Therapeutic Potential of Hibiscus sabdariffa Linn. in Attenuating Cardiovascular Risk Factors

Cardiovascular diseases (CVDs) represent a broad spectrum of diseases afflicting the heart and blood vessels and remain a major cause of death and disability worldwide. CVD progression is strongly associated with risk factors, including hypertension, hyperglycemia, dyslipidemia, oxidative stress, inflammation, fibrosis, and apoptosis. These risk factors lead to oxidative damage that results in various cardiovascular complications including endothelial dysfunctions, alterations in vascular integrity, the formation of atherosclerosis, as well as incorrigible cardiac remodeling. The use of conventional pharmacological therapy is one of the current preventive measures to control the development of CVDs. However, as undesirable side effects from drug use have become a recent issue, alternative treatment from natural products is being sought in medicinal plants and is gaining interest. Roselle (Hibiscus sabdariffa Linn.) has been reported to contain various bioactive compounds that exert anti-hyperlipidemia, anti-hyperglycemia, anti-hypertension, antioxidative, anti-inflammation, and anti-fibrosis effects. These properties of roselle, especially from its calyx, have relevance to its therapeutic and cardiovascular protection effects in humans. This review summarizes the findings of recent preclinical and clinical studies on roselle as a prophylactic and therapeutic agent in attenuating cardiovascular risk factors and associated mechanisms.


Introduction
Cardiovascular diseases (CVDs) present a significant burden to populations across the globe. Despite encouraging advances in prevention and treatments, CVDs remain a major cause of death and disability worldwide and relentlessly continue to grow over the years. More than 85 million people are living with CVDs in Europe [1], while more than half of all adults in the United States have some form of CVDs [2]. On the global scale, it was estimated that CVDs killed 17.9 million people in 2019, representing 32% of global deaths. Out of these deaths, 85% were due to heart attack and stroke [3]. Mitigating risk factors leading to the development of CVDs would be the best strategy for reducing CVD-related deaths. In fact, the World Health Organization (WHO) reported that reducing risk factors

Origin and Beneficial Use of Roselle
Roselle, also known as red sorrel, is a tropical shrub of the Malvaceae family, particularly found in tropical regions such as India, Malaysia, Indonesia, Thailand, China, the Caribbean, Central America, West Africa, Asia, Brazil, Australia, Hawaii, and the Philippines [15]. Roselle has an acidic taste close to that of cranberry fruits. There are about 300 species of roselle that have been identified all over the world. This plant is commonly planted as an ornamental plant. However, due to the overwhelming utilization of roselle tree parts, including its calyx, leaves, stems, and seeds, in industries, roselle has now gained interest among researchers for its beneficial effects. Figure 1 shows a roselle plant.
Roselle has been traditionally used for decades as it has enormous benefits, mainly in medicinal and culinary aspects. The most common part of roselle that is widely used is the calyx. Fresh or dried calyces are used in the preparation of tea, either hot or cold, as well as fermented beverages, wines, jams, jellies, ice cream, aromatic agents, natural colorants, flavoring agents, and cakes [16]. Apart from that, roselle seeds are also commonly used to make oil, coffee substitutes, and are roasted and ground into a powder for Pharmaceuticals 2023, 16, 807 3 of 25 seasoning in soups and sauces [17]. Roselle is a plant valued worldwide because all of its parts have medicinal uses thanks to various bioactive compounds that have significant effects, such as antioxidative, anti-inflammatory, anti-fibrosis, and anti-apoptosis effects, in attenuating various diseases. For decades, roselle has been traditionally used as folk medicine to treat urinary tract infections, toothaches, colds and fever, coughs, indigestion, and wounds, and to lower body temperature. Recent ethnomedicinal studies have shown that roselle consumption can be used as an anti-cancer, anti-microbial, anti-hyperlipidemic, anti-hyperglycemic, and anti-hypertensive treatment [14,18,19]. Roselle also has been widely used to detoxify aflatoxicosis [20]. Roselle has been proven to combat aflatoxicosis by attenuating toxicity-induced oxidative stress in the heart, which further limits CVD events. Roselle has been traditionally used for decades as it has enormous benefits, mainly in medicinal and culinary aspects. The most common part of roselle that is widely used is the calyx. Fresh or dried calyces are used in the preparation of tea, either hot or cold, as well as fermented beverages, wines, jams, jellies, ice cream, aromatic agents, natural colorants, flavoring agents, and cakes [16]. Apart from that, roselle seeds are also commonly used to make oil, coffee substitutes, and are roasted and ground into a powder for seasoning in soups and sauces [17]. Roselle is a plant valued worldwide because all of its parts have medicinal uses thanks to various bioactive compounds that have significant effects, such as antioxidative, anti-inflammatory, anti-fibrosis, and anti-apoptosis effects, in attenuating various diseases. For decades, roselle has been traditionally used as folk medicine to treat urinary tract infections, toothaches, colds and fever, coughs, indigestion, and wounds, and to lower body temperature. Recent ethnomedicinal studies have shown that roselle consumption can be used as an anti-cancer, anti-microbial, anti-hyperlipidemic, anti-hyperglycemic, and anti-hypertensive treatment [14,18,19]. Roselle also has been widely used to detoxify aflatoxicosis [20]. Roselle has been proven to combat aflatoxicosis by attenuating toxicity-induced oxidative stress in the heart, which further limits CVD events.

