Pharmacological basis for the medicinal use of polyherbal formulation and its ingredients in cardiovascular disorders using rodents

Cardiovascular disorders (CVDs) are huge disease burden worldwide [1]. Hypertension, dyslipidemia and endothelial dysfunctions are the major pathologies of commonly prevailing CVDs [2]. Hypertension is a silent killer, which is known to cause around 9.8 million deaths annually [3]. Pakistan is one of the South Asian countries where around 18 million people at the age of fifteen and above are living with hypertension. Out of these, around 70% population is even unaware about the disease status, while only 3% successfully control blood pressure with regular medication [4‐6]. Multiple antihypertensive therapeutic agents are available to combat this deadly disorder, however, their high cost and life-long use of the medication along with associated multiple adverse effects ultimately results in patient unaffordability and poor-compliance [7]. The current strategy to defeat chronic non-communicable disorders is changing throughout the world. The use of alternative approaches, particularly medicinal remedies and supplements [8, 9], have become popular over the past three decades. Around 80% of people worldwide rely on natural products [10] for therapeutic purpose. Treating chronic disorders like hypertension or diabetes, the herbal remedies are considered relatively safe, affordable and easily accessible. The relative safety of natural products when used for longer duration might be because of the presence of synergistic and/or side effects neutralizing constituents inbuilt in medicinal herbs [11]. In our settings due to cultural acceptance, people prefer to use herbal remedies for the treatment of various chronic ailments like gastro-intestinal [12], respiratory tract [13], endocrine (diabetes) [14] and cardiovascular disorders [15]. Despite using individual herbs to treat different ailments, there are multiple evidences supporting therapeutic use of compound formulations in the treatment of hypertension and dyslipidemia. Some of the combined formulations like Sharbat Abresham, Hab Fishar for hypertension, Arq-Sheer-Morakkab as cardiotonic, Muffrahe-Barid-Sada and Arq-Gazar Ambari as anti-palpitant [16] are used in indigenous system of medicine in Pakistan. POL₄, an indigenous polyherbal formulation containing four edible medicinal herbs (Cichorium intybus L., Gymnema sylvestre R. Br., Nigella sativa L., and Trigonella foenum graecum L.) is also popular for its medicinal use in cardiometabolic disorders, however, no study is available to rationalize its medical use in cardiometabolic disorders like hypertension.

The ingredients of POL₄: C. intybus (family: Asteraceae) commonly called Chicory or Kasni, G. sylvestre (family: Asclepiadaceae) known as Gurmar booti, N. sativa (family: Ranunculaceae) referred as Black cumin or Kalonji and T. foenum graecum (family: Fabaceae) vernacularly called as Fenugreek or Methi are popular for their medicinal use as cardiotonic, antihypertensive and cadio-depressant [17]. C. intybus (coumarins, sterols, tannins, triterpene cichoridiol, lupeol, sesquiterpene glycoside, caffeic acid and cichotyboside) [18], G. sylvestre (gymnemasides anthraquinone, stigmasterol, betaine, quercitol and gymnemagenin) [19], N. sativa (thymoquinone, α-pinene, p-cymnene, carvacrol, terpineol, nigellicimine and nigellidine) [20] and T. foenum graecum (trigonelline, cholin, gentian, carpaine and 4-hydroxyisoleucine and diosgenin) [21] have been found enriched with variety of phytochemicals. Most of the determined phytochemicals on the part of the components of POL₄ are also known for their diverse health benefits including their effectiveness in cardiovascular disorders [22‐27]. Though detailed studies are lacking to justify the cardiovascular benefits of POL₄ and its ingredients, the available data is providing some support to individual components {C. intybus [27, 28], G. sylvestre [19, 29] and T. foenum graecum [30, 31]} for their utility in cardiovascular disorders with exception of N. sativa which has been studied widely. Despite the availability of ample data on N. sativa and its constituents (thymoquinone, α-pinene and p-cymene) for their effectiveness in hypertension [24‐26, 32‐34], the effect of its seed extract with holistic picture on vascular and atrial preparations is yet to be determined for its modulating role on vascular resistance and cardiac output. This study has been designed to provide an evidence to the medicinal use of POL₄ and its ingredients in cardiovascular disorders like hypertension with a prime objective to determine the blood pressure lowering effects and the possible insight into mechanism(s). While the secondary objective was to compare the efficacy of ingredients with the parent formulation to justify the use of this formulation in hypertension and/or to suggest modification in its current composition. The in-vivo assays were carried out in rat as per their similarity with human cardiovascular system, which are commonly employed worldwide to determine antihypertensive efficacy of the test compounds [35‐37], while isolated tissue experiments were conducted in rat (aorta) and guinea-pig (atria) tissues.

