Introduction

Vascular diseases refer to medical conditions that narrow blood vessels [1]. It is well documented that the major risk factors leading to these disorders include hypertension, obesity, atherosclerosis, and inflammatory diseases [2]. Recent update shows significant rise in numbers of deaths from vascular diseases [3•], and so, there is an increasing interest in natural products to protect the cardiovascular system [4••]. Garlic (Allium sativum L.) has historical significance as a folk medicine in many cultures around the world for prevention and treatment of several diseases from ancient times to date. Its consumption has been dated back over 6000 years ago in Ayurvedic medicine, over 3000 years ago in ancient Chinese medicine and the ancient Egyptian Codex Ebers [5]. Nowadays, garlic is a popular natural medicine that has demonstrated potential health benefits, therapeutic effects, and almost no adverse effects [6•]. The major garlic components such as OSCs and polyphenols are responsible for the garlic biological activities which include benefits in cardiovascular protection [7, 8], anti-inflammatory properties [9], nephroprotective role [10], neuroprotective effects [11], as well as protection against mitochondrial damage [12]. The present review summarizes the recent findings of garlic’s bioactive component effects on inflammation, atherosclerosis, obesity, and mitochondria dysfunction, from in vitro and in vivo studies to preclinical and clinical trials.

Garlic’s Active Constituents: the Organosulfur Compounds

Garlic has been recognized for taste and therapeutic properties, mainly due to its high content of organosulfur compounds (OSCs) [13]. Garlic bulbs accumulate sulfur-compounds as γ-glutammil peptides which can be hydrolyzed and oxidized to form alk(en)yl-L-cysteine sulfoxides (ACSOs), being alliin the majority. Once garlic tissue is disrupted, volatile sulfur compounds are formed by the action of alliinase. As a result, a wide range of unstable OSCs is synthesized. The first products of the reaction are highly reactive sulfenic acids, which self-condensed to generate thiosulfinates (Fig. 1). These compounds are responsible for the characteristic flavor and pungency of Allium genus and ‘allicin’ is the resulting thiosulfinate of alliin hydrolysis. Allicin is the major OSC in raw garlic; its levels can vary between 3 and 37 mg g−1 in dry weight [14]. It has been proposed to be the main active metabolite and responsible for most of the biological activities of garlic and can be consumed mainly as fresh crushed garlic. In turn, thiosulfinates are unstable and participate in a cascade of non-enzymatic reactions to produce a broad array of compounds such as (poly)sulfides, ajoenes, vinyldithiines (VD), among others (Fig. 1). Different garlic culinary processes or preparations allow to OSC transformations. Stir-fried garlic possess (poly)sulfides, ajoenes, vinyldithiines (VD), and boiled garlic contain SAC, allicin, ajoene, poysulfides (VD) [15]. Meanwhile, garlic oil macerate is the principal dietary source of VD [16••, 17]. From the above, it follows that each garlic by-product shows a different OSC profile (Table 1).

Fig. 1
figure 1

Scheme of organosulfur compound synthesis and transformation pathways

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Table 1: Sulfur-compound profile in different garlic preparations and by-products.
Full size table

Further in parallel, ɤ-glutamylcysteines are also converted to S-allylcysteine (SAC) via other pathway than the alliin/allicin pathway. This sulfur-amino acid is initially present in garlic, although it is not a major compound. Garlic product enriched in SAC is prepared as aged garlic extract (AGE), which is made through an aqueous or hydro alcoholic maceration of garlic cloves for long periods of time [18]. SAC is the most active bioavailable compound in garlic and is a major contributor to garlic’s health benefits.

OSCs present in garlic and its preparations can be classified into four categories:

  • Sulfur amino acid derivatives such as SAC, which are stable and odorless. These compounds are present in whole cloves of the garlic bulb and AGE.

  • Thiosulfinates such as allicin, which are unstable and highly reactive compounds. They are present in crushed cloves and give the typical aroma and flavor of fresh garlic.

  • Ajoene and vinyldithiines, which are formed when allicin is kept at a moderate temperature in a lipophilic medium. They are present in stir-fried garlic and garlic macerated oil.

