Natural Products in Cardiovascular Diseases: The Potential of Plants from the Allioideae Subfamily (Ex-Alliaceae Family) and Their Sulphur-Containing Compounds

Cardiovascular diseases (CVDs) are the leading cause of mortality worldwide and, together with associated risk factors such as diabetes, hypertension, and dyslipidaemia, greatly impact patients’ quality of life and health care systems. This burden can be alleviated by fomenting lifestyle modifications and/or resorting to pharmacological approaches. However, due to several side effects, current therapies show low patient compliance, thus compromising their efficacy and enforcing the need to develop more amenable preventive/therapeutic strategies. In this scenario, medicinal and aromatic plants are a potential source of new effective agents. Specifically, plants from the Allioideae subfamily (formerly Alliaceae family), particularly those from the genus Allium and Tulbaghia, have been extensively used in traditional medicine for the management of several CVDs and associated risk factors, mainly due to the presence of sulphur-containing compounds. Bearing in mind this potential, the present review aims to gather information on traditional uses ascribed to these genera and provide an updated compilation of in vitro and in vivo studies validating these claims as well as clinical trials carried out in the context of CVDs. Furthermore, the effect of isolated sulphur-containing compounds is presented, and whenever possible, the relation between composition and activity and the mechanisms underlying the beneficial effects are pointed out.


Introduction
Cardiovascular diseases (CVDs) continue to lead mortality rates worldwide [1], accounting for nearly 18 million annual deaths, primarily due to coronary heart disease and stroke [2]. Unfortunately, these numbers tend to increase as several non-modifiable and modifiable risk factors associated with the onset and development of these disorders are also escalating. While non-modifiable risk factors such as aging, gender, genetic predisposition, family history of heart-related problems and ethnicity cannot be altered [3][4][5], modifiable risk factors are changeable. These include hypertension, dyslipidaemia, diabetes, obesity, smoking, alcohol misuse, unhealthy diet, sedentary lifestyle, and psychosocial factors [6] and are recognised as relevant targets to manage CVDs. For example, the IN-TERHEART case-control study pointed out that 90% of acute myocardial infarction cases are due to these risk factors and that controlling or eliminating them per se could lead to a drastic decrease in CVD mortality [7,8]. Indeed, due to their huge impact on CVDs, these risk factors are included in the World Health Organisation (WHO) target list that aims to reduce their prevalence by 2025 [9]. The negative impact of CVDs is further fuelled by the fact that 60% of patients fail to correctly adhere to the therapeutic regimen [10], mainly due to the cost of CVD therapies [11]. Therefore, new therapeutic interventions and/or preventive strategies with fewer side effects are mandatory, with aromatic and medicinal plants emerging as promising agents to manage both CVDs and associated risk factors. In fact, herbal medicines are relevant sources of bioactive molecules, used by ca. 80% of the world's population in basic health care [12]. Moreover, many of these medicinal plants have already been used in the treatment of chronic and acute conditions including CVDs [13][14][15][16] and are part of the Mediterranean-style diet with proven beneficial effects on cardiovascular risk factors [17], as pointed out in several meta-analysis and critical reviews [18][19][20][21][22][23]. Interestingly, these effects are associated with the increased consumption of fruit, vegetables, spices, garlic, and onions [24]. Overall, the preventive/therapeutic potential of aromatic and medicinal plants is mainly attributed to the presence of secondary metabolites [25] including phenolic compounds, terpenes, alkaloids, and organosulfur compounds [26]. Organosulfur compounds are widely found in plants from the Allioideae subfamily (ex-Alliaceae family) and, together with extracts or raw bulbs from these plants, are widely reported for their medicinal properties [27]. Therefore, bearing in mind the bioactive potential of these plants, a systematised review gathering information on the effects of sulphur-containing extracts/compounds on major CVD risk factors, namely hypertension and dyslipidaemia/diabetes, is presented. Additionally, whenever reported, the mechanisms underlying the observed effects are referred to and the relation between composition and activity pointed out. To achieve this, a bibliographic search was conducted using Pubmed, Scopus and Google scholar databases, combining the keywords "Allium", "Tulbaghia", "Alliaceae" or "Allioideae" with "cardiovascular", "diabetes", "obesity", "dyslipidaemia", "hypertension" or "vasorelaxation". Studies published over the last 20 years that had an available DOI were considered.