Bioactive Compounds of Roselle
Roselle is a plant that is rich in bioactive components, especially its calyx parts. Diverse phytochemical studies have validated that the major bioactive compounds found in roselle calyx are phenolic acids, flavonoids, anthocyanins, and organic acids, which are

Bioactive Compounds of Roselle
Roselle is a plant that is rich in bioactive components, especially its calyx parts. Diverse phytochemical studies have validated that the major bioactive compounds found in roselle calyx are phenolic acids, flavonoids, anthocyanins, and organic acids, which are responsible for many biological activities [21][22][23]. Figure 2 demonstrates the bioactive compounds found in roselle.
Anthocyanins are the vital compounds that contribute to the bright color of roselle calyx and are rich in delphinidin-3-sambubioside, and cyanidin-3-sambubioside; meanwhile, delphinidin-3-glycoside and cyanidin-3-glycoside are present in smaller amounts [24,25]. Furthermore, several flavonoids have been identified in roselle calyx and leaves. Frequently reported flavonoids that are found abundantly are quercetin-3-glucoside, methyl epigallocatechin, myricetin, quercetin, rutin, and kaempferol [26]. Other types of flavonoids present in roselle include sabdaritrin, hibiscitrin, gossytrin, gossypitrin, luteolin, and pelargonidic acid [27,28]. In some studies, phenolic acid was noted to be present in roselle calyx, stems, and leaves. Protocatechuic and chlorogenic acids are essential phenolic acids and are present in higher amounts in roselle than other phenolic acids. Other phenolic acids in roselle are caffeic acid and gallic acid. Previous research has reported that total phenolic compounds were the most abundant in the calyx, followed by leaves and stems [29,30]. Anthocyanins are the vital compounds that contribute to the bright color of roselle calyx and are rich in delphinidin-3-sambubioside, and cyanidin-3-sambubioside; meanwhile, delphinidin-3-glycoside and cyanidin-3-glycoside are present in smaller amounts [24,25]. Furthermore, several flavonoids have been identified in roselle calyx and leaves. Frequently reported flavonoids that are found abundantly are quercetin-3-glucoside, methyl epigallocatechin, myricetin, quercetin, rutin, and kaempferol [26]. Other types of flavonoids present in roselle include sabdaritrin, hibiscitrin, gossytrin, gossypitrin, luteolin, and pelargonidic acid [27,28]. In some studies, phenolic acid was noted to be present in roselle calyx, stems, and leaves. Protocatechuic and chlorogenic acids are essential phenolic acids and are present in higher amounts in roselle than other phenolic acids. Other phenolic acids in roselle are caffeic acid and gallic acid. Previous research has reported that total phenolic compounds were the most abundant in the calyx, followed by leaves and stems [29,30].
Moreover, roselle calyx has an acidic taste due to the high content of organic acid. Among the organic acids, hibiscus acid is the main compound that exists in roselle calyx, followed by citric acid, hydroxycitric acid, malic acid, and tartaric acid. Meanwhile, oxalic acid and ascorbic acid are found in smaller proportions compared to other organic acids [31,32]. Figure 2 demonstrates the bioactive compounds found in roselle. Roselle extract has also shown the presence of other secondary metabolites, including tannins, saponins, alkaloids, and glycosides that are responsible for contributing to biological activities. Several non-polar compounds also have been identified in roselle, particularly in seeds. The compounds include vitamin A (retinol), vitamin D, and vitamin E (α-tocopherol) [30].
The amount of each chemical constituent may vary depending on the condition used during the roselle extraction procedure. For instance, roselle polyphenol-rich extract contains hibiscus acid, chlorogenic acids, gallic acid, and quercetin, but no anthocyanin is detected in the extract [33]. This might be due to the high temperature used in the extraction process, which might cause anthocyanins to degrade. Additionally, total phenolic content was higher in the ethanol extract of roselle than in the aqueous extract [34]. When roselle calyx was extracted with citric acid, it was found that the total anthocyanin content was higher than when it was extracted with acidified ethanol. However, the same study found that the total phenolic content was higher when extracted with citric acid than acid- Moreover, roselle calyx has an acidic taste due to the high content of organic acid. Among the organic acids, hibiscus acid is the main compound that exists in roselle calyx, followed by citric acid, hydroxycitric acid, malic acid, and tartaric acid. Meanwhile, oxalic acid and ascorbic acid are found in smaller proportions compared to other organic acids [31,32]. Figure 2 demonstrates the bioactive compounds found in roselle. Roselle extract has also shown the presence of other secondary metabolites, including tannins, saponins, alkaloids, and glycosides that are responsible for contributing to biological activities. Several non-polar compounds also have been identified in roselle, particularly in seeds. The compounds include vitamin A (retinol), vitamin D, and vitamin E (α-tocopherol) [30].
The amount of each chemical constituent may vary depending on the condition used during the roselle extraction procedure. For instance, roselle polyphenol-rich extract contains hibiscus acid, chlorogenic acids, gallic acid, and quercetin, but no anthocyanin is detected in the extract [33]. This might be due to the high temperature used in the extraction process, which might cause anthocyanins to degrade. Additionally, total phenolic content was higher in the ethanol extract of roselle than in the aqueous extract [34]. When roselle calyx was extracted with citric acid, it was found that the total anthocyanin content was higher than when it was extracted with acidified ethanol. However, the same study found that the total phenolic content was higher when extracted with citric acid than acidified ethanol extract [35]. Nevertheless, the condition used for extraction should be considered to target the desired compounds from roselle extract.