On account of the medicinal use of POL₄ and its the ingredients in cardiometabolic disorders like diabetes and associated hypertension [17], when tested in anesthetized rats, POL₄ and its ingredients attenuated the mean arterial pressure (MAP) in a dose-dependent manner. Interestingly, the parent formulation was found the most effective followed by Ns.Cr ≅ Ci.Cr > Tfg.Cr > Gs.Cr. Our findings on the part of N. sativa are in line with earlier report on its antihypertensive efficacy [32]. However, this study is reporting for the first time, the blood pressure lowering efficacy of C. intybus and G. sylvestre in normotensive rats. These findings are also providing a rationale to their medicinal use as cardiotonic or hypotensive [17, 19]. The blood pressure lowering effects are also in agreement with the prior findings related to vasorelaxant mechanisms on the part of C. intybus [27] and T. foenum graecum [30, 31], which might be playing a pivotal role in overall effectiveness of these plants in hypertension. On the contrary, Preuss et al. [44] reported that G. sylvestre did not show antihypertensive effect in spontaneously hypertensive rats, however, we did find its efficacy but with varying pattern in normotensive rats which needs further investigations using multiple models and species to attest or oppose its therapeutic utility in hypertension.

Blood pressure is the product of cardiac output and peripheral resistance [45]. An increase in any of these two determinants can develop hypertension. Antihypertensive drugs target both components either alone or in combination. The preferred approach would be to cause a predominantly reduction in peripheral vascular resistance along with cardiac output.

To explore the effect of parent formulation and its components on vascular resistance, in pre-contracted rat aortic preparations, POL₄ and its ingredients caused inhibition of K⁺ (80 mM)-induced contractions, where Ci.Cr was found the most potent followed by Ns.Cr > Tfg.Cr > Gs.Cr ≅ POL₄. Based on inhibitory effects of test material(s) against K⁺ (80 mM)-induced contractions, to further attest the presence of Ca⁺⁺ antagonist like activity on the part of test material(s), the concentration-response curves (CRCs) of Ca⁺⁺ were constructed in the absence and presence of different concentrations of POL₄ and its ingredients, respectively. All produced a non-parallel rightward shift in CRCs of Ca⁺⁺ with suppression of the maximum effect, similar to activity pattern of verapamil, a known Ca⁺⁺ antagonist [46]. Against phenylephrine (P.E)- induced contraction, C. intybus and T. foenum graecum showed complete relaxation, however, POL₄, G. sylvestre and N. sativa showed initially vasoconstriction followed by relaxation at higher tested concentrations. The inhibitory potential of POL₄ and its ingredients against K⁺ (80 mM) and P.E signify their ability to interfere with Ca⁺⁺ entry through voltage-dependent channels (VDCs) and receptor-operated channels (ROCs). This is evident as K⁺ (80 mM) and P.E (1 μM) mediate contraction by influx of extracellular Ca⁺⁺ through VDCs and ROCs, respectively, resulting in increased intracellular Ca⁺⁺ levels trigging sustained contractions [43, 47]. Based on observed (vasoconstrictor and/or vasodilator) effects of test material(s) on P.E-induced contractions, the parent formulation and its ingredients were studied for the presence of vaso-stimulant activity on the baseline status of isolated rat aortic preparations. All exhibited vasoconstriction with varying efficacy, being Gs.Cr the most potent followed by Ns.Cr > POL₄ > Ci.Cr), similar to the effect of phenylephrine. However, T. foenum graecum was devoid of any vasoconstriction, similar to the effect of verapamil. When these effects were studied in the presence of phentolamine, an α- adrenergic antagonist, all showed partial sensitivity to phentolamine except C. intybus which was found completely phentolamine-sensitive. These data suggest the presence of multiple types of vasoconstrictor constituents like phentolamine sensitive and insensitive, which needs further studies to characterize. It was interesting to note that despite the presence of vasoconstrictor constituents both in parent formulation and most of its ingredients, these did not increase blood pressure in anaesthetized rats, which might be because of the dominant role of endogenous mediators in intact body systems offering blockade to vasoconstrictor constituents. On the other hand, the co-existence of strong vasorelaxant and cardio depressant elements in the parent formulation and its ingredients may not be allowing the expression of vasoconstrictor elements to dominate and cause hypertension in the in vivo system.

