Journal of Ethnopharmacology 153 (2014) 400–407

Contents lists available at ScienceDirect

Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep

Antihypertensive activities of the aqueous extract of Kalanchoe pinnata (Crassulaceae) in high salt-loaded rats Orelien Sylvain Mtopi Bopda a,n, Frida Longo b, Thierry Ndzana Bella d, Protais Marcellin Ohandja Edzah b, Germain Sotoing Taïwe a,c, Danielle Claude Bilanda d, Esther Ngo Lemba Tom b, Pierre Kamtchouing d, Theophile Dimo d a

Department of Zoology and Animal Physiology, Faculty of Science, University of Buea, P.O. Box 63 Buea, Cameroon Department of Biological Sciences, ENS, University of Yaounde, P.O. Box 3805 Yaoundé, Cameroon c Institut National de la Santé et de la Recherche Médicale, Unité 836, LabEx Ion Channels, Science and Therapeutics, Grenoble Institut de Neurosciences, Université Joseph Fourier, Chemin Fortuné Ferrini, Site santé de la Tronche, P.O. Box 170, 38042 Cedex 9, Grenoble, France d Department of Animal Biology and Physiology, University of Yaounde I, P.O. Box 812, Yaounde, Cameroon b

art ic l e i nf o

a b s t r a c t

Article history: Received 23 November 2013 Received in revised form 30 January 2014 Accepted 18 February 2014 Available online 26 February 2014

Ethnopharmacological relevance: The leaves of Kalanchoe pinnata (Crassulaceae) are used in Cameroon folk medicine to manage many diseases such as cardiovascular dysfunctions. In this work, we aimed to evaluate the activities of aqueous leaf extract of Kalanchoe pinnata on the blood pressure of normotensive rat (NTR) and salt hypertensive rats (SHR), as well as its antioxidant properties. Materials and methods: Hypertension was induced in rats by oral administration of 18% NaCl for 4 weeks. For the preventive study, three groups of rats received 18% NaCl solution and the plant extract at 25 mg/ kg/day, 50 mg/kg/day or 100 mg/kg/day by gavage. Two positive control groups received 18% NaCl solution and either spironolactone (0.71 mg/kg/day) or eupressyl (0.86 mg/kg/day) by gavage for 4 weeks. At the end of this experimental period, systolic arterial pressure (SAP), diastolic arterial pressure (DAP) and heart rate (HR) were measured by the invasive method. Some oxidative stress biomarkers (reduced glutathione (GSH), superoxide dismutase (SOD), nitric monoxide (NO) were evaluated in heart, aorta, liver and kidney. NO level was indirectly evaluated by measuring nitrite concentration. Results: Kalanchoe pinnata extract prevented significantly the increase of systolic and diastolic arterial pressures in high salt-loaded rats (SHR). In SHR, concomitant administration of Kalanchoe pinnata at 25, 50 and 100 mg/kg/day significantly prevented the increase in blood pressure by 32%, 24% and 47% (for SAP); 35%, 33% and 56% (for DAP), respectively. No significant change was recorded in heart rate of those rats. The plant extract improved antioxidant status in various organs, but more potently in aorta. Thus, antioxidant and modulatory effects of Kalanchoe pinnata at the vasculature might be of preponderant contribution to its overall antihypertensive activity. Conclusion: The work demonstrated that the concomitant administration of high-salt and the aqueous extract of Kalanchoe pinnata elicits prevention of salt-induced hypertension in rat. This antihypertensive activity is associated with an improvement of antioxidant status. Overall, results justify and support the use of Kalanchoe pinnata as antihypertensive medicine. & 2014 Elsevier Ireland Ltd. All rights reserved.

Chemical compounds studied in this article: Sodium chloride (PubChem CID: 5234) Spironolactone (PubChem CID: 5833) Eupressyl (PubChem CID: 5639) Urethane (PubChem CID: 5641) Heparin (PubChem CID: 25244225) Sulfanilamide (PubChem CID: 5333) N-(1-Naphthyl) ethylenediamine dihydrochloride (CID: 15106) Orthophosphoric acid (CID: 1004) Keywords: Kalanchoe pinnata Salt hypertensive rat Antihypertensive Antioxidant

1. Introduction

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; DAP, diastolic arterial pressure; GSH, reduced glutathione; HR, heart rate; NO, nitric monoxide; NO2
, nitrite; NTR, normotensive rats; SAP, systolic arterial pressure; SHR, salt hypertensive rats; SOD, superoxide dismutase n Corresponding author. Tel.: þ 237 75 07 21 74. E-mail addresses: [email protected] (O.S.M. Bopda), [email protected] (F. Longo), [email protected] (T.N. Bella), [email protected] (P.M.O. Edzah), [email protected] (G.S. Taïwe), [email protected] (D.C. Bilanda), [email protected] (E.N.L. Tom), [email protected] (P. Kamtchouing), [email protected] (T. Dimo). http://dx.doi.org/10.1016/j.jep.2014.02.041 0378-8741 & 2014 Elsevier Ireland Ltd. All rights reserved.

Hypertension is currently increasing in world population. In 2000, hypertension already affected 26.4% of the overall adult population worldwide, and this ratio was predicted to be increased by about 60% in 2025 (Kearney, 2005). Strongly concerned are economically developing countries (Kearney, 2005; Balde et al., 2006; Fezeu et al., 2010) like Cameroon. In 2003, a prevalence of 25.6% and 23.1% was reported respectively in male and female subjects from Cameroonian urban area (Kamadjeu et al., 2006). Parts of the reasons of increase in the hypertension prevalence are

