Plants and Mushrooms as Possible New Sources of H2S Releasing Sulfur Compounds

Abstract

Hydrogen sulfide (H₂S), known for many decades exclusively for its toxicity and the smell of rotten eggs, has been re-discovered for its pleiotropic effects at the cardiovascular and non-cardiovascular level. Therefore, great attention is being paid to the discovery of molecules able to release H₂S in a smart manner, i.e., slowly and for a long time, thus ensuring the maintenance of its physiological levels and preventing “H₂S-poor” diseases. Despite the development of numerous synthetically derived molecules, the observation that plants containing sulfur compounds share the same pharmacological properties as H₂S led to the characterization of naturally derived compounds as H₂S donors. In this regard, polysulfuric compounds occurring in plants belonging to the Alliaceae family were the first characterized as H₂S donors, followed by isothiocyanates derived from vegetables belonging to the Brassicaceae family, and this led us to consider these plants as nutraceutical tools and their daily consumption has been demonstrated to prevent the onset of several diseases. Interestingly, sulfur compounds are also contained in many fungi. In this review, we speculate about the possibility that they may be novel sources of H₂S-donors, furnishing new data on the release of H₂S from several selected extracts from fungi.

1. Introduction

Hydrogen sulfide (H₂S) has been known for many decades for its toxicity and the smell of rotten eggs. Hydrogen sulfide poisoning usually occurs by inhalation; local irritant effects result in direct irritation to the eyes, causing conjunctival injection initially and corneal injury eventually. When inhaled, it leads to pulmonary injury presenting as hemorrhagic pulmonary edema. Hydrogen sulfide inhibits mitochondrial cytochrome oxidase by making a complex bond to the ferric moiety of the protein, therefore arresting aerobic metabolism. Once it enters the bloodstream and passes the blood–brain barrier, neurotoxic effects can be seen, namely dizziness, seizure, coma, and ultimately death. In brief, hydrogen sulfide is a known pulmonary irritant and asphyxiant that primarily causes respiratory and neurological clinical manifestations when inhaled. High concentrations (more than 700 ppm) have the potential to cause sudden death [1,2].

However, in the last twenty years, it has been discovered as the third endogenously produced gasotransmitter, in addition to nitric oxide and carbon monoxide [3].

The importance of characterizing and discovering new sources to be used as supplements of H₂S or developing new chemical entities endowed with H₂S-donor properties is due to the pleiotropic role of this gaseous molecule. Indeed, H₂S is involved in the regulation of numerous pathophysiological functions: it intervenes in the anti-inflammatory process [4], in oxidative stress [5], neuro-modulation [6], vaso-regulation [7], protection from ischaemia/reperfusion damage after myocardial infarction [8] and insulin resistance [9]. Furthermore, recent studies have shown that an abnormal metabolism and altered pathways of H₂S are related to the development of various cardiovascular and non-cardiovascular diseases (Figure 1) [10,11,12].

Numerous synthetically derived molecules have been developed, and the discovery of new interesting scaffolds able to release H₂S in biological environments, including thioureas [13], isothiocyanates [14,15,16], thiazolydindione [17], arylthioamide [18], and imminothioether derivatives [19], have highlighted the concrete usefulness of using these molecules for the treatment of several pathological conditions. Moreover, H₂S-donor moieties have also been used for developing hybrid drugs upon merging the chemical portion able to release H₂S to drugs already used in the clinic [20,21,22,23,24,25,26,27]. This strategy allows us to exploit the beneficial effect of H₂S and the well characterized pharmacological effect of the native drug.

In addition to synthetic molecules, the observation that plants containing sulfur compounds share the same pharmacological properties with H₂S led to characterize naturally derived compounds as H₂S donors. Polysulfuric compounds occurring in plants belonging to the Alliaceae family were the first to be characterized as H₂S donors, followed by isothiocyanates derived from vegetables belonging to the Brassicaceae family [28,29]. This important discovery paved the way to consider these plants as nutraceutical tools and their daily consumption has been demonstrated to prevent the onset of several diseases [30]. Sulfur compounds are also contained in many fungi. However, to date no data about their possible H₂S releasing properties have been reported.

