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Chemopreventive functions and molecular mechanisms of garlic organosulfur compounds Cite this: Food Funct., 2014, 5, 833
Phoebe Zapanta Trio,a Sixiang You,bc Xi He,b Jianhua He,b Kozue Sakaoc and De-Xing Hou*abc Garlic (Allium sativum L.) has long been used both for culinary and medicinal purposes by many cultures. Population and preclinical investigations have suggested that dietary garlic intake has health benefits, such as lowering the risk of esophageal, stomach and prostate cancers. Extensive studies from laboratory and animal models have revealed that garlic has a wide range of biological activities, and garlic organosulfur compounds (OSCs) are responsible for the biological activities. However, the presence and potency of garlic OSCs vary with respect to the mode of garlic preparation and extraction. Cooked or processed garlic products showed different kinds of garlic OSCs, some of which are highly Received 10th October 2013 Accepted 29th January 2014
unstable and instantly decomposed. These facts, possibly gave paradoxical results on the garlic effects. In this review, we first summarized the biotransformation processes of garlic alliin into different garlic OSCs as well as the garlic OSCs compositions from different garlic preparations. Next, we reviewed the
DOI: 10.1039/c3fo60479a
chemopreventive functions and molecular mechanisms focusing on the anti-inflammation, antioxidation,
www.rsc.org/foodfunction
anti-diabetes and anticancer activity behind different garlic OSCs.
Introduction Since ancient times and until today, garlic (Allium sativum) has been a part of people's lives either as a culinary spice, therapeutic agent against common diseases, cleansing aid or energy booster for athletes and sports enthusiasts. In Biblical times, the Jewish diet was already composed of garlic.1 Even in ancient Greece as well as ancient Rome, consumption of garlic was believed to give courage to soldiers and sailors, protect the skin from toxins, and aid respiratory ailments.2 Furthermore, garlic in Asia served as a food preservative, a remedy for indigestion and fever, and an antimicrobial agent.3 Garlic is predominantly composed of organosulfur compounds (OSCs), giving a pungent smell and a spicy taste. The major sulphur-containing compounds in garlic are S-allyl-Lcysteine sulfoxide (alliin) and g-glutamyl-S-allyl-L-cysteine (GSAC).4 Alliin is a natural compound present in garlic and forms complexes with allinase, a lyase enzyme released from crushing, cutting and grinding of garlic bulb (Fig. 1).5 The resulting complex is unstable and undergoes dehydration in the presence of a cofactor pyridoxal-phosphate, thereby, yielding a
a
Course of Biological Science and Technology, United Graduate School of Agricultural Sciences, Faculty of Agriculture, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan. E-mail: [email protected]; Fax: +81 99 285 8649; Tel: +81 99 285 8649
b
The Engineering Research Center of Feed Safety and Efficient Utilization of Education Ministry, Hunan Agriculture University, Changsha, Hunan 410128, P.R.China
c Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan
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reactive intermediate, sulfenic acid, pyruvic acid and ammonia. Sulfenic acid is an unstable organic compound at room temperature and undergoes self-condensation resulting in diallyl thiosulnate (allicin).6 However, allicin is a highly unstable compound, and easily decomposes into dithiins, ajoenes and allyl suldes.7 Likewise, allyl suldes are also reduced into allyl mercaptan and allyl persulde.8 Whereas in vivo, alliin can react with glutathione (GSH) to produce S-allylmercaptoglutathione (SAMG), or with L-cysteine to produce S-allylmercaptocysteine (SAMC) (Fig. 1). Moreover, in human red blood cells, garlic-derived organic polysuldes, e.g., diallyl disulde (DADS) and diallyl trisulde (DATS), undergo nucleophilic substitution at the a-carbon upon reacting with the exofacial thiol-containing molecules, yielding S-allyl-glutathione and allyl perthiol.9 Then, allyl perthiol undergoes nucleophilic substitution at the S-atom, producing allyl glutathione disulde and hydrogen sulde (H2S). Allyl glutathione disulde further undergoes nucleophilic substitution at the a-carbon forming additional H2S.9 Polysuldes bioconversion into H2S is benecial in preventing the progression of cardiac hypertrophy and heart attack. It is facilitated by intracellular glutathione in the presence of glucose (Fig. 1). The existence and potency of garlic bioactive constituents vary with respect to its mode of preparation and extraction.10 During the processing, such as cutting or crushing, hundreds of OSCs are produced in a short span of time.11 Ingestion of raw garlic introduces allicin, which is metabolized into allyl mercaptan and DADS, while cooked garlic mostly delivers DADS and DATS and small amounts of other allyl mono-, di-, and polysuldes.12 Garlic
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Fig. 1 Biotransformation processes of alliin from garlic into different garlic OSCs. Alliin is a naturally present compound in garlic and forms complex with allinase, a lyase enzyme released from the crushing, cutting and grinding of the garlic bulb. As reacting with allinase, reactive intermediates including sulfenic acid, pyruvic acid and ammonia are yielded (a). Sulfenic acid is an unstable organic compound which undergoes self-condensation resulting to allicin (b). Allicin is easily decomposes into dithiins (c), ajoenes (d) and allyl sulfides (e) which further reduce into allyl mercaptan and allyl persulfide (f). In vivo, alliin can react with GSH to produce SAMG (g), or with L-cysteine to produce SAMC (h). Human red blood cells can convert garlic-derived DADS and DATS into hydrogen sulfide (H2S) (i).
essential oil, obtained by subjecting raw garlic to steam distillation, produces between 0.2% and 0.5% of DADS and DATS, but without any traces of water-soluble OSCs and allicin (Fig. 2).11,13 In fact, no traces of DADS and DATS are found in in vivo experiments, only oxidized forms of S-allyl methylsuldes alone.14 Conversely, dehydrated garlic powder is obtained by crushing, drying and pulverizing raw garlic. It contains 1% alliin, which is lower than the expected due to dehydration process.11 Oil maceration of garlic paves way to conversion of alliin into unstable OSCs, (e.g., dithiins, ajoenes and allyl suldes).15 Aged garlic extract is prepared by soaking whole or sliced garlic into water and alcohol mixture for a certain period of time. It mostly contains water-soluble OSCs, such as S-allylcysteine (SAC) and Sallylmercaptocysteine (SAMC). In vivo studies indicated the presence of water-soluble garlicderived OSCs in plasma, liver and kidney aer oral consumption; but absence of oil-soluble OSCs in blood and urine even
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aer taking ample amounts of garlic.16 Because of these paradoxical reports, there is no doubt that it still continues to captivate the attention of the scientic communities; even with over 3000 publications dedicated to garlic's bioactivities, health benets and disease-preventive action. Along with the potencies of garlic bioactive functions, we highlight the chemopreventive functions of garlic OSCs focusing in anti-inammation, antioxidation, anti-diabetes and anticancer in a variety of cell types, and compile the molecular evidences for these functions of oil and water-soluble OSCs in vitro and in vivo.
Antioxidant activity and underlying mechanisms Glutathione modulation and phase II enzymes activation Reactive oxygen species (ROS) are chemically reactive oxygencontaining molecules that impose damaging effects on
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Fig. 2 The presence and potency of garlic OSCs in the mode of preparation and extraction. In fresh garlic bulb (a), alliin is the major OSC. Garlic essential oil (b), obtained by subjecting raw garlic to steam distillation, contains allyl sulfides (DADS, DATS and AMTS). Garlic powder (c), obtained by crushing drying or pulverizing, contains alliin and allyl sulfides. Garlic macerates (d) by grinding whole garlic cloves contain allyl sulfides, dithiins and ajoenes. Aged garlic extract (AGE) (e) prepared by soaking whole or sliced garlic into water and alcohol mixture for a certain period of time mostly contains water-soluble OSCs such as SAC and SAMC.