Cardiovascular Risk Factors and Their Management
Over 70% of CVD cases and deaths in the world were attributed to risk factors [36]. Annual reports on regional and national CVDs and risk-related burdens may serve as the basis for developing effective strategies for CVD prevention [37]. Therefore, preventing and limiting the risk factors is an important step that should be undertaken to effectively reduce CVD-related mortality and morbidity [38]. Researchers have conducted extensive studies in order to find the best pharmacotherapy agents for CVD management by targeting risk factors, especially hypertension, diabetes mellitus, and dyslipidemia [39]. Moreover, focusing on the mechanisms that induce the occurrence of risk factors and progression of CVDs targeting oxidative stress, inflammation, fibrosis, and apoptosis is crucial to successfully control and limit CVD development [40][41][42]. CVDs are a chain of events initiated by related risk factors and progress through numerous pathophysiological pathways and processes to the development of end-stage cardiovascular disorder [43]. CVD risk factors, such as dyslipidemia, hypertension, and hyperglycemia, are known to promote oxidative stress and initiate a cascade of events, including inflammatory response, fibrosis, and apoptosis [44]. These mechanisms prompt endothelial damage, vascular stiffness, atherosclerosis, myocardial ischemia, and cardiomyocyte hypertrophy, which eventually lead to vascular and cardiac remodeling that culminates in CVDs [45,46].
Oxidative stress plays a crucial role in the progression of CVDs. Hyperglycemia results in the activation of alternative pathways of glucose metabolism, triggering the overproduction of reactive oxygen species (ROS) [47]. Moreover, hyperglycemia induces excessive fatty acid oxidation that leads to the derangement of free fatty acid generation [48]. Apart from that, hyperlipidemic conditions inaugurate beta-oxidation that promotes the increment in the electron transport chain and eventually accelerates mitochondrial dysfunction [49]. ROS generation can also be contributed by the activation of reduced nicotinamide adenine dinucleotide phosphate oxidase (NOX) derived from the stimulation of the reninangiotensin-aldosterone system (RAAS) by hyperlipidemic and hyperglycemic conditions. The upregulation of NOX hampers the production of nitric oxide (NO) through the downregulation of endothelial nitric oxide synthase (eNOS) and further induces hypertension and promotes ROS generation [49]. Moreover, aflatoxicosis also can promote ROS production as aflatoxin can inhibit metabolic systems' homeostasis and further effectuate oxidative stress [50,51].
The overproduction of ROS exceeds endogenous antioxidant capacity, leading to oxidative stress and the activation of inflammation, fibrosis, and apoptosis [52,53]. Oxidative stress further triggers inflammation by activating nuclear factor kappa B (NFκB), which upregulates proinflammatory genes and cytokine production and provokes the upregulation of transforming growth factor beta (TGF-β) and connective tissue growth factor (CTGF) that triggers the profibrotic response, which further causes extracellular matrix (ECM) accumulation, collagen deposition, and, in the end, fibrosis [54]. Oxidative stress, inflammation, and mitochondrial dysfunction trigger the cascade of apoptosis by enhancing the Bax/Bcl ratio, which prompts the activation of cytochrome c and caspase 3 that eventually lead to apoptosis [55]. These mechanisms ultimately cause cardiac hypertrophy, cardiac fibrosis, endothelial dysfunction, vascular stiffness, myocardial ischemia, and, ultimately, CVDs. Therefore, targeting oxidative stress, inflammation, fibrosis, and apoptosis in hypertension, dyslipidemia, and hyperglycemia that eventually contribute to CVDs remains a primary strategy in controlling and managing the development of CVDs. Figure 3 illustrates the association of risk factors and the mechanisms involved in the progression of CVDs.
Generally, the pharmacological management of CVDs involves controlling and managing the risk factors. Pharmacological therapy has shown explicitly that risk factor reduction decreases the risk of morbidity and mortality. For instance, treating blood pressure levels and lipid levels, particularly low-density lipoprotein (LDL) cholesterol, to the recommended levels has consistently been shown to reduce CVD events [56][57][58]. Managing normal glycemic levels by employing anti-hyperglycemic drugs helps reduce the risk of microvascular diseases such as retinopathy and nephropathy [59] in the short term while reducing risks for myocardial infarction and stroke [60,61]. In clinical trials, anti-hypertensive treatment, including diuretics, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers, and calcium channel blockers, have effectively lowered blood pressure [62].  Hyperglycemia reduces glucose oxidation and activates alternative pathways of AGE accumulation, polyol, hexosamine, and PKC pathways that contribute to ROS. Hyperlipidemia also contributes to the elevation of ROS production via lower HDL, enhances LDL that eventually promotes an increase in free fatty acid accumulation and activates beta-oxidation, and induces an increment in electron transfer chain and mitochondrial dysfunction. Both hyperglycemia and hyperlipidemia promote RAAS activation and lead to the upregulation of NOX and then the downregulation of eNOS, which lead to a reduced NO level. This will cause vasoconstriction and hypertension. The upregulation of NOX further contributes to ROS generation. The overproduction of ROS hampers endogenous antioxidants and causes oxidative stress. Oxidative damage leads to the activation of NFkB and promotes the elevation of proinflammatory cytokines (IL-6, IL-1β, TNF-α, ICAM, VCAM) and Hyperglycemia reduces glucose oxidation and activates alternative pathways of AGE accumulation, polyol, hexosamine, and PKC pathways that contribute to ROS. Hyperlipidemia also contributes to the elevation of ROS production via lower HDL, enhances LDL that eventually promotes an increase in free fatty acid accumulation and activates beta-oxidation, and induces an increment in electron transfer chain and mitochondrial dysfunction. Both hyperglycemia and hyperlipidemia promote RAAS activation and lead to the upregulation of NOX and then the downregulation of eNOS, which lead to a reduced NO level. This will cause vasoconstriction and hypertension. The upregulation of NOX further contributes to ROS generation. The overproduction of ROS hampers endogenous Despite a lot of conventional pharmacotherapy agents having been used for more than two decades, many studies have been conducted extensively in order to provide alternative treatment in the management of CVDs. Among the candidates, natural products have been proposed and widely investigated for their promising therapeutic effects, mainly on CVD risk factors. There exists broad consensus based on evidence from past studies that the consumption of natural products is rising around the world due to their proven effectiveness and because they can delay the progression of CVDs and are claimed to be safe to use [63].