This review aims to describe the pharmacological effects of sulfur compounds present in plants belonging to the Alliaceae and Brassicaceae families and provide new data on the H₂S-releasing properties of fungi extracts.

2. Hydrogen Sulfide Endogenous Production

In mammals, the endogenous production of this gasotransmitter is ensured by several enzymes, that are differently expressed in tissues, suggesting its crucial role in the control of homeostasis in numerous systems. Recently, H₂S has been reported to modulate target proteins through S-sulfhydration reactions, which are a post-translational modification of the hydrosulfuryl groups of cysteine residues responsible for the activation/inhibition of specific proteins [31,32].

H₂S is mainly bio-synthetized using two cytosolic enzymes: cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS), and one mitochondrial enzyme: 3-mercaptopyruvate sulfur-transferase (MST), using a shared substrate, the amino acid L-cysteine [33]. Interestingly, CBS is mainly but not exclusively expressed in the central nervous system (CNS), while CSE is recognized as the enzyme responsible for the endogenous production of H₂S in the cardiovascular system. More recently, additional/alternative biosynthetic pathways have been described; indeed, H₂S can be generated by cysteinyl-tRNA-synthetase enzyme and by selenium-binding protein 1, although their contribution is still unclear [34]. The wide distribution of biosynthetic pathways for the endogenous production of H₂S accounts for its ubiquitous and pleiotropic behavior.

With regards to metabolic processes, H₂S can follow different pathways: firstly, H₂S is a reducing molecule, and it is involved in reducing reactive oxygen species (ROS). Oxidation of H₂S represents the main catabolic function at the mitochondrial level, where H₂S is quickly metabolized to sulfite and sulfate species due to the involvement of quinone oxidoreductase, rhodanese, and sulfur dioxygenase [32].

3. Overview of Hydrogen Sulfide and Its Therapeutic Potential in Oxidative Stress-Based Pathologies

3.1. Oxidative Stress

It is well-known that oxygen is an indispensable molecule, but because of its structure, it can lead to the generation of highly unstable intermediates known as ROS [35]. When the ROS level exceeds the neutralizing capability of endogenous anti-oxidant systems, the tissue is exposed to oxidative stress. The accumulation of ROS is implicated in mitochondrial dysfunction of the cells due to oxidation of mitochondrial lipids leading to a decrease in the membrane potential and finally apoptosis [36]. In this regard, the nuclear transcription factor erythroid 2 (Nrf-2) is the main regulator of the anti-oxidant defense mechanisms within the cell. Nrf-2 modulates the gene expression of anti-oxidant and cytoprotective enzymes and is inhibited by Kelch-like ECH-associated protein 1 (Keap1). For this reason, Nrf-2 represents a possible promising pharmacological target to decrease cellular oxidative stress [37].

Although many studies have demonstrated direct H₂S scavenging action, the physiological concentration of H₂S is in the low nanomolar range, significantly lower than that of other endogenous anti-oxidant molecules [38]. Consequently, the anti-oxidant effects described for H₂S comprise the up-regulation of anti-oxidant defense systems [39].

H₂S is known for its ability to protect from oxidative stress by inducing an S-sulfhydration reaction of the Keap1 protein, in cysteine-151 residue (Figure 2B). This reaction induces a conformational change in Keap1 and its consequent dissociation from Nrf-2, which translocates into the nucleus. Here, Nrf-2 binds to promoters containing the anti-oxidant response element (ARE) gene sequence, favoring the expression of ARE-dependent genes encoding for major anti-oxidant enzymes, such as glutathione reductase, Catalase (CAT), Superoxide dismutase (SOD), and for non-enzymatic anti-oxidants, such as glutathione (GSH) and Thioredoxin 1 (Trx-1) [40].

3.2. Inflammation

H₂S promotes a significant anti-inflammatory effect mainly by inhibiting the pro-inflammatory transcription factor Nuclear factor kappaB (NF-kB) and preventing mast cell degranulation [41,42]. In a model of induced inflammatory response in pulmonary artery endothelial cells, H₂S inhibits the inhibitor of κβ kinase (IKκβ) enzyme activity through S-sulfhydration of the Cysteine 179 residue, thereby preventing NF-kB translocation into the nucleus [43,44] (Figure 2A).