biological macromolecules such as lipids, proteins and DNA.17 In response, cells develop a protective scheme to cope with the damage in the form of endogenous antioxidants.18 Dietary supplementation of garlic juice in alloxan-induced diabetic rats normalized thiobarbituric acid reactive substances and glutathione S-transferase (GST) activity in plasma, liver, testes, brain and kidney.19 Whereas in DMBA-induced buccal pouch carcinogenesis model, garlic inhibited lipid peroxidation by increasing reduced GSH, glutathione peroxidase (GPx) and GST activities.20 When garlic-derived allicin was supplemented to mice, it markedly lowered intracellular ROS with respect to the untreated group.21 This ROS lowering effect is modulated by various gene expressions.22 Microarray analysis further revealed that allicin upregulated phase II detoxifying enzymes (e.g., thioredoxin reductase 1 and 2, heme oxygenase 1 (HO-1) and glutamate L-cysteine ligase (Glcl)) while blocking ROS-dependent pathway activation (e.g., ERK1/2, JNK1/2 and AKT).21,22 Although allicin is easily degraded in the blood, it could still sustain the inhibition of OH radical formation.8 This is due to its rapid interaction with other thiol-containing molecules like L-cysteine and glutathione upon passing through the cellular membrane.8,23 As a result, new metabolites, SAMC and S-allylmercaptoglutathione (SAMG), are formed.24,25 The resulting metabolites serve as mediators in prolonging the antioxidant activity of allicin. SAMG is a strong antioxidant molecule,
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serving as an inhibitor of OH radicals and lipid peroxides.8 It induces glutathione production signicantly more than allicin.22 This could indicate that most of the allicin is modied by thiol proteins into SAMG upon penetrating the phospholipid membrane, and glutathione can dissociate from SAMG. Like allicin and SAMG, aged garlic extract-derived water-soluble OSCs also exhibit potent ROS scavenging effects resulting to the increase of super oxide dismutase (SOD), catalase (CAT) and GPx activities.26–30 For example, SAC, formed during the aging process from g-glutamyl-S-allylcysteine in the presence of g-glutamyl-transferase, exhibits cell protective function from LDL-induced injury via removal of peroxides.31 It is coupled by inhibition of intracellular glutathione depletion and NF-kB activation.32 In human T lymphocytes tumor necrosis factor a (TNF-a) and H2O2-induced cells, SAC also protects cells from ROS-induced injury via attenuation of NF-kB activity.33 However, substitution of allyl group with benzyl or propyl ameliorated its scavenging capacity.34 Therefore, SAC exhibits a cell protective mechanism against ROS by scavenging peroxides and modulating the intracellular glutathione cycle and NFkB pathway, despite the cell variability. Also, this indicates the effect of the allyl group attached to the sulfur atom of the cysteinyl backbone is towards detoxication. Meanwhile, in rat liver, oil-soluble garlic OSCs (e.g., diallyl sulde (DAS), DADS, and DATS) were found to lower oxidative
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stress by signicantly increasing enzymatic activities of glutathione reductase (GRd) and GST while reducing GPx activity.35 Subsequently, DAS, DADS, and SAC delayed the oxidation of human LDL and plasma via inhibiting the loss of CAT and GPx activities.36 Specically, DAS and DADS signicantly hindered superoxide formation from hypoxanthine and xanthine by inhibiting xanthine oxidase.37 In vivo, DAS supplementation induced pulmonic enzyme CAT, GST, GPx, and GRd enzyme activities which correlated with the increase of HO-1, CAT, SOD, GPx, and NAD(P)H:quinone oxidoreductase 1 (NQO1) mRNA expressions.38 This increase suggests that DAS could induce antioxidant enzymes in vivo via oral administration. Nitric oxide (NO), as a signaling molecule, mediates biochemical processes via chemical reactions that control accumulation of reactive nitrogen species and alteration of protein functions.39 A human intervention study indicated that fresh garlic ingestion increased plasma NO level as much as 2–8 folds aer 2–4 hours, respectively.40 The extract, usually containing a high amount of allicin, tends to exhibit an antioxidant property via mediating the NO production in endothelial cells.41 Thus, modulation of the NO signaling cascade protects the vascular tissues from superoxide formation, and prevents atherosclerosis.42 Furthermore, aqueous garlic homogenate showed an increase of antioxidant enzyme activities in isoproterenol-induced mice; but it tends to lose its ability to enhance antioxidant enzymes when NO was blocked.43 This follows that garlic compounds also exhibit inhibitory action against ROS through increasing of NO levels. Similarly, SAC also showed antioxidative activity against H2O2-induced oxidative stress via NO/cGMP signaling pathway in placental explants and TEV-1 cells.44 SAC treatment was able to revert to normal level NO, cGMP and eNOS reduced by H2O2-induction.
Modulation of the Nrf2-ARE pathway Nuclear factor-E2-related factor 2 (Nrf2), a transcription factor, is the main protein that complexes with MafK to bind with antioxidant response element (ARE) to initiate gene transcription. Nrf2 also mediates the expression of several phase II metabolizing enzymes such as HO-1, GST, NQO1, and Glcl.45 In colon cancer cells, garlic induced Nrf2 nuclear translocation leading to transactivation of the antioxidant enzymes; thereby, promoting apoptosis.46 It prevents cognitive dysfunction due to ageing.47 It also inhibits cardiac hypertrophy and dysfunction by enhancing NQO1 and g-GSC gene expressions via Nrf2 induction.48 Similarly, DAS, DADS and DATS are found to upregulate the expressions of antioxidant enzymes including HO-1 and NQO1 via Nrf2-mediated ARE activation in hepatic cells, and the potency looks dependent to the number of sulfur atoms and the corresponding allyl group.49 In gentamicininduced nephrotoxicity on rat models, DAS exhibited its renal protective role via the induction of antioxidant enzymes and suppression of inammatory cytokines which are attributed to Nrf2 activation followed by subsequent nuclear translocation via the ERK/p38 signaling pathway (Fig. 3).38,50 Therefore, DAS and DATS also downregulate ROS-induced NF-kB and MAPK signalings to exert the crosstalk with anti-inammatory activity.
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Review
Anti-inflammatory effects and mechanisms Inhibition of inammatory mediators Inammation is a complex biological response to harmful stimuli, infection or injury. Macrophages are usually activated to release various proinammatory molecules in response to inammatory stimuli. Recent epidemiological investigations have indicated that the dietary intake of garlic may reduce the risk of different types of malignant cancers by suppressing inammatory processes.5,51,52 Cellular experiments demonstrated that SAC inhibited NO production and iNOS expression.53 Even extract from aged red garlic could suppress NO production and iNOS expression in lipopolysaccharide (LPS)-activated RAW264.7 cells.54 DADS also suppressed the production of NO and PGE2 in LPS-stimulated BV2 microglia at a concentration-dependent manner.55 In addition, Liu et al. have compared the inhibitory effect of DATS, DADS and DAS on the suppression of iNOS expression and NO production.56 They found that the order of the inhibitory effect was DATS > DADS > DAS, suggesting that it is related to the number of sulfur atoms of the OSCs. Our group recently screened the inhibitory effects of most OSCs including GSAC, alliin, SAC, DAS, DADS, DATS, and allicin on cyclooxygenase 2 (COX-2) production in LPS-activated RAW264.7 cells, and found that DATS is the most effective inhibitor.57 OSCs have also been reported to attenuate proinammatory cytokines to exert an anti-inammatory effect. Allicin is observed to attenuate TNFa-induced proinammatory cytokines and chemokines.58 DADS suppressed LPS-induced proinammatory cytokines such as IL-1b and TNF-a in LPSactivated BV2 microglia cells.55 In our lab, we introduced the multi-plex technology to investigate the effect of DATS on the productions of 24 kinds of cytokines in LPS-activated mouse RAW264.7 cells, and found that DATS could suppress the production of IL-6, IL-10, IL-12, KC, MCP-1, and TNF-a among 24 kinds of cytokines.57 Modulation of the cellular signal pathways Accumulated data demonstrated that the NF-kB pathway mediates the expression of iNOS, COX-2 and various proinammatory cytokines. DAS is found to attenuate the activation of NF-kB.59 Whereas, DADS inhibited activation and translocation of p65 into the nucleus and repressed other transcription factors, e.g., c-Jun and c-fos.60 On the other hand, allicin could also suppress the degradation of IkB.58 In our laboratory, we have demonstrated that DATS effectively suppressed phosphorylation and the degradation of IkB in RAW264.7 cells, and attenuated the translocation of p65 into nuclear membrane. Moreover, DATS inhibited the phosphorylation of LPS-induced IKKa/b, an upstream kinase of IkB kinase.57 These data suggest that the downregulation of the IKKmediated NF-kB signaling pathway is involved in the inhibition of the LPS-induced inammation of DATS. Mitogen-activated protein kinases (MAPKs) including JNK, p38 and ERK play critical roles in inammatory responses to stimulate
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Fig. 3 The schematic mechanisms of the antioxidative activities of garlic OSCs. The mechanisms at least involve OSCs reacting with intracelluar glutathione to produce the thiol derivative, GSSA, since allicin could easily penetrate the cellular membrane and it readily reacts with the most abundant non-protein thiol in the mammalian system (a). OSCs modulate Nrf2-ARE pathway to enhance the expressions of antioxidative enzymes or protein genes (b), and also downregulate ROS-induced NF-kB and MAPK signalings to exert the crosstalk with anti-inflammatory activity (c) (also see Fig. 4).
the production of cytokines and inammatory mediators.61 Thus, the inhibition of MAPKs is considered a promising therapeutic strategy for chronic inammatory diseases such as rheumatoid arthritis, psoriasis, inammatory bowel disease, and chronic obstructive pulmonary disease. Several lines of study have demonstrated that DADS suppressed LPS-induced MAPKs signaling to attenuate inammation responses.55 Even, allicin inhibited cancer proliferation by inhibiting the ERK signaling pathway.62 In detail, DADS and DATS suppressed JNK and ERKmediated AP-1 activation and induced p class GST protein and mRNA expressions.63 DATS signicantly attenuated AP-1 activation and COX-2 expression via modulation of JNK or Akt signaling pathway.64 Moreover, it also inhibited the phosphorylation of TAK1 which is the common upstream regulator of both IKKa/b and MAPK signaling.55,57 Aside from that, it inhibited the phosphorylation of AKT1, which acts as an upstream protein kinase to activate IKK.65,66 These signaling data suggested that the inhibition of AKT1/TAK1-mediated MAPK and NF-kB pathways are involved in the inhibition of chemical-induced inammatory responses by OSCs (Fig. 4). Inhibition of inammation in vivo The in vivo anti-inammatory effects of DATS were conrmed in a mouse paw edema model induced by LPS.57 Mice were divided
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into three groups: control, LPS, and LPS plus DATS. DATS was injected i.p. (50 mg kg 1 body weight) for 4 days before LPS treatment. LPS was injected i.p. (1 mg kg 1 body weight), and the paw thickness was measured using a digital caliper before and every hour aer LPS treatment until 6 h. The results showed that DATS decreased the edema by 30.0% and 61.3%, compared with LPS alone aer 1 h and 6 h, respectively. Simultaneously, the serum levels of IL-6, MCP-1 and TNF-a induced by LPS were signicantly reduced in the mice group pretreated with DATS. The data conrmed in vivo the anti-inammatory effect of DATS.