Roselle in the Attenuation of Cardiovascular Diseases Risk Factors
Roselle is known as a plant that provides many health benefits, particularly as a source of antioxidants, and can serve as an alternative treatment for various diseases. Accumulated evidence has proven that roselle, mainly its calyx, contains many phytochemical compounds that are considered to be influential in providing various health benefits, especially as having anti-hyperlipidemic, anti-hyperglycemic, anti-hypertensive, antioxidative, anti-inflammatory, and anti-fibrosis effects. Hence, the utilization of roselle as an alternative treatment is crucial to alleviate CVD risk factors.

Anti-Hypertensive
Hypertension leads to CVD, which eventually causes death. A study reported that the consumption of 100, 200, and 400 mg/kg of aqueous extract of roselle leaves that contain saponins, tannins, flavonoids, alkaloids, phenols, and steroids for 6 weeks daily reduced systolic and diastolic blood pressure, mean arterial blood pressure, and heart rate in salt-induced hypertensive rats [64]. Furthermore, roselle aqueous extract that was mixed with several other plants, including chamomile, marjoram, and doum, successfully showed a significant reduction in blood pressure in N(gamma)-nitro-L-arginine methyl ester (L-NAME)-induced hypertensive animal models [65]. The same study suggested that the biological activities contributed by roselle were due to the presence of high concentrations of saponins, flavonoids, alkaloids, phenols, and tannins.
Nicotine is one of the culprits leading to hypertension. However, in one study, aqueous extracts of roselle treatments significantly decreased heart rate in a nicotine-induced heart damage model. The same study reported no difference in blood pressure. However, the treatment group showed a reduction trend in blood pressure [66]. In another study, the consumption of 100 mg/kg of roselle aqueous extract utilization rich in delphinidin-3-O-sambubioside and cyanidin-3-O-sambubioside for 28 days was revealed to effectively prevent the deterioration of cardiac systolic and diastolic function, coronary flow, as well as cardiac output in a diet-induced obesity rat model [13]. In a past study, roselle aqueous extract significantly ameliorated myocardial infarction-induced cardiac systolic and diastolic dysfunction, improved left ventricular developed pressure, improved isovolumetric relaxation, and attenuated angiotensin II-induced cardiomyocyte hypertrophy that eventually promoted hypotensive effects in animal models [67]. The same study indicated that these biological effects were due to the presence of ascorbic acid, chlorogenic acid, and caffeic acid in the roselle aqueous extract. Impaired vasorelaxation is a crucial event in the pathogenesis of hypertension.
In a diabetic cardiomyopathy model, polyphenol-rich extract of 100 mg/kg roselle supplementation for 8 weeks daily was shown to elevate left ventricular developed pressure and improve coronary flow, suggesting that roselle improves cardiac contractility and relaxation rate, which indicates that roselle has potential as a cardioprotective agent [68]. In another study, roselle polyphenol-rich extract containing a high concentration of flavonoids and phenolic acids was demonstrated to lower systolic function to the normal level, reduce heart rate, and improve coronary blood flow; all these positive effects were elicited by the pharmacological agonists for L-type Ca 2+ channel, ryanodine receptor, B-adrenergic receptor, and sarcoendoplasmic reticulum calcium transport ATPase (SERCA) blocker, which were all abolished by roselle polyphenol extract in isolated rat heart [33].
One of the common pathways contributing to impaired vasorelaxation is an injury to the endothelium since it cannot produce relaxing factors such as nitric oxide. A previous study revealed that roselle aqueous extract supplementation with a dose of 100 mg/kg containing protocatechuic acid, hibiscus acid, delphinidin-3-sambubioside, cyanidin-3sambubioside, and kaempferol-3-O-sambubioside for 28 days was found to restore vasorelaxation in aortic rings and exerted a vasodilatory effect by activating potassium channels, which causes hyperpolarization in vascular smooth muscle cells which then successfully lower systolic blood pressure in nicotine-induced vascular endothelial dysfunction [25]. Polyphenol-rich extract of roselle has been suggested to lower systolic and diastolic blood pressure mediated by the promotion of diuresis and ACE inhibitor activity of aorta in diabetic rat models given 100 mg/kg for 8 weeks, and it is believed to be mediated by delphinidin-3-O-sambubioside and cyanidin-3-O-sambubioside [69].
In an ex vivo study, treatment using methanolic extract of roselle calyx at a concentration of 10 ng/mL to 1 mg/mL in hypertensive rats was able to induce a vasodilator effect in isolated aortas through the activation of the nitric oxide/cGMP-relaxant pathway as well as the inhibition of Ca 2+ influx, which explains the blood pressure-lowering effect by roselle [70]. Apart from that, the combination treatment of roselle with olive in hydro alcoholic powder with doses of 125, 250, and 500 mg/kg for 4 weeks resulted in a decrease in both systolic and diastolic blood pressure, reversed the L-NAME-induced suppression in serum nitric oxide, lowered the elevated ACE activity, enhanced eNOS gene and protein expression of the heart, and reduced aortic media thickness in a hypertensive rat model [71].