  • Allyl (poly) sulfides such as diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS). They are present in steam-distilled garlic oil, which is used as seasoning in foods due to its strong aroma.

The bioactive compounds achieve their activities within the gastrointestinal tract (in situ). These compounds need to be bioaccessible but not necessarily be absorbed into the body. Among hydrophilic OSCs, SAC is the most bioavailable compound. Their absorption rates are higher than 90% [19], and alliin is up to 16.5% bioavailable with oral intake of 60 mg kg−1 after 4 h [20]. Although allicin is the most studied garlic thiosulfinate, in biological environments, it is quite unstable, reacting quickly with thiol groups of proteins and/or conjugating with small molecules such as glutathione or cysteine [21]. Awareness of the bioaccessibility of the different OSCs would help to understand the bioavailability of garlic’s bioactive compounds.

OSCs in Inflammation and Atherosclerosis

Immune responses, which include different cell types such as monocytes, macrophages, neutrophils, and lymphocytes, are implicated in the development of atheroma plaques [22••]. Moreover, inflammatory biomarkers such as high-sensitivity C-reactive protein (hsCRP) and interleukin-6 (IL-6) could also be considered as predictors of cardiovascular events, just as conventional lipoprotein-cholesterol levels [23•, 24]. A common nexus between atherosclerosis and inflammation is undoubtedly oxidative stress. Large evidence indicates how traditional risk factors may translate into oxidative stress and contribute to atherogenesis [25•]. As result of steady research, it seems that a better control of inflammation and oxidative stress could mean a promising therapy for reducing cardiovascular disease (CVD) and atherosclerosis [23•].

Particularly, in inflammatory diseases, garlic is associated with the decrease of both inflammatory mediators and reactive oxygen species (ROS) generation and is involved in the cellular immune response, acting as an immunomodulator [26]. The onset and progression of atherosclerosis are closely linked to chronic low-grade systemic inflammation, recognized as an important mechanism in atherogenesis [27, 28]. The atherosclerotic process is accelerated not only by the release of inflammatory chemokines and cytokines, but also by the generation of ROS, partly responsible for endothelial dysfunction and vascular smooth muscle cell proliferation [29]. Therefore, therapeutic approaches currently being used for atherosclerotic vascular disease should exert pleiotropic anti-inflammatory and anti-oxidative effects.

In a recently published review, the anti-inflammatory actions of garlic are summarized. Authors focalized on the action of aged garlic extract (AGE) and allicin (diallyl thiosulfinate), one of the major constituent of garlic, and they concluded that, as far as they concerned, garlic could inhibit inflammation mostly by affecting inflammatory mediators such as nitric oxide (NO), tumor necrotic factor alpha (TNF-α), and interleukin (IL)-1 [4••].

Allicin was also found to mitigate lipopolysaccharide (LPS)-induced vascular injury in endothelial cells and this effect may be closely related to the capacity of reduce oxidative stress, modulate nuclear factor E2-related factor 2 (Nrf2) activation, and impair inflammatory response by decreasing TNF-alpha and IL-8 production [12].

Although OSC bioactivities have been extensively studied, there is less information about their bioaccessibility. Recent research from our group has shown that, after cooking, garlic still contains bioactive compounds and they show antioxidant and anti-inflammatory activities. The main OSCs found in cooked garlic were ajoene, 2-vinyl-4H-1,3-dithiin (2-VD), diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), and interestingly, these bioactive OSCs could be absorbed at a moderate rate. [30••]. We also reported that allicin, DAS, and 2VD permeated less than 5% through the intestine, and only the 20% of 2VD, DAS, and DADS could remain unreacted after half hour. Finally, the garlic compound that showed better permeability and stability was 2VD and would be the most stable compound in the early assimilation stages [30••]. 2VD exhibited inhibitory platelet aggregation and hypolipidemic activity associated with cardiovascular disease and inflammation by a mechanism that may be related to the sulfhydryl signaling pathway [31]. Our group is actually developing a comprehensive study of the different compound derived from garlic, as well as their biological effects. So far, we observed that 2VD exhibits antiproliferative, antimigratory, and antioxidant activities by enhancing the adaptive ability of vascular smooth muscle cell obtained from mesenteric arteries, to restore dynamic homeostasis after angiotensin II disturbance (Torres-Palazzollo et al., in preparation). Our finding suggests that garlic intake containing 2VD would contribute to ameliorate inflammation and atherogenesis. Nonetheless, further studies will be needed to elucidate the 2VD action mechanisms.