O.S.M. Bopda et al. / Journal of Ethnopharmacology 153 (2014) 400–407

the changes in population lifestyle, which include a diet rich in salt, sugar and high fat processed foods as well as sedentary behaviour. Since the proportion of hypertensive people worldwide is predicted to rise up dangerously, the detection, prevention, treatment and control of this burden status must be a top priority (Kearney, 2005). However, because the cost of modern drug therapy is prohibitive, many patients turn to traditional herbal medicine for the management of Hypertension (Adjanohoun et al., 1996; Dimo et al., 2007; Bopda et al., 2007). Thus scientific validation and rationalisation of medicinal plants need to be intensified. Kalanchoe pinnata, also named Bryophyllum pinnatum, belongs to the family Crassulaceae whose species possess an array of medicinal effects. They are used in folk medicine as stomach pain relief, against gastritis, diarrhoea, bilharzias, dysmenorrhoea, liver disorders, fever, female infertility, genitourinary infections, snake and scorpio bites, leprosy, cough, asthma, kidney stones, arthritis, cardiovascular diseases and general tiredness (Hutchings et al., 1996; Van Wyk et al., 1997; Quazi Majaz et al., 2011). Furthermore, pharmacological activities (antidiabetic, antioxidant, antineoplastic, immunomodulatory, antilipidaemic, antiallergic, antiviral, antitumoral, hepatoprotective and antithrombotic) of Kalanchoe pinnata have been reported (Supratman et al., 2001; Yadav and Dixit, 2003; Quazi Majaz et al., 2011). In some parts of the south, west and centre Provinces of Cameroon, the folk medical practice considers the leaves of Kalanchoe pinnata as a useful remedy against diabetes and hypertension. Phytochemical analysis of Kalanchoe pinnata leaf extracts showed the presence of alkaloids, saponins, tanins, sterols, cardiac glycosides, flavonoids, vitamins, and mineral salts (Supratman et al., 2001; Muzitano et al., 2006; Quazi Majaz et al., 2011). It has been reported for long now that excess load of salt (NaCl) leads to hypertension with increase of peripheral resistance (Guyton, 1989; Dimo et al., 1999; Badyal et al., 2003; Bopda et al., 2007), as well as oxidative stress (Kitiyakara et al., 2003; Banday et al., 2007). In this work, we aimed to evaluate the antihypertensive activities of aqueous leaf extract of Kalanchoe pinnata in normotensive Wistar rats (NTR) and in salt hypertensive rats (SHR), as well as its antioxidant properties.

2. Materials and methods 2.1. Animals Three months old male albinos Wistar rats, weighting between 180 and 250 g were used. They were carefully handled according to guidelines for the care and use of laboratory animals approved by the Japanese Pharmacological Society (1987) and the International Guiding Principles for Biomedical Research Involving Animals developed by the Council for International Organizations of Medical Sciences (CIOMS, 1985). Animals were raised in the Animal House of ENS, University of Yaoundé I in plastic cages, under standard light (12-hour day/night natural cycle) and temperature (25 1C). Rats were fed with standard diet and water ad-libitum.

401

Normotensive rats were randomly divided into seven groups of five animals each. One group, neutral control, received tap water and served as normotensive rats (NTR), one group received 18% NaCl. Salt-induced hypertension was obtained by administration of 18% NaCl (1 mL/100 gbw) to normotensive Wistar rats using a gastric pipe. Gavage was done daily, between 08:00 and 09:30 a.m., for 4 weeks. Rats with systolic blood pressure level above 140 mmHg and/or diastolic blood pressure level above 90 mmHg were considered hypertensive. Three groups received 18% NaCl solution and the plant extract at 25 mg/kg/day, 50 mg/ kg/day or 100 mg/kg/day by gavage. Two positive control groups received 18% NaCl solution and either spironolactone (0.71 mg/kg) or eupressyl (0.86 mg/kg) by gavage. At the end of this experimental period, systolic arterial pressure (SAP), diastolic arterial pressure (DAP) and heart rate (HR) were measured. Biochemical parameters were also evaluated in heart, aorta, liver and kidney. Table 1 shows the sharing of rats in the various groups. 2.2. Plant material and extraction Fresh leaves of Kalanchoe pinnata were harvested in Yaoundé (Cameroon), in July 2010, and authenticated at the South western Cameroon Herbarium, Limbe, where a specimen has been deposited (Voucher Number SCA2770). The material (2 kg) was pounded by means of porcelain laboratory pounding cup, and then mixed with water (3 L). This step was followed by filtration and lyophilisation. A 59.2 g (yield of 2.96%) of dried powder was obtained. The aqueous extract was dissolved in a given volume of distilled water, giving rise to the stock solution (1 g/mL) for subsequent use. 2.3. Effects of Kalanchoe pinnata on blood pressure and heart rate During the experiment, weight and food intake (weekly) and water consumption (daily) were assessed. At the end of the 4-weeks treatment, we evaluated the effect of aqueous extract of Kalanchoe pinnata on salt-induced hypertension. Rats were anaesthetised using an intraperitoneal injection of 15% urethane (1.5 g/kg). The trachea was exposed and cannulated to facilitate spontaneous respiration. A polyethylene catheter (PE 50) was inserted into the right femoral vein and a bolus injection of 10% heparin (0.1 mL/100 gbw) was immediately administered. The invasive method was used for the recording of cardiovascular parameters. To that effect, a catheter connected to a pressure transducer was inserted into the left carotid artery. The transducer was coupled with a Biopac Student Lab MP35 hemodynamic recorder and a computer. The Biopac Student Lab MP35 is a multipurpose device that helps to record simultaneously the systolic blood pressure (SAP), diastolic blood pressure (DAP) and heart rate (HR). Values were always recorded after 30–60 min stabilisation (Dimo et al., 2003). 2.4. Effects of Kalanchoe pinnata on biochemical parameters At the end of the treatment with Kalanchoe pinnata extract, heart, aorta, liver and kidney were dissected out and homogenised in Mc Even solution for heart and aorta or in Tris–HCl 50 mM

Table 1 Allocation of rats for various treatments. Groups

NTR

Treatment Water (10 mL/kg/ day)

SHR

Kalanchoe pinnata (25 mg/kg)

NaCl 18% NaCl 18%þ Kalanchoe (10 mL/kg/day) pinnata (25 mg/kg/day)

Kalanchoe pinnata (50 mg/kg)

Kalanchoe pinnata (100 mg/kg)

Spironolactone (0.71 mg/kg)

Eupressyl (0.86 mg/kg)

NaCl 18% þKalanchoe pinnata (50 mg/kg/day)