Anti-Hyperlipidemic
Several studies have shown that anti-hyperlipidemic treatment by roselle to reduce the level of cholesterol, especially targeting LDL cholesterol, can effectively prevent the risk of CVDs [72]. Aqueous extract of roselle supplementation of 100 mg/kg for 28 days significantly reduced the level of LDL cholesterol, plasma leptin, cholesterol, and triacylglycerol in diet-induced obesity rat models [13]. Moreover, alloxan-induced diabetic rats supplemented with methanolic extract of roselle seed in doses of 200 and 400 mg/kg for 2 weeks had considerably lowered concentrations of triglyceride, LDL cholesterol, and total cholesterol and increased high-density lipoprotein (HDL) cholesterol concentrations [73]. The same study revealed that the presence of tannins, saponins, glycosides, steroids, phlobatannins, flavonoids, phenols, and alkaloids contribute to the hypolipidemic effects of roselle methanolic extracts. Similarly, the administration of 100 mg/kg of polyphenol-rich extract of roselle for 4 weeks to type I diabetic rats significantly reduced plasma triglyceride, total cholesterol, and LDL cholesterol while increasing the level of HDL cholesterol [69,74].
A study by Farombi and Ige [75] revealed that the utilization of 100 and 200 mg/kg of ethanolic extract of roselle for 4 weeks demonstrated a better anti-hyperlipidemia effect compared to the dyslipidemia therapeutic drug, Lovastatin, in alloxan-induced diabetic rats. The same study demonstrated that roselle exhibited a decrease in cholesterol by Pharmaceuticals 2023, 16, 807 9 of 25 29%, very low-density lipoprotein (VLDL) cholesterol by 36%, LDL cholesterol by 40%, and atherogenic index by 32% compared to 5%, 23%, 28%, and 31% in rats treated with Lovastatin, respectively. Moreover, ethanolic extract of roselle leaves and calyxes containing anthocyanins, glycosides, protocatechuic acid, and hydroxycitric acid has been reported to lower total cholesterol, triglyceride, LDL, and VLDL cholesterol while increasing the level of serum HDL cholesterol in induced hyperlipidemic rats [76]. The same study hypothesized that the decrease in LDL cholesterol when consuming 500 mg/kg of ethanolic extract of roselle for 30 days is due to the capacity to inhibit the intestinal absorption of cholesterol, interfere with lipoprotein production, and induce hepatic LDL receptors' expression, which could lead to the excessive removal of LDL cholesterol from the blood and increase the degradation and catabolism of cholesterol from the body.
Similarly, the consumption of polyphenol-rich extract of roselle significantly declined the serum level of LDL, triglyceride, and total cholesterol, as well as elevated HDL cholesterol levels in diabetic rat models [68]. On top of that, the utilization of roselle aqueous extract has been shown to reduce the atherogenic index, the coronary risk index, and LDL/HDL cholesterol significantly in type I diabetic rat models [12]. On the other hand, polyphenol extract and aqueous extract of roselle combined with a high-fat diet given to hamsters decreased cholesterol and triglyceride levels and suppressed the expression of fatty acid synthesis in hepatocytes, as well as lipid synthesis [77]. The same study indicates that the decrease in lipid content is due to the activation of 5' adenosine monophosphate-activated protein kinase (AMPK) and the reduction in sterol regulatory element binding 1 (SREBP-1), thus inhibiting the expression of fatty acid synthase and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase.