H₂S also plays a regulatory role in macrophages, which are traditionally classified into two subgroups: M1, which are classically activated macrophages, are differentiated by the action of Th1 cytokines, and M2, which are alternatively activated macrophages, are differentiated by the action of Th2 cytokines, such as IL-4 or IL-13 [45].

M1s produce pro-inflammatory cytokines, and are important for antibacterial defense, while M2 macrophages mainly produce anti-inflammatory mediators and are involved in the anti-inflammatory and repair response [46]. H₂S promotes the polarization of macrophages toward M2, thus involving an anti-inflammatory effect (Figure 2D) [47].

3.3. Immune System

H₂S is also involved in the regulation of the immune system, playing a pivotal role in the regulation of neutrophils. Interestingly, neutrophils express all three major enzymes responsible for H₂S production, although recent studies have failed to measure detectable activity of CBS in the homogenate of human neutrophils [48]. Even if the mechanisms are complex, research data suggest that H₂S inhibits the adhesion of neutrophils [48]. The molecular mechanisms by which H₂S reduces leukocyte migration and adhesion in various experiments are different: activation of different classes of potassium channels [49], induction of heme oxygenase-1 [50], and downregulation of CD11b expression [51]. However, in the presence of a strong pro-inflammatory stimulus, H₂S stimulates neutrophil tissue adhesion and infiltration [52] through the upregulation of adhesion receptors, such as intercellular adhesion molecule 1 (ICAM-1) and P-selectin [53]. It is not contradictory with data reported earlier: under a septic condition, an increase in leukocyte infiltration is desirable since the organism is compromised.

Studies performed under microbial infection conditions showed that inhibition of H₂S biosynthesis increased the capacity for bacterial migration (and thus reduced the rate of killing) as fewer mycobacteria were internalized in the acidic vesicles of the macrophages [54]. This suggests that the biosynthesis of H₂S by the host organism is necessary for proper macrophage activity. In fact, when neutrophils were co-cultured with E. coli HB101 in the presence of H₂S for 24 h, the elimination of bacteria was more efficient than in the absence of H₂S [32].

3.4. Cardiovascular and Metabolic Systems

At the vascular level, H₂S induces most of its effects by S-sulfhydration of proteins, such as ion channels, enzymes, and receptors, which thereby undergo a conformational change responsible for their activation or inhibition [55,56].

One of the first reported mechanisms of action underlying H₂S-induced vasodilation consists of the activation of ATP-sensitive potassium (KATP) channels with the subsequent hyperpolarization of vascular smooth muscle cells [57]. Subsequently, it was shown that another important mechanism of action responsible for H₂S-induced vasodilation is the activation of voltage-dependent potassium channels belonging to the Kv7 family [58]. H₂S is also able to inhibit 5-phosphodiesterase (5-PDE) enzyme and has also been described as an endothelium-derived hyperpolarizing factor (EDHF) (Figure 2C) [59]. Several studies shed light on the cardioprotection played by this gasotransmitter against several kinds of damage, including the ischemia-reperfusion, diabetic cardiomyopathy, myocardial infarction, and drug-induced cardiotoxicity [60,61]. Although, multiple targets seem to be implicated, among which the regulation of nitric oxide (NO) levels [62], the up-regulation of 5’ adenosine monophosphate-activated protein kinase (AMPK) and Nrf-2 [63], the stimulation of mitochondrial ATP-sensitive potassium (mitoKATP) channels has been probably the most investigated target [64].

Moreover, H₂S is considered a master regulator of systemic metabolism, indeed through the S-sulfhydration of the insulin receptor substrate 1 (IRS-1), it may maintain insulin sensitivity and, upon direct S-sulfhydration at the cysteine139 site, it may increase peroxisome proliferator-activated receptor γ (PPARγ) activity, thereby changing glucose into triglyceride storage in adipocytes (Figure 2E) [65,66].

