Chronic Administration of Aqueous Extract of Hibiscus sabdariffa. Enhances Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase Activities of Rat Heart

Abstract

The effect of oral administration of an aqueous extract of Hibiscus sabdariffa. Linn petals on Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase activities of rat heart have been investigated in Sprague-Dawley rats. The extract was administered orally in doses of 25.0 and 50.0 mg/kg body weight for 28 days. Results showed that Hibiscus sabdariffa. treatment led to significant increases (p < 0.001) of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase at both doses. Cardiac weight index of rats treated with H. sabdariffa. at a dose of 50 mg/kg body weight was significantly reduced (p < 0.01) compared with those of the control and rats treated with H. sabdariffa. at a lower dose. Administration of the extract at both doses did not show any signs of cardiotoxicity and hepatotoxicity as judged by biochemical “marker” enzymes (alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase) activities in plasma, heart, and liver of rats. These results demonstrate that aqueous extract of H. sabdariffa. enhances cardiac Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase activities and supports the public belief that H. sabdariffa. may be a safe cardioprotective agent.

Keywords:

• Aqueous extract
• Ca²⁺-Mg²⁺-ATPase
• Hibiscus sabdariffa
• Na⁺-K⁺-ATPase

Introduction

The aqueous extract of the petals of Hibiscus sabdariffa. Linn (Malvaceae), a local soft drink material and medicinal herb, is usually used effectively in native medicines against hypertension, pyrexia, and liver disorders. It has been reported that water extract of H. sabdariffa. has a blood pressure–lowering effect in rats (Adegunloye et al., 1996; Odigie et al., 2003) and in humans (Haji-Faraji & Haji-Tarkhani, 1999). Studies have also shown that the aqueous extract of H. sabdariffa. reduced contractile responses to norepinephrine by stimulating vascular Na⁺-K⁺-ATPase activity (Adegunloye et al., 1993), and by reducing Ca²⁺ influx and intracellular store release (Owolabi et al., 1995) in rat vascular smooth muscle.

The reduction of Na⁺-K⁺-ATPase activity has been shown to enhance myocardial contractility due to increased intracellular Ca²⁺ concentration (Godfraind, 1985). Cardiac cell damage and death after ischemia have been associated with cellular Ca²⁺ overload (Steenbergen et al., 1990). It has also been suggested that depressed Na⁺-K⁺-ATPase (Blaustein, 1996) or depressed Ca²⁺-Mg²⁺-ATPase (M'Buyamba et al., 1994) activities play a pathophysiological role in the genesis of cardiovascular diseases. The current study was designed to examine the effect of chronic administration of H. sabdariffa. on Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase activities. In addition, we evaluated the cardiotoxicity and hepatotoxicity effects of the extract using cellular “marker” enzymes.

Materials and Methods

Animals

A total of 18 Sprague-Dawley rats of both sexes weighing between 110 and 140 g were obtained from the Animal Breeding Unit of the Department of Biochemistry, University of Ilorin (Ilorin, Nigeria). All rats were maintained on a standard rat chow and tap water ad libitum..

Extraction

Fresh samples of the petals of H. sabdariffa. were collected from Maiduguri (Borno State, Nigeria) and botanically authenticated by Prof. F.A. Oladele of the Department of Botany, University of Ilorin. The preparation of the plant material was as previously described (Adegunloye et al., 1993; Odigie et al., 2003; Olatunji et al., 2005). The extract was filtered and the residue discarded. The filtrate was evaporated to dryness and the resulting powder was stored in capped bottles until needed (Adegunloye et al., 1993; Obiefuna et al., 1993; Olatunji et al., 2005). The extract was dissolved in distilled water to make stock solutions of 25 and 50 mg/mlfor lower and higher doses.

Administration of aqueous extracts of petals of H. sabdariffa.

The animals were randomly distributed into three groups of six. Group I received an appropriate amount of distilled water (vehicle) per day by gavage for 28 days. Groups II and III were given 25 and 50 mg/kg orally by gavage, respectively, for 28 days. The vehicle or H. sabdariffa. treatment was stopped 24 h before the end of the experiment in order to study the long-term effects of H. sabdariffa. without the involvement of the effects of acute administration.

Tissue preparation

The animals were sacrificed by cervical dislocation at the end of the experimental period. Blood was collected from the jugular vein into lithium heparinized sample bottles. The plasma was obtained after centrifugation at 3000 rpm for 10 min, stored at 4°C, and used within 12 h of collection (Adebayo et al., 2003). The heart and liver were quickly excised, cleared of connective tissue, and transferred into the ice-cold 0.25 M sucrose solution. The cardiac weight index (HW/BW) was calculated by dividing the heart weight (HW) by the body weight (BW). The heart and liver were sectioned before being homogenized in ice-cold 0.25 M sucrose solution (1:5, w/v) as previously reported (Adebayo & Malomo, 2002; Adebayo et al., 2003). The homogenates were kept frozen overnight.