Anti-Hyperglycemic
Roselle has significant anti-hyperglycemic effects. Aqueous extracts of red and white roselle were reported to reduce hyperglycemia through the inhibition of α-amylase and α-glucoside in an in vitro study as the enzymes responsible for carbohydrate hydrolysis, thus attenuating the occurrence of chronic diseases, mainly DM and CVDs [78]. Moreover, according to a study by Mardiah and colleagues [79], roselle aqueous extract given to type I diabetic rats at a dose of 288 mg/200 g body weight daily for 20 days reduced blood glucose due to the improvement of the number of β cells producing insulin in pancreatic cells. A study by Ajani et al. [12] proved that roselle aqueous extract was able to reduce the percentage of glucose in the blood in type I diabetic models given 200 and 500 mg/kg of the extract for 28 days.
Additionally, diabetic mice supplemented with 200, 400, and 600 mg/kg of ethanolic roselle calyx extract demonstrated a significant reduction in the level of blood glucose. The anti-hyperglycemic effect of roselle is attributed to bioflavonoids which mimic insulin by reducing blood glucose levels but also stimulate the secretion of insulin and the formation of glycogen in muscles [80]. Additionally, the administration of 0.1 and 1.0 g/kg daily of roselle ethanolic extract to streptozotocin-induced diabetic rats for 6 weeks significantly decreased fasting blood glucose levels, improved glucose tolerance, enhanced the basal release of insulin, and stimulated the regeneration of islets of Langerhans in the pancreas [81].
The methanolic seed extract of roselle decreased blood plasma glucose a week after diabetes induction, suggesting that roselle enhances the regeneration of β pancreatic cells [73]. Mashi et al. [82] reported that the antioxidant phenols and flavonoids might be responsible for the inhibition of blood glucose via insulin modulation, while El-Barky et al. [83] instead supported that saponin can induce the secretion of insulin from the pancreas or restore the insulin response or both to reduce plasma glucose. Furthermore, polyphenol-rich extract of roselle in diabetic rats significantly reduced plasma glucose [69].

Antioxidant
Roselle is well known for its antioxidant properties [29]. Morales-Luna et al. [84] reported that red and white roselle contains various phytochemical compounds, including organic acids (hibiscus acid), phenolic acids, anthocyanin, and flavonoids, with the capacity to inhibit the damaging effect of oxidative stress. For instance, aqueous extract of roselle fed to obese rats with myocardial infarction led to a significant reduction in oxidative stress by downregulating NOX2 and 8-isoprostane gene expression, and enhanced superoxide dismutase (SOD) enzyme activity and glutathione (GSH) concentration [13]. This is due to the ability of anthocyanins, phenolic acid, and flavonoids to inhibit oxidative stress by scavenging free radicals such as ROS and reactive nitrogen species (RNS) [84,85].
Furthermore, 100 kg/mL of aqueous roselle extract exhibited in vitro and in vivo the attenuation of cardiac oxidative stress by decreasing the gene expression of oxidative stress (NOX2 and 8-isoprostane level) and increasing the level of antioxidants (SOD and GSH level) in post-myocardial infarction rats [67]. Previously, Ramalingam and colleagues [86] revealed that supplementation with aqueous extract of roselle for 28 days prevented nicotine-induced myocardial oxidative stress by lowering the malonaldehyde (MDA) level, as well as by restoring activities of Cu/Zn-SOD, GSH content, and the GSH/GSSG ratio.
MDA, an oxidative marker of lipid peroxidation, correlates with the level of serum total cholesterol, triglyceride (TG), and LDL cholesterol [87], and extract leaves and calyces of roselle were proven to inhibit lipid peroxidation in vitro [76]. Moreover, atherosclerotic rabbit models that were fed delphinidin-3-O-glucoside from roselle in doses of 10 and 20 mg/kg for 12 weeks showed inhibited oxidative stress via the upregulation of GSH-PX and SOD1 mRNA levels [88].
Additionally, polyphenol-rich extract of roselle in diabetic rats significantly promoted the production of antioxidant markers SOD, GSH, and catalase (CAT) and attenuated the production of heart lipid peroxidation (TBARS), advanced oxidation protein product (AOPP), and MDA in the DM heart [69,74].
Mitochondrial damage is one of the key triggers that induce oxidative damage [9]. Previously, polyphenol-rich extract of roselle prevented mitochondrial-induced oxidative damage in DM hearts by decreasing the complex I activity of the mitochondrial respiratory chain [74]. Moreover, a decrease in mitochondrial structural damage and an increase in the mitochondrial antioxidant defense system in endothelial cells induced by DM was noted in diabetic rats fed a polyphenol-rich extract of roselle (Mohammed Yusof et al., 2018). The inhibition of lipid peroxidation and oxidative damage is associated with roselle's Fe 2+ chelating and OH − radical scavenging properties and roselle's reinforcement of the "mitochondrial" antioxidant system [74,78]. It can be concluded that roselle serves as a potent cardiovascular protective agent by attenuating ROS overexpression [69].

Anti-Inflammatory
Inflammation plays a critical role in the genesis, progression, and manifestation of CVD. Nevertheless, while safely modulating inflammation using targeted therapeutics remains a challenge, the results from past studies that utilize roselle as an alternative treatment demonstrate that targeting inflammation may offer the potential to reduce the risk for CVDs. A recent study conducted by Sun et al. [88] revealed that the supplementation of delphinidin-3-O-glucoside, a bioactive compound in roselle calyx, was able to reduce inflammation by downregulating the mRNA levels of interleukin (IL)-6, vascular cell adhesion molecule 1 (VCAM-1), and NF-κB in atherosclerotic rabbit models.
Moreover, the inflammation in a nicotine-induced cardiac injury animal model was successfully attenuated after being supplemented with aqueous roselle extract by preserving intracellular lactate dehydrogenase (LDH) activity in heart tissue [86]. Moreover, the utilization of 100 mg/kg roselle aqueous extract for 7 days has been proven to alleviate inflammation by attenuating the elevation of plasma troponin-T, IL-6, and IL-10, as well as downregulating IL-10 in rat models of myocardial infarction [89].
Previously, 500 mg/kg of a methanolic leaf extract from roselle fed to carrageenaninduced inflammation rats successfully decreased inflammation by decreasing inflamed paw diameter [90]. Similarly, a significantly higher inhibition of inflammation was also noted using roselle seed oil or extract compared to indomethacin on carrageenan-induced inflammation rats, indicating that the inhibition can be attributed to roselle's ability to either inhibit the synthesis, release, or action of the histamine inflammatory mediators or antagonize the activity of the mediators after release and inhibit cyclooxygenase, lipoxygenase enzymes, and mast cell degranulation [91]. The same study stipulated that these biological effects were contributed by the presence of phenolic compounds, flavonoids, saponins, linalool, and alkaloids.
Furthermore, supplementation of petroleum ether extract of roselle seed greatly decreased the paw's chronic and acute inflammation in a dose-dependent manner, inhibiting the inflammatory response of carrageenan in edema and the formation of granular tissue [92]. Roselle is also rich in the anti-inflammatory agent ascorbic acid and in antioxidant polyphenols that can reduce oxidation and promote the increase in the anti-inflammatory cytokine (IL10), therefore inhibiting proinflammatory cytokines (IL6 and tumor necrosis factor α (TNFα)) [93,94]. Hence, roselle possesses great antioxidant effects, which can inhibit inflammation in the cardiovascular and provide a cardioprotective effect.