Importance of Allioideae Species in Cardiovascular Diseases
In the following sections, both the relevance and potential of plants from the Allioideae subfamily are described. First, the traditional uses ascribed to these plants in several ethnobotanical surveys is shown in order to highlight their importance in local health care systems. Then, studies validating some of these effects are systematised, considering preclinical approaches and clinical trials. The effect of isolated sulphur-containing compounds is also presented, and whenever possible, the relation between composition and activity is discussed, thus opening new avenues for further investigations in the field.

Traditional Uses of Allioideae
A plethora of traditional uses are ascribed to Allioideae plants or plant-based preparations, as summarised in Table 1. Plants' scientific and common names are included as well as the region of use. In addition, the plant part or preparations used (with reference to the preparation method and posology, when known) and beneficial effects on the cardiovascular system are pointed out. Overall, the majority of the studies focus on the genus Allium, with only a few studies reporting the effects of two species from the Tulbaghia genus. In traditional preparations, the plant bulb is commonly used (7/11 total studies), with leaves (1/11), aerial parts (1/11) or whole plants (1/11) referred to in much less often. In addition, the use of a combination of plants is frequent and, therefore, this information is also provided. A list of abbreviations, used throughout the table, is provided at the end of the table.

Pre-Clinical Studies Validating the Cardioprotective Effects of Allioideae
Given the importance of Allioideae plants in the management of CVDs and associated risk factors in ethnopharmacological studies, we next compile several pre-clinical studies validating these effects. First, the effect of plants or their extracts is pointed out ( Table 2) followed by the effect of isolated sulphur-containing compounds (Table 3) and then clinical trials.

The Effect of Plant Parts or Extracts
In Table 2, studies reporting the beneficial effects of plant parts or extracts is presented with reference to the species name, the plant part/extract used (with reference to the preparation method and concentration), the study model and the main findings regarding the effect observed in the cardiovascular system. Unless stated, a daily administration was used. Studies are grouped considering the cardiovascular disease and/or risk factor assessed with plants organised in alphabetical order of their scientific name. A list of abbreviations, used throughout the table, is provided at the end of the table.
Plants from this subfamily are rich in cysteine sulfoxide derivatives, such as alliin [132], which, by the action of alliinase, are converted into thiosulfinates, e.g., allicin, which in turn are instable and change into organosulfur compounds like ajoene [133]. Thiosulfinates are considered to be the main class of compounds responsible for the biological activities reported for plants from Allioideae subfamily [134]. Accordingly, several studies have assessed the role of alliinase activity on the effect of the extracts. Indeed, the antihypertensive effect of onions (Allium cepa) is lost or is much weaker upon boiling [81]. Similarly, the antiplatelet aggregation potential of these bulbs is also compromised, since longer heating times in either a conventional oven or microwave led to a pro-aggregatory effect rather than the expected anti-aggregatory potential [109]. Additionally, with the loss of alliinase activity, the vasorelaxant properties of Allium sativum, were abolished in aortic rings pre-contracted with phenylephrine [95]. Similar dependency on alliinase activity was reported for the hypolipidemic activity of A. sativum where long heating times or microwave heating compromised this effect [135].
On the other hand, the antidyslipidaemic and antidiabetic effects of A. sativum seem to depend on the PI3K/Akt/Nrf2 [87] or IGFIR/PI3K/Akt [67,68] pathways since, upon treatment, activation of these pathways is observed.
In order to better disclose the putative factors underlying the hypolipidemic effect of A. hookeri, a metabolomic analysis on the serum of hamsters consuming a high-fat diet and administered A. hookeri powder orally was carried out. The authors found 25 putative markers which could explain the lipid-lowering effect of this species, with phosphatidylcholines, lysophosphatidylcholines and lysophosphatidylethanolamines the most common targets. Furthermore, the metabolism for glycerophospholipids was increased in the treated group [57].