To determine cardio-suppressant activity contributing in attenuating cardiac output, when the parent formulation and its ingredients were tested on spontaneously beating atrial preparations of guinea-pigs, POL₄ and three of its ingredients showed exclusively negative inotropic and chronotropic effects similar to the effect of verapamil. However, N. sativa showed mild cardiac stimulation in terms of an increase in the force of contraction, which is also in support to its medicinal use as cardiac stimulant in the form of a formulation (Lubub-Al-Asrar) [16]. The probable mechanism likely to be involved in cardio-depressant activities seems Ca⁺⁺ antagonist pathway, while mild cardiac stimulant effect on the part of N. sativa needs further exploration to confirm. The standard Ca⁺⁺ antagonist like verapamil which is known to lower blood pressure [48, 49] partly by causing cardiac suppression via inhibition of inward slow current at the time of action potential [50]. The observed cardiac inhibitory effect of the test material(s) might be attenuating cardiac output leading to a decrease in the blood pressure. Our findings are in line with the cardiac inhibitory effect of medicinal plants causing reduction in the cardiac output, thus resulting in decreased blood pressure [15, 43, 51‐53]. In summary, it appears that nature has composed the opposing mechanisms not allowing the vasodilator effect to cross the desired limit thus overriding excessive drop in blood pressure, which has usually been observed when using standard antihypertensive drugs [45]. The co-existence of components with combating activities have been found common in many plants like Viola odorata [43], Carthamus oxycantha [47], Curcuma long [54], Acorus calamus [55] and piperine, an alkaloid of Piper longum and Piper nigrum [56] and is also in agreement with the concept that natural products in their original form possess synergistic and/or side effects nullifying combinations [11]. Varied distribution of total phenolic and flavonoid in POL₄ and its constituents is also providing strength to their cardiovascular benefits as phenolic and flavonoids are known for their effectiveness in cardiovascular disorders [50, 57, 58]. On the other hand, these findings suggest that excluding G. sylvestre from POL₄ formulation may result in a better product with improved efficacy in treating hypertension.

This study indicates that POL₄ and its ingredients possess blood pressure lowering effect predominantly mediated through inhibition of Ca⁺⁺ influx via membranous Ca⁺⁺ channels and receptor (α-adrenergic) operated pathways. The additional presence of vasoconstrictor agents (partially phentolamine-sensitive) in parent formulation possibly because of G. sylvestre and N. sativa may be meant to suppress the excessive hypotension, a known adverse effect associated with the use of antihypertensive drugs when used at higher doses and/or for longer duration. Thus, this study provides a rationale to the medicinal use of POL₄ and its ingredients in cardiovascular disorders like hypertension.