OSCs and Obesity

Overweight and obesity are defined as abnormal or excessive fat accumulation that presents a risk to health. Body fat distribution is important because abdominal obesity, rather than other fat depots, is strongly associated to metabolic syndrome (MetS), together with hypertension, atherogenic dyslipidemia, hyperglycemia, and prothrombotic and proinflammatory conditions [32]. Worldwide rise of obesity is most likely a result of complex interactions between genetic factors and obesogenic environments. Environmental factors are likely to be major contributors to the obesity epidemic and include behavioral, social, economic factors and fundamentally the ample access to energy-rich foods and low physical activity levels [33]. However, overweight and obesity can be largely preventable and one way is changing dietary habits. In this sense, certain foods offer strong medicinal and protective qualities [34]. Thus, garlic is one of the most important vegetables with bioactive properties, including its by-products [13]. Evidence from in vitro, animal, and human research has shown that garlic or its sulfur-containing compounds have beneficial effects on obesity and MetS. An anti-adipogenic effect of garlic-derived compounds inhibiting 3T3-L1 adipocyte differentiation in vitro through AMPK activation, acetyl CoA carboxylase-1 (ACC-1) inhibition and carnitine palmitoyltransferase (CPT-1) upregulation [35] or through ERK activation was reported [36].

More recently, allicin was shown to potentially prevent obesity and associated metabolic disorders by enhancing the expression of brown adipocyte-specific genes, including UCP-1, through KLF15 signal cascade [37••]. Many studies have been published in relation to the effects of garlic (in various forms of supplementation) and its derivatives on diverse animal models of obesity. Oral administration of fermented garlic extract during 8 weeks showed anti-obesity effects by reducing body weight, adiposity, triglyceride, and total cholesterol levels and suppressing adipogenesis in diet-induced obese mice [38]. A reduction of body weight and liver protection from damage in obese rats treated with garlic oil was also reported, and interestingly, authors established that garlic administration via acupuncture was more effective than oral administration [39].

Raw garlic homogenate could attenuate multiple abnormalities of MetS such as body weight increased, blood glucose, dyslipidemia and restored the activity of lactate dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G6PDH) and catalase in liver of fructose-induced MetS in rats [40]. It was also seen that insulin sensitivity associated to MetS and obesity is improved by garlic and its OSCs in animal models [41,42,43].

Liver lipotoxicity is a consequence of obesity [44]. Thus, garlic extract oil and DADS were shown to protect, dose-dependently, obese mice with long-term high-fat diet-induced non-alcoholic fatty liver disease (NAFLD), from lipid accumulation, inflammation, and oxidative damage by ameliorating lipid metabolic disorders and oxidative stress [45••]. Also, SAC orally administrated to neonatal rats attenuates neonatal fructose-induced programming for hepatic lipid accumulation in adulthood, but not against visceral obesity induced by a high fructose diet [41]. In the same way, aged garlic extract improved abnormal fat accumulation and insulin resistance, prevented triglyceride hepatic accumulation, and also improved gut microbiota in insulin-resistant mice [46]. In addition, some synergistic effects were found in the literature. It was reported that DATS and its combination with a synthetic anti-obesity drug Orlistat reduced body weight, ameliorated lipid profile, and restored liver function parameters on high-fat diet-induced obese rats [47]. Other garlic derivative, DADS, was shown to potentiate the anti-obesity effect of green tea in high-fat/high-sucrose diet-induced obesity [48].

