Anti-Inflammatory and Vasorelaxant Effects Induced by an Aqueous Aged Black Garlic Extract Supplemented with Vitamins D, C, and B12 on Cardiovascular System

Multiple studies demonstrated biological activities of aged black garlic, including anti-inflammatory, antioxidant, and cardioprotective effects. We aimed to investigate the protective effects of an aged black garlic water extract (ABGE) alone or in association with multivitamins consisting of combined Vitamins D, C, and B12, on mouse heart specimens exposed to E. coli lipopolysaccharide (LPS). Moreover, we studied the hydrogen sulphide (H2S) releasing properties and the membrane hyperpolarization effect of the Formulation composed by ABGE and multivitamins, using Human Aortic Smooth Muscle Cells (HASMCs). ABGE, vitamins D and C, and the Formulation suppressed LPS-induced gene expression of cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α, interleukin (IL)-6, nuclear factor-kB (NF-kB), and inducible nitric oxide synthase (iNOS) on mouse heart specimens. The beneficial effects induced by the extract could be related to the pattern of polyphenolic composition, with particular regard to gallic acid and catechin. The Formulation also increased fluorescence values compared to the vehicle, and it caused a significant membrane hyperpolarization of HASMCs compared to ABGE. To conclude, our present findings showed that ABGE, alone and in association with multivitamins, exhibited protective effects on mouse heart. Moreover, the Formulation increased intracellular H2S formation, further suggesting its potential use on cardiovascular disease.


Introduction
Cardiovascular disease (CVD) represents the main cause of mortality all over the world. In this context, it has been demonstrated that inflammation and oxidative stress play a pivotal role in the development of CVD such as heart failure, acute coronary syndromes, atherosclerosis, and hypertension [1][2][3][4]. In agreement, an association between alterations in levels of pro-inflammatory and pro-oxidant markers and CVD has been found [5]. Various herbal extracts, particularly in pharmacological associations, were shown to be effective in decreasing the burden of inflammation and oxidative stress [6][7][8][9]. Moreover, hydrogen sulfide (H 2 S), a gaseous molecule, is critically involved in CVD. To this end, several preclinical and clinical studies suggested its protective role in heart failure, myocardial infarction, and hypertension [10].
The beneficial properties of garlic in CVD have been hypothesized to be related to polyphenolic compounds and SAC [22]. Multiple studies showed significant differences in the total phenolic content of black and fresh garlic. In particular, the content in phenolic compounds is 5-8-times higher in black garlic than that in fresh garlic [23].
The maintenance of cardiovascular health is also attributed to vitamins, such as vitamin B 12, vitamin C, and vitamin D [24].
In particular, vitamin D supplementation improves the cardiac function [25], while vitamin C supplementation, thanks to its antioxidant properties, is effective in the prevention or treatment of several cardiovascular diseases [26]. Furthermore, vitamin B12 deficiency promotes the onset of different CVD, including myocardial infarction, stroke, and other circulatory health problems [27].
The present study aims to investigate the potential antioxidant and anti-inflammatory effects of an ABG water extract (ABGE), alone or in association with multivitamins consisting of the combined Vitamin D, C, and B12 Formulation on mouse heart specimens exposed to Escherichia coli lipopolysaccharide (LPS), a known proinflammatory agent. In this context, we evaluated gene expression of various biomarkers involved in inflammation and oxidative stress, including cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-α, interleukin (IL)-6, nuclear factor-kB (NF-kB), and inducible nitric oxide synthase (iNOS). In addition, we studied the H 2 S releasing properties and the membrane hyperpolarization effect of ABGE, as well as the Formulation composed by ABGE and multivitamins using Human Aortic Smooth Muscle Cells (HASMCs).
The ABGE was also investigated in order to identify and quantify the polyphenolic content using high-performance liquid chromatography coupled with a photo diode array detector (HPLC-DAD) analytical method.

Preparation of ABGE
ABG cloves were supplied as dried material by il Grappolo S.r.l. (Soliera, Modena, Italy). Preparation of ABGE was performed as previously reported [28,29]. The detailed protocol is enclosed as supplementary materials.

Total Polyphenol Content of ABGE
Total polyphenol content was determined according to the Folin-Ciocalteu method, as described in Savini et al. (2017) [30] with some modifications. The detailed protocol related to total polyphenol content of ABGE is described in the Supplementary Materials Section.

