Antihypertensive effects of the hydro-ethanol extract of Senecio serratuloides DC in rats

One in three adults worldwide has hypertension (HTN) and the prevalence has been shown to increase with age [1]. Hypertension is a growing public health concern in sub-Saharan countries where it was previously not reported. In this population, HTN is characterized by a rapid onset, poor control and an early onset of target organ damage [2]. Poor control of HTN contributes enormously to the burden of cardiovascular diseases (CVDs) and associated morbidity and mortality. Indeed, a relevant systemic meta-analysis showed that every 10 mmHg reduction of systolic blood pressure (BP) was accompanied by a significant decrease in the risk for CVD [3]. Several classes of anti-hypertensive medications have been developed with the aim of reducing BP and consequently the associated risks [4]. However, affordability and availability of pharmaceutical antihypertensive medications are important challenges especially in rural African communities thus affecting compliance with treatment regiments. Importantly, reported side effects of pharmaceutical drugs [5] and the belief that plant medicines are less toxic are contributing to the preference of plant extracts over pharmaceutical drugs [6].

In rural South African communities, medicinal plants are used for BP management. S. serratuloides DC, is an Asteraceae commonly found in areas of South Africa which receive summer rainfalls. S. serratuloides is a perennial which grows from a woody root/stem, has serrated leaves and tiny yellow flowers which are clustered at the terminals. It is used in folk medicine for treatment of various ailments [7, 8]. A recent ethnobotanical survey of medicinal plants used for self-medication by lay people of Maputaland and Northern Kwazulu-Natal, South Africa, showed that S. serratuloides was one of the most commonly used plants for treatment of HTN [9, 10]. Furthermore, S. serratuloides was reportedly used in combination with several medicinal plants to prepare concoctions for hypertension treatment [11]. Importantly none of the plant combinations which included S. serratuloides had been previously described. Other authors from the same region indicated that S. serratuloides was among the most used plants for respiratory infections [12] thus confirming the fact that this plant has extensive uses. Although S. serratuloides is widely used in the South African traditional medicine, it’s medicinal properties have not received much scientific attention. Extracts of this plant are reported to have antioxidant, analgesic, anti-inflammatory and wound healing activities [13, 14]. Due to the efficacy of the plant extract in treating various diseases it is traditionally referred to as the ‘two day cure’ plant. Although there is no scientific report on the use of S. serratuloides in the treatment of hypertension, another member of the Genus, Senecio nutans which is native to South America has demonstrated antihypertensive and hypotensive effects in rats [15]. The health benefits of the Senecios may be associated with their rich flavonoid and phenol contents [16]. Indeed, studies have demonstrated the usefulness of plant flavonoids in the prevention of atherosclerosis and hyperlipidemia [17] though these properties have not been evaluated for S. serratuloides.

Even though S. serratuloides is popular in South African traditional medicine, only its antimicrobial, analgesic and anti-inflammatory/wound healing properties have been investigated [13, 14]. Its potential usefulness in the management of any of the chronic diseases of lifestyle has not been studied. To our knowledge, this is will be the first study reporting on the antihypertensive effects of S. serratuloides. In this study we used L-NAME to induce hypertension. L-NAME is an inhibitor of nitric oxide synthase activity and consequently the synthesis of nitric oxide (NO). Chronic administration of L-NAME results in generalized reduction of NO resulting in endothelial dysfunction which is observed in the early phase of hypertension [18]. L-NAME induced hypertension is associated with activated sympathetic tone and generalized vasoconstriction [19]. This experimental model of hypertension has several similarities with human hypertension in the African population who demonstrate increased sympathetic activation, salt sensitivity and decreased NO dependent vasodilation [20] and high rate of target organ damage [21]. Therefore, the aim of the study was to investigate the safety and antihypertensive properties of the hydroethanol extract of S. serratuloides (HESS) in the L-NAME-induced hypertensive rat model and the effect of treatment on selected target organs.

Acute toxicity of HESS was greater than 5000 mg/kg. According to the toxicity guidelines by Konaté et al. [32], pharmacological substances with LD₅₀ less than 5 mg/kg are classified as highly toxic substances, those with LD₅₀ between 5 mg/kg and 5000 mg/kg are classified as moderately toxic substances while those with LD₅₀ > 5000 mg/kg are not toxic. Therefore the hydroethanolic extract of S. serratuloides whose LD₅₀ > 5000 mg/kg was considered non-toxic and safe for consumption.

