Garlic

Allium sativum (of the Allioideae subfamily) is a food product known as Garlic in the same genus as onion plants (allium) alongside some other common food products such as onion, garlic, chive, leek, and rakkyo. The genera contains above 500 different plants, and while in the past it was placed in the Liliaceae family it now resides as the largest genera in the Amaryllidaceae family.^[1][2][3] There are some other vegetables that bear the name of garlic but are different species, and this includes Tulbaghia violacea (Sweet Garlic) and Allium ursinum (Wild Garlic).^[4]

There are two main varieties (variants or var.) of garlic named either ophioscorodon (full designation is Allium sativum var. ophioscorodon), which is also called hardnecked garlic or purple striped garlic, and sativum (literally Allium sativum var. sativum), which is also called creole garlic or artichoke garlic.^[1][5] Other variants include Voghiera (large bulbed Italian variety),^[6] Spring Garlic (pinkish and with a milder taste),^[7]

Garlic is a plant known as Allium sativum, which is a plant related to both onions and leeks, and it can be found in a wide variety of 'variants' (variations of the typical vegetable), which are specifically cultivated for their taste properties or yield

Garlic appears to have historical usage for being a medicinal food product as the Babylonians, Egyptians, Phoenicians, Vikings, Chinese, Greeks, and Romans have reported usage of garlic^[8][9] for intestinal disorders, flatulence, worms, respiratory infections, skin diseases, wounds, symptoms of aging, and a variety of other purposes (as a general prophylactic).^[8] It can be traced back about 6,000 years with known cultivation 5,000 years ago in India for medicinal purposes and recorded usage in China (3,000 years ago) and Egypt (1,550 BC)^[10][11] and appears to have been given to slaves to increase their ability to do more labour^[10] and was given to Grecian athletes and is sometimes referred to as the first performance enhancing supplement.^[11]

It also appears to be quite a popular supplement, with survey research in 2002 (USA) suggesting that 3.76% of the population used garlic supplements^[12] and in Australia (2007) this number was 10.7%, with 29.8% of those using garlic supplements in Australia (3.18% of the population) using it for the treatment of colds.^[13]

Traditional usage of garlic tends to be catered towards intestinal health and longevity, but it has a fair bit of traditional usage in bolstering the defenses of the body (this can be interpreted as either the immune system, anti-infective properties, or actual physical enhancement) and there are limited reports of its cardiovascular benefits

When looking at the garlic bulb itself (overall vegetable product) it tends to contain:

• A water content of around 65%^[10]
• A carbohydrate content of around 28% (which is mostly fructans)^[10]
• Protein at around 2% (mostly alliinase and glycoproteins) and 1.2% free amino acids^[10]
• 2.3% organosulfur compounds (commonly seen as the main bioactives)^[10]
• 1.5% dietary fiber^[10]

Garlic bioactives are somewhat unique in the vegetable, as there are two main groups of molecules that exist in the actual clove prior to processing; alliin (S-allylcysteine sulfoxide) and the Glutamyl-S-allylcysteine molecules. These two classes are some of the organosulfur compounds mentioned above, and aside from being relatively balanced (unless otherwise processed) they make up the majority of the organosulfurs.^[14]

When the clove is mechanically disturbed (chewing, slicing, crushing) then alliin turns into allicin via alliinase and then allicin spontaneously creates all manner of bioactives and gives off some hydrogen sulfide (H₂S) in the process. When the clove ages, the Glutamyl-S-allylcystiene molecules slowly lose their glutamyl moieties and it increases levels of S-allylcysteine (SAC) among some other similar cysteine prodrugs.

Garlic contains two main classes of molecules, which spontaneously form a wide variety of bioactives. This includes alliin (main sulfur containing compound in fresh garlic), which converts into allicin via the above pathway, and the glutamyl-S-allylcysteine, which gradually form S-allylcysteine during the aging process (which can then form SAMC and SMC)

The known bioactives of garlic are:

• Alliin (S-allylcysteine sulfoxide) as one a pool of bioactives at 10mg/g fresh weight and 30mg/g dry weight of raw garlic^[14] and of which 70-80% degrades into Allicin (Diallyl thiosulfinate; not present in the garlic initially^[8][15]) and is then further degraded into the Diallyl sulfides, the Ajoene molecule^[16] and the