Enzymes assays

Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase activities were assayed by spectrophotometrically measuring the amount of inorganic phosphate liberated following incubation of the tissue extract with disodium ATP (Sigma, England) as in previous studies (Okolie & Uanseoje, 2000; Adebayo & Malomo, 2002). Alkaline phosphatase (ALP; EC 3.1.3.1), aspartate aminotransferase (AST; EC 2.6.1.1), and alanine aminotransferase (ALT; EC 2.6.1.2) activities were determined in plasma, heart, and liver by standard methods (Wang et al., 2000; Adebayo et al., 2003) using assay kits supplied by Quimica Clinica Aplicada (Amposta, Spain).

Statistical analysis

Results are expressed as means ± SEM of measurements. Significance was determined by analysis of variance (ANOVA). p values less than 0.05 were taken as significant.

Results

Effects of Hibiscus sabdariffa. on body weight and heart weight

Chronic administration of H. sabdariffa. at both doses did not affect the body weight (BW) significantly. However, the cardiac mass index (HW/100 g BW) was significantly reduced (p < 0.01) in H. sabdariffa.–treated rats at 50.0 mg/kg as compared with vehicle-treated rats. Cardiac mass index in rats treated with 50.0 mg/kg of H. sabdariffa. was significantly lower than that of the rats treated with 25.0 mg/kg (Table 1).

Table 1 Effect of Hibiscus sabdariffa. on body weight and cardiac mass index.^a.

Treatment	Body weight (g)	Cardiac mass index (g/100 g)
Control	180.6 ± 17.3	0.37 ± 0.02
25 mg/kg	189.2 ± 10.9	0.36 ± 0.01
50 mg/kg	188.9 ± 21.0	0.30 ± 0.01

^a.Values are presented as mean ± SEM for 6 rats per group.

*Significantly different at p < 0.01 compared with other groups.

The effects of Hibiscus sabdariffa. on Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase activities

Table 2 shows activities of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase in the heart of three groups of rats. The administration of H. sabdariffa. at both doses significantly increased (p < 0.001) activities of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase when compared with those of the control hearts. The activities of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase were not significantly greater (p > 0.05) in the hearts of rats treated with 50.0 mg/kg than those treated with H. sabdariffa. at 25.0 mg/kg.

Table 2 The activities of cation ATPases (µmol P_i mg⁻¹h⁻¹) in the hearts of rats treated with Hibiscus sabdariffa..^a.

	Control	25 mg/kg	50 mg/kg
Na^+-K^+-ATPase	6.6 ± 0.4	14.4 ± 0.7	16.9 ± 1.9
Ca^2+-Mg^2+-ATPase	1.7 ± 0.4	10.2 ± 3.3	12.8 ± 3.2

^a.Values are presented as mean ± SEM for 6 rats per group.

*Significantly different at p < 0.001 compared with control group.

The effect of Hibiscus sabdariffa. on ALP, AST, and ALT activities in plasma, heart, and liver of rats

The administration of H. sabdariffa. at 25.0 and 50.0 mg/kg did not produce any significant effect on the activities of AST and ALT in plasma, heart, and liver when compared with the control values (Tables 3 and 4). The activity of ALP in the heart of rats treated with H. sabdariffa. at both doses were significantly increased (p < 0.01) while the activities of ALP in the plasma and liver were not significantly affected (Table 5).

Table 3 Effect of Hibiscus sabdariffa. on alanine aminotransferase (ALT) activity.^a.

	Control	25 mg/kg	50 mg/kg
Plasma	12.9 ± 2.9	15.2 ± 4.8	14.3 ± 3.4
Heart	251.1 ± 33.3	176.7 ± 28.9	222.5 ± 18.8
Liver	1124.4 ± 29.9	1195.6 ± 42.2	1160.1 ± 51.5

^a.Values are presented as mean ± SEM for 6 rats per group.

Table 4 Effect of Hibiscus sabdariffa. on aspartate aminotransferase (AST) activity.^a.

	Control	25 mg/kg	50 mg/kg
Plasma	92.0 ± 10.5	111.5 ± 15.5	99.3 ± 14.4
Heart	1628.8 ± 49.5	1728.3 ± 142.2	1673.5 ± 73.5
Liver	1322.2 ± 105.5	1392.5 ± 129.7	1432.1 ± 126.1

^a.Values are presented as mean ± SEM for 6 rats per group.