Anti-diabetic effects and mechanisms Hypoglycemic effects Garlic has long been hypothesized to exhibit anti-diabetic activity but discrepancies of its hypoglycaemic activity still exist.67–70 Animal model studies showed that garlic extracts using different organic solvents could lower the fasting blood glucose level of diabetic rabbits, and improved oral glucose tolerance of both normal and diabetic animals.71 Similarly, Matthew and Augusti showed that garlic-derived allicin could improve oral glucose tolerance of mild but not severe alloxaninduced diabetes mellitus (DM) rats.67 Nevertheless, studies
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Fig. 4 Potential mechanisms of the anti-inflammatory activity of garlic OSCs. DATS and DADS can downregulate TAK1-mediated MAPK and NFkB signalings, and TRIF-mediated IRF3 pathway to suppress the expression of inflammatory enzymes such as COX-2, iNOS and inflammatory factors including IL-6, MCP-1 and TNF-a to attenuate inflammation.
using aqueous garlic, fresh garlic, and garlic oil continued to support that garlic has a blood glucose lowering potential.19,72,73 Conversely, ethanol extracted-steamed garlic showed no blood glucose lowering effects in normal rats with varied blood sugar concentrations.74 Whereas, garlic powder revealed a negative result in streptozotocin-induced diabetic rats.75 The contrasting results of these studies could be attributed by the differences in terms of available OSCs in the two garlic preparations. Garlic powder mostly contains alliin while steamed garlic contains allyl and methyl sulde derivatives.11 Thus, the allyl and methyl suldes might have a higher potential to lower glucose over alliin. Despite of these, garlic remained the most commonly used vegetable in the United States as a natural remedy for type 2 DM.76 Additionally, garlic-based formulation, allicor, when taken in conjunction with dietary treatments and sulfonyl urea derivative was found to have more effective metabolic control for type 2 DM.77 This follows that garlic could act synergistically with other anti-diabetic drugs. Though the contrasting results may have posed an incomprehensible hypoglycemic property of garlic, it should not be overlooked that the prevailing studies utilized different garlic preparations and extraction procedures, sample concentrations,
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experimental designs and treatment durations. These factors may have contributed to the disparateness. Also, the variability of the animal model with a complicated genetic and environmental background may also inuence the effects of garlic.
Anti-diabetic mechanisms Garlic OSCs could either act as an insulin secretagogue or insulin-sensitizer to exert anti-diabetic action.78 Matthew and Augusti observed the insulin-mimetic activity of allicin in alloxan-induced diabetic rabbits.67 Chang and Johnson observed a similar effect via the increase in the peripheral insulin's concentration of steamed garlic-fed non-diabetic rats.74 Henceforth, the account of Augusti and Sheela also proved that garlic OSCs could act as an insulin secretagogoue when they studied the effect of alliin in alloxan-induced diabetic rats for a month.79 The administration of alliin to diabetic rats alleviated the condition of the animals in the same degree as compared with glibenclamide and insulin supplementation. Furthermore, in vitro experiment using alliin as an insulinsecretagogue revealed that it has the ability to stimulate insulin secretion from b-cells isolated in normal rats.79 Therefore, alliin
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possibly accomplished its insulin-independent therapeutic effect by amplifying the antioxidant activity and lipolytic enzymes. The results supported the previous assumption of Matthew and Augusti that the possible increase of the peripheral insulin-like activity is either due to the direct stimulation of the compound on pancreatic b-cells or the indirect release of the gastrointestinal hormones related to the secretion of insulin in the pancreas.67 Moreover, a recent single-blind intervention study on type 2 DM-affected patients conrmed the blood glucose lowering activity of garlic tablets as an insulin-secretagogue.80 A garlic tablet which contains a high amount of alliin was found to lower signicantly the fasting blood sugar level of type 2 DM-affected individuals in a dose and time dependent manner.81,82 This result is in agreement with the previous human studies conducted on healthy individuals.83,84 The probable mechanism for this action is that allicin reacts with insulin-inactivating-sulfahydryl group compounds; thereby, preventing the inactivation of insulin. This mechanism might be feasible since OSCs could modify the protein function by interacting with the sulfahydryl domain. August and Matthew demonstrated the insulin-sensitizing of allicin in normal rats through the increased a-glucan phosphorylase activity in liver and inhibited action of glucose-6phosphatase.85 Hence, it is probable that allicin has a stimulating action on endogenous insulin, and a modulating function on the breakdown of glucose-6-phosphate into glucose and free phosphate. The insulin sensitivity potential of garlic oil and its constituent compound, DATS, were also observed in streptozotocin-induced diabetic rats. Liu et al. found out that garlic compounds could signicantly improve the insulin resistance as determined by a homeostasis model and the rst-order rate constant of glucose disappearance, implying that garlic may enhance the peripheral insulin sensitivity.86 These results partly illustrate the probability of garlic compounds as an insulinsensitizer in the lowering of blood glucose.
into the in vitro model did not potentiate a positive response. Zhang et al. revealed that tumor cell inhibition depends on allicin concentration and treatment time.90 Miron et al. further strengthened the previous study by performing an in situ approach of allicin generation.91 Introduction of alliin to a system having allinase conjugated with a monoclonal antibody directed towards targeted tumor cells, promoted conversion of alliin into allicin, and exhibited its anti-proliferative effects in a specic location without harming the surrounding healthy tissue.91 Recently, allicin was also identied as the most active compound of garlic water extract responsible for the anti-cancer activity in colon cancer cells.92 DAS, DADS, and DATS have been demonstrated to have anticancer activities in animal models induced by a variety of chemical carcinogens.93–95 In a transgenic adenocarcinoma of mouse prostate (TRAMP) cancer model, the oral gavage of DATS at 1–2 mg per day for 13 weeks signicantly inhibited the progression to poorly differentiated carcinoma and pulmonary metastasis multiplicity.96 Though it showed no signicant effect on apoptosis induction, angiogenesis or natural killer and dendritic cell function, it revealed tumor inhibition by reduction of Ki-67 and synaptophysin proteins, and increase of securin and cyclin B1 levels.96 Thus, DATS administration prevents the progression of invasive carcinoma and lung metastasis. In a PC-3 human prostate cancer xenogra and CT26 allogra tumor mice models, DATS induced Bax and Bak protein expressions with reduced hemoglobin level and volume respectively.97,98 The promotion of a chemically induced-mouse skin tumor was also found to be inhibited by DAS and DADS.99 Although a lower DATS dosage (i.e., 5 mmol) did not affect the tumor incidence, it reduced DMBA-initiated and TPA-promoted mouse skin tumor multiplicity by 25.63%. A higher dosage of DATS (i.e., 25 mmol) reduced both the incidence and the multiplicity of papillomas by 22.22% and 71.08%, respectively.100
Anticancer effects and mechanisms
Cell cycle arrest
Cancer is a disease with a complicated biology which stems from uncontrolled cell growth, and studies have revealed that garlic OSCs could modulate the different phases of carcinogenesis.87
When cells are exposed to stress, signal transduction pathways known as check points are switched on in the G1/S or G2/M phase, leading to cell cycle arrest.101 One of the earliest reports on OSCs treatment of cancer cells leading to cell cycle arrest was on allicin-treated leukemic cells where allicin caused cell cycle blockage of the S/G2/M boundary phase and the induction of apoptosis.102 A subsequent study on human colon cancer cells, HCT-15, showed that SAMC also inhibited cell growth at the G2/M phase in the cell cycle.103 Similarly, DADS treatment caused cell accumulation in the G2/M phase coupled with the reduction of the cells in G1 and S phases, and Cdk1 kinase activity.104 Cdk1 kinase could interact with cyclin B1 to form Cdk1-cyclin B1 complex which dictates transition of cells from G2 to M phase in the cell cycle.105 The complex formation is dependent on the binding of the catalytic subunit, Cdk1, to a regulatory unit, cyclin B1.106 Henceforth, the suppression of Cdk1 kinase activity due to DADS is attributed to the increase of cyclin B1 expression, Cdk1-cyclin B1 complex formation reduction, Cdk1 hyperphosphorylation inactivation, and
Tumor growth inhibition Di Paolo and Carruthers were the rst to study the tumor growth inhibition property of garlic.88 Some studies followed including the individual administration of garlic powder and extract containing alliin–allinase and alliin alone, respectively, in hepatic cells. A negative effect on tumor growth inhibition was found but extract administration supplemented with the powder showed signicant inhibition even at very low concentration.89 Results suggest that if alliin is already within the cell and allinase enzyme is introduced to the system, allinase became activated and could promote the breakdown of alliin into biologically active OSCs derivatives, e.g., allicin. Meanwhile, mixing of garlic powder and extract before introducing
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Cdc25C protein level reduction.107 The DADS-induced G2/M phase cell arrest is not cell-type specic as it behaved alike in lung, prostate and gastric cancer cell lines (e.g., A549, PC-3 and MGC-803).108–110 Another study, using DATS on BaP-induced MCF-10A cells, showed that it promoted cell cycle arrest. Pretreatment with DATS was the most effective attenuator of BaP-induced S/G2/M phase shis.111 DATS pretreatment, and DATS and BaP cotreatment shied cells to the G1 and G2/M phases, respectively. Pretreatment with DATS prior to carcinogenic induction prevented damaged DNA replication; thus, allowing DNA repair whereas cotreating cells with DATS and BaP induced G2/M arrest, allowing the repair of damaged chromosomes. The results demonstrated that DATS pretreatment is more effective in inhibiting BaP-induced ROS. Similar results were also obtained in DATS-treated human colon cancer cells, HCT-15 and DLD-1.112 Treatment with DATS increased arrested cells at G2/M phase and caspase-3 activity, and disrupted microtubule network formation of cells and microtubules fragments at the interphase. In human liver tumor (J5) and prostate cancer cells (PC-3), studies on the effect of DAS, DADS and DATS on cell cycle arrest were also done. Data indicated that garlic allyl compounds arrested cell cycle via modulation of cyclin and Cdks protein expressions in human liver tumor cells.113 DATS-treated cells showed the highest increase of cyclin B1 and decrease of Cdk7 protein expressions whereas DAS had the lowest contribution. Since Cdk7 is essential in the activation of Cdk1 which promotes the entering of cells in M phase from G2 phase, this suggests that DATS prevents G2/M phase transition of damaged cells by upregulating cyclin B1 and Cdk7 supression. Similarly, DADS treatment arrested cells at the G2/M phase and caused an increase in cyclin B1 expression accompanied by the decrease of Cdk1 kinase activity in prostate cancer cells.109 In PC-3 and DU145 human prostate cancer cells, DATS causes degradation of ferritin thus increasing the levels of chelatable iron and leading to ROS generation.114,115 DATS-induced ROS generation results in down-regulation of Cdc25C and Cdk1 expressions in prostate cancer cells. The mechanisms at least involved the accumulation of inactive (Tyr15 phosphorylated) Cdk1/cyclin B1 kinase. DATS also causes ATR/Chk1-dependent prometaphase arrest in cancer cells that may involve ROS-dependent DNA double strand breaks (DNA–DSB). The Chk1 activation correlates with inactivation of anaphase-promoting complex/cyclosome (APC/C). Moreover, DATS causes JNK andERK1/2-dependent phosphorylation of Bcl-2 leading to reduced interaction between Bax and Bcl-2, which initiates mitochondria-mediated caspase activation and apoptosis in PC-3 and DU145 cells. DATS also inactivates Akt resulting in phosphorylation and mitochondrial translocation of proapoptotic protein Bad in prostate cancer cells.