NaCl 18% þKalanchoe pinnata (100 mg/kg/day)

NaCl 18%þ Spironolactone (0.71 mg/kg/day)

NaCl 18%þ Eupressyl (0.86 mg/kg/day)

402

O.S.M. Bopda et al. / Journal of Ethnopharmacology 153 (2014) 400–407

buffer solution for liver and kidney (20%, w/v). Blood levels of creatinine, bilirubin (Bartels et al., 1972), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (Reitman and Frankel, 1957) were assayed. Tissue levels of reduced glutathione (GSH) and superoxide dismutase activity (SOD) were assayed using colorimetric method as described by Jollow et al. (1974) and Misra and Fridovich (1972), respectively. Nitric oxide (NO) level was indirectly assayed by measuring the concentration of nitrite (NO2
), its stable metabolite, using Griess reagent method (Green et al., 1982; Ikeda et al., 2003; Patil et al., 2009). Nitrite contents of tissues were determined by mixing 0.5 mL of homogenate with an equal volume of Griess reagent (1% sulfanilamide and 0.1% naphthyl ethylenediamine dihydrochloride in 2.5% orthophosphoric acid). The reading of the absorbance was done at 546 nm. Nitrite levels were expressed in mmoles per milligram of protein.

change of heart rate, compared to NTR. In SHR, heart rate increased nonsignificantly (P 40.05) by 9% (from 310 720 to 3397 24 BPM), as compared to NTR. Also, from the value of 310 720 BPM in NTR, heart rate still remained at 314 715 BPM, after a 4-week treatment with NaCl (18%) plus Kalanchoe pinnata (100 mg/kg/day). Similar results were recorded in animal group administered with NaCl (18%) plus spironolactone (0.71 mg/kg/ day) but not in that which received NaCl (18%) plus eupressyl (0.86 mg/kg/day) (Table 2) Data are given as mean 7 SEM of the heart rate in various groups of rat. n ¼5.nP o0.05 vs. Normotensive Rats (NTR). During four weeks, NTR received only tap water, SHR received only NaCl (18%), while other groups received Kalanchoe pinnata extract (25–100 mg/kg/day), spironolactone (0.71 mg/kg/day) or eupressyl (0.86 mg/kg/day) in addition to NaCl (18%).

2.5. Statistical analysis

3.3. Effects of Kalanchoe pinnata on some biochemical parameters

Results are expressed as mean 7SEM. One-way analysis of variance (ANOVA) followed by Dunnett's test was used for statistical evaluation. P values less than 0.05 were considered significant.

3. Results 3.1. Effects of Kalanchoe pinnata on body weight and water consumption After four weeks of treatment with NaCl (18%) and NaCl (18%) plus Kalanchoe pinnata extract (25–100 mg/kg), no significant difference was observed between body weights in normotensive (NTR), hypertensive untreated (SHR) and hypertensive treated rats. Likewise, no significant variation was observed between estimated food consumption in various groups (data not shown). Conversely, Salt-loaded Hypertensive Rats (SHR) showed increase of water consumption, compared to Normotensive Rats (NTR). From a volume of 11 71 mL/day/rat in NTR, water consumption rose up to 31 72 mL/day/rat in SHR, corresponding to a 182% increase (P o0.001). Kalanchoe pinnata (25–100 mg/kg/day) prevented this increase of water consumption. In rats administered with the doses 25, 50 and 100 mg/kg/day, water consumption dropped respectively to 13 72, 14 72 and 12 71 mL/day/rat, corresponding to a significant (P o0.001) reduction of 58%, 55% and 61% respectively, compared to SHR. Similar results were obtained in rats treated with NaCl (18%) plus reference drugs (spironolactone, 0.71 mg/kg/day; eupressyl, 0.86 mg/kg/day) (Fig. 1). 3.2. Effects of Kalanchoe pinnata on arterial pressure and heart rate Subchronic administration of NaCl (18%) to rats for four weeks increases systolic arterial pressure (SAP), diastolic arterial pressure (DAP) and heart rate (HR). SAP and DAP significantly varied from 12475 mmHg (NTR) to 156 78 mmHg (SHR) and from 897 4 mmHg (NTR) to 13575 mmHg (SHR), corresponding to 26% (Po 0.01) and 52% (P o0.001) increases, respectively. In SHR, concomitant administration of Kalanchoe pinnata at 25, 50 and 100 mg/kg/day significantly prevented the increase in blood pressure by 32%, 24% and 47% (for SAP); 35%, 33% and 56% (for DAP), respectively. It is worth noting that at 25 and 50 mg/kg/day, SAP value was reduced, significantly (Po 0.05), below NTR level. Similar results were observed in reference animal groups which received simultaneously NaCl (18%) and either spironolactone (0.71 mg/kg/day) or eupressyl (0.86 mg/kg/day) (Fig. 2). Treatment with NaCl (18%) and NaCl (18%) plus Kalanchoe pinnata extract (25–100 mg/kg) did not display any significant