HPLC-DAD-MS Analysis of Phenolic Compounds
The extract was analyzed for phenol quantitative determination using a reversed-phase HPLC-DAD-MS in gradient elution mode [31]. The details of the analysis are reported in Supplementary Materials (Tables S1 and S2).

Ex Vivo Studies
Adult C57/BL6 male mice (3-month-old, weight 20-25 g) were housed in Plexiglas cages (2-4 animals per cage; 55 × 33 × 19 cm) and maintained under standard laboratory conditions (21 ± 2 • C; 55 ± 5% humidity) on a 14/10 h light/dark cycle, with ad libitum access to water and normal laboratory chow (RMH-B diet, Arie Blok animal feed, Woerden, the Netherlands). Housing conditions and experimentation procedures were strictly in agreement with the European Community ethical regulations (EU Directive no. 63/2010) on the care of animals for scientific research. According to the recognized principles of "Replacement, Refinement and Reduction in Animals in Research", heart specimens were obtained as residual material from vehicle-treated animals randomized in our previous experiments, approved by the local ethical committee ('G. d'Annunzio' University, Chieti, Italy) and Italian Health Ministry (Project no. 885/2018-PR).
Extraction of total RNA was performed from the heart specimens using TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA), in agreement with the manufacturer's protocol. Contaminating DNA was removed using 2 units of RNase-free DNase 1 (DNA-free kit, Ambion, Austin, TX, USA). Determination of gene expression of COX-2, IL-6, NF-kB, TNF-α, and iNOS was performed by quantitative real-time PCR using TaqMan probebased chemistry, as previously reported [7,35]. The detailed protocol is described in the Supplementary Materials Section.

Cell Line
HASMCs were cultured in Medium 231 (Life Technologies, Carlsbad, CA, USA) supplemented with a Smooth Muscle Growth Supplement (SMGS, Life Technologies, Carlsbad, CA, USA) and 1% of 100 units/mL penicillin and 100 mg/mL streptomycin (Sigma Aldrich, St. Louis, MO, USA) in tissue culture flasks at 37 • C in a humidified atmosphere and 5% CO 2 , as previously described [36,37]. Cells were split 1:2 twice a week and used until passage 18.

Evaluation of H 2 S Release on HASMCs
After 24 h, to allow cell attachment, the medium was replaced and cells were incubated for 30 min in the buffer standard (HEPES 20 mM, NaCl 120 mM, KCl 2 mM, CaCl 2 ·2H 2 O 2 mM, MgCl 2 ·6H 2 O 1 mM, Glucose 5 mM, pH 7.4, at room temperature), as previously described [36,37]. The detailed experimental procedure is reported in the Supplementary Materials Section.

Evaluation of the Membrane Hyperpolarizing Effects on HASMCs
After 24 h to allow cell attachment, the medium was replaced and cells were incubated for 1 h in the buffer standard containing the bisoxonol dye bis-(1,3-dibutylbarbituric acid) DiBac4(3) (Sigma Aldrich, St. Louis, MO, USA) 2.5 µM [38]. NS1619 (Sigma-Aldrich, St. Louis, MO, USA) 10 µM, a BK Ca channel opener, was used as a reference drug. The ABGE (1-100 µg/mL), or the Formulation and the vitamins alone (Vitamin B12 1 µg/mL, Vitamin C 10 µg/mL and Vitamin D 1 µg/mL), were added to the cells, and the trends of fluorescence were followed for 35 min. The relative fluorescence decrease, linked to hyperpolarizing effects, was recorded every 2.5 min and was calculated as previously reported [38]. Six different experiments (n = 6) were performed.

Statistical Analysis
The data were analyzed by the licensed software GraphPad Prism version 6.0 (Graphpad Software Inc., San Diego, CA, USA). Analysis of means ± SEM for each experimental group was performed by one-way analysis of variance (ANOVA), followed by either the Newman-Keuls multiple comparison post hoc test or by the Bonferroni post hoc test [39]. The level of significance was set to 0.05. The Tukey-Kramer's Honest Significant Difference (HSD) test was used to compare the mean polyphenol contents of the extracts.

Total Polyphenol Content of ABGE
The ABGE provided a yield equal to 21.91 mg GAE/g DM in phenolic components [extraction yields of polyphenolic compounds obtained in ABGE (mg GAE/g DM): means ± SEM, 21.91 ± 1.07]. In our experiments, the ABGE showed a yield comparable to those reported by Najman et al. [35] (2021). Water extracts from conventional and organic black garlic have shown a content in polyphenolic components between 13.64 and 17.24 mg GAE/g DM [40]. In particular, a higher content in polyphenols was shown in black compared to fresh garlic, which was suggested to be dependent on various factors, including the garlic aging process (time, temperature, and relative humidity) [40].