Anti-Fibrosis
Fibrosis plays a fundamental role in the pathogenesis of heart failure and CVD. A body of growing evidence from the literature has demonstrated that roselle possesses protective effects against fibrosis. In the heart, extensive deposition of collagen and extracellular matrix end up stiffening and altering the physiological function of the heart. However, roselle aqueous extract treatment for 28 days ablated the deposition of myocardium collagen I and collagen III, and showed the ability to reduce the enlargement of cardiomyocytes in myocardial infarction rat models [67]. A previous study that was conducted by Si et al. [25] has shown that roselle treatments were able to reduce cardiac interstitial collagen accumulation, indicated by the reduction in B-type natriuretic peptide (BNP) gene expression, which is a cardiac fibrosis biomarker. Therefore, the reduction in cardiac fibrosis and BNP gene expression by roselle could improve ventricular compliance and relaxation in diet-induced obese animal models with myocardial infarction.
Furthermore, aqueous roselle extract has exerted anti-fibrotic activity by ameliorating the increase in collagen III gene expression as well as reduced collagen deposition in the heart with myocardial infarction [89]. Moreover, the combination treatment of roselle and olive with the doses of 125, 250, and 500 mg/kg for 4 weeks successfully attenuated extensive collagen fiber deposition and markedly reduced myocardial fibrosis in hypertensive animal models [71]. All of this accumulating evidence has displayed that roselle can be used as adjuvant therapy in ameliorating hypertension and later attenuating the progression of CVD. Table 2 summarizes the potential of roselle in the attenuation of CVD risk factors. Decreased blood pressure and heart rate in nicotine-exposed rats.
[66]  Reduced the concentration of α-amylase and α and β-glucosidase in a concentrationdependent manner, inhibited glucosidase, and disrupted hydrolysis and the release of glucose.