3.3.1. Effects on some oxidative stress biomarkers Fig. 3 shows that reduced glutathione (GSH) level was significantly (Po0.01) decreased in aorta of SHR by 76% (from 5075 to 1277 mmol/L), as compared to NTR. Kalanchoe pinnata extract (25– 100 mg/kg/day) significantly reduced that drop (i.e. increased GSH level) in the aorta of SHR. At the higher dose of 100 mg/kg/day the extract even permitted a total recovery of glutathione level. The level of glutathione was enhanced from 1277 to 5374 mmol/L, corresponding to an increase of 341%, as compared to SHR (Po0.001). Similar results were observed with spironolactone (0.71 mg/kg/day). No significant decrease of the level of glutathione was noticed in other organs of SHR (heart, liver and kidney) However, the plant extract, at the dose of 25 mg/kg/day, increased the level of that biomarker, especially in heart and kidney. In heart, the increase was by 107% (from 1572 to 3176 mmol/L, Po0.05), meanwhile in kidney it was 93% (from 1574 to 2971 mmol/L, Po0.05), as compared to SHR. Similar results were observed with spironolactone (0.71 mg/kg/day), but not with eupressyl (0.86 mg/ kg/day). Salt-loaded animals exhibited a significant decrease in aorta, heart, liver and kidney SOD activities, as compared to NTR. Kalanchoe pinnata extract (25–100 mg/kg/day) reduced that fall (i.e. increase SOD activity) in all organs of SHR. At the higher dose of 100 mg/kg/day the rise of SOD activity in aorta, heart, liver and kidney corresponded to 103% (from 29 73 to 59 73 U/mg proteins, Po 0.001), 71% (from 21 72 to 36 75 U/mg proteins, P4 0.05), 86% (from 227 3 to 41 73 U/mg proteins, P o0.001) and 76% (from 21 71 to 37 73 U/mg proteins, P o0.01), respectively. Similar results were observed with spironolactone (0.71 mg/kg/day) and eupressyl (0.86 mg/kg/day) in all organs (Fig. 4). Fig. 5 shows that in salt-loaded hypertensive rats, nitrite (NO2
) levels were significantly (P o0.001) decreased in aorta, liver and kidney but not in heart, as compared to NTR. In aorta and kidney, Kalanchoe pinnata extract at the moderate dose of 50 mg/kg/day displayed a more potent effect. In fact, Kalanchoe pinnata (50 mg/ kg/day) significantly (Po 0.01) increased the concentration of NO2
, in both organs. The level of the biomarker rose by 395% in aorta and 23% in kidney, compared to SHR. Similar results were observed with spironolactone (0.71 mg/kg/day) in both organ types. 3.3.2. Effects on creatinine, bilirubin, AST and ALT levels Salt-loaded rats displayed a significant (P o0.001) increase in blood levels of creatinine and bilirubin, as compared to NTR. Kalanchoe pinnata extract (25, 50 and 100 mg/kg/day) significantly reduced the rise of concentration of creatinine, meanwhile only

Water consump on (mL/day/rat)

O.S.M. Bopda et al. / Journal of Ethnopharmacology 153 (2014) 400–407

35 30 25 20 15 10 5 0

403

Animal groups

Fig. 1. Effects of Kalanchoe pinnata extract on water consumption in different groups of rat. Each bar represents the mean7 SEM, n¼ 5. nPo 0.001 vs. Normotensive Rats (NTR), ♯P o 0.001 vs. Salt-loaded Hypertensive Rats (SHR). During four weeks, NTR received only tap water, SHR received only NaCl (18%), while other groups received Kalanchoe pinnata extract (25–100 mg/kg/day), spironolactone (0.71 mg/kg/day) or eupressyl (0.86 mg/kg/day) in addition to NaCl (18%).

Arterial pressure (mmHg)

200 DAP (mmHg)

175 150

**

SAP (mmHg)

***

## ##

125

###

##

*

*

100

###

###

##

##

##

* ###

75

***

50 25

Animal group

0

Fig. 2. Effects of Kalanchoe pinnata extract on arterial pressure in different groups of rat. Each bar represents the mean 7 SEM, n¼5. nP o0.05, nnPo 0.01 and nnnP o0.001 vs. Normotensive Rats (NTR). ♯♯Po 0.01 and ♯♯♯Po 0.001 vs. Salt Hypertensive Rats (SHR). During four weeks, NTR received only tap water, SHR received only NaCl (18%), while other groups received Kalanchoe pinnata extract (25–100 mg/kg/day), spironolactone (0.71 mg/kg/day) or eupressyl (0.86 mg/kg/day) in addition to NaCl (18%).

Table 2 Effect of Kalanchoe pinnata extract on the heart rate in different groups of rat. SHR

Kalanchoe pinnata (25mg/kg)

Animal group

NTR

HR (BPM)

3107 20 339 7 24 3357 8

Kalanchoe pinnata (50mg/kg)

Kalanchoe pinnata (100mg/kg)

Spironolactone (0.71mg/kg)

Eupresssyl (0.86mg/kg)

329 724

3147 15

366 718

4017 23n

Data are given as mean7 SEM of the heart rate in various groups of rat. n¼5. nPo 0.05 vs. Normotensive Rats (NTR). During four weeks, NTR received only tap water, SHR received only NaCl (18%), while other groups received Kalanchoe pinnata extract (25–100 mg/kg/day), spironolactone (0.71 mg/kg/day) or eupressyl (0.86 mg/kg/day) in addition to NaCl (18%).

the dose of 25 mg/kg/day had such effect on bilirubin level. At that lower dose of 25 mg/kg/day the reduction corresponded to 56% (P o0.001) and 44% (Po 0.01) for creatinine and bilirubin, respectively. Similar results were observed in animals treated with NaCl (18%) and spironolactone (0.71 mg/kg/day) (Fig. 6). Fig. 7 shows that in SHR, AST level increased significantly (Po0.01) while ALT level increased nonsignificantly, compared to NTR. Kalanchoe pinnata extract at the higher dose of 100 mg/kg/day reduced AST level from 0.05370.003 to 0.02270.003 IU/L, corresponding to a 58% reduction (Po0.01). Similar results were observed in both positive control groups.

4. Discussion This study investigated for the first time the effects of aqueous leaf extract of Kalanchoe pinnata on salt-loaded hypertension. The outcome is that the aqueous extract of Kalanchoe pinnata induces an antihypertensive activity in salt-loaded hypertensive rats (SHR). Moreover, the antihypertensive effect of the extract is associated with antioxidant properties in hypertensive rats. In our study, hypertension was induced by administering 18% NaCl to normotensive rats. At the first look, this dose may seem too high, but it is worth noting that between administration of salt

O.S.M. Bopda et al. / Journal of Ethnopharmacology 153 (2014) 400–407

GSH Concentration (mmol/L)

404

NTR SHR K. pinnata (25mg/kg) K. pinnata (50mg/kg) K. pinnata (100mg/kg) Spironolactone (0.71mg/kg) Eupresssyl (0.86mg/kg)

100 90 80 70 60 50 40 30 20 10 0

Organ type Aorta

Heart

Liver

Kidney

SOD activity (Units/mg proteins)

Fig. 3. Effects of Kalanchoe pinnata extract on reduced glutathione concentration in different groups of rat. Each bar represents the mean7 SEM, n ¼5. nP o 0.05,nnPo 0.01 and nnnPo 0.001 vs. Normotensive Rats (NTR). ♯Po 0.05, ♯♯Po 0.01 and ♯♯Po 0.001 vs. Salt Hypertensive Rats (SHR). During four weeks, NTR received only tap water, SHR received only NaCl (18%), while other groups received Kalanchoe pinnata extract (25–100 mg/kg/day), spironolactone (0.71 mg/kg/day) or eupressyl (0.86 mg/kg/day) in addition to NaCl (18%).