HPLC-DAD-MS Analysis
The retention times, m/z ratio, as well as quantity (µg/mL) of the investigated phenolic compounds in ABGE are reported in Table 1. In this context, a total of 12 compounds were identified at a wavelength of 254 nm. Gallic acid (#1) and catechin (#4) were the prominent phytochemicals, as shown in Figure 1.
Results are only in part comparable to those reported in the literature [41]. A study performed by Moreno-Ortega and collaborators [42] (2020) has found an increase in phenolic compounds, such as gallic acid and epigallocatechin gallate, in black compared to fresh garlic. In addition, it is well known that each cultivar expresses a different analytes content dependent on cultivation methods. Different studies have confirmed that the bioactive compounds of ABG possess a wide range of pharmacological activities, such as hypolipidemic, anticancer, and cardiovascular effects [43], which have been suggested to be mainly due to its anti-inflammatory and antioxidant properties.

Toxicological and Pharmacological Studies
In the first series of experiments, we tested the effects of the ABGE (1-100 µg/mL) on the viability of cardiomyoblast (H9c2) cells. The experiments have been conducted both in basal conditions and after LPS-treatment for inducing an inflammatory status, in vitro. ABGE (1-100 µg/mL) did not alter H9c2 cell viability in basal conditions (Figure 2a). On the other hand, when H9c2 cells were treated with LPS, their viability was reduced, but ABGE (1-100 µg/mL) was able to revert the cytotoxicity (Figure 2b). In particular, preclinical and clinical evidence has demonstrated that inflammation and oxidative stress play a crucial role in various CVD, including hypertension, fibrosis, diastolic dysfunction, left ventricular hypertrophy, heart failure, and ischemia/reperfusion damage [44].
Therefore, we investigated the protective effects induced by ABGE (1-100 µg/mL) in mouse heart specimens stimulated with LPS, which represents a validated model to study the modulatory activities of herbal extracts and drugs on inflammatory pathways and oxidative stress [33,34]. In particular, we evaluated the effects of ABGE (1-100 µg/mL) on pro-inflammatory and pro-oxidant mediators, such as COX-2, TNF-α, IL-6, NF-kB, and iNOS mRNA levels on isolated LPS-stimulated heart specimens, by RT-PCR analysis. This demonstrates the involvement of NF-κB in the transcription of various proinflammatory cytokines, such as TNF-α, and IL-6 [45], whose involvement in mediating cardiac dysfunction is well known [46].
In our ex vivo model, we observed that ABGE (10 and 100 µg/mL) significantly inhibited all markers investigated without showing a dose-dependent relationship (Figure 3a-e). In this context, polyphenol compounds have been suggested to induce cardioprotective effects by inhibiting oxidative stress and inflammation, as confirmed by a recently published study [7,[47][48][49][50][51]. In particular, the beneficial activities induced by ABGE could be related to the pattern of polyphenolic composition, with particular regard to gallic acid and catechin. Accordingly, BenSaad and collaborators [52] (2017) reported that gallic acid inhibited LPS-induced prostaglandin E 2 and IL-6 production in RAW264.7 cells. Gallic acid was hypothesized to be able to exert a protective effect on rat liver mitochondria by reducing oxidative stress induced by bisphenol A in ex vivo studies [53]. In addition, gallic acid pretreatment decreased levels of cardiac marker enzymes, including troponin T, which has been hypothesized to be involved in the myocardial damage reduction in rats [54]. Cardioprotective activities of catechins are also well known [55]. In particular, catechin administration attenuated coronary heart disease in a rat model by suppressing inflammation [56]. Moreover, catechin, as well as being known for its antioxidant activities, has been described as an anti-inflammatory agent, being able to inhibit COX-2 expression [57,58].
Furthermore, black garlic was found to exert stronger antioxidant activity than fresh garlic, as confirmed by in vivo and in vitro experiments [41].
On the basis of these results, we performed a second series of experiments, aimed at evaluating the effects of the Formulation [ABGE (100 µg/mL) + Vitamin B12 (1 µg/mL) + Vitamin C (10 µg/mL) + Vitamin D (1 µg/mL)] on the viability of LPS-pretreated and not LPS-pretreated H9c2 cells. The results were compared with vitamins alone [Vitamin B12 (1 µg/mL), Vitamin C (10 µg/mL), and vitamin D (1 µg/mL)]. Our findings showed that the Formulation and the vitamins alone did not modify H9c2 cell viability in basal conditions (Figure 4a). In addition, the Formulation and the vitamins alone were able to contrast the cytotoxicity induced by LPS in H9c2 cells (Figure 4b). Thereafter, we investigated the effects induced by the Formulation and the vitamins alone on COX-2, TNF-α, NF-kB, IL-6, and iNOS mRNA levels in mouse heart specimens treated with LPS.
As shown in Figure 5a-e, vitamins C and D as well the Formulation reduced gene expression of almost all markers tested in our ex vivo study. In particular, the Formulation was more effective than vitamins alone in blunting LPS-induced gene expression of IL-6, TNF-α, and NF-kB. Recent studies reported that vitamin D represents one of the mediators playing a pivotal role in the pathogenesis of CVD [59]. In agreement, vitamin D supplementation was able to decrease inflammation and oxidative stress [59,60], confirming its pivotal role in heart tissue. In particular, TNF-α and IL-6 secretion was decreased by vitamin D in monocytes and macrophages [61]. Vitamin D also exerts various potent antioxidant effects by downregulating intracellular oxidative stress-related protein oxidation, lipid peroxidation, and DNA damage [62].
Similarly, an inverse correlation between vitamin C supplementation and the risk of CVD has been suggested in various observational studies [63,64]. In this regard, the antioxidant effects of vitamin C have been shown to be involved in both prevention and treatment of CVD [65]. In particular, Ellulu [62] (2017) showed that vitamin C protected against oxidative stress via its effect on nitric oxide release as well as alleviating inflammation by down-regulating IL-6, TNF-α, and NF-kB mRNA levels [66]. As for vitamin B12, its deficiency can cause hyperhomocysteinemia, an independent risk factor for CVD [27]. Moreover, an association between vitamin B12 deficiency and increased incidence of inflammation and associated metabolic complications has been demonstrated by a number of studies [67,68]. Our present findings showed that vitamin B12 decreased LPS-induced gene expression of NF-kB, IL-6, and iNOS. In agreement, Birch and collaborators [69] (2009) showed that vitamin B12 decreased NF-kB levels, which could represent a signaling molecule of vitamin B12 deficiency. Moreover, vitamin B12 was able to suppress IL-6 production, in vitro. Weinberg et al. [70] (2009) also reported that vitamin B12 is involved in the modulation of NOS function and NO synthesis in vivo.