Apoptosis Malfunction of apoptosis leads to tumor initiation, progression or metastasis. In vitro, allicin inhibits cell proliferation and promotes mitochondrial apoptosis in a concentration-dependent manner. Specically, induction of apoptosis by allicin
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happened via upregulation of the Bax/Bcl-2 ratio, induction of mitochondrial membrane potential decrease, and activation of caspase-3, -12 and cytochrome c.116,117 Whereas, garlic hexane extract induced apoptosis through the mediation of ROS production, resulting in the decrease of mitochondrial membrane potential and antiapoptotic proteins, Bcl-2 and BclxL, followed by switching on caspase-9 and caspase-3 activities.118 The extract also activated caspase-8 and reduced the expression of BID, a proapoptotic Bcl-2 protein member of BH-3 domain. Also, DAS, DADS and DATS have been demonstrated to induce apoptosis in PC-3 cell model by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay.119 DATS exhibited the strongest activity followed by DADS and DAS, where apoptotic cells increased by 9, 4.3 and 2.6-folds, respectively, as compared with the control. Moreover, DATS increases Bax/Bcl-2 ratio, promotes collapse of the mitochondrial membrane potential, and inhibits ROS release.120 Aside from that, knockout of Bax and Bak proteins revealed apoptosis is controlled by Bax/Bak and not by Bcl-2 or Bcl-xL. On the contrary, DADS treatment in human breast cancer cells induced cell death via upregulation of Bcl-2, downregulation of Bcl-xL and activation of caspase-3.121 The ability of garlic to induce mitochondrial apoptosis depends upon the number of sulde atoms of the OSCs, as demonstrated in colon cancer cell experiment.122 Additionally, allicin triggered the mitochondrial release of cytochrome c via upregulation of Bax and Fas proteins and further prompted the activities of caspase-3, -8 and -9 in adenocarcinoma cells, SG7901.90 These data suggest that garlic OSCs induced apoptosis not only via the mitochondrial pathway but also through the death receptor pathway. Recently, diallyl tetrasulde (DATTS) was synthesized from allyl mercaptan and disulfur dichloride because the anti-cancer activity of the diallyl polysufanes likely increases with the length of the sulfur chain.123 Cancer cells (HCT116) were found to be less resistant against DATTS treatment as it exhibited continuous down-regulation of cell viability as compared to normal cells (ARPE-19). Mechanistic evaluation using cancer cells showed that DATTS induced cell arrest at G2/M phases leading to apoptosis via release of the cytochrome c into the cytoplasm, caspase 3 cleavage, PARP cleavage and induction of endoplasmic reticulum stress signalling pathway.123 In contrast, DATTS treatment on normal cells did not lead to apoptosis. This varying action could be attributed to the difference in the recovery of the thiol status in normal cells and cancer cells by DATTS. Although DATTS induce ROS in both normal cells (ARPE-19) and cancer cells (HCT116), it recover normal thiol concentration in normal cells faster than that in cancer cells, thus, normal cells is more resistant against DATTS-induced oxidative imbalance and apoptosis. Taken together, garlic OSCs induced apoptosis in cancer cells through the downregulation of proapoptotic Bcl-2 protein family and the upregulation of Bax, which promotes mitochondrial membrane collapse. The collapse of mitochondrial membrane enhances the release of cytochrome c from the mitochondria into the cytosol, and promotes the formation of Apaf-ATP-procaspase-9 complex which stimulates the activity of caspase-3 and -9 (Fig. 5).
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Autophagy
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Autophagy acts as either a tumor suppressor by inhibiting the building up of damaged proteins and organelles, or as a mechanism of cell survival promoting established tumors growth.124 Allicin induced p53-mediated autophagy in HepG2 cells by reducing cytoplasmic p53 and Bcl-2 levels, modulating the PI3K/mTOR signaling pathway and increasing the AMPK/ TSC2 and Beclin-1 expressions.125
Remarkable and conclusions Population investigations and extensive studies from laboratory and animal models have shown that garlic has a wide range of biological activities including antioxidant, anti-inammatory, antidiabetic and anticancer activities. The mechanisms underlying the biological activities have been partially claried as the
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ability of garlic to scavenge ROS, inhibit lipoprotein oxidation, induce endogenous antioxidant enzymes expressions, suppress inammation, lower glucose levels, inhibit tumor growth, promote apoptosis, and arrest cell cycle. The OSCs in garlic have been identied as bioactive compounds responsible for the biological activities of garlic. It was noticed that many OSCs are absent in intact garlic but are produced in cooked or processed garlic products. It is clear that the primary OSCs in intact garlic are GSAC, which are hydrolyzed and oxidized into alliin during storage.126 In intact garlic, bioactive DADS and DATS are not available. They are the decomposed product of allicin, which is catalyzed by alliinase, an enzyme released during crushing or chopping garlic process.7 The presence of different bioactive OSCs in intact garlic and cooked or processed garlic products are considered as the major reasons for the paradoxical reports in the previous studies of
Multiple molecular mechanisms of garlic OSCs towards anticancer activities. OSCs such as DATS, DADS and allicin can cause apoptosis in cancer cells by mitochondrial-mediated caspase activation pathways (a). Allicin, DATS and SAMC also can cause cell cycle arrest by downregulating cdc25B and cdc25C that results in the inactivation of Cdk1, and induces G2/M phase arrest (b). Allicin may induce p53-mediated autophagy by reducing cytoplasmic p53 and Bcl-2 levels, modulating the PI3K/mTOR signaling pathway and increasing the AMPK/TSC2 and Beclin-1 expressions (c).
Fig. 5
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intact garlic and its OSCs. For example, in a chemical experiment the thiol-derivative of allicin, SAMG, exhibited long-term antioxidant activity as compared with allicin which indicates that the allicin derivative is more stable when it comes to antioxidant potential. However, it cannot be disregarded that allicin could have been transformed into another compound upon prolonged standing and this compound might posses a different bioactivity. Other than that, discrepancies between garlic water-soluble OSCs and its oil-soluble counterparts have been observed. Water-soluble compounds are more potent cell cycle arrest and apoptosis inducer in hepatic cells as compared to its lipophilic derivatives.127 Nonetheless, it is still difficult to pinpoint which water-soluble OSCs are of the highest potencies for these chemopreventive effects because few studies are available on garlic transformations during metabolic processing, especially in hepatic cells. Conversely, in breast cancer cells, oil-soluble OSCs are more effective suppressors of cancer cell growth than the water-soluble OSCs.128 Therefore, we should not overlook the different garlic preparation and extraction procedures, sample concentration, experimental design and treatment duration because these factors contribute to the disparateness. Also, we must not disregard the fact that garlic compounds have different biological actions, potency and mechanisms in different cell lines and organ tissues. To further fully estimate the biological activities of garlic, rst of all, we have to standardize the garlic preparation conditions to obtain almost all of the bioactive OSCs. It is known that allinase is the key enzyme that facilitates the transformation of cysteine sulfoxides to thiosulnates, though cooking could inactivate allinase.129 Reports further showed that allicin could readily pass through the cellular membrane and reacts with thiol-containing molecules rapidly, indicating that it can be converted into thiolcontaining derivatives. Thus, it is recommended that letting garlic stand for ten minutes aer chopping or crushing before cooking should be primary point to get bioactive garlic compounds.5,130 Moreover, to shed light on this contradicting issues, it is recommended to perform a step by step metabolic study to determine which water-soluble molecules are involved in a specic signal transduction modulation, e.g., for cell arrest and apoptosis. This process could be just one way to solve the mystery of the different bioactivities of garlic OSCs.
Conflict of interest The authors have declared no conict of interest.
Abbreviations ARE CAT Chk COX-2 DADS DAS DATS DATTS
Antioxidant response element Catalase Checkpoint kinase Cyclooxygenase-2 Diallyl disulde Diallyl sulde Diallyl trisulde Diallyl tetrasulde
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DM Glcl GPx GRd GSAC GSH GSSG GST HO-1 iNOS LPS MAPK NFkB
Diabetes mellitus Glutamate-L-cysteine ligase Glutathione peroxidase Glutathione reductase g-Glutamyl-S-allyl-L-cysteine Glutathione Glutathione disulde Glutathione-S-transferase Heme oxygenase 1 Inducible nitric oxide synthase Lipopolysaccharide Mitogen-activated protein kinase Nuclear factor kappa-light-chain-enhancer of activated B cells NO Nitric oxide NQO1 NAD(P)H:quinone oxidoreductase Nrf2 Nuclear factor-E2-related factor 2 OSCs Organosulfur compounds PGE2 Prostaglandin E2 ROS Reactive oxygen species SAC S-Allylcysteines SAMC S-Allylmercaptocysteine SAMG S-Allylmercaptoglutathione SOD Superoxide dismutase TNFa Tumor necrosis factor a
Acknowledgements This work was supported by the Hunan Agriculture University grant for 100 Intelligent Projects, and the fund of the Scholar Research of Kagoshima University (to D. X. Hou).