NTR SHR K. pinnata (25mg/kg) K. pinnata (50mg/kg) K. pinnata (100mg/kg) Spironolactone (0.71mg/kg) Eupresssyl (0.86mg/kg)

100 90 80 70 60 50 40 30 20 10 0

Organ type Aorta

Heart

Liver

Kidney

Fig. 4. Effects of Kalanchoe pinnata extract on superoxide dismutase activity in different groups of rat. Each bar represents the mean 7SEM, n¼5. nnPo 0.01 and nnnPo 0.001 vs. Normotensive Rats (NTR). ♯♯Po 0.01 and ♯♯Po 0.001 vs. Salt Hypertensive Rats (SHR). During four weeks, NTR received only tap water, SHR received only NaCl (18%), while other groups received Kalanchoe pinnata extract (25–100 mg/kg/day), spironolactone (0.71 mg/kg/day) or eupressyl (0.86 mg/kg/day) in addition to NaCl (18%).

NO2-concentration (mmol/mg proteins)

140 120

##

**

100

## ##

** **

* * * *** *** **

#

***

#

***

NTR SHR K. pinnata (25mg/kg) K. pinnata (50mg/kg) K. pinnata (100mg/kg) Spironolactone (0.71mg/kg) Eupresssyl (0.86mg/kg)

80 ##

60 40

***

#

* 20 0 Aorta

Heart

Liver

Kidney

Fig. 5. Effects of Kalanchoe pinnata extract on nitrite (NO2
) concentration in different groups of rat. Each bar represents the mean 7SEM, n¼ 5. nP o 0.05, nnPo 0.01 and nnn P o0.001 vs. Normotensive Rats (NTR). ♯P o0.05 and ♯♯Po 0.001 vs. Hypertensive Rats (SHR). During four weeks, NTR received only tap water, SHR received only NaCl (18%), while other groups received Kalanchoe pinnata extract (25–100 mg/kg/day), spironolactone (0.71 mg/kg/day) or eupressyl (0.86 mg/kg/day) in addition to NaCl (18%).

solutions, rats had access to normal water ad-libitum. This model presents as advantages the accuracy of dosage, the monitoring of precise time for salt ingestion by animals and the fast (4 weeks) occurrence of hypertension in animals. Neither in NaCl-loaded hypertensive rats nor in NaCl plus extract-treated rats was observed any significant change of body weight and food consumption. Conversely, salt treatment caused increase of water consumption

in rats. It is worth noticing that they also urinated consequently. This diuretic behaviour accounts for an obvious homoeostatic need of their organism. High blood pressure is a major risk for stroke, coronary diseases and renal dysfunction. Excess salt accumulation in internal medium is well known to cause hypertension (Bayorh et al., 2004; Banday et al., 2007; Bopda et al., 2007) frequently associated with increase in oxidative stress (Bayorh et al., 2004;

Blood levels (mg/mL) of creatinine and bilirubin

O.S.M. Bopda et al. / Journal of Ethnopharmacology 153 (2014) 400–407

16.00 15.50 15.00 14.50 14.00 13.50 13.00 12.50 12.00 11.50 11.00 10.50 10.00 9.50 9.00 8.50 8.00 7.50 7.00 6.50 6.00 5.50 5.00 4.50 4.00 3.50 3.00 2.50 2.00 1.50 1.00 0.50 0.00

405

Creatinine (mg/mL) Bilirubin (mg/mL)

###

###

Animal group

Blood levels (IU/L) of AST and ALT

Fig. 6. Effects of Kalanchoe pinnata extract on creatinine and bilirubin levels in different groups of rat. Each bar represents the mean 7SEM, n¼ 5. nnPo 0.01and nnnPo 0.001 vs. Normotensive Rats (NTR). ♯P o 0.05, ♯♯Po 0.01 and ♯♯♯Po 0.001 vs. Salt Hypertensive Rats (SHR). During four weeks, NTR received only tap water, HSR received only NaCl (18%), while other groups received Kalanchoe pinnata extract (25–100 mg/kg/day), spironolactone (0.71 mg/kg/day) or eupressyl (0.86 mg/kg/day) in addition to NaCl (18%).

0.070 0.060 0.050 #

**

**

**

**

AST

*

ALT

*

0.040 0.030

##

##

##

0.020 0.010 0.000

Animal group

Fig. 7. Effects of Kalanchoe pinnata extract on AST and ALT levels in different groups of rat. Each bar represents the mean7 SEM, n¼5. nPo 0.05 and nnPo 0.01 vs. Normotensive Rats (NTR). ♯Po 0.05 and ♯♯Po 0.01 vs. Salt Hypertensive Rats (SHR). During four weeks, NTR received only tap water, SHR received only NaCl (18%), while other groups received Kalanchoe pinnata extract (25–100 mg/kg/day), spironolactone (0.71 mg/kg/day) or eupressyl (0.86 mg/kg/day) in addition to NaCl (18%).