Evaluation of H 2 S Release in HASMCs
H 2 S has been suggested to be able to modulate many pathways related to cardiovascular pathophysiology [71]. In addition, it is one of the most important biological mediators involved in different pathological processes, where inflammation plays a predominant role, including CVD [72]. H 2 S is known to be critically involved in garlic-induced cardioprotective effects [73][74][75]. In this context, H 2 S was shown to play a key role in preventing the progression of cardiac hypertrophy to heart failure [76]. Considering the inhibitory effects induced by both ABGE (1-100 µg/mL) and the Formulation [ABGE (100 µg/mL) supplemented with Vitamin B12 (1 µg/mL) + Vitamin C (10 µg/mL) + Vitamin D (1 µg/mL)] on the investigated markers of inflammation and oxidative stress in our study, we also evaluated their potential effects on H 2 S releasing properties using cultured HASMCs. DADS (300 µM) was used as a known H 2 S releasing molecule and significantly increased the fluorescence index, thus indicating the intracellular H 2 S formation (Figure 6a,b). We showed that ABGE did not determine significant H 2 S formation into the cells with respect to the vehicle (Figure 6a,b). Our findings are in agreement with those of Leitao et al. (2022) [77], showing that improvement of microvascular reactivity induced by aged garlic extract was not mediated by H 2 S in older adults at CVD risk. Interestingly, the Formulation significantly increased fluorescence values compared to the vehicle, reflecting the H 2 S formation inside the cells. In this context, we speculate that the presence of the vitamins into the Formulation allows the garlic extract to more easily cross the cell membrane and release H 2 S. In this regard, B vitamins could act as cofactors of enzymes playing a key role in the sulfur network and modulate H 2 S production [78]. Accordingly, Wilinski et al. [79] (2012) showed that vitamin D increased H 2 S levels in a number of mouse organs, including the heart [79]. membrane and release H2S. In this regard, B vitamins could act as cofactors of enzymes playing a key role in the sulfur network and modulate H2S production [78]. Accordingly Wilinski et al. [79] (2012) showed that vitamin D increased H2S levels in a number of mouse organs, including the heart [79]. An important finding of our study is that the Formulation tested has increased the release of H2S, suggesting its potential role on CVD, including hypertension, thanks to its vasodilatation action [80]. In addition to its vasoprotective effects, H2S could be critically An important finding of our study is that the Formulation tested has increased the release of H 2 S, suggesting its potential role on CVD, including hypertension, thanks to its vasodilatation action [80]. In addition to its vasoprotective effects, H 2 S could be critically involved in the pathogenesis of hypertension-related vascular dysfunction through its effects on blood pressure regulation, too, as well as inflammation [81][82][83][84].