Treatment with L-NAME resulted in decreased HDL and increased LDL/TG/VLDL thus corroborating the findings of Salam et al. [33] who showed that L-NAME treatment had an adverse effect on lipid profiles in treated rats. L-NAME treatment raised the concentration of LDL which in turn was capable of interfering with eNOS activity. Under normal conditions, eNOS is associated with cholesterol-enriched caveolae in endothelial cells, where its activity can be carefully regulated [34]. However, in hyperlipidemia, LDL, especially oxLDL negatively affects the activity and sub-cellular distribution of eNOS hence leading to a decrease in NO bioavailability [35, 36]. On the other hand, HDL causes activation of eNOS within the caveolae, with the resultant generation of NO [37]. Our findings showed that HESS significantly improved lipid profiles in a dose dependent manner in L-NAME treated animals indicating that HESS inhibited hypertension by reducing inactivation of eNOS by LDL and promotion of its activity by improved HDL. This was confirmed by its ability to prevent L-NAME induced decrease in serum NO levels. In the heart and kidneys however, the concentration of NO in L-NAME groups was comparable to normotensive control. This finding was consistent with previous studies by Adaramoye et al. [38] and Berkban et al. [39] who also observed normal levels of NO in heart and kidney of L-NAME treated rats. Abdel-Raham et al. [40] proposed that eNOS protein expression may be up-regulated in L-NAME treated rats as a counter-regulatory mechanism to compensate for increased BP. On the other hand Luvarà et al. [41] showed that chronic L-NAME administration was associated with the induction of iNOS expression both at mRNA and protein level. Inducible NOS-derived NO is implicated in the pathogenesis of tissue injury [42] probably through the formation of peroxynitrite and ROS suggesting the pathology observed in cardiac tissue of L-NAME treatment group despite the NO availability. However this finding was inconsistent with Talas et al. [43] who reported decrease in NO concentration in heart and kidney after L-NAME treatment for 15 days. This divergence in findings may be due to differences in duration of studies, dose of L-NAME administered and/or route of administration. In our finding, CPT improved NO availability in the absence of HDL improvement. Similarly, Bernátová et al. [44] showed improved tissue specific NOS activity with CPT at the higher dose of 100 mg/kg, contrary to that reported by Pechanova et al. [45] in which a similar dose of CPT failed to reverse L-NAME induced NO depletion. We have no explanation for this difference at the present moment but it may be worthwhile to investigate CPT effects in a dose response manner.

In this study L-NAME suppressed serum Ang II production thus confirming the finding of Johnson and Freeman [46] who showed that L-NAME decreases serum Ang II concentration. The ability of HESS to maintain serum Ang II at normal levels suggested that it decreased BP by maintaining a balance between Ang II and NO. Endothelial cells generate both Ang II and NO which both have antagonistic effects on vascular smooth muscle cell [47]. Therefore, a balance in concentration of the two molecules is important for maintaining homeostasis.

L-NAME-induced increase in mineralocorticoid receptor concentration in the kidney observed in this study was in line with previous reports that indicated that L-NAME treatment activated the renal renin angiotensin system (RAS) [48] while cardiac hypertrophy was also observed in L-NAME treated rats [49]. These findings suggested the existence of systemic as well as renal RAS thus corroborating studies by Giani et al. [46] which confirmed that L-NAME treated rats are a model of low plasma renin hypertension. These researchers further showed that L-NAME induced a marked activation of the renal RAS in wild-type mice which was absent in the angiotensin converting enzyme (ACE) mutant mice. Angiotensin converting enzyme is the target of ACE inhibitors, which are important medications in the treatment of both L-NAME induced HTN and human essential HTN. The endothelial lining of the lungs is considered the main source of ACE hence it is believed that ACE inhibitors lower BP by inactivating endothelial ACE [50]. However, findings from this study and the fact that many hypertensive patients have normal or low renin levels [51, 52] contradicted this belief. In addition, ACE inhibitors still reduce BP in patients with normal plasma angiotensin II levels [48] further suggesting that the source of ACE may not be limited to the endothelium. Furthermore, besides endothelial ACE, large amounts of ACE are synthesized locally in tissues particularly in the kidney, with potentially broad influences on renal function and ultimately BP [53]. Previous reports by Quiroz et al. [54] and Graciano et al. [48] showed that L-NAME increased renal abundance of several local RAS components, including angiotensinogen, ACE, and the AT1 receptor. Consequently, several authors proposed that intra-renal RAS activation may play a major role in the development of HTN and renal injury, even when there is no clear evidence of increased systemic RAS [46, 55]. This may be due to L-NAME–driven renal inflammation and oxidative stress which can override the physiologic regulation of the local renal RAS and induce its activation [42, 50]. A similar mechanism may explain the effects of L-NAME in cardiac tissue. The role of HESS in treating HTN was further supported by its ability to prevent cardiac hypertrophy and L-NAME induced increase in mineralocorticoid receptor concentration in the kidney in a dose dependent manner. The cardio-protective effect of HESS may have been through inhibition of inflammation and oxidative stress and hence preventing the elevation of RAS components in the heart. Indeed, this plant has been shown to have anti-inflammatory, anti-antioxidant [13] and wound healing effects [14].