Wilcox, 2005; Banday et al., 2007) and hardening of blood vessels (Guyton, 1989; Dimo et al., 1999; Badyal et al., 2003; Bopda et al., 2007). In the present study, NaCl (18%) induced a significant increase in systolic and diastolic blood pressure. Our results demonstrated that the concomitant subchronic (four weeks) administration of aqueous extract of Kalanchoe pinnata led to reduction of the increase in both systolic and diastolic arterial blood pressure. The higher dose of 100 mg/kg/day appeared to be the most potent and the effect of extract on blood pressure was

comparable to that induced by the α1-adrenoceptor antagonist, eupressyl, and aldosterone antagonist, spironolactone (Lechat et al., 1990; Givertz, 2001). From the above, one might promptly hypothesise that the mechanism involved in this antihypertensive effect could be either vasodilation or increase of natriuresis and diuresis. Antihypertensive plant extracts act, in general, either by cardiodepression, increase of diuresis or vasorelaxation (Corallo et al., 1997; Dimo et al., 2007; Bopda et al., 2007, 2011; Tom et al., 2011; Nguelefack-Mbuyo et al., 2012). Because Kalanchoe pinnata extract

406

O.S.M. Bopda et al. / Journal of Ethnopharmacology 153 (2014) 400–407

did not modify heart rate, we suggest that organs targeted by Kalanchoe pinnata extract should be primarily kidney and blood vessels. From our results, more can be known with regard to mechanism. An important aspect to consider here is the reduction of oxidative stress by the plant extract. Indeed, it is well-established that high salt consumption is associated to increased tissue production of reactive oxygen species (ROS) (Bayorh et al., 2004; Wilcox, 2005; Banday et al., 2007). Lower activity of antioxidants has been reported in various animal models of hypertension (Zhou et al., 2002; Bayorh et al., 2004; Seifi et al., 2010). Accordingly, our results showed that several biomarkers (reduced glutathione, superoxide dismutase and nitrite) of oxidative stress are impaired in rats on high salt diet. Reduced glutathione (GSH) is an endogenous and ubiquitous antioxidant produced in eukaryote's cells, which contributes in maintaining the integrity of cell against oxidation (Stein et al., 1989; Zhou et al., 2002). Our data reported a decrease in GSH levels in aorta of high salt-hypertensive rats. This drop was well corrected by plant extract. The plant extract might also be able to enhance the basal level of GSH in normotensive and hypertensive rats, as exemplified by heart and kidneys recorded values. The activity of superoxide dismutase (SOD) was measured. SOD is the enzyme that catalyzes the dismutation of the O2
free radical in water and hydrogen peroxide (Faraci and Didion, 2004; Wang et al., 2006). Our data revealed decreased SOD activity in all organs of hypertensive rats. Long lasting stress due to subchronic administration of salt to rats induces a massive and toxic amount of oxygen active species, leading to destruction of SOD; which therefore undergoes a drop of its concentration. Indeed, salt intake enhances generation of O2
accompanied by enhanced expression and activity of NADPH oxidase with diminished expression of SOD in the vasculature and kidneys; this contributes in the pathogenesis of several models of hypertension (Kitiyakara et al., 2003). The fact that subchronic treatment with the aqueous extract of Kalanchoe pinnata significantly prevented the fall in SOD level, as the case was for GSH too, confirmed antioxidant properties of the plant extract. In addition to the above, the level of NO2
, a stable metabolite of NO (Ikeda et al., 2003) was investigated. We recorded a drop of that biomarker in all organs of high salt hypertensive rats, except heart. NO is not as stable as NO2
, but is the active biomarker in the vessels. NO is a major down-regulator of vascular tone in humans (Calver et al., 1993; Corvol, 1993; Nguelefack-Mbuyo et al., 2012). In animal models of salt-sensitive hypertension, the increase in blood pressure after salt loading is characterized by reduced NO production (Bayorh et al., 2004; Banday et al., 2007). Interaction of ROS, particularly superoxide with NO, leads to the production of peroxynitrite, which is a highly cytotoxic reactive compound (Zhou et al., 2002). Interest is focused on overexpression of NADPH oxidase components, or underexpression of SOD, as potential prohypertensive mechanisms in models of salt or mineralocorticoid-induced hypertension. Since SOD catalyzes the conversion of O2
to H2O2, the dominant effect of extracellular and intracellular SOD activity normally is to metabolize O2
and thereby prevent hypertension (Wilcox, 2005). There is no consensus on whether oxidative stress in animal models such as excess salt hypertensive rats is a cause or a consequence of hypertension, but at least it is now an agreement that prevention of oxidative stress contributes to a drop of blood pressure in such hypertensive rats (Wilcox, 2005). Thus antioxidant properties of Kalanchoe pinnata might represent other strong mechanisms involved in its antihypertensive activity. However, because NO activity contributes to antihypertensive effects by vasodilation (Corvol, 1993; Nguelefack-Mbuyo et al., 2012) it will be very interesting to carry out in vitro experiments using vascular rings, to assess the endothelium-dependent vasoreactivity of high salt hypertensive rats treated with Kalanchoe pinnata aqueous extract.

In addition to prooxydants/antioxydants disturbances due to NaCl 18% consumption, disturbance of the production or bioavailability of AST, a hepatic enzyme, was also recorded. High salt intake induced increase of plasma AST, as a footprint of an increase of cytolysis. Concomitant administration of NaCl 18% and extract (25 and 100 mg/ kg/day) permitted to reduce significantly the increase of AST, in hypertensive rats. This result suggests that plant extract might partially protect various tissues, including hepatic tissues, from high salt-induced injuries. Prolonged administration of NaCl 18% induced a significant increase of plasma creatinine. This is likely to be caused by an alteration of glomerular filtration (Hadj-Aïssa et al., 1994). A decrease of glomerular filtration by at least 50% can induce a hypercreatinemia (Kingdom et al., 2003). As Kalanchoe pinnata extract prevented that hypercreatinemia, it might prevent any eventual disruption of glomerular filtration. Plasma level of bilirubin was also significantly increased in NaCl 18% loaded rats. This variation was inhibited by Kalanchoe pinnata extract (25 mg/ kg/day), suggesting that Kalanchoe pinnata up-regulates red blood cells production and/or bioavailability. These protective activities are consistent with antioxidant properties of the plant extract. Moreover, these results while arguing in favour of a very low toxicity of Kalanchoe pinnata aqueous extract, when used at therapeutic doses, also confirm hepatoprotective properties of the plant leaves as reported by Yadav and Dixit (2003), Quazi Majaz et al. (2011) and Biswas et al. (2011). The presence of compounds such as flavonoids and polyphenols in Kalanchoe pinnata (Harlalka et al., 2007; Biswas et al., 2011) argues in favour of its displayed hepatoprotective, antioxidant and antihypertensive properties.