Table 5 Effect of Hibiscus sabdariffa. on alkaline phosphatase (ALP; IU/l) activity.^a.

	Control	25 mg/kg	50 mg/kg
Plasma	228.4 ± 17.0	232.2 ± 16.8	239.4 ± 24.6
Heart	1448.1 ± 94.2	2267.7 ± 186.7	2135.0 ± 104.2
Liver	2630.3 ± 105.7	2191.7 ± 233.4	2532.5 ± 149.7

^a.Values are presented as mean ± SEM for 6 rats per group.

*Significantly different at p < 0.01 compared with control group.

Discussion

The main finding of the current study was that chronic administration of an oral daily dose of petal extract of H. sabdariffa. enhanced Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase activities of rat heart. The results also showed that administration of H. sabdariffa. led to reduced cardiac mass index. These results are in the same line of thought with an in vitro. study (Adegunloye et al., 1993) that showed that H. sabdariffa. caused vasodilatation of rat aortic rings by enhancing vascular smooth muscle Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase activities.

Inhibition of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase activities has been reported to be one of the major mechanisms responsible for hypertension (Blaustein, 1996). Ca²⁺-Mg²⁺-ATPase activity of the vascular smooth muscle has been demonstrated to decrease in vascular disease (M' Buyamba et al., 1994). Altered cellular handling of Na⁺, Ca²⁺, and Mg²⁺ have been shown in human and experimental hypertension to be associated with cardiac cell damage and death (Resnick et al., 1984; Steenbergen et al., 1990). The increased activities of Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase in the hearts of the rats chronically treated with H. sabdariffa. suggest that one of the mechanisms whereby H. sabdariffa. prevents development of hypertension and cardiac hypertrophy (Haji-Faraji & Haji-Takhani, 1999; Odigie et al., 2003) may be attributed to enhancement of activities of cardiac Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase.

Cardiac muscle contractility and hypertrophy has been linked with increased intracellular Ca²⁺ and Na⁺, and sarcolemial depolarization (Steenbergen et al., 1990). Enhanced Na⁺-K⁺-ATPase activity has been shown to cause hyperpolarization, resulting in a secondary decrease in intracellular Ca²⁺, which in turn reduces myocardial contractility hypertrophy (Perez-Vizcaino et al., 1999). Indeed in vitro. studies have shown that H. sabdariffa. attenuates vascular contractile response to norepinephrine by stimulating Na⁺-K⁺-ATPase activity and reducing Ca²⁺ influx (Adegunloye et al., 1993; Owolabi et al., 1995), leading to decreased cytoplasmic Ca²⁺ concentration. Because Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase are membrane-bound enzymes, it is not unreasonable to suggest that the enhancement of the activity of the enzymes in the heart tissue of the H. sabdariffa.–treated animals may at least in part be due to the improved membrane integrity/stabilization by H. sabdariffa. as previously reported by Adegunloye et al. (1993).

The activities of ALP, AST, and ALT in plasma, heart, and liver were used as biochemical “marker” enzymes of heart and liver damage. Alkaline phosphatase is a “marker” enzyme for the plasma membrane (Adebayo et al., 2003) while AST and ALP are cytosolic “marker” enzymes (Wang et al., 2000). All these “marker” enzymes were used to assess the integrity of plasma membrane, cytosolic activity, cell lysis, or death (Olagunju et al., 2000). The increase in the activity of ALP in the heart without any change in the plasma level of ALP is suggestive of enhancement of the enzyme molecules by the plant extract or activation of the enzyme molecules in situ., which in turn may lead to greater availability of the phosphate group for oxidative phosphorylation required for increased production of ATP. Increased production of ATP will lead to enhanced activity of cation ATPase, which ensures relaxation of muscle (Okolie & Uanseoje, 2000). Activities of AST and ALT in the plasma, heart, and liver were not significantly affected by the extract, whereas the activity of ALP in the heart of rat treated with H. sabdariffa. at both doses was significantly increased (Tables 3, 4, and 5). These results are in consonance with a recent in vitro. study (Wang et al., 2000). The findings provide evidence that administration of aqueous extract of H. sabdariffa. for 28 days may have cardioprotective effects that are not associated with deleterious effects on the heart or liver cell cytoplasm and/or membrane integrity.

In conclusion, this study demonstrates that chronic administration of aqueous extract of H. sabdariffa. enhances Na⁺-K⁺-ATPase and Ca²⁺-Mg²⁺-ATPase activities in rats hearts and supports the public belief that the water extract of H. sabdariffa. is a safe and useful cardioprotective agent.