Based on the numerous beneficial effects that can be mediated by this gasotransmitter, great attention is being paid to the discovery of molecules able to release H₂S in a smart manner, i.e., slowly and for a long time, thus ensuring the maintenance of its physiological levels and preventing “H₂S-poor” diseases.

4. Naturally Derived H₂S-Donors

4.1. Polysulfides from the Alliaceae Family

Traditional medicine suggests daily consumption of garlic (Allium sativum L.) to maintain the physiological values of systolic and diastolic pressure, and several studies support the effects of garlic extracts and its polysulfides as cardiovascular-protective compounds [67,68,69]. The most abundant polysulfides present in garlic and in other edible species from the Alliaceae family, such as chives, onion, and shallot, are allyl sulfide derivatives, including diallyl disulfide and diallyl trisulfide, produced by the cleavage of odorless molecules, including S-alk(en)yl cysteine sulphoxide precursors due to the activity of alliinase enzyme. These secondary metabolites are S-methyl cysteine sulphoxide, S-allyl cysteine sulphoxide (known as alliin), S-propyl cysteine sulphoxide, and S-transprop-1-enyl cysteine sulphoxide [70]. Consequently, after the disruption of cells (upon cutting or cooking), alliinase enzyme, present in vacuoles, it is released and the reaction responsible for the formation of typical polysulfides occurs (Figure 3). The enrichment in organosulfur compounds is closely correlated with the aging of the vegetable; interestingly, it has been reported that reactive organosulfur compounds, such as allicin, are converted to their stable isoforms, such as S-allyl cysteine [71].

H₂S release is the main mechanism accounting for the vascular effects of polysulfides. The vasorelaxant effects following the administration of garlic extract in rat aortic rings were associated with a release of H₂S in the isolated organ bath [72]. Finally, the exact mechanism through which H₂S is released from polysulfides has also been described. They demonstrated that the functional group present in organic thiols, such as L-cysteine or glutathione, can promote a nucleophilic attack on the carbon atom in α-position to the allyl group and then trigger the rupture of the molecule, ultimately favoring the release of H₂S (Figure 4) [72].

In addition, other beneficial effects of garlic are related to the release of H₂S, including metabolic ones. Garlic shows potential beneficial effects in T2D [73,74,75]; this vegetable, and its derivatives are endowed with antihyperglycemic effects in genetic animal models of diabetes and in humans, thus preventing cardiovascular complications [76]. Consistently, garlic improves insulin sensitivity and the associated metabolic syndrome in animal models, and its derivatives reduce both insulin resistance and blood glucose in streptozotocin- and alloxan-induced diabetes [77].

Based on the putative targets identified for H₂S, it has been hypothesized that the contribution of garlic in the maintenance of glucose homeostasis or lipid profile is due—at least in part—to the release of H₂S, and several putative targets have been identified. Interestingly, polysulfides upregulated and S-sulfhydrated PPARγ and sirtuin 3 (SIRT-3) in cardiomyocytes and such a mechanism was relevant for assuring a prevention of diabetic cardiomyopathy in murine model [78].

4.2. Isothiocyanates from the Brassicaceae Family

The discovery about the H₂S releasing properties of the Alliaceae family paved the way for a great breakthrough in the research on plant species containing sulfur compounds, as a nutraceutical approach to be employed at early stages of several diseases. In this scenario, the Brassicaceae family is rich in glucosinolates and sulfur compounds that are metabolized into isothiocyanates because of the activation of myrosinase when these plants are cut or shredded [79]. Since there is an overlap between physio/biological effects evoked by isothiocyanates and those exerted by H₂S (as reported above), the H₂S-releasing properties of some natural isothiocyanates were evaluated [29]. Most of the compounds generated appreciable release of H₂S following incubation with cysteine. This behavior is due to the nucleophilic reaction of the thiol group of L-cysteine with the isothiocyanate moiety. This characteristic makes these molecules “smart H₂S donors” since they can donate the gaseous transmitter only in a biological environment. Recently, the chemical mechanism explaining the generation of H₂S from isothiocyanates has been clarified: the reaction between L-cysteine and isothiocyanates resulted in the generation of H₂S due to the generation of dithiocarbamic derivatives between L-cysteine and the isothiocyanate moiety. Intramolecular cyclization leads to the generation of dihydrothiazole derivatives and the formation of H₂S (Figure 5) [80].