5. Conclusion This study demonstrates the antihypertensive effects of aqueous extract of the leaves of Kalanchoe pinnata in salt hypertensive rats. Our results provide justification and support for the use of this extract as traditional medicine against hypertension in Cameroon. The effect on blood pressure might be partly due to the improvement of the antioxidant status in hypertensive organisms. As the antioxidant mechanism seems to be emphasised at the level of the aorta, in vitro investigations of the endotheliumdependent vasodilation properties of Kalanchoe pinnata should be strongly envisaged.

Acknowledgements The authors are very grateful to Prof. Georges Chuyong (H.O.D., Botany and Plant Physiology, University of Buea) and Miss MarieClaire Veranso (Ph.D. student in the same Department) for their assistance in the preliminary identification of the plant. References Adjanohoun, J.E., Aboubakar, N., Dramane, N.N., 1996. Contribution to Ethnobotanical and Floristic Studies in Cameroon. National Centre for Production of School Tools, Benin p. 19. Badyal, D.K., Lata, H., Dadhich, A.P., 2003. Animal models of hypertension and effect of drugs. Indian J. Pharmacol. 35, 349–362. Balde, M.D., Balde, N.M., Kaba, M.L., Diallo, I., Diallo, M.M., Kake, A., Bah, D., Camara, A., 2006. Hypertension artérielle: épidémiologie et anomalies métaboliques au Foutah-Djallon en Guinée. Mali Méd. 3, 19–21. Banday, A.A., Muhammad, A.B., Fazili, F.R., Lokhandwala, M., 2007. Mechanisms of oxidative stress-induced increase in salt sensitivity and development of hypertension in Sprague-Dawley rats. Hypertension 49, 664–671. Bartels, H., Bohmer, M., Heierli, C., 1972. Serum creatinine determination without protein precipitation. Clinica Chimica Acta 37, 193–197.

O.S.M. Bopda et al. / Journal of Ethnopharmacology 153 (2014) 400–407

Bayorh, M.A., Ganafa, A.A., Socci, R.R., Silvestrov, N., Abukhalaf, I.K., 2004. The role of oxidative stress in salt-induced hypertension. Am. J. Hypertens. 17 (1), 31–36. Biswas, S.K., Chowdhury, A., Das, J., Zahid Hosen, S.M., Uddin, R., Rahaman, M.S., 2011. Literature review on pharmacological potentials of Kalanchoe pinnata (Crassulaceae). Afr. J. Pharm. Pharmacol. 5 (10), 1258–1262. Bopda, M.O.S., Dimo, T., Nguelefack, T.B., Dzeufiet, D., Rakotonirina, S.V., Kamtchouing, P., 2007. Effect of Brillantaisia nitens Lindau (Acanthaceae) methylene chloride/methanol leaf extract on rat arterial blood pressure and heart rate. Pharmacol. Online 1, 495–510. Bopda, M.O.S., Dimo, T., Tonkep, S.I., Zapfack, L., Zeufiet, D.D., Kamtchouing, P., 2011. Cardiodepression as a possible mechanism of the hypotensive effects of the methylene chloride/methanol leaf extract of Brillantaisia nitens Lindau (Acanthaceae) in rats. Afr. J. Biotechnol. 10 (72), 16393–16401. Calver, A., Collier, J., Vallance, P., 1993. Nitric oxide and the control of human vascular tone in health and disease. Eur. J. Med. 2, 48–53. Corallo, A., Foungbe, S., Davy, M., Cohen, Y., 1997. Cardiovascular pharmacology of aqueous extract of the leaves of Bridelia atroviridis Muell. Arg. (Euphorbiaceae) in the rat. J. Ethnopharmacol. 57, 189–196. Corvol, P., 1993. L'endothélium, plaque tournante de la vasomotricité et de la trophicité de la paroi artérielle. Médecine/Sciences 9 (10), 1031–1033. Dimo, T., Nguelefack, T.B., Kamtchouing, P., Dongo, E., Rakotonirina, A., Rakotonirina, S.V., 1999. Effets hypotensifs de l'extrait au méthanol de Bidens pilosa Linn. chez le rat hypertendu. C. R. Acad. Sci. 32, 323–329. Dimo, T., Nguelefack, T.B., Tan, P.V., Yewah, M.P., Dongo, E., Rakotonirina, S.V., Kamanyi, A., Bopelet, M., 2003. Possible mechanisms of action of the neutral extract from Bidens pilosa L. leaves on the cardiovascular system of anaesthetized rats. Phytotherapy Res. 17, 1135–1139. Dimo, T., Bopda Mtopi, O.S., Nguelefack, T.B., Kamtchouing, P., Zapfack, L., Asongalem, E.A., Dongo, E., 2007. Vasorelaxant effect of Brillantaisia nitens Lindau (Acanthaceae) extracts on isolated rat vascular smooth muscle. J. Ethnopharmacol. 111 (1), 104–109. Faraci, F.M., Didion, S.P., 2004. Vascular protection: superoxide dismutase isoforms in the vessel wall. Artheriosclerosis Thrombose and Vascular Biology 24, 1367–1373. Fezeu, L., Kengne, A.P., Balkau, B., Awah, P.K., Mbanya, J.C., 2010. Ten-year change in blood pressure levels and prevalence of hypertension in urban and rural Cameroon. J. Epidemiol. Community Health 64 (4), 360–365. Givertz, 2001. Manipulation of the renin-angiotensin system. Circulation 104, 14–18. Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannenbaum, S.R., 1982. Analysis of nitrate, nitrite, and [15N]-nitrate in biological fluids. Anal. Biochem. 126 (1), 131–138. Guyton, A.C., 1989. Traité de physiologie médicale. Edion (Eds.), Paris, 46–52, 148–163, 220–223. Hadj-Aïssa, A., Cochat, P., Dubourg, L., Wright, C., Pozet, N., 1994. La mesure de la function rénale chez l'enfant. Archives of Pediatrics 1, 273–280. Harlalka, G.V., Patil, C.R., Patil, M.R., 2007. Protective effect of Kalanchoe pinnata pers. (Crassulaceae) on Gentamycin-induced nephrotoxicity in rats. Indian J. Pharmacol. 39 (4), 201–205. Hutchings, A., Scott, A.H., Lewis, G., Cunninghan, A., 1996. Zulu Medicinal Plants, An Inventory. University of Natal Press, Pietermarizburg, South Africa. Ikeda, U., Takahashi, M., Shimada, K., 2003. C-reactive protein directly inhibits nitric oxide production by cytokine-stimulated vascular smooth muscle cells. J. Cardiovasc. Pharmacol. 42 (5), 607–611. Jollow, D., Mitchell, L., Zampaglione, N., Gillete, J., 1974. Bromobenze induced liver necrosis: protective role of glutathione and evidence for 3,4-bromebenzenoxide as the hepatotoxic intermediate. Pharmacology 11, 151–169. Kamadjeu, R.M., Edwards, R., Atanga, J.S., Unwin, N., Kiawi, E.C., Mbanya, J.C., 2006. Prevalence, awareness and management of hypertension in Cameroon: findings