The Effect of Isolated Sulphur-Containing Compounds
In this section, the effect of isolated sulphur-containing compounds found in the Allioideae subfamily is presented. Then, a composition-activity relation is discussed in order to bring attention to potential active extracts. Table 3 systematises the main studies performed in these compounds, with the compound name, chemical structure, study model used, and the main findings of the study pointed out. Additionally, whenever reported, the route of administration and concentration used is highlighted. A list of abbreviations, used throughout the table, is provided at the end of the table. Plants from this subfamily are rich in cysteine sulfoxide derivatives, such as alliin [132], which, by the action of alliinase, are converted into thiosulfinates, e.g., allicin, which in turn are instable and change into organosulfur compounds like ajoene [133]. Thiosulfinates are considered to be the main class of compounds responsible for the biological activities reported for plants from Allioideae subfamily [134]. Accordingly, several studies have assessed the role of alliinase activity on the effect of the extracts. Indeed, the antihypertensive effect of onions (Allium cepa) is lost or is much weaker upon boiling [81]. Similarly, the antiplatelet aggregation potential of these bulbs is also compromised, since longer heating times in either a conventional oven or microwave led to a pro-aggregatory effect rather than the expected anti-aggregatory potential [109]. Additionally, with the loss of alliinase activity, the vasorelaxant properties of Allium sativum, were abolished in aortic rings pre-contracted with phenylephrine [95]. Similar dependency on alliinase activity was reported for the hypolipidemic activity of A. sativum where long heating times or microwave heating compromised this effect [135].
On the other hand, the antidyslipidaemic and antidiabetic effects of A. sativum seem to depend on the PI3K/Akt/Nrf2 [87] or IGFIR/PI3K/Akt [67,68] pathways since, upon treatment, activation of these pathways is observed.
In order to better disclose the putative factors underlying the hypolipidemic effect of A. hookeri, a metabolomic analysis on the serum of hamsters consuming a high-fat diet and administered A. hookeri powder orally was carried out. The authors found 25 putative markers which could explain the lipid-lowering effect of this species, with phosphatidylcholines, lysophosphatidylcholines and lysophosphatidylethanolamines the most common targets. Furthermore, the metabolism for glycerophospholipids was increased in the treated group [57].