Currently, isothiocyanates are attracting increasing interest mainly for their pharmacological effects at the cardiovascular level. In this regard, a recent study evaluated the vascular effects of erucin related to its H₂S-donating properties. In this study, the authors demonstrated that erucin releases H₂S in human aortic smooth muscle cells (HASMCs) in a concentration-dependent manner. Furthermore, a significant antihypertensive effect was reported in spontaneously hypertensive rats after erucin intraperitoneal administration, recording systolic pressure values similar to normotensive rats [81].

Moreover, erucin also promotes protective effects in oxidative stress and LPS-induced damage in endothelial and vascular cells, and at cardiac level [82,83,84].

Finally, an emerging role of H₂S is the regulation of glucose metabolism: several studies have been performed on Brassicaceae as a possible nutraceutical approach in an early stage of diabetes or combination with standard therapy. Consumption of Alliaceae and Brassicaceae can significantly improve blood glucose control in patients with diabetes, concluding that edible plants that contain sulfur compounds could be useful for treating type 2 diabetes (T2D). Furthermore, supplementation with edible plants containing sulfur compounds could significantly enhance the effect of standard therapy on glucose control [85].

Male balb/c mice fed for 10 weeks with a high-fat diet and supplemented with an extract obtained from the seeds of Eruca sativa Mill., rich in glucoerucin, showed a reduction in the body weight gain and improvement of glucose homeostasis. Interestingly, a decrease in white adipose tissue and the size of adipocytes was also observed. In addition, the extract improved adipocyte metabolism by enhancing the activity of citrate synthase and reducing triglyceride levels in mice fed a high-fat diet. Worthy of note, a diet rich in Brassicaceae has no adverse effects: the most common adverse events are mild gastrointestinal issues (heartburn, flatulence, stomach discomfort, increased defecation) and hot flashes [85].

Another isothiocyanate capable of releasing H₂S is moringin. This molecule derives from Moringa oleifera Lam., a plant belonging to the Moringaceae family that is widely used in the traditional medicine for the treatment of stomach pain, ulcers, vision defects, joint pain and as a digestive [86]. Specifically, the authors analyzed changes in the content of glucosinolates and isothiocyanates in different moringa tissues and measured the H₂S-releasing properties with the lead acetate assay, a method that takes advantage of the high affinity of divalent lead with H₂S to form a black precipitate of lead sulfide (PbS). The authors showed that moringa seeds and leaves had a significant amount of total glucosinolates that can be converted to isothiocyanates (mainly benzyl isothiocyanate) by the action of myrosinase [82].

4.3. Organosulfur Compounds Present in Mushrooms

Edible mushrooms have been widely used in cooking, but in recent years, their consumption for their medical benefits has increased. In this regard, similar to Alliaceae and Brassicaceae vegetables, the main areas of use are cardio-metabolic disorders and the regulation of the immune system. A recent systematic review highlights that the consumption of mushrooms—as part of a healthy dietary pattern—is associated with a reduced risk of mortality for cardiovascular diseases, suggesting benefits on cardiovascular and metabolic systems. It has been proposed that these benefits are correlated to hypolipidemic and hypoglycemic effects and anti-oxidants and anti-inflammatory activities [87,88,89,90]. More recently, a putative role in slowing down aging has also been considered and associated with the ability to influence the expression of hallmarks of senescence, including Nrf-2 and FOXOs [91]. Moreover, some authors supposed the role of supplementation with mushrooms to support the treatment of the early stages of neurodegenerative diseases or anti-cancer because of the high anti-oxidant and anti-inflammatory capacities [92,93,94]. Finally, with regard to the regulation of immune system, mushrooms are reported to stimulate cell surface receptor activity, enhancing the activity of natural killer cells, neutrophils and macrophages, responsible for anti-viral and anti-tumor responses [94].