407

of the 2003 Cameroon burden of diabetes baseline survey. J. Human Hypertens. 20, 91–92. Kearney, P.M., 2005. Global burden of hypertension: analysis of worldwide data. Lancet 365, 217–223. Kingdom, E.J., Knight, D.K., Irwin, A.G.M., Powis, S.H., Burns, A., Hilson, A.J.W., Black, C.M., 2003. Calculated glomerular filtration rate is a useful screening tool to identify scleroderma patients with renal impairment. Rheumatology 42, 26–33. Kitiyakara, C., Chabrashvili, T., Chen, Y., Blau, J., Karber, A., Aslam, S., Welch, W.J., Wilcox, C.S., 2003. Salt intake, oxidative stress, and renal expression of NADPH oxidase and superoxide dismutase. journal of American Society of Nephrology 14 (11), 2775–2782. Lechat, P., Calvo, F., Cremoux, de P., Giroud, J.P., Lagier, G., Lechat, Ph., Rouviex, B., Wer, S., 1990. Pharmacologie Médicale, Ed. Masson, France p. 446. Misra, H., Fridovich, I., 1972. The role of superoxide anion in the autoxidation of epinephrine to adrenochrome and a simple assay for superoxide dismutase. J. Biol. Chem. 247, 3170–3175. Muzitano, M.F., Tinoco, L.W., Guette, C., Kaiser, C.R., Rossi-Bergmann, B., Costa, S.S., 2006. The antileishmanial activity assessment of unusual flavonoids from Kalanchoe pinnata. Phytochemistry 67 (18), 2071–2078. Nguelefack-Mbuyo, E.P., Dongmo, A.B., Nguelefack, T.B., Kamanyi, A., Kamtchouing, P., Dimo, T., 2012. Endothelium/nitric oxide mediates the vasorelaxant and antihypertensive effects of the aqueous extract from the stem bark of Mammea africana Sabine (Guttiferae). Evidence-Based Complement. Altern. Med. , http: //dx.doi.org/10.1155/2012/961741 Patil, S.M., Kadam, V.J., Ghosh, R., 2009. In vitro antioxidant activity of methanolic extract of stem bark of gmelina arborea roxb. (verbenaceae). Int. J. PharmTech Res. 1 (4), 1480–1484. Quazi Majaz, A., Tatiya, A.U., Khurshid, M., Nazim, S., Siraj, S., 2011. The miracle plant (Kalanchoe pinnata): a phytochemical and pharmacological review. Int. J. Res. Ayurveda Pharm. 2 (5), 1478–1482. Reitman, S., Frankel, S., 1957. A colorimetric determination of serum glutamic oxalo-acetatic and glutamic pyruvic transaminase. Am. J. Clin. Pathol. 28, 56–63. Seifi, B., Kadkhodaee, M., Karimian, S.M., Zahmatkesh, M., Xu, J., Soleimani, M., 2010. Evaluation of renal oxidative stress in the development of DOCA-salt induced hypertension and its renal damage. Clin. Exp. Hypertens. 32 (2), 90–97. Stein, H.J., Esplugues, J., Whittle, B.J.R., 1989. Direct cytotoxic effect of oxygen free radicals on the gastric mucosa. Surgery 106, 318–324. Supratman, U., Fujita, T., Akiyama., K., Hayashi, H., Murakami, A., Sakai, H., Koshimizu, K., Ohigashi, H., 2001. Anti-tumor promoting activity of bufadienolides from Kalanchoe pinnata and K. daigremontiana x tubiflora. Biosci. Biotechnol. Biochem. 65 (4), 947–956. Tom, E.N.L., Demougeot, C., Bopda Mtopi, O.S., Dimo, T., Dzeufiet, P.D.D., Bilanda, D.C., Girard, C., Berthelot, A., 2011. The aqueous extract of Terminalia superba (Combretaceae) prevents glucose-induced hypertension in rats. J. Ethnopharmacol. 133, 828–833. Van Wyk, B.E., Van Oudtshoorn, B., Gericke, N., 1997. Medical Plants of South Africa, 1st ed. Briza publications, Pretoria. Wilcox, C.S., 2005. Oxidative stress and nitric oxide deficiency in the kidney: a critical link to hypertension? Am. J. Physiol. – Regul. Integr. Comp. Physiol. 289, 913–935. Wang, Y., Chen, A.F., Wang, D.H., 2006. Enhanced oxidative stress in kidneys of saltsensitive hypertension: role of sensory nerves. Am. J. Physiol. 291 (6), 3136–3143. Yadav, N.P., Dixit, V.K., 2003. Hepatoprotective activity of leaves of Kalanchoe pinnata. J. Ethnopharmacol. 86 (2), 197–200. Zhou, X.J., Vaziri, N.D., Wang, X.Q., Silva, F.G., Laszik, Z., 2002. Nitric oxide synthase expression in hypertension induced by inhibition of glutathione synthase. J. Pharmacol. Exp. Ther. 300 (3), 762–767.