The Effect of Isolated Sulphur-Containing Compounds
In this section, the effect of isolated sulphur-containing compounds found in the Allioideae subfamily is presented. Then, a composition-activity relation is discussed in order to bring attention to potential active extracts. Table 3 systematises the main studies performed in these compounds, with the compound name, chemical structure, study model used, and the main findings of the study pointed out. Additionally, whenever reported, the route of administration and concentration used is highlighted. A list of abbreviations, used throughout the table, is provided at the end of the table. Plants from this subfamily are rich in cysteine sulfoxide derivatives, such as alliin [132], which, by the action of alliinase, are converted into thiosulfinates, e.g., allicin, which in turn are instable and change into organosulfur compounds like ajoene [133]. Thiosulfinates are considered to be the main class of compounds responsible for the biological activities reported for plants from Allioideae subfamily [134]. Accordingly, several studies have assessed the role of alliinase activity on the effect of the extracts. Indeed, the antihypertensive effect of onions (Allium cepa) is lost or is much weaker upon boiling [81]. Similarly, the antiplatelet aggregation potential of these bulbs is also compromised, since longer heating times in either a conventional oven or microwave led to a pro-aggregatory effect rather than the expected anti-aggregatory potential [109]. Additionally, with the loss of alliinase activity, the vasorelaxant properties of Allium sativum, were abolished in aortic rings pre-contracted with phenylephrine [95]. Similar dependency on alliinase activity was reported for the hypolipidemic activity of A. sativum where long heating times or microwave heating compromised this effect [135].
On the other hand, the antidyslipidaemic and antidiabetic effects of A. sativum seem to depend on the PI3K/Akt/Nrf2 [87] or IGFIR/PI3K/Akt [67,68] pathways since, upon treatment, activation of these pathways is observed.
In order to better disclose the putative factors underlying the hypolipidemic effect of A. hookeri, a metabolomic analysis on the serum of hamsters consuming a high-fat diet and administered A. hookeri powder orally was carried out. The authors found 25 putative markers which could explain the lipid-lowering effect of this species, with phosphatidylcholines, lysophosphatidylcholines and lysophosphatidylethanolamines the most common targets. Furthermore, the metabolism for glycerophospholipids was increased in the treated group [57].
Although the compounds presented in Table 3 are commonly found in plants from the Allioideae subfamily, there are others that, despite being found in lower amounts, have been assessed for their cardioprotective effect. For example, the antidyslipidaemic effects reported for garlic (A. sativum) seem to be due to the capacity of S-allyl cysteine, N-acetyl-Sallyl cysteine, alliin, allixin, and allylmercaptocysteine to suppress low-density lipoprotein oxidation since all these compounds were able to reduce LDL oxidation induced by copper (II) [60]. Additionally, S-methylcysteine sulfoxide in high cholesterol-fed rats, was able to reduce the levels of total cholesterol, triglycerides and phospholipids. Furthermore, this compound reduced the activity of lipoprotein lipase without affecting the activity of other lipogenic proteins, while decreasing the levels of free fatty acids. In addition, the excretion of bile acids and sterols was enhanced in the treated group [154].
Regarding the vascular protective effect of garlic, it seems that allithiamine (vitamin B analogue found in garlic) might play a relevant role. Indeed, the presence of this compound in HUVEC growing in high glucose conditions showed a lower level of advanced glycation end products as well as a lower inflammatory profile when compared to high glucose-only treated cells. In addition, this compound also showed a very potent antioxidant potential [156]. Moreover, 2-vinyl-4H-1,3-dithiin, an organosulfur compound found in macerated garlic oil or in stir-fried garlic, decreased spontaneously hypertensive rat's vascular smooth muscle cells proliferation and cell migration and arrested cell cycle at G2 phase. Furthermore, it decreased reactive oxygen species production induced by angiotensin II [157]. Also, diallyl disulphide and diallyl trisulphide have been reported for their capacity to induce neovasculogenesis via PI3K/Akt pathway activation [68,141]. In addition, reduction of cell death dependent on death receptor and mitochondria is also reported for both compounds [68]. Furthermore, for diallyl trisulphide, the promotion of neovasculogenesis is also attributed to a decrease in the microRNA 221 [68,141]. This compound also activated Nrf2 via the PI3K/Akt pathway [147] and induced the release of hydrogen sulphide by cystathionine-γ-lyase [148] using in vitro conditions mimicking diabetes. The reported effects for ajoene might be due to its capacity to inhibit protein prenylation, particularly that dependent on protein farnesyltransferase and protein geranylgeranyltransferase type I [136].
Some studies also assessed the activity of synthetic derivatives of naturally occurring sulphur-containing compounds. A study compared the antihypercholesterolaemic properties of diallyl disulphide analogues and showed that all the tested analogues lowered serum and hepatic levels of several lipids, including low-density lipoprotein while increasing those of high-density lipoprotein. The authors suggested that this lipid-lowering effect is due to the modulation of the 3-hydroxy-3-methylglutaryl-CoA reductase activity since a decrease in mRNA levels with a concomitant inactivation of sterol regulatory element-binding protein-2 and cyclic adenosine monophosphate response element-binding protein is observed [158]. Another study assessed the antihypertensive and vasorelaxant properties of five synthetic derivatives of diallyl disulphide. The results showed that all analogues were able to decrease systolic blood pressure in the N ω -nitro-L-arginine methyl ester-induced hypertensive animal model. Similarly, all compounds restored the antioxidant defences as observed by an increase in the activity of glutathione peroxidase, glutathione and superoxide dismutase with concomitant decrease in malondialdehyde and protein carbonyl levels. Furthermore, nitric oxide metabolites and cyclic guanosine monophosphate levels were restored by all the analogues, while the activity of angiotensin-converting enzyme was decreased [159].
The effect of sulphur-containing compounds on the pharmacodynamic and pharmacokinetics of other drugs was also assessed. Indeed, it was reported that the oral co-consumption of diallyl trisulphide and nifedipine led to a higher maximum concen-tration and area under the curve, thus suggesting that the compound might affect the gastrointestinal metabolism of nifedipine, since no effect on the pharmacokinetics was observed when nifedipine was given intravenously [160].