Undoubtedly in these properties, an important contribution is due to the presence of polysaccharides (including β-glucans), enzymes, nucleic acids and other bioactive compounds, including ergosterol and monacolin k [92,93,94]; indeed, usually, the phyto-complex is considered to be responsible for a wide spectrum of beneficial effects; nevertheless today investigations on the mechanisms of action are poor, and a deeper exploration seems to be necessary.

Exploring further bioactive compounds endowed with putative healthy properties, a peculiar characteristic of these vegetables is their distinctive flavor, and the scent is critical in the determination of their quality and popularity. Indeed, a wide variety of volatile organic compounds has been described, and their content, along with non-volatile organic compounds, renders each edible mushroom unique, but at the same time, it can influence medicinal properties. Interestingly, among the volatile organic compounds, organic sulfur compounds have been described [95]. Their recognized precursors are L-cysteine and methionine, which can be converted into thioheterocycles, thioethers, thiols and thiophenes in enzymatic and non-enzymatic ways [96]. A peculiar organic sulfur compound typical of mushrooms and not present in other kinds of vegetable and nonvegetable cells is ergothioneine, an amino acid bearing a cyclized thioureidic group that participates in the health-promoting activity of mushrooms [97].

One of the most studied mushrooms is Lentinula edodes (well-known as shiitake). It is rich in sulfurs; indeed, the unique aroma of the mushroom is due to the presence of lenthionine (1,2,3,5,6-pentathiepane), a cyclic sulfur compound [98,99]. In addition to its sensorial properties, lenthionine is endowed with biological effects, including antibiotic and anti-aggregation properties [100]; therefore, great attention has been paid to it.

From a biosynthetic perspective, lenthionine derives from lentinic acid in a two-step enzymatic reaction. In particular, lentinic acid is activated upon the elimination of its γ-glutamyl moiety due to the action of γ-glutamyl transpeptidase, producing an L-cysteine sulfoxide derivative, which then undergoes α,β-elimination, catalyzed by cysteine sulfoxide lyase (better known as alliinase, present in Alliaceae vegetables), resulting in a highly reactive sulfenic acid intermediate. The sulfenic acid is then rapidly condensed to form thiosulfinate, which is often further transformed into other sulfur compounds, including lenthionine [101]. Drying conditions, probably due to the Maillard reaction, may deeply influence the organic volatile composition, particularly the content of organic sulfur compounds. The concentration of organic sulfur compounds in shiitake increased in the early stages of drying (about 0.5–1.5 h), and the most representative compounds were dimethyl trisulfide, thioanisole, and lenthionine [102]. In the middle stages of drying the organic sulfur compounds increased with the esters; while in the final stages (4–12 h) their concentration decreased. Interestingly, the sulfur perception as a negative impression of food was hypothesized; nevertheless, it could be interesting from a health point of view [103].

Currently, to the best of our knowledge, no organic sulfur compounds described in shiitake have been described as H₂S donors; on the other hand, poor information is currently available on the content of organic sulfur compounds in other edible mushrooms. Interestingly, our preliminary studies suggest it is a potentially new source of sulfur compounds endowed with H₂S-donor properties.

5. Analysis of H₂S Release from Mushroom Extracts

Using an amperometric approach [13,104], we screened a few extracts of mushrooms (listed in Table 1) to explore their ability to release H₂S.

Surprisingly, the extracts of Lentinula edodes Berk., Ganoderma lucidum Curtis and Polyporus umbellatus, tested using an amperometric method at a concentration of 1 mg/mL in the presence and absence of L-cysteine (4 mM), showed a cysteine-dependent release of H₂S. In detail, in the presence of this amino acid, Lentinula edodes was able to release about 2.5 μM of H₂S, while Ganoderma lucidum released 2.1 μM of H₂S after 30 min incubation and Polyporus umbellatus 2 μM. In the absence of cysteine, there was no increase in the concentration of H₂S (Table 1 and Figure 6).

Three other extracts showed an H₂S cysteine-mediated release but reached lower H₂S levels: Inonotus obliquus P. Karst., which showed a release of 1.8 μM, Agaricus subrufescens Peck., with a release of 1.6 μM and Cordyceps sinensis Berk. with a release of 1.2 μM. For these extracts, the release of H₂S in the absence of cysteine did not reach the instrument detection threshold (Table 1).