Evaluation of Membrane Hyperpolarization of HASMCs
In addition, the effects of ABGE and the Formulation were evaluated on the membrane potential of cultured HASMCs. We showed that ABGE (1-100 µg/mL) did not modify membrane hyperpolarization. On the other hand, we showed that the Formulation caused a significant membrane hyperpolarization of HASMCs compared to ABGE (1-100 µg/mL) (Figure 7).  These results seem to suggest that the hyperpolarization is a consequence of the ability of the Formulation to release H2S. Indeed, it is well known that compounds able to release H2S, also called as H2S-donors, exhibited the property to induce vascular smooth muscle hyperpolarization through the activation of different subtypes of potassium channels [36,85].
Experimental and clinical studies showed that ABG was able to exert beneficial effects on cardiometabolic alterations, which are usually related to metabolic syndrome [86,87]. Accordingly, Amor and collaborators [22] (2019) showed an improvement of metabolic syndrome following ABG treatment in rats [22]. In this context, the aging process was suggested to enhance the activity of bioactive compounds, including Sallylcysteine and S-allylmercaptocysteine, whose cardioprotective effects are well known [88,89]. It is also well known that black garlic shows a reduced content of allicin when subjected to high temperatures during the production phase [87]. Moreover, Bradley and collaborators [73] (2016) suggested that allicin and alliin could not be the main bioactive These results seem to suggest that the hyperpolarization is a consequence of the ability of the Formulation to release H 2 S. Indeed, it is well known that compounds able to release H 2 S, also called as H 2 S-donors, exhibited the property to induce vascular smooth muscle hyperpolarization through the activation of different subtypes of potassium channels [36,85].
Experimental and clinical studies showed that ABG was able to exert beneficial effects on cardiometabolic alterations, which are usually related to metabolic syndrome [86,87]. Accordingly, Amor and collaborators [22] (2019) showed an improvement of metabolic syndrome following ABG treatment in rats [22]. In this context, the aging process was suggested to enhance the activity of bioactive compounds, including S-allylcysteine and S-allylmercaptocysteine, whose cardioprotective effects are well known [88,89]. It is also well known that black garlic shows a reduced content of allicin when subjected to high temperatures during the production phase [87]. Moreover, Bradley and collaborators [73] (2016) suggested that allicin and alliin could not be the main bioactive compounds involved in the cardioprotective effects induced by aged garlic.
The content of phytochemicals in garlic has also been reported to be dependent on environmental, genetic, and agronomic factors [90].
Interestingly, gallic acid was found able to induce hyperpolarization of the cell membranes and excitation of muscles by binding to glutamate-gated chloride channels [91]. Furthermore, catechins have also been suggested to display inhibitory effects on voltagedependent Ca 2+ channels involving, albeit partially, membrane hyperpolarization deriving from the opening of K + channels [92]. Finally, we hypothesized that the beneficial effects of the Formulation are due to the presence of gallic acid, catechin, and vitamins.
In conclusion, our results showed that ABGE, alone and in association with multivitamins consisting of combined Vitamins D, C, and B12, exhibited protective effects, as confirmed by the inhibitory activities on multiple inflammatory and oxidative stress-related pathways on mouse heart specimens exposed to LPS. These effects could be related, at least in part, to the ABGE content in polyphenolic compounds, with particular regards to gallic acid and catechin. Moreover, the Formulation increased intracellular H 2 S formation, and caused a significant membrane hyperpolarization of HASMCs, further suggesting its potential use on CVD. In this context, we speculate that the presence of the vitamins in the Formulation allows the garlic extract to more easily cross the cell membrane and release H 2 S. However, further studies using independent experimental paradigms are necessary to accurately evaluate the in vivo activity.

Data Availability Statement:
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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