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REVIEW
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Chemopreventive functions and molecular mechanisms of garlic organosulfur compounds Cite this: Food Funct., 2014, 5, 833
Phoebe Zapanta Trio,a Sixiang You,bc Xi He,b Jianhua He,b Kozue Sakaoc and De-Xing Hou*abc Garlic (Allium sativum L.) has long been used both for culinary and medicinal purposes by many cultures. Population and preclinical investigations have suggested that dietary garlic intake has health benefits, such as lowering the risk of esophageal, stomach and prostate cancers. Extensive studies from laboratory and animal models have revealed that garlic has a wide range of biological activities, and garlic organosulfur compounds (OSCs) are responsible for the biological activities. However, the presence and potency of garlic OSCs vary with respect to the mode of garlic preparation and extraction. Cooked or processed garlic products showed different kinds of garlic OSCs, some of which are highly Received 10th October 2013 Accepted 29th January 2014
unstable and instantly decomposed. These facts, possibly gave paradoxical results on the garlic effects. In this review, we first summarized the biotransformation processes of garlic alliin into different garlic OSCs as well as the garlic OSCs compositions from different garlic preparations. Next, we reviewed the
DOI: 10.1039/c3fo60479a
chemopreventive functions and molecular mechanisms focusing on the anti-inflammation, antioxidation,
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anti-diabetes and anticancer activity behind different garlic OSCs.
Introduction Since ancient times and until today, garlic (Allium sativum) has been a part of people's lives either as a culinary spice, therapeutic agent against common diseases, cleansing aid or energy booster for athletes and sports enthusiasts. In Biblical times, the Jewish diet was already composed of garlic.1 Even in ancient Greece as well as ancient Rome, consumption of garlic was believed to give courage to soldiers and sailors, protect the skin from toxins, and aid respiratory ailments.2 Furthermore, garlic in Asia served as a food preservative, a remedy for indigestion and fever, and an antimicrobial agent.3 Garlic is predominantly composed of organosulfur compounds (OSCs), giving a pungent smell and a spicy taste. The major sulphur-containing compounds in garlic are S-allyl-Lcysteine sulfoxide (alliin) and g-glutamyl-S-allyl-L-cysteine (GSAC).4 Alliin is a natural compound present in garlic and forms complexes with allinase, a lyase enzyme released from crushing, cutting and grinding of garlic bulb (Fig. 1).5 The resulting complex is unstable and undergoes dehydration in the presence of a cofactor pyridoxal-phosphate, thereby, yielding a
a
Course of Biological Science and Technology, United Graduate School of Agricultural Sciences, Faculty of Agriculture, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan. E-mail: [email protected]; Fax: +81 99 285 8649; Tel: +81 99 285 8649
b
The Engineering Research Center of Feed Safety and Efficient Utilization of Education Ministry, Hunan Agriculture University, Changsha, Hunan 410128, P.R.China
c Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Korimoto 1-21-24, Kagoshima 890-0065, Japan
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reactive intermediate, sulfenic acid, pyruvic acid and ammonia. Sulfenic acid is an unstable organic compound at room temperature and undergoes self-condensation resulting in diallyl thiosulnate (allicin).6 However, allicin is a highly unstable compound, and easily decomposes into dithiins, ajoenes and allyl suldes.7 Likewise, allyl suldes are also reduced into allyl mercaptan and allyl persulde.8 Whereas in vivo, alliin can react with glutathione (GSH) to produce S-allylmercaptoglutathione (SAMG), or with L-cysteine to produce S-allylmercaptocysteine (SAMC) (Fig. 1). Moreover, in human red blood cells, garlic-derived organic polysuldes, e.g., diallyl disulde (DADS) and diallyl trisulde (DATS), undergo nucleophilic substitution at the a-carbon upon reacting with the exofacial thiol-containing molecules, yielding S-allyl-glutathione and allyl perthiol.9 Then, allyl perthiol undergoes nucleophilic substitution at the S-atom, producing allyl glutathione disulde and hydrogen sulde (H2S). Allyl glutathione disulde further undergoes nucleophilic substitution at the a-carbon forming additional H2S.9 Polysuldes bioconversion into H2S is benecial in preventing the progression of cardiac hypertrophy and heart attack. It is facilitated by intracellular glutathione in the presence of glucose (Fig. 1). The existence and potency of garlic bioactive constituents vary with respect to its mode of preparation and extraction.10 During the processing, such as cutting or crushing, hundreds of OSCs are produced in a short span of time.11 Ingestion of raw garlic introduces allicin, which is metabolized into allyl mercaptan and DADS, while cooked garlic mostly delivers DADS and DATS and small amounts of other allyl mono-, di-, and polysuldes.12 Garlic
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Fig. 1 Biotransformation processes of alliin from garlic into different garlic OSCs. Alliin is a naturally present compound in garlic and forms complex with allinase, a lyase enzyme released from the crushing, cutting and grinding of the garlic bulb. As reacting with allinase, reactive intermediates including sulfenic acid, pyruvic acid and ammonia are yielded (a). Sulfenic acid is an unstable organic compound which undergoes self-condensation resulting to allicin (b). Allicin is easily decomposes into dithiins (c), ajoenes (d) and allyl sulfides (e) which further reduce into allyl mercaptan and allyl persulfide (f). In vivo, alliin can react with GSH to produce SAMG (g), or with L-cysteine to produce SAMC (h). Human red blood cells can convert garlic-derived DADS and DATS into hydrogen sulfide (H2S) (i).
essential oil, obtained by subjecting raw garlic to steam distillation, produces between 0.2% and 0.5% of DADS and DATS, but without any traces of water-soluble OSCs and allicin (Fig. 2).11,13 In fact, no traces of DADS and DATS are found in in vivo experiments, only oxidized forms of S-allyl methylsuldes alone.14 Conversely, dehydrated garlic powder is obtained by crushing, drying and pulverizing raw garlic. It contains 1% alliin, which is lower than the expected due to dehydration process.11 Oil maceration of garlic paves way to conversion of alliin into unstable OSCs, (e.g., dithiins, ajoenes and allyl suldes).15 Aged garlic extract is prepared by soaking whole or sliced garlic into water and alcohol mixture for a certain period of time. It mostly contains water-soluble OSCs, such as S-allylcysteine (SAC) and Sallylmercaptocysteine (SAMC). In vivo studies indicated the presence of water-soluble garlicderived OSCs in plasma, liver and kidney aer oral consumption; but absence of oil-soluble OSCs in blood and urine even
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aer taking ample amounts of garlic.16 Because of these paradoxical reports, there is no doubt that it still continues to captivate the attention of the scientic communities; even with over 3000 publications dedicated to garlic's bioactivities, health benets and disease-preventive action. Along with the potencies of garlic bioactive functions, we highlight the chemopreventive functions of garlic OSCs focusing in anti-inammation, antioxidation, anti-diabetes and anticancer in a variety of cell types, and compile the molecular evidences for these functions of oil and water-soluble OSCs in vitro and in vivo.
Antioxidant activity and underlying mechanisms Glutathione modulation and phase II enzymes activation Reactive oxygen species (ROS) are chemically reactive oxygencontaining molecules that impose damaging effects on
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Fig. 2 The presence and potency of garlic OSCs in the mode of preparation and extraction. In fresh garlic bulb (a), alliin is the major OSC. Garlic essential oil (b), obtained by subjecting raw garlic to steam distillation, contains allyl sulfides (DADS, DATS and AMTS). Garlic powder (c), obtained by crushing drying or pulverizing, contains alliin and allyl sulfides. Garlic macerates (d) by grinding whole garlic cloves contain allyl sulfides, dithiins and ajoenes. Aged garlic extract (AGE) (e) prepared by soaking whole or sliced garlic into water and alcohol mixture for a certain period of time mostly contains water-soluble OSCs such as SAC and SAMC.