A further group could be identified using extracts with thiol-independent H₂S-donor properties: Grifola frondosa Dicks. which, both in the presence and absence of cysteine, reached values of approximately 1.1 μM and Hericum erinaceus Bull. which reached values of approximately 1.2 μM (Table 1).

Finally, the last group could be identified by including those extracts devoid of H₂S-donor properties, either in the presence or absence of cysteine: the extracts of Pleurotus ostreatus Jacq., Coprinus comatus O.F. Müll., Auricularia auricula Hook. f., Poria cocos F.A. Wolf and Phellinus igniarius L. did not reach the threshold of instrumental detectability for the release of H₂S.

Based on quantitative analysis, all mushroom extracts contained significant levels of ergothioneine. Agaricus subrufescens Peck., Pleurotus ostreatus Jacq., Coprinus comatus O.F. Müll., Lentinula edodes Berk., and Grifola frondosa Dicks. appear to be the extracts with the highest ergothioneine content, far exceeding 200 μg per gram of extract. Nevertheless, the levels of the amino acid detected did not correlate with the release of H₂S; therefore, we supposed that it did not significantly contribute to this property. Furthermore, using amperometric analysis, commercial ergothioneine incubated at a concentration of 1 mM in the presence and absence of cysteine showed a very modest release of H₂S, amounting to 0.38 μM. This result suggests that despite the molecule’s chemical–physical potential, it is not capable of donating H₂S, probably because cyclic thiourea does not exhibit the same reactivity as the previously tested thioureas [13].

The other organosulfur compound previously described in mushrooms is lenthionine. Therefore, we evaluated the release of H₂S by testing the isolated compound at a concentration of 1 mM. In the presence of cysteine, the release of H₂S from lenthionine reached very high concentrations (approximately 30 μM after 30 min of incubation) (Table 1). In contrast, in the absence of cysteine, lenthionine showed no release of H₂S, suggesting that the cysteine-derived thiols can interact with lenthionine, thus leading to the hydrolysis of the molecule and release of H₂S. Although this compound has already been described in the literature, this is the first time it has been identified as an H₂S donor. Based on these results, it is possible to hypothesize that Lentinula edodes owes H₂S-donor properties—at least in part—due to the presence of lenthionine. However, to our knowledge, this sulfur compound has not been identified in the other mushroom extracts that emerged from our screening as potential novel sources of H₂S; therefore, it might be challenging to further characterize the content of organosulfur compounds in mushroom extracts that have been tested.

6. Conclusions

Alliaceae and Brassicaceae families were confirmed to be the main natural sources of organosulfur compounds, endowed with pleiotropic beneficial effects that reflect—under numerous aspects—those exerted by H₂S.

In addition, among the vegetables in the Alliaceae and Brassicaceae families, mushrooms emerged as a new putative source of organosulfur compounds endowed with H₂S-donor properties, and this hypothesis paves the way for a re-analysis of the health benefits of these vegetables and for the exploration of a new field of study in which the gasotransmitter is implicated.

These original data presented in this review represent a novelty in the scenario of research on naturally derived H₂S donors. In this regard, a qualitative and quantitative analysis of the organosulfur compounds contained in the selected mushroom extracts will have to be carried out in order to associate the release of H₂S with specific chemical entity/ies. It will be the first step in discovering new natural sources of H₂S and studying further medicinal or nutraceutical applications of these vegetables.

Author Contributions

Conceptualization, L.T. and V.C. (Valentina Citi); methodology, V.C. (Valentina Citi); data curation, V.C. (Valentina Citi); writing—original draft preparation, L.T. and V.C. (Valentina Citi); writing—review and editing, V.C. (Vincenzo Calderone) and M.P.; supervision, V.C. (Vincenzo Calderone) and L.T.; funding acquisition, L.T. and V.C. (Vincenzo Calderone). All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

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Figures and Table

Figure 1. H2S-related disorders.