The beneficial effects of garlic and their OSCs on obesity are well documented in many randomized clinical trials. A recent meta-analysis reported by Darooghegi Mofrad et al. [49] suggests that garlic supplementation seems to reduce waist circumference but not body weight and body mass index (BMI). In a parallel, double-blind, placebo-controlled, randomized study, 6 weeks of aged garlic extract consumption modulated immune cell distribution, prevented the increase of serum TNF-α and IL-6 concentrations, and reduced blood LDL concentration in adults with obesity. No effect was reported regarding BMI or waist circumference (WC) reduction [50]. Consumption of raw crushed garlic during 4 weeks significantly reduced components of MetS including WC, blood pressure, fasting blood glucose, and improved lipid profile, though no changes on BMI of patients with MetS were reported [51].

Although the lack of evidence in reducing body weight or BMI in clinical trials, many studies succeeded in ameliorate diseases associated to obesity. Thus, after 12 weeks of garlic supplementation, overweight and obese women with osteoarthritis decreased resistin levels, a pro-inflammatory adipokine and reduced the pain compared with placebo patients in a randomized, double-blind, placebo-controlled, parallel-design trial [52]. Matsumoto et al. reported in a prospective randomized double-blind study that consumption of aged garlic extract reduced low-attenuation plaque in patients with MetS [53].

It turns out clearly that more investigations on OSCs (both preclinical and clinical) would be very seasonable to fully define anti-obesogenic effects of garlic’s derivate compounds and thus use them properly and safety in preventing MetS.

OSCs and Mitochondria Dysfunction

Increased oxidative stress linked to inflammation involves mitochondrial dysfunction and mitochondria emerge as a critical and interlocutory piece of cardiovascular pathological events [54•, 55]. Consequently, mitochondrial uncoupling could be used to treat several human diseases, such as obesity, cardiovascular diseases, or neurological disorders. In this context, OSCs might target the cornerstone of pathological processes underlying cardiovascular disorders like inflammation, oxidative stress, and mitochondrial dysfunction [50, 56]. An unprecedented work by Tilli et al. showed that modulation of mitochondrial function associated with the use of OSCs was mainly due to the induction of the mitochondria-dependent apoptosis route [57•]. Consistently, unpublished results from our laboratory demonstrated a neuroprotective effect linked to OSCs due to a decrease in oxidative stress/fibrosis/apoptosis associated with reduction of angiotensin type 1 receptor (AT1) and mitochondrial edema, as well as prevention of dilated crests. Similar results were shown in adrenal and renal tissues of streptozotocin-induced diabetic rats with down-regulation of AT1 expression [58]. Likewise, in chronic kidney disease, allicin was reported to exert similar protection as AT1 inhibitor losartan, and in silico bioinformatics analyses supported the concept that allicin and losartan could interact with AT1 receptor by a similar mechanism [59].

Angiotensin-oxidative stress axis linked to mitochondrial dysfunction is a critical site of mechanistic interactions in obesity and insulin resistance [60], MetS [61], and inflammatory process [62]. Consistent with mitochondrial ultrastructural findings, Wang et al. demonstrated that NADPH oxidase activity was significantly decreased and heat shock protein 70 (Hsp70) was induced as a result of allicin treatment [63]. In the same way, allicin protective effect was demonstrated in spinal cord neurons model from glutamate-induced oxidative stress through regulating the Hsp70 pathway [64]. Hence, mitochondrial dysfunction is the final consequences of oxidative stress, over-activation of the renin–angiotensin–aldosterone system (RAAS) linked to the NADPH oxidase activity [65], as well as loss of protective response mediated by Hsp70 [66].

OSCs increase Hsp as part of its antioxidant protective effect [67]; in fact, our laboratory has previously discussed the potential protective role of Hsp70 linked to functional foods in atherosclerosis [68]. In close connection, the mitochondrial integrity is coordinated by multiple signaling pathways, including vitagenes—genes involved in preserving cellular homeostasis—that encode for Hsp32, Hsp70, as well as sirtuin protein systems [69]. Sirtuins are implicated in multiple cellular processes like apoptosis, inflammation, stress resistance, and—of special interest for this review—mitochondrial biogenesis [70]. To highlight, OSCs activate sirtuin 3 to prevent cardiac oxidative stress and mitochondrial dysfunction [71]. Moreover, OSCs improve mitochondrial function by activating a wide array of signaling pathways such as Nrf2. Thus, another mechanism to consider is the co-induction of both Hsp70 and Nrf2-dependent antioxidant genes as a coordinated adaptive cytoprotective system on the oxidative stress modulation [72]. Indeed, OSCs upregulate antioxidant proteins including Nrf2 and heme oxygenase-1 (HO-1) [73] and reduce inflammatory response by inhibiting nuclear factor κB (NF-κB) activation [74].