biological macromolecules such as lipids, proteins and DNA.17 In response, cells develop a protective scheme to cope with the damage in the form of endogenous antioxidants.18 Dietary supplementation of garlic juice in alloxan-induced diabetic rats normalized thiobarbituric acid reactive substances and glutathione S-transferase (GST) activity in plasma, liver, testes, brain and kidney.19 Whereas in DMBA-induced buccal pouch carcinogenesis model, garlic inhibited lipid peroxidation by increasing reduced GSH, glutathione peroxidase (GPx) and GST activities.20 When garlic-derived allicin was supplemented to mice, it markedly lowered intracellular ROS with respect to the untreated group.21 This ROS lowering effect is modulated by various gene expressions.22 Microarray analysis further revealed that allicin upregulated phase II detoxifying enzymes (e.g., thioredoxin reductase 1 and 2, heme oxygenase 1 (HO-1) and glutamate L-cysteine ligase (Glcl)) while blocking ROS-dependent pathway activation (e.g., ERK1/2, JNK1/2 and AKT).21,22 Although allicin is easily degraded in the blood, it could still sustain the inhibition of OH radical formation.8 This is due to its rapid interaction with other thiol-containing molecules like L-cysteine and glutathione upon passing through the cellular membrane.8,23 As a result, new metabolites, SAMC and S-allylmercaptoglutathione (SAMG), are formed.24,25 The resulting metabolites serve as mediators in prolonging the antioxidant activity of allicin. SAMG is a strong antioxidant molecule,
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serving as an inhibitor of OH radicals and lipid peroxides.8 It induces glutathione production signicantly more than allicin.22 This could indicate that most of the allicin is modied by thiol proteins into SAMG upon penetrating the phospholipid membrane, and glutathione can dissociate from SAMG. Like allicin and SAMG, aged garlic extract-derived water-soluble OSCs also exhibit potent ROS scavenging effects resulting to the increase of super oxide dismutase (SOD), catalase (CAT) and GPx activities.26–30 For example, SAC, formed during the aging process from g-glutamyl-S-allylcysteine in the presence of g-glutamyl-transferase, exhibits cell protective function from LDL-induced injury via removal of peroxides.31 It is coupled by inhibition of intracellular glutathione depletion and NF-kB activation.32 In human T lymphocytes tumor necrosis factor a (TNF-a) and H2O2-induced cells, SAC also protects cells from ROS-induced injury via attenuation of NF-kB activity.33 However, substitution of allyl group with benzyl or propyl ameliorated its scavenging capacity.34 Therefore, SAC exhibits a cell protective mechanism against ROS by scavenging peroxides and modulating the intracellular glutathione cycle and NFkB pathway, despite the cell variability. Also, this indicates the effect of the allyl group attached to the sulfur atom of the cysteinyl backbone is towards detoxication. Meanwhile, in rat liver, oil-soluble garlic OSCs (e.g., diallyl sulde (DAS), DADS, and DATS) were found to lower oxidative
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stress by signicantly increasing enzymatic activities of glutathione reductase (GRd) and GST while reducing GPx activity.35 Subsequently, DAS, DADS, and SAC delayed the oxidation of human LDL and plasma via inhibiting the loss of CAT and GPx activities.36 Specically, DAS and DADS signicantly hindered superoxide formation from hypoxanthine and xanthine by inhibiting xanthine oxidase.37 In vivo, DAS supplementation induced pulmonic enzyme CAT, GST, GPx, and GRd enzyme activities which correlated with the increase of HO-1, CAT, SOD, GPx, and NAD(P)H:quinone oxidoreductase 1 (NQO1) mRNA expressions.38 This increase suggests that DAS could induce antioxidant enzymes in vivo via oral administration. Nitric oxide (NO), as a signaling molecule, mediates biochemical processes via chemical reactions that control accumulation of reactive nitrogen species and alteration of protein functions.39 A human intervention study indicated that fresh garlic ingestion increased plasma NO level as much as 2–8 folds aer 2–4 hours, respectively.40 The extract, usually containing a high amount of allicin, tends to exhibit an antioxidant property via mediating the NO production in endothelial cells.41 Thus, modulation of the NO signaling cascade protects the vascular tissues from superoxide formation, and prevents atherosclerosis.42 Furthermore, aqueous garlic homogenate showed an increase of antioxidant enzyme activities in isoproterenol-induced mice; but it tends to lose its ability to enhance antioxidant enzymes when NO was blocked.43 This follows that garlic compounds also exhibit inhibitory action against ROS through increasing of NO levels. Similarly, SAC also showed antioxidative activity against H2O2-induced oxidative stress via NO/cGMP signaling pathway in placental explants and TEV-1 cells.44 SAC treatment was able to revert to normal level NO, cGMP and eNOS reduced by H2O2-induction.
Modulation of the Nrf2-ARE pathway Nuclear factor-E2-related factor 2 (Nrf2), a transcription factor, is the main protein that complexes with MafK to bind with antioxidant response element (ARE) to initiate gene transcription. Nrf2 also mediates the expression of several phase II metabolizing enzymes such as HO-1, GST, NQO1, and Glcl.45 In colon cancer cells, garlic induced Nrf2 nuclear translocation leading to transactivation of the antioxidant enzymes; thereby, promoting apoptosis.46 It prevents cognitive dysfunction due to ageing.47 It also inhibits cardiac hypertrophy and dysfunction by enhancing NQO1 and g-GSC gene expressions via Nrf2 induction.48 Similarly, DAS, DADS and DATS are found to upregulate the expressions of antioxidant enzymes including HO-1 and NQO1 via Nrf2-mediated ARE activation in hepatic cells, and the potency looks dependent to the number of sulfur atoms and the corresponding allyl group.49 In gentamicininduced nephrotoxicity on rat models, DAS exhibited its renal protective role via the induction of antioxidant enzymes and suppression of inammatory cytokines which are attributed to Nrf2 activation followed by subsequent nuclear translocation via the ERK/p38 signaling pathway (Fig. 3).38,50 Therefore, DAS and DATS also downregulate ROS-induced NF-kB and MAPK signalings to exert the crosstalk with anti-inammatory activity.
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Anti-inflammatory effects and mechanisms Inhibition of inammatory mediators Inammation is a complex biological response to harmful stimuli, infection or injury. Macrophages are usually activated to release various proinammatory molecules in response to inammatory stimuli. Recent epidemiological investigations have indicated that the dietary intake of garlic may reduce the risk of different types of malignant cancers by suppressing inammatory processes.5,51,52 Cellular experiments demonstrated that SAC inhibited NO production and iNOS expression.53 Even extract from aged red garlic could suppress NO production and iNOS expression in lipopolysaccharide (LPS)-activated RAW264.7 cells.54 DADS also suppressed the production of NO and PGE2 in LPS-stimulated BV2 microglia at a concentration-dependent manner.55 In addition, Liu et al. have compared the inhibitory effect of DATS, DADS and DAS on the suppression of iNOS expression and NO production.56 They found that the order of the inhibitory effect was DATS > DADS > DAS, suggesting that it is related to the number of sulfur atoms of the OSCs. Our group recently screened the inhibitory effects of most OSCs including GSAC, alliin, SAC, DAS, DADS, DATS, and allicin on cyclooxygenase 2 (COX-2) production in LPS-activated RAW264.7 cells, and found that DATS is the most effective inhibitor.57 OSCs have also been reported to attenuate proinammatory cytokines to exert an anti-inammatory effect. Allicin is observed to attenuate TNFa-induced proinammatory cytokines and chemokines.58 DADS suppressed LPS-induced proinammatory cytokines such as IL-1b and TNF-a in LPSactivated BV2 microglia cells.55 In our lab, we introduced the multi-plex technology to investigate the effect of DATS on the productions of 24 kinds of cytokines in LPS-activated mouse RAW264.7 cells, and found that DATS could suppress the production of IL-6, IL-10, IL-12, KC, MCP-1, and TNF-a among 24 kinds of cytokines.57 Modulation of the cellular signal pathways Accumulated data demonstrated that the NF-kB pathway mediates the expression of iNOS, COX-2 and various proinammatory cytokines. DAS is found to attenuate the activation of NF-kB.59 Whereas, DADS inhibited activation and translocation of p65 into the nucleus and repressed other transcription factors, e.g., c-Jun and c-fos.60 On the other hand, allicin could also suppress the degradation of IkB.58 In our laboratory, we have demonstrated that DATS effectively suppressed phosphorylation and the degradation of IkB in RAW264.7 cells, and attenuated the translocation of p65 into nuclear membrane. Moreover, DATS inhibited the phosphorylation of LPS-induced IKKa/b, an upstream kinase of IkB kinase.57 These data suggest that the downregulation of the IKKmediated NF-kB signaling pathway is involved in the inhibition of the LPS-induced inammation of DATS. Mitogen-activated protein kinases (MAPKs) including JNK, p38 and ERK play critical roles in inammatory responses to stimulate
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Fig. 3 The schematic mechanisms of the antioxidative activities of garlic OSCs. The mechanisms at least involve OSCs reacting with intracelluar glutathione to produce the thiol derivative, GSSA, since allicin could easily penetrate the cellular membrane and it readily reacts with the most abundant non-protein thiol in the mammalian system (a). OSCs modulate Nrf2-ARE pathway to enhance the expressions of antioxidative enzymes or protein genes (b), and also downregulate ROS-induced NF-kB and MAPK signalings to exert the crosstalk with anti-inflammatory activity (c) (also see Fig. 4).
the production of cytokines and inammatory mediators.61 Thus, the inhibition of MAPKs is considered a promising therapeutic strategy for chronic inammatory diseases such as rheumatoid arthritis, psoriasis, inammatory bowel disease, and chronic obstructive pulmonary disease. Several lines of study have demonstrated that DADS suppressed LPS-induced MAPKs signaling to attenuate inammation responses.55 Even, allicin inhibited cancer proliferation by inhibiting the ERK signaling pathway.62 In detail, DADS and DATS suppressed JNK and ERKmediated AP-1 activation and induced p class GST protein and mRNA expressions.63 DATS signicantly attenuated AP-1 activation and COX-2 expression via modulation of JNK or Akt signaling pathway.64 Moreover, it also inhibited the phosphorylation of TAK1 which is the common upstream regulator of both IKKa/b and MAPK signaling.55,57 Aside from that, it inhibited the phosphorylation of AKT1, which acts as an upstream protein kinase to activate IKK.65,66 These signaling data suggested that the inhibition of AKT1/TAK1-mediated MAPK and NF-kB pathways are involved in the inhibition of chemical-induced inammatory responses by OSCs (Fig. 4). Inhibition of inammation in vivo The in vivo anti-inammatory effects of DATS were conrmed in a mouse paw edema model induced by LPS.57 Mice were divided
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into three groups: control, LPS, and LPS plus DATS. DATS was injected i.p. (50 mg kg 1 body weight) for 4 days before LPS treatment. LPS was injected i.p. (1 mg kg 1 body weight), and the paw thickness was measured using a digital caliper before and every hour aer LPS treatment until 6 h. The results showed that DATS decreased the edema by 30.0% and 61.3%, compared with LPS alone aer 1 h and 6 h, respectively. Simultaneously, the serum levels of IL-6, MCP-1 and TNF-a induced by LPS were signicantly reduced in the mice group pretreated with DATS. The data conrmed in vivo the anti-inammatory effect of DATS.