Clinical Trials
The importance of plants from the Allioideae subfamily is also validated by a small number of clinical trials. However, some contradictory results have been reported that may be related to the different doses used, duration of the treatment and/or association with other compounds. For example, in a small placebo-controlled and double-blind trial, firefighters were given four tablets containing 300 mg/table of aged garlic extract and 30 mg/table of coenzyme Q10 for up to 1 year. The results showed that the consumption improved their vascular elasticity and endothelial function [161]. In another study, the consumption of 1200 mg of this extract daily for 4 weeks followed by 4 weeks of washout had no effect on several parameters assessed such as glycated haemoglobin A1c, blood pressure, total cholesterol, triglycerides and high-density lipoprotein, and did not prevent endothelial dysfunction, oxidative stress or inflammation in patients with type 2 diabetes with high cardiovascular risk [162]. Furthermore, the administration of aged garlic extract (250 mg) supplemented with vitamins B12 and B6, folic acid and L-arginine daily for a 12-month period increased the ratio between brown and white epicardial adipose tissues with concomitant increase in the temperature-rebound index, while decreasing homocysteine levels and preventing the progression of coronary artery calcification [163]. In patients with coronary artery calcification and increased cardiovascular disease risk, the consumption of 2400 mg of aged garlic extract daily for 1 year inhibited the progression of the calcification. Regarding secondary outcomes, the extract decreased interleukin-6 levels as well as the glucose levels and blood pressure [164]. The same concentration increased cutaneous microcirculation in diabetic patients, thus suggesting that aged garlic extract might promote wound healing in these patients [165]. Overall, it seems that longer treatment durations (up to 1 year) have better outcomes.
Regarding other extracts, the consumption of 125 mL of red wine extract of onion twice daily for 10 weeks by healthy individuals showed hypocholesterolaemic, antioxidant and anti-inflammatory effects [166]. Additionally, the consumption of 300 mg of A. sativum standardised powder for 8 weeks by patients undergoing haemodialysis decreased the absolute values for oxidised low-density lipoprotein and homocysteine. In addition, the powder significantly ameliorated the values of calcium, triglycerides, oxidised low-density lipoprotein and homocysteine [167]. The consumption of quercetin-rich A. cepa extract daily for 6 weeks decreased systolic blood pressure in hypertensive individuals when compared to the placebo group [168]. The consumption of A. cepa peel extract twice daily for 12 weeks improved the flow-mediated dilation as well as the number of circulating endothelial progenitor cells in healthy overweight and obese patients. Indeed, the rate of patients with endothelial dysfunction decreased from 26% to 9% after extract administration [169].
Concerning CVD risk factors, some studies have been performed, such as the Tehran Lipid and Glucose study that assessed the effect of dietary consumption of A. sativum and A. cepa in cardiometabolic risk factors (body mass index, waist circumference, systolic blood pressure, diastolic blood pressure, fasting plasma glucose, triglycerides-to-high-density lipoprotein ratio, insulin, creatinine, estimated glomerular filtration rate and creatinine clearance) for 6 years. The results showed that high consumption of these vegetables led to a 64% reduction in CVD outcomes, as well as a lower incidence of chronic kidney disease and hypertension while no association was made with type 2 diabetes. Furthermore, it improved TG levels and creatine clearance [170].
Although the majority of the reported clinical trials are conducted using a small cohort of patients and are usually single-centre studies, they highlight the potential of plants from the Allioideae subfamily in the management of CVDs and associated risk factors. Nevertheless, these effects should be validated in more complete clinical trials with access to bigger multicentre cohorts to account for the genetic polymorphisms which impact the activity of drug metabolising enzymes leading to altered pharmacokinetics [171].
Drug interactions between conventional drugs or between these and herbal medicines are common, with both beneficial and detrimental effects reported. For example, the consumption of capsules containing 0.5 g of A. macrostemon bulb extract powder (three times a day) for eight weeks by patients undergoing baseline therapy for unstable angina, led to lower oxidised low-density lipoprotein and plasminogen activator inhibitor-1 level, while increasing plasminogen activity [172]. Moreover, in patients undergoing simvastatin therapy, supplementation with fenugreek and garlic for 8 weeks significantly reduced total cholesterol, triglycerides, non-high-density lipoprotein and low-density lipoprotein levels and increased those of high-density lipoprotein [173]. On the other hand, care must be taken with antiplatelet drugs, particularly warfarin and aspirin, as a simultaneous consumption of garlic or onion with these drugs can increase the risk of bleeding [174,175]. This interaction is attributed to their capacity to decrease platelet adhesion and aggregation, by inhibiting plasminogen activating factor and fibrinogen receptors and by decreasing thromboxane X 2 synthesis [176]. In addition, garlic consumption is known to inhibit CYP3A4, the enzyme responsible for warfarin metabolism [175].

Final Remarks
The present review sheds light on the potential of plants from the Allioideae subfamily in the management of CVDs and associated risk factors. Traditional uses of some of these species are widely recognised, with garlic (Allium sativum) and onions (Allium cepa) being the most common. Additionally, pre-clinical studies and clinical trials validating their beneficial potential are frequent, thus confirming their importance. Nevertheless, other species such as A. jacquermontii, A. rotundum and Tulbaghia alliacea, despite being used in traditional remedies in some regions, lack scientific validation while other plants have undergone clinical trials but with no beneficial effects on the cardiovascular system.
Regarding CVD risk factors, plants from the Allioideae subfamily showed promising antiplatelet aggregation, antidiabetic, and dyslipidaemic effects, and were able to exert protection against atherosclerotic events.
Overall, we gathered information on both the tapped and untapped potential of plants belonging to the Allioideae subfamily, by highlighting scientific gaps as well as wellvalidated effects that pave the way for the development of new preventive/therapeutic approaches for CVDs.

Conflicts of Interest:
The authors declare no conflict of interest.