To further elucidate the signaling pathways decoupled concerning mitochondrial dysfunction, and consequently, propose therapeutic alternatives, Zhang et al. remarkably identify crucial several signaling pathways convergent which include NF-κB, FOXO, mTOR, Nrf-2, Klotho/fibroblast growth factor 23 (FGF23), and sirtuins [75••]. In brief, mTOR inhibition with enhanced sirtuin and Klotho signaling contributes to the mitochondrial integrity. Likewise, the transcription factor FOXO is strongly associated with mitochondrial functionality and longevity [76]. Of particular interest, OSCs modulate all these cell-signaling pathways by phosphatidylinositol 3-kinase (PI3K)-AKT axis linked to activation of sirtuin-1 signaling, which activate cellular stress-response pathways resulting in the upregulation of neuroprotective gene products [77].

Interestingly, OSCs attenuate brain mitochondrial dysfunction and cognitive deficit in obese-insulin resistant rats [78], and significantly protect neuronal injury preserving mitochondrial function, mitochondrial biogenesis, and mitochondrial fission/fusion balance in a Parkinson’s disease model induced by 6-hydroxydopamine [79•]. The latest findings suggest that OSCs, and specifically, its interaction with the multiple signaling pathways altered during mitochondrial dysfunction, represent an attractive therapeutic alternative.

OSCs: for Both Prevention and Treatment

The therapeutic potential of OSCs has been studied in numerous pathologies, and these compounds have not only shown favorable effects on several clinically relevant risk factors but also have been useful in the treatment of patients with atherosclerosis-related diseases [7]. Many observational studies and randomized controlled trials (RCT) recently published reported the effectiveness of garlic preparations on atherosclerosis, inflammation, and obesity [1, 8, 49, 80••, , 81]—pathologies of interest in this review. Among the different supplements, aged garlic extract (AGE) accounts for the most studied product associated with health benefits [31]. A study showed that short-term treatment with garlic powder (2 tablets/day during 3 months) could improve the endothelial function enhancing brachial flow-mediated dilation in patients with coronary artery disease [82]. Moreover, after 12 months of treatment, consumption of AGE (250 mg), supplemented with vitamin B6 (12.5 mg), vitamin B12 (100 μg), folate (300 μg), and L-arginine (100 mg), reduced the rate progression of epicardial, pericardial, periaortic, and subcutaneous adipose tissue volume, decreasing a metabolic risk factor associated with the severity of coronary artery calcification [83].

In another study, patients with uncontrolled hypertension were treated with AGE (1.2 g/day), and after 12 weeks, these patients reduced peripheral and central blood pressure and improved arterial stiffness, inflammation, and other cardiovascular markers [84]. High blood pressure has been associated to gut dysbiosis, with a significant decrease in microbial richness and diversity, and an increased inflammatory status. In a 3-month randomized controlled double-blind trial (RCT), Ried et al. found that Kyolic-aged-garlic-extract (1.2 g/day, 12 weeks) was effective in reducing blood pressure in patients with uncontrolled hypertension and could reduce inflammation and improve arterial stiffness, and gut microbial profile [85]. In addition, AGE (2–4 g /day, 12 months) was shown to reduce low-attenuation plaque in coronary arteries of patients with MetS [53]. Further research is needed to evaluate whether AGE has the ability to stabilize vulnerable plaque and decrease adverse cardiovascular events.