Anti-diabetic effects and mechanisms Hypoglycemic effects Garlic has long been hypothesized to exhibit anti-diabetic activity but discrepancies of its hypoglycaemic activity still exist.67–70 Animal model studies showed that garlic extracts using different organic solvents could lower the fasting blood glucose level of diabetic rabbits, and improved oral glucose tolerance of both normal and diabetic animals.71 Similarly, Matthew and Augusti showed that garlic-derived allicin could improve oral glucose tolerance of mild but not severe alloxaninduced diabetes mellitus (DM) rats.67 Nevertheless, studies
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Fig. 4 Potential mechanisms of the anti-inflammatory activity of garlic OSCs. DATS and DADS can downregulate TAK1-mediated MAPK and NFkB signalings, and TRIF-mediated IRF3 pathway to suppress the expression of inflammatory enzymes such as COX-2, iNOS and inflammatory factors including IL-6, MCP-1 and TNF-a to attenuate inflammation.
using aqueous garlic, fresh garlic, and garlic oil continued to support that garlic has a blood glucose lowering potential.19,72,73 Conversely, ethanol extracted-steamed garlic showed no blood glucose lowering effects in normal rats with varied blood sugar concentrations.74 Whereas, garlic powder revealed a negative result in streptozotocin-induced diabetic rats.75 The contrasting results of these studies could be attributed by the differences in terms of available OSCs in the two garlic preparations. Garlic powder mostly contains alliin while steamed garlic contains allyl and methyl sulde derivatives.11 Thus, the allyl and methyl suldes might have a higher potential to lower glucose over alliin. Despite of these, garlic remained the most commonly used vegetable in the United States as a natural remedy for type 2 DM.76 Additionally, garlic-based formulation, allicor, when taken in conjunction with dietary treatments and sulfonyl urea derivative was found to have more effective metabolic control for type 2 DM.77 This follows that garlic could act synergistically with other anti-diabetic drugs. Though the contrasting results may have posed an incomprehensible hypoglycemic property of garlic, it should not be overlooked that the prevailing studies utilized different garlic preparations and extraction procedures, sample concentrations,
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experimental designs and treatment durations. These factors may have contributed to the disparateness. Also, the variability of the animal model with a complicated genetic and environmental background may also inuence the effects of garlic.
Anti-diabetic mechanisms Garlic OSCs could either act as an insulin secretagogue or insulin-sensitizer to exert anti-diabetic action.78 Matthew and Augusti observed the insulin-mimetic activity of allicin in alloxan-induced diabetic rabbits.67 Chang and Johnson observed a similar effect via the increase in the peripheral insulin's concentration of steamed garlic-fed non-diabetic rats.74 Henceforth, the account of Augusti and Sheela also proved that garlic OSCs could act as an insulin secretagogoue when they studied the effect of alliin in alloxan-induced diabetic rats for a month.79 The administration of alliin to diabetic rats alleviated the condition of the animals in the same degree as compared with glibenclamide and insulin supplementation. Furthermore, in vitro experiment using alliin as an insulinsecretagogue revealed that it has the ability to stimulate insulin secretion from b-cells isolated in normal rats.79 Therefore, alliin
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possibly accomplished its insulin-independent therapeutic effect by amplifying the antioxidant activity and lipolytic enzymes. The results supported the previous assumption of Matthew and Augusti that the possible increase of the peripheral insulin-like activity is either due to the direct stimulation of the compound on pancreatic b-cells or the indirect release of the gastrointestinal hormones related to the secretion of insulin in the pancreas.67 Moreover, a recent single-blind intervention study on type 2 DM-affected patients conrmed the blood glucose lowering activity of garlic tablets as an insulin-secretagogue.80 A garlic tablet which contains a high amount of alliin was found to lower signicantly the fasting blood sugar level of type 2 DM-affected individuals in a dose and time dependent manner.81,82 This result is in agreement with the previous human studies conducted on healthy individuals.83,84 The probable mechanism for this action is that allicin reacts with insulin-inactivating-sulfahydryl group compounds; thereby, preventing the inactivation of insulin. This mechanism might be feasible since OSCs could modify the protein function by interacting with the sulfahydryl domain. August and Matthew demonstrated the insulin-sensitizing of allicin in normal rats through the increased a-glucan phosphorylase activity in liver and inhibited action of glucose-6phosphatase.85 Hence, it is probable that allicin has a stimulating action on endogenous insulin, and a modulating function on the breakdown of glucose-6-phosphate into glucose and free phosphate. The insulin sensitivity potential of garlic oil and its constituent compound, DATS, were also observed in streptozotocin-induced diabetic rats. Liu et al. found out that garlic compounds could signicantly improve the insulin resistance as determined by a homeostasis model and the rst-order rate constant of glucose disappearance, implying that garlic may enhance the peripheral insulin sensitivity.86 These results partly illustrate the probability of garlic compounds as an insulinsensitizer in the lowering of blood glucose.
into the in vitro model did not potentiate a positive response. Zhang et al. revealed that tumor cell inhibition depends on allicin concentration and treatment time.90 Miron et al. further strengthened the previous study by performing an in situ approach of allicin generation.91 Introduction of alliin to a system having allinase conjugated with a monoclonal antibody directed towards targeted tumor cells, promoted conversion of alliin into allicin, and exhibited its anti-proliferative effects in a specic location without harming the surrounding healthy tissue.91 Recently, allicin was also identied as the most active compound of garlic water extract responsible for the anti-cancer activity in colon cancer cells.92 DAS, DADS, and DATS have been demonstrated to have anticancer activities in animal models induced by a variety of chemical carcinogens.93–95 In a transgenic adenocarcinoma of mouse prostate (TRAMP) cancer model, the oral gavage of DATS at 1–2 mg per day for 13 weeks signicantly inhibited the progression to poorly differentiated carcinoma and pulmonary metastasis multiplicity.96 Though it showed no signicant effect on apoptosis induction, angiogenesis or natural killer and dendritic cell function, it revealed tumor inhibition by reduction of Ki-67 and synaptophysin proteins, and increase of securin and cyclin B1 levels.96 Thus, DATS administration prevents the progression of invasive carcinoma and lung metastasis. In a PC-3 human prostate cancer xenogra and CT26 allogra tumor mice models, DATS induced Bax and Bak protein expressions with reduced hemoglobin level and volume respectively.97,98 The promotion of a chemically induced-mouse skin tumor was also found to be inhibited by DAS and DADS.99 Although a lower DATS dosage (i.e., 5 mmol) did not affect the tumor incidence, it reduced DMBA-initiated and TPA-promoted mouse skin tumor multiplicity by 25.63%. A higher dosage of DATS (i.e., 25 mmol) reduced both the incidence and the multiplicity of papillomas by 22.22% and 71.08%, respectively.100
Anticancer effects and mechanisms
Cell cycle arrest
Cancer is a disease with a complicated biology which stems from uncontrolled cell growth, and studies have revealed that garlic OSCs could modulate the different phases of carcinogenesis.87
When cells are exposed to stress, signal transduction pathways known as check points are switched on in the G1/S or G2/M phase, leading to cell cycle arrest.101 One of the earliest reports on OSCs treatment of cancer cells leading to cell cycle arrest was on allicin-treated leukemic cells where allicin caused cell cycle blockage of the S/G2/M boundary phase and the induction of apoptosis.102 A subsequent study on human colon cancer cells, HCT-15, showed that SAMC also inhibited cell growth at the G2/M phase in the cell cycle.103 Similarly, DADS treatment caused cell accumulation in the G2/M phase coupled with the reduction of the cells in G1 and S phases, and Cdk1 kinase activity.104 Cdk1 kinase could interact with cyclin B1 to form Cdk1-cyclin B1 complex which dictates transition of cells from G2 to M phase in the cell cycle.105 The complex formation is dependent on the binding of the catalytic subunit, Cdk1, to a regulatory unit, cyclin B1.106 Henceforth, the suppression of Cdk1 kinase activity due to DADS is attributed to the increase of cyclin B1 expression, Cdk1-cyclin B1 complex formation reduction, Cdk1 hyperphosphorylation inactivation, and
Tumor growth inhibition Di Paolo and Carruthers were the rst to study the tumor growth inhibition property of garlic.88 Some studies followed including the individual administration of garlic powder and extract containing alliin–allinase and alliin alone, respectively, in hepatic cells. A negative effect on tumor growth inhibition was found but extract administration supplemented with the powder showed signicant inhibition even at very low concentration.89 Results suggest that if alliin is already within the cell and allinase enzyme is introduced to the system, allinase became activated and could promote the breakdown of alliin into biologically active OSCs derivatives, e.g., allicin. Meanwhile, mixing of garlic powder and extract before introducing
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Cdc25C protein level reduction.107 The DADS-induced G2/M phase cell arrest is not cell-type specic as it behaved alike in lung, prostate and gastric cancer cell lines (e.g., A549, PC-3 and MGC-803).108–110 Another study, using DATS on BaP-induced MCF-10A cells, showed that it promoted cell cycle arrest. Pretreatment with DATS was the most effective attenuator of BaP-induced S/G2/M phase shis.111 DATS pretreatment, and DATS and BaP cotreatment shied cells to the G1 and G2/M phases, respectively. Pretreatment with DATS prior to carcinogenic induction prevented damaged DNA replication; thus, allowing DNA repair whereas cotreating cells with DATS and BaP induced G2/M arrest, allowing the repair of damaged chromosomes. The results demonstrated that DATS pretreatment is more effective in inhibiting BaP-induced ROS. Similar results were also obtained in DATS-treated human colon cancer cells, HCT-15 and DLD-1.112 Treatment with DATS increased arrested cells at G2/M phase and caspase-3 activity, and disrupted microtubule network formation of cells and microtubules fragments at the interphase. In human liver tumor (J5) and prostate cancer cells (PC-3), studies on the effect of DAS, DADS and DATS on cell cycle arrest were also done. Data indicated that garlic allyl compounds arrested cell cycle via modulation of cyclin and Cdks protein expressions in human liver tumor cells.113 DATS-treated cells showed the highest increase of cyclin B1 and decrease of Cdk7 protein expressions whereas DAS had the lowest contribution. Since Cdk7 is essential in the activation of Cdk1 which promotes the entering of cells in M phase from G2 phase, this suggests that DATS prevents G2/M phase transition of damaged cells by upregulating cyclin B1 and Cdk7 supression. Similarly, DADS treatment arrested cells at the G2/M phase and caused an increase in cyclin B1 expression accompanied by the decrease of Cdk1 kinase activity in prostate cancer cells.109 In PC-3 and DU145 human prostate cancer cells, DATS causes degradation of ferritin thus increasing the levels of chelatable iron and leading to ROS generation.114,115 DATS-induced ROS generation results in down-regulation of Cdc25C and Cdk1 expressions in prostate cancer cells. The mechanisms at least involved the accumulation of inactive (Tyr15 phosphorylated) Cdk1/cyclin B1 kinase. DATS also causes ATR/Chk1-dependent prometaphase arrest in cancer cells that may involve ROS-dependent DNA double strand breaks (DNA–DSB). The Chk1 activation correlates with inactivation of anaphase-promoting complex/cyclosome (APC/C). Moreover, DATS causes JNK andERK1/2-dependent phosphorylation of Bcl-2 leading to reduced interaction between Bax and Bcl-2, which initiates mitochondria-mediated caspase activation and apoptosis in PC-3 and DU145 cells. DATS also inactivates Akt resulting in phosphorylation and mitochondrial translocation of proapoptotic protein Bad in prostate cancer cells.