Figure 2. The figure schematically shows the main pharmacological activities of hydrogen sulfide, (A) H2S and inflammation: through the sulfhydration of IKkB, H2S inhibits the dissociation between Nf-kB and IkB, impeding the translocation of Nf-kB into the nucleus and promoting anti-inflammatory effects. (B) H2S and oxidative stress: H2S sulfhydrates Keap1 allowing the dissociation of Nrf-2 which translocates into the nucleus and promotes the expression of anti-oxidant species. Furthermore, H2S acts as a direct scavenger of ROS. (C) H2S and vasodilation: H2S induces the opening of potassium channels and inhibits PDE5, leading to hyperpolarization of vascular cells and increase in NO levels. (D) H2S and immune system: H2S facilitates the differentiation in M2 polarized macrophages, which produce anti-inflammatory mediators. (E) H2S and metabolism: H2S sulfhydrates PPAR- γ, increasing insulin sensitivity and lipid synthesis. Abbreviations: Nf-kB: nuclear factor kappa-light-chain-enhancer of activated B cells; IkB: IkappaB kinase; IKkB: inhibitor of nuclear factor kappa-B kinase subunit beta; P: phosphate; Nrf-2: Nuclear factor erythroid 2-related factor 2; Keap1: Kelch-like ECH-associated protein 1; ARE: anti-oxidant responsive elements; KATP: ATP-sensitive potassium channel; Kv7: voltage-gated potassium channels; PDE5: Phosphodiesterase Type 5; PPAR—γ: Peroxisome proliferator-activated receptor gamma.

Figure 3. Schematic representation of the biosyntethic reactions leading to the formation of allylsulfur compounds in Alliaceae vegetables.

Figure 4. Mechanism of the release of H2S from allylsulfur compounds present in Alliaceae vegetables. The reaction is triggered by a nucleophilic attack of organic thiols (here represented by glutathione (GSH)) that promotes the formation of H2S and perthiols, which generate another molecule of H2S and glutathione disulfide (GSSG).

Figure 5. Mechanism of the release of H2S from organic isothiocyanates (ITCs) compounds present in Brassicaceae vegetables. The reaction is triggered by the nucleophilic attack of thiols (here represented by cysteine) and leads to the formation of an instable intermediate, that spontaneously decomposes in H2S and aminic derivatives.

Figure 6. Amperometric recordings of H2S release after the incubation of Lentinula edodes (a), Ganoderma lucidum (b) and Polyporus umbellatus (c) 1 mg/mL in the presence or absence of L-Cysteine. Data are expressed as mean ± SEM.

Table 1

Maximum H₂S release after the incubation of the mushroom extracts or ergothioneine and lenthionine.

Tested Item	Ergothioneine Content (µg/g)	H2S Release in the Presence of L-Cys 4 mM (μM)	H2S Release in theAbsence of L-Cys (μM)
Ganoderma lucidum	39.1 ± 0.2	2.1 ± 0.2	n.d.
Hericum erinaceus	184.6 ± 0.9	1.2 ± 0.2	1.2 ± 0.3
Lentinula edodes	348.3 ± 1.2	2.5 ± 0.1	n.d.
Grifola frondose	205.1 ± 3.4	1.1 ± 0.2	1.0 ± 0.2
Polyporus umbellatus	25.7 ± 0.8	2.0 ± 0.6	n.d.
Auricularia auricola	4.24 ± 0.1	n.d.	n.d.
Inonotus obliquus	0.3 ± 0.0	1.8 ± 0.1	n.d.
Agaricus subrufescens	479.3 ± 4.6	1.6 ± 0.4	n.d.
Poria cocos	5.4 ± 0.3	n.d.	n.d.
Pleurotus ostreatus	477.7 ± 2.7	n.d.	n.d.
Coprinus comatus	450.2 ± 4.8	n.d.	n.d.
Cordyceps sinensis	5.9 ± 0.1	1.2 ± 0.4	n.d.
Ergothioneine	n.d.	n.d.	n.d.
Lenthionine	n.d.	32 ± 4.3	n.d.

Abbreviations: L-Cys: L-Cysteine; n.d.: not detected.

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