Another study performed in obese individuals found that AGE (400 mg/day, 3 months) favorably modifies endothelial biomarkers associated with cardiovascular risk such as hsCRP, LDL cholesterol, plasminogen activator inhibitor 1 (PAI-1), and total antioxidant status (TAS), suggesting that AGE can be used to overwhelm chronic inflammation in obese subjects [86]. Accordingly, a supplement of aged garlic (3.6 g/day, 6 weeks) was found to modulate immune cell distribution, prevent the increase of serum IL-6 and TNF-α concentrations, and reduce LDL cholesterol in adults with obesity, indicating beneficial effects of aged garlic in preventing the development of chronic diseases associated with low-grade systemic inflammation in adults with obesity [87]. Moreover, significant improvements in adipokines and leptin were found in overweight men and women underwent exercise and diet, and also, an adiponectin increase was found in the group which was additionally taking a dietary supplement containing garlic, capsaicin, raspberry ketone, caffeine, and Citrus aurantium (4 capsules/day, 8 weeks), indicating a better regulation of metabolic status [88].

The risk of cardiovascular disease and atherosclerosis progression in women is significantly increased after menopause, probably due to the decline of estrogen levels. It was reported that isoflavonoid-rich herbal preparation, containing tannins from garlic powder, may decrease the progression of existing atherosclerotic plaques and even suppress the formation of new ones in post-menopausal women after 12 months of treatment [89]. However, more recently, the same group has found no major improvement in atherosclerosis prevention, despite alleviation of climacteric syndrome using another phytoestrogen-rich natural preparation containing garlic powder [90].

Despite many studies have confirmed the effectiveness of AGE to reduce endothelial dysfunction, vascular inflammation and oxidative stress in a non-diabetic population, in high risk cardio-vascular patients with type 2 diabetes, 4-week treatment with AGE (1.2 g/day, 4 weeks) did not significantly improve the aforementioned risk factors nor insulin resistance [91], which leads to think that it will be necessary more studies to assess the long-term benefit of AGE in population with severe cardiovascular risk factors.

AGE is not the only way to incorporate garlic’s health beneficial OSCs, notwithstanding aged garlic preparations reduce pungent taste and smell and gastrointestinal discomfort caused by raw garlic [31]. Raw crushed garlic (100 mg/kg twice/day, 4 weeks) has shown beneficial effects on components of metabolic syndrome including waist circumference, systolic and diastolic blood pressure, triglycerides, fasting blood glucose, and significantly increased serum HDL cholesterol [51]. In a crossover feeding trial, the intake of raw crushed garlic (5 g) in a single meal can modify expression of immunity- and cancer-related genes in whole blood of humans [92]. Additionally, after a single acute dose of sofrito sauce consumption (240 g/70 kg), containing tomato, olive oil, onion, and garlic, a significant decrease in the inflammatory biomarkers hsCRP and TNF-α was observed [93].

A recent meta-analysis conducted to summarize results regarding the effects of garlic supplementation on serum inflammatory biomarkers in adults showed that garlic supplementation reduced serum concentrations of hsCRP, TNF-α, IL-6, but failed on changing serum leptin and adiponectin levels [49]. Regarding serum inflammatory markers, another randomized clinical trial suggested that 400 mg of standardized garlic extract administration twice a day for 8 weeks resulted in a significant reduction of IL-6 and erythrocyte sedimentation rate [94].

Overall, these studies highlight the need for adherence to a more Mediterranean-style, anti-inflammatory dietary pattern by increasing the consumption of anti-inflammatory foods, spices, and herbs and decreasing the consumption of pro-inflammatory foods in order to promote healthy lifestyle [95].

Conclusions

Garlic’s bioactive components include sulfur-containing compounds, such as allicin, alliin, ajoene, 2-vinyl-4H-1,3-dithiin (2-VD), diallyl sulfide (DAS), diallyl disulfide (DADS), diallyl trisulfide (DATS), and S-allyl-cysteine. Vast evidence and studies, including RTC, confirm garlic and some of its bioactive components, exert numerous and different biological functions, such as antioxidant, anti-inflammatory, immunomodulator, and mitochondrial protector, which lead to great health benefits. What would be missing is a profound study about OSC bioavailability and bioaccesibility, especially after ingesting different garlic forms (raw, aged, allicin, macerated garlic oil, etc.). In addition, the effects of processing garlic and the release into the bloodstream of derivate bioactive compounds should be evaluated to profoundly illustrate their way of action, as well as to determine the molecular mechanisms responsible for the numerous beneficial effects that OSCs have for human health.