Clinical Studies
Roselle consumption has great potential to contribute to better human health. There is significant accumulating evidence in preclinical studies that corroborate roselle as a potent candidate for limiting CVD progression by targeting the risk factors and mechanisms involved [12,67]. In this regard, several clinical studies have been carried out to determine the effectiveness of roselle in enhancing human health, mainly by targeting CVD risk factors.
Some human trials have shown that roselle has exhibited positive effects in intervening cardiovascular risk factors. Recently, Harmili et al. [97] reported that the utilization of roselle as an intervention for hypertension patients for 7 days with a dose of 2 g every day has been shown to reduce systolic and diastolic blood pressure. Similar studies that were conducted on diabetic patients with mild hypertension have shown similar effects when consuming 240 mL of roselle infusion for a month and have revealed a decline in the systolic and diastolic blood pressure to the normal level [98]. In a quasi-experimental design study, the consumption of roselle flower tea twice a day for 2 weeks among hypertensive patients was able to reduce systolic and diastolic blood pressure [99].
A study has evaluated the effects of roselle flowers in the treatment of hypertension among postpartum mothers, and it was shown that consumption of 10 g brewed roselle with 200 mL of water together taken with anti-hypertensive drugs was able to reduce systolic and diastolic blood pressure when comparing before and after the intervention period [100]. Another previous study that was conducted in a double-blind, randomized controlled trial on sixty diabetic patients with mild hypertension who consumed roselle sour tea reported decreased systolic blood pressure and mean pulse pressure, highlighting the potential of roselle as an anti-hypertensive treatment [101].
Moreover, in a randomized, double-blind clinical trial, a standardized herbal medicinal product containing 1200 mg roselle that was consumed by hypertensive patients for 8 weeks led to a reduction in blood pressure, lowered TG, and reduced mean serum renin and ACE [102]. A non-randomized quasi-experimental study empirically corroborated that roselle has favorable cardiovascular effects in hypertensive patients, as 4 weeks of roselle ingestion with the dose of 250 mL every day was proven to bring down pulse pressure and heart rate to normal levels, and led to the regression of left ventricular hypertrophy [103]. Furthermore, 15 g of roselle infusion administered orally to people with hypertension for four weeks in a randomized clinical-case study was shown to have a lowering effect on arterial systolic and diastolic blood pressure and suppressed LDL levels, reduced cholesterol levels, enhanced HDL levels, and diminished TG levels [104]. This suggests that roselle is a potent candidate for hypertensive treatment.
Previously, a study that was conducted by Asgary et al. [105] in a double-blind placebocontrolled clinical trial showed that the daily consumption of 500 mg of roselle calyx powder for 4 weeks among forty adult patients with metabolic syndrome (hypertension, dyslipidemia, hyperglycemia) was able to significantly reduce serum TG and systolic blood pressure. Continuing with another randomized, double-blind, placebo-controlled trial, 300 mL of jelly drink containing polyphenol-rich roselle calyx extract given to forty-three adults with dyslipidemia for 8 weeks was demonstrated to decrease the levels of LDL-C, TG, TNFα, and MDA, and enhance the GSH level [87]. Hence, this study proved that roselle consumption could prevent CVD due to its polyphenolic content by exerting antioxidant and anti-inflammatory effects.
Additionally, Abubakar et al. [106] reported that a 250 mL polyphenol-rich roselle drink containing 7.5 g of roselle calyx powder, with 311 mg of gallic acid and 150 mg of anthocyanins, decreased postprandial systolic and diastolic blood pressure in subjects aged 47 to 49 with a cardiovascular risk of 1-10% after 4 h of consumption relative to baseline in a randomized, controlled, single-blinded, acute, and cross-over study. The same study also indicated that roselle consumption reduced serum glucose, plasma insulin, serum TG, and C-reactive protein (CRP) levels, and induced a significant improvement in the antioxidant response curve and no significant changes in arterial stiffness. The roselle extract improved postprandial vascular function and suggested a useful dietary strategy to reduce endothelial dysfunction and cardiovascular risk.
A study by Boushehri et al. [107] on the efficacy of sour roselle tea consumption in reducing selected CVD risk factors, involving about 362 participants in a systematic review and meta-analysis of randomized clinical trials, reported that sour roselle tea consumption significantly reduces fasting plasma glucose, systolic blood pressure, and diastolic blood pressure. Although drinking sour roselle tea had no significant influence on serum levels of triacylglycerol, total cholesterol, and HDL cholesterol, there was a tendency for a considerable reduction in LDL cholesterol serum concentrations. Another systematic review and meta-analysis on randomized controlled trials using roselle as an intervention for lipid profiles, blood pressure, and fasting plasma glucose levels in adult populations by Ellis et al. [108] indicated that roselle exerted stronger effects on an induced reduction in systolic and diastolic blood pressure and lowered LDL levels compared with other teas and a placebo.
Many clinical trials have been carried out to evaluate the effect of roselle in attenuating cardiovascular risk factors. No adverse effects were reported during the clinical trials.
However, clinical studies exhibited some limitations, and the number of clinical studies is also still limited. Furthermore, treatment with roselle has addressed certain issues and highlighted why it is enigmatic to pursue clinical studies for large-scale clinical applications. For example, there is a lack of data related to the bioavailability of roselle phytochemicals during gastrointestinal digestion, which is crucial for maximizing the effects of roselle in alleviating CVD risk factors. Additionally, there is still limited research on the safety profile of roselle and the side effects, adverse effects, and toxicity associated with its consumption. Nowadays, the available roselle products on the market are not standardized. The isolation of bioactive compounds from roselle is time-consuming and costly, which impedes rosellebased product production on a large scale. Moreover, the stability of roselle-based products always faces obstacles regarding the stability of the physicochemical properties of the product, requiring a long process to access a proper evaluation of the stability to improve the product quality as well as increase both safety and efficacy. Thus, further studies are required in the future to validate the clinical efficacy of roselle in humans, mainly in alleviating cardiovascular risk factors, before proceeding with its large-scale clinical application. Table 3 summarizes the clinical studies that use roselle as treatment. Figure 4 proposes a summary of the role of roselle in attenuating CVD by targeting its risk factors and the mechanisms involved.    Roselle was able to alleviate the progression of CVD by targeting its risk factors, especially hyperglycemia, dyslipidemia, and hypertension, as well as the involved mechanisms, including oxidative stress, inflammation, fibrosis, and apoptosis. The green line indicates the activity of roselle meanwhile the black color line indicates the mechanism that lead to cardiovascular diseases.

Conclusions
The risk factors of CVD, especially hyperglycemia, hypertension, and hyperlipidemia, are the main culprits that induce the development of CVD via oxidative stress, inflammation, fibrosis, and apoptosis mechanisms. Roselle has been studied for decades due to its promising effects in limiting the CVD progression by exhibiting anti-hyperglycemic, anti-hyperlipidemic, anti-hypertensive, antioxidative, anti-inflammatory, anti-fibrosis, and anti-apoptosis effects in preclinical studies and clinical studies. The main metabolites in roselle that play a significant role in these biological effects are anthocyanins, phenolic acids, flavonoids, organic acids, saponins, and tannins. However, most of the studies that were conducted do not highlight which compound found from each type of extract shows potent results in alleviating CVD risk factors. Hence, future studies should consider this matter so that the biological properties of roselle can be fully utilized. Furthermore, limited studies have been conducted to develop roselle as a nutraceutical product. Therefore, this review can provide knowledge about roselle for developing nutraceuticals in the future, mainly targeting CVDs.

Conflicts of Interest:
The authors declare no conflict of interest. The funder has no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.