosemide at 20 mg/kg used as reference molecule was noted, with a volumetric urinary excretion of 2.31%. The results obtained, with reference molecule, make it possible to validate the study model of diuresis in a situation of water overload used in the present study. Similar results have been obtained with extracts of food and/or medicinal plants. This is the case of Cymbopogon densiflorus ⁷ and Piper glabratum ¹⁵. In addition, the aqueous and hydroethanol extracts at the two doses (75 and 150 mg/kg) used caused an increase in the urinary excretion of Na+, K+ and Cl- with an alkanization (pH> or = 7) of the urine and this alkanization is significant for the batch of rats treated with the hydroethanolic extract at a dose of 150 mg/kg. Consequently, the diuretic activity of these extracts may involve a mechanism other than those of Furosemide and Spironolactone because these diuretics cause marked natriuresis and acidification of the urine (pH<7) ¹⁶. On the urinary excretion of calcium and magnesium, only furosemide (20 mg/kg) and the hydroethanolic extract at a dose of 150 mg/kg significantly increase the urinary elimination of these ions (of Ca²⁺ and of Mg²⁺). However, it is known that Anse diuretics act on the ascending loop of Henle by blocking the co-transporter sodium-potassium-chlorine (Na⁺/K⁺/2Cl^-). Inhibition of active transport of NaCl causes an associated increase in urinary excretion of Ca²⁺ and Mg^{2+ 11}. As a result, the hydroethanol extract at a dose of 150 mg/kg would behave like an Anse diuretic (Furosemide) because the latter led to an associated increase in the urinary excretion of Ca²⁺ and Mg²⁺. Concerning the diuretic action and the diuretic index, the aqueous and hydroethanol extracts at a dose of 150 mg/kg showed a diuretic action and a high diuretic index approaching those of furosemide (20 mg/kg) and higher than those of Spironolactone (50 mg/kg). The natriuretic index represents the ratio (Na⁺/K⁺). If (Na⁺/K⁺)>1, indicates a satisfactory natriuresis (sodium eliminated more than potassium) if If (Na⁺/K⁺)>2 indicates a favorable natriuresis and if it is greater than ten, a sparing activity of potassium (K⁺), and yet the salidiuretic index (Na⁺+K⁺+Cl^-) provides information on the elimination of both water and sodium ¹¹. Consequently, the aqueous extract and the essential oil at the two doses (75 and 150 mg/kg) tested showed a satisfactory natriuretic index accompanied by a saludiuretic activity like spironolactone (50 mg/kg). However, the hydroethanolic extract at both doses (75 and 150 mg/kg) showed a favorable natriuretic index accompanied by a saludiuretic activity like furosemide (20 mg/kg). No test product showed a potassium-sparing effect. These results are similar to those found with other previous studies on diuretic plants, which showed a satisfactory and favorable natruiretic index. No potassium-sparing activity, this is the case of Netsanet et al. ^{13, 15, 17}. The index of carbonic anhydrase (Cl^-/ Na⁺+ K⁺) inhibition in a test substance can predict the diuretic mechanism modalities of IAC effect. If the ratio (Cl^-/ Na⁺+ K⁺)> 1 or between 0.8 and 1, it excludes the effect of IAC, but if it is less than 0.8, it is considered to have a strong activity of IAC ¹⁸. In this study, only rats treated with the hydroethanolic extract at a dose of (150 mg/kg) showed an inhibiting effect on carbonic anhydrase. However, the inhibition of carbonic anhydrase leads to the inhibition of the concomitant reabsorption of sodium bicarbonates and chlorine at the level of the proximal tubule. Through this mechanism, the hydroethanolic extract also has a diuretic effect ⁷. Thus, it is possible to suggest that the hydroethanolic extract could, unlike Furosemide, Spironolactone and the other extracts studied, exert its diuretic effect by a third mechanism. Similar results were found with other medicinal plants, Piper glabratum, Cymbopogon densiflorus, Clerodendrum myricoides respectively ^{7, 8, 15}. The renin-angiotensin-aldosterone system (RAAS) is very important in the regulation of bodily fluids and plays a major role in the control of water and sodium homeostasis. Aldosterone plays a key role in the reabsorption of Na⁺ and water thus raising blood pressure ¹⁹. In this study, the hydroethanolic extract at two doses (75 and 150 mg/kg) reduced the plasma concentration of aldosterone in the treated rats and this reduction is significant for the rats which received the hydroethanolic extract at the dose of 150 mg. /kg as those treated with Spironolactone (50 mg/kg) compared to control rats (0.9% NaCl + distilled water). However, like furosemide (20 mg/kg), the aqueous extract and the essential oil (75 and 150 mg/kg) do not influence the plasma concentration of aldosterone compared respectively to the controls (NaCl 0.9% + l distilled water) and control NaCl 0.9% + DMSO (5 mL/kg). The reduction in the plasma concentration of aldosterone observed with the hydroethanolic extract suggested a probable interaction on the RAAS with the bioactive compounds in the extract thus inhibiting the synthesis of aldosterone either by inhibition of the synthesis of renin or either by inhibition of the angiotensin converting enzyme, which would justify this reduction. These results are similar to those found by Zhiyong and al ¹⁹ who showed that the ethanolic extract of Lagopsis supina suppressed the action of RAAS by reducing the plasma aldosterone concentration in rats while the fractions of the same extract had no effect on aldosterone production.

5. Conclusion

In conclusion, all the extracts used (aqueous, hydroethanolic extracts and of the essential oil of P. guineense bark) have diuretic properties and these are important for the aqueous and hydroethanolic extracts at a dose of 150 mk/kg and low for essential oil at two doses (75 and 150 mk/kg). They increase the elimination of sodium and water (natriuretic and saludiuretic effects). At a dose of 150 mk/kg, the hydroethanolic extract basifies the urinary pH, inhibits the activity of carbonic anhydrase and decreases the plasma concentration of aldosterone in rats. From these facts, the hydroethanolic extract would act both like Furosemide and Spironolactone. However, the aqueous extract and the essential oil have no effect on urinary pH, carbonic anhydrase activity and plasma aldosterone concentration in rats, but they increase sodium and water secretion. this shows that the aqueous extract and the essential oil would act according to the same mechanism as furosemide.

References