Apoptosis Malfunction of apoptosis leads to tumor initiation, progression or metastasis. In vitro, allicin inhibits cell proliferation and promotes mitochondrial apoptosis in a concentration-dependent manner. Specically, induction of apoptosis by allicin
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happened via upregulation of the Bax/Bcl-2 ratio, induction of mitochondrial membrane potential decrease, and activation of caspase-3, -12 and cytochrome c.116,117 Whereas, garlic hexane extract induced apoptosis through the mediation of ROS production, resulting in the decrease of mitochondrial membrane potential and antiapoptotic proteins, Bcl-2 and BclxL, followed by switching on caspase-9 and caspase-3 activities.118 The extract also activated caspase-8 and reduced the expression of BID, a proapoptotic Bcl-2 protein member of BH-3 domain. Also, DAS, DADS and DATS have been demonstrated to induce apoptosis in PC-3 cell model by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay.119 DATS exhibited the strongest activity followed by DADS and DAS, where apoptotic cells increased by 9, 4.3 and 2.6-folds, respectively, as compared with the control. Moreover, DATS increases Bax/Bcl-2 ratio, promotes collapse of the mitochondrial membrane potential, and inhibits ROS release.120 Aside from that, knockout of Bax and Bak proteins revealed apoptosis is controlled by Bax/Bak and not by Bcl-2 or Bcl-xL. On the contrary, DADS treatment in human breast cancer cells induced cell death via upregulation of Bcl-2, downregulation of Bcl-xL and activation of caspase-3.121 The ability of garlic to induce mitochondrial apoptosis depends upon the number of sulde atoms of the OSCs, as demonstrated in colon cancer cell experiment.122 Additionally, allicin triggered the mitochondrial release of cytochrome c via upregulation of Bax and Fas proteins and further prompted the activities of caspase-3, -8 and -9 in adenocarcinoma cells, SG7901.90 These data suggest that garlic OSCs induced apoptosis not only via the mitochondrial pathway but also through the death receptor pathway. Recently, diallyl tetrasulde (DATTS) was synthesized from allyl mercaptan and disulfur dichloride because the anti-cancer activity of the diallyl polysufanes likely increases with the length of the sulfur chain.123 Cancer cells (HCT116) were found to be less resistant against DATTS treatment as it exhibited continuous down-regulation of cell viability as compared to normal cells (ARPE-19). Mechanistic evaluation using cancer cells showed that DATTS induced cell arrest at G2/M phases leading to apoptosis via release of the cytochrome c into the cytoplasm, caspase 3 cleavage, PARP cleavage and induction of endoplasmic reticulum stress signalling pathway.123 In contrast, DATTS treatment on normal cells did not lead to apoptosis. This varying action could be attributed to the difference in the recovery of the thiol status in normal cells and cancer cells by DATTS. Although DATTS induce ROS in both normal cells (ARPE-19) and cancer cells (HCT116), it recover normal thiol concentration in normal cells faster than that in cancer cells, thus, normal cells is more resistant against DATTS-induced oxidative imbalance and apoptosis. Taken together, garlic OSCs induced apoptosis in cancer cells through the downregulation of proapoptotic Bcl-2 protein family and the upregulation of Bax, which promotes mitochondrial membrane collapse. The collapse of mitochondrial membrane enhances the release of cytochrome c from the mitochondria into the cytosol, and promotes the formation of Apaf-ATP-procaspase-9 complex which stimulates the activity of caspase-3 and -9 (Fig. 5).
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Autophagy
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Autophagy acts as either a tumor suppressor by inhibiting the building up of damaged proteins and organelles, or as a mechanism of cell survival promoting established tumors growth.124 Allicin induced p53-mediated autophagy in HepG2 cells by reducing cytoplasmic p53 and Bcl-2 levels, modulating the PI3K/mTOR signaling pathway and increasing the AMPK/ TSC2 and Beclin-1 expressions.125
Remarkable and conclusions Population investigations and extensive studies from laboratory and animal models have shown that garlic has a wide range of biological activities including antioxidant, anti-inammatory, antidiabetic and anticancer activities. The mechanisms underlying the biological activities have been partially claried as the
Food & Function
ability of garlic to scavenge ROS, inhibit lipoprotein oxidation, induce endogenous antioxidant enzymes expressions, suppress inammation, lower glucose levels, inhibit tumor growth, promote apoptosis, and arrest cell cycle. The OSCs in garlic have been identied as bioactive compounds responsible for the biological activities of garlic. It was noticed that many OSCs are absent in intact garlic but are produced in cooked or processed garlic products. It is clear that the primary OSCs in intact garlic are GSAC, which are hydrolyzed and oxidized into alliin during storage.126 In intact garlic, bioactive DADS and DATS are not available. They are the decomposed product of allicin, which is catalyzed by alliinase, an enzyme released during crushing or chopping garlic process.7 The presence of different bioactive OSCs in intact garlic and cooked or processed garlic products are considered as the major reasons for the paradoxical reports in the previous studies of
Multiple molecular mechanisms of garlic OSCs towards anticancer activities. OSCs such as DATS, DADS and allicin can cause apoptosis in cancer cells by mitochondrial-mediated caspase activation pathways (a). Allicin, DATS and SAMC also can cause cell cycle arrest by downregulating cdc25B and cdc25C that results in the inactivation of Cdk1, and induces G2/M phase arrest (b). Allicin may induce p53-mediated autophagy by reducing cytoplasmic p53 and Bcl-2 levels, modulating the PI3K/mTOR signaling pathway and increasing the AMPK/TSC2 and Beclin-1 expressions (c).
Fig. 5
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intact garlic and its OSCs. For example, in a chemical experiment the thiol-derivative of allicin, SAMG, exhibited long-term antioxidant activity as compared with allicin which indicates that the allicin derivative is more stable when it comes to antioxidant potential. However, it cannot be disregarded that allicin could have been transformed into another compound upon prolonged standing and this compound might posses a different bioactivity. Other than that, discrepancies between garlic water-soluble OSCs and its oil-soluble counterparts have been observed. Water-soluble compounds are more potent cell cycle arrest and apoptosis inducer in hepatic cells as compared to its lipophilic derivatives.127 Nonetheless, it is still difficult to pinpoint which water-soluble OSCs are of the highest potencies for these chemopreventive effects because few studies are available on garlic transformations during metabolic processing, especially in hepatic cells. Conversely, in breast cancer cells, oil-soluble OSCs are more effective suppressors of cancer cell growth than the water-soluble OSCs.128 Therefore, we should not overlook the different garlic preparation and extraction procedures, sample concentration, experimental design and treatment duration because these factors contribute to the disparateness. Also, we must not disregard the fact that garlic compounds have different biological actions, potency and mechanisms in different cell lines and organ tissues. To further fully estimate the biological activities of garlic, rst of all, we have to standardize the garlic preparation conditions to obtain almost all of the bioactive OSCs. It is known that allinase is the key enzyme that facilitates the transformation of cysteine sulfoxides to thiosulnates, though cooking could inactivate allinase.129 Reports further showed that allicin could readily pass through the cellular membrane and reacts with thiol-containing molecules rapidly, indicating that it can be converted into thiolcontaining derivatives. Thus, it is recommended that letting garlic stand for ten minutes aer chopping or crushing before cooking should be primary point to get bioactive garlic compounds.5,130 Moreover, to shed light on this contradicting issues, it is recommended to perform a step by step metabolic study to determine which water-soluble molecules are involved in a specic signal transduction modulation, e.g., for cell arrest and apoptosis. This process could be just one way to solve the mystery of the different bioactivities of garlic OSCs.
Conflict of interest The authors have declared no conict of interest.
Abbreviations ARE CAT Chk COX-2 DADS DAS DATS DATTS
Antioxidant response element Catalase Checkpoint kinase Cyclooxygenase-2 Diallyl disulde Diallyl sulde Diallyl trisulde Diallyl tetrasulde
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DM Glcl GPx GRd GSAC GSH GSSG GST HO-1 iNOS LPS MAPK NFkB
Diabetes mellitus Glutamate-L-cysteine ligase Glutathione peroxidase Glutathione reductase g-Glutamyl-S-allyl-L-cysteine Glutathione Glutathione disulde Glutathione-S-transferase Heme oxygenase 1 Inducible nitric oxide synthase Lipopolysaccharide Mitogen-activated protein kinase Nuclear factor kappa-light-chain-enhancer of activated B cells NO Nitric oxide NQO1 NAD(P)H:quinone oxidoreductase Nrf2 Nuclear factor-E2-related factor 2 OSCs Organosulfur compounds PGE2 Prostaglandin E2 ROS Reactive oxygen species SAC S-Allylcysteines SAMC S-Allylmercaptocysteine SAMG S-Allylmercaptoglutathione SOD Superoxide dismutase TNFa Tumor necrosis factor a
Acknowledgements This work was supported by the Hunan Agriculture University grant for 100 Intelligent Projects, and the fund of the Scholar Research of Kagoshima University (to D. X. Hou).
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