Vasodilation and anti-hypertension effects of an orthogonal stimulus response optimised combines formulation of eupatorin, sinensetin and 3-hydroxy 5 6 7 4 tetramethoxyflavone in a rat models

Yam, Mun Fei (2026) Vasodilation and anti-hypertension effects of an orthogonal stimulus response optimised combines formulation of eupatorin, sinensetin and 3-hydroxy 5 6 7 4 tetramethoxyflavone in a rat models. PhD thesis, UTAR.

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Abstract

Modern medicine forms the backbone of current healthcare, providing a diverse array of synthetic and semi-synthetic compounds for both diagnosis and treatment. Designed to mimic or enhance the action of natural molecules, these compounds primarily target specific pathways within the body to alleviate symptoms or provide therapeutic benefits. Over the years, pharmaceutical development has been largely guided by the “single compound, single-target” approach. While this paradigm has been effective in developing treatments that address distinct molecular pathways, it has limitations when dealing with the increasingly prevalent multifactorial diseases seen today. Complex conditions, such as metabolic syndrome, emerge from a blend of genetic, environmental, and lifestyle factors, which are challenging to manage with single-target therapies. These limitations call for a reevaluation of current therapeutic strategies and invite an exploration of alternative approaches. As society's health landscape shifts, there is an increasing interest in exploring alternative therapeutic options that are believed to have better suited to the complexities of contemporary diseases. Botanical medicines, often incorporating multiple active compounds, offer a holistic approach that potentially addresses several pathways simultaneously. Traditional medicines, especially those compounds derived from plants, have a long history of empirical use for treating complex conditions in various approach. One such medicinal plant, Orthosiphon stamineus Benth. (Lamiaceae) (commonly known as Java tea), has been traditionally used in Southeast Asia for its diuretic, anti-inflammatory, and antihypertensive properties. This plant has gained attention as a potential adjunct in modern antihypertensive regimens, attributed to its rich phytochemical profile, which includes flavonoids like sinensetin, eupatorin, and 3’-hydroxy-5,6,7,4’-tetramethoxyflavone (TMF). These compounds, known for their vasodilatory effects, may provide a synergistic impact on blood pressure regulation, making O. stamineus a promising candidate for further research. According to the theory of Chinese Material Medica, O. stamineus shares similar characteristics with Polyporus umbellatus (Zhuling) and Plantago asiatica (Che Qian Zi), as all three promote urination and clear dampness, which is essential for hypertension associated with phlegm-dampness, where fluid retention contributes to hypertension. Additionally, its cooling nature helps to moderate excess heat or yang, making it suitable for hypertension patterns involving liver fire or liver yang rising. By clearing heat and supporting fluid metabolism, O. stamineus can aid in reducing blood pressure, particularly in cases where dampness and heat are predominant. Previous studies have revealed that the chloroform fraction of O. stamineus exhibits substantial vasodilatory effects on isolated rat aorta in vitro. These effects were notably greater than the combined vasodilatory effects of its main components—sinensetin, eupatorin, and TMF—when tested individually. Such findings suggest that a synergistic interaction occurs when these compounds are administered together, possibly enhancing the therapeutic efficacy of the plant extract. The present study aimed to elucidate the interactions among sinensetin, eupatorin, and TMF and to identify the optimal combination ratio that maximizes their vasodilatory effect. To achieve this, in vitro aortic ring assays from Sprague-Dawley (SD) rats were utilized for vasodilation study. Using an orthogonal stimulus–response compatibility approach, various ratios of the three compounds were systematically tested to identify the most efficacious combination. Once the optimal ratio was established, the antihypertensive effect and mechanism of action of the optimum ratio were investigated. In this study, aortic rings from spontaneously hypertensive rats (SHRs) and SD rats were used to examine the vasodilation mechanisms. The antihypertensive effect of optimum ratio was also assessed through repeated-dose administration in SHRs. In the experiments, the combined formulation designated as G28 (comprising eupatorin, sinensetin, and TMF in the ratios of EC₂₀:EC₁₅:EC₅) exhibited a significant concentration-dependent vasodilatory response, with a maximum response (RMAX) of 119.05 ± 3.29% and an EC₅₀ of 6.78 ± 0.70 µg/mL. Interestingly, the vasorelaxant effects of G28 were more pronounced in SHR aortic rings than in SD rat aortic rings, suggesting that the formulation has heightened potency under hypertensive conditions. This may indicate that the compounds within G28 are more effective in the pathological environment of hypertension, which could be due to altered receptor sensitivity or changes in signaling pathways associated with vascular reactivity in hypertensive states. The mechanistic studies revealed that the vasorelaxant effects of G28 were partially endothelium-dependent, as removing the endothelium reduced the vasodilatory response in both SHR and SD aortic rings. Further investigations into the specific pathways involved indicated that G28’s action was independent of the nitric oxide (NO)/soluble guanylate cyclase (sGC)/cyclic guanosine monophosphate (cGMP) pathway, as evidenced by its retained efficacy in the presence of inhibitors such as L-NAME, methylene blue, and ODQ. However, the presence of potassium channel blockers, including glibenclamide, BaCl₂, 4-AP, and TEA, significantly attenuated the vasorelaxant effect, implicating the involvement of potassium channels in the formulation’s mechanism of action. In addition, G28 appeared to exert its effects through modulation of intracellular calcium (Ca²⁺) dynamics. Specifically, G28 antagonized voltage-operated calcium channels (VOCC) and inositol triphosphate receptors (IP3R), which are critical regulators of Ca²⁺ influx in vascular smooth muscle cells. By reducing intracellular Ca²⁺ concentrations, G28 further contributes to the relaxation of vascular smooth muscle. In vivo assessments of G28-treated groups (15 mg/kg, 30 mg/kg, and 60 mg/kg) demonstrated significant reductions in the blood pressure of SHRs in a time-dependent manner over 21 days of oral treatment. The initial systolic blood pressure (SBP) of G28-treated groups (15 mg/kg, 30 mg/kg, and 60 mg/kg) were documented at 210.94±5.31 mmHg, 213.23±5.31 mmHg, and 205.98±2.04 mmHg, respectively, and decreased substantially to 189.79±4.17 mmHg (P<0.05), 189.40±2.87 mmHg (P<0.05), and 180.75±1.67 mmHg (P<0.001), respectively, following the 21 days of oral treatment compared to the negative control. The initial diastolic blood pressure (DBP) levels of the G28-treated groups (15 mg/kg, 30 mg/kg, and 60 mg/kg) were 152.67±9.34 mmHg, 154.56±9.09 mmHg, and 139.46±4.64 mmHg, respectively, before treatment, and ended up at 133.83±4.68 mmHg, 138.25±3.12 mmHg, and 129.67±3.12 mmHg after 21 days of treatment. Similarly, the initial mean arterial blood pressure (MAP) values for G28 treated groups were 171.73±7.95 mmHg, 173.75±7.82 mmHg, and 160.33±4.11 mmHg, which decreased to 140.56±3.60 mmHg (P<0.01), 152.13±4.41 mmHg, and 146.56±2.13 mmHg (P<0.05), respectively, compared to the negative control. In conclusion, this investigation delineates the vasodilatory potential of G28, a novel formulation comprising eupatorin, sinensetin, and TMF at specific ratios. G28 exhibits a remarkable capacity to activate NO/sGC/cGMP signaling pathways while concurrently initiating the AC/cAMP/PKA signaling cascade by enhancing PGI2 production in vascular endothelium. Additionally, G28 elicits hyperpolarizing currents by activating potassium channels (KCa, KV, KATP, and Kir). G28 also inhibits vasoconstriction, achieved through antagonism of VOCC and IP3R. Although these mechanisms observed in SD rat aorta were consistent, some variations were noted in SHRs, underscoring the impact of altered vascular physiology in the hypertensive state. It is essential to recognize that ex vivo rat aortic ring assays, while informative, offer only a limited perspective on the intricate mechanisms underlying G28-induced vasodilation. Thus, further exploration using molecular and histochemical methodologies is necessary to unravel protein expression and delineate the precise mechanisms of G28-induced vasodilation. The in vivo antihypertensive results underscore G28's potential as a candidate for further research in developing novel therapeutic strategies for hypertension, given its complex modulation of vasodilation mechanisms and blood pressure regulatory pathways. Keywords: Sinensetin; eupatorin; 3’-hydroxy-5,6,7,4’-tetramethoxyflavone; aortic ring; antihypertension; vascular tone; orthogonal stimulus-response Subject Area： RM1-950 Therapeutics. Pharmacology Subject Area： RM300-666 Drugs and their actions Subject Area： RS1-441 Pharmacy and materia medica Subject Area： RS153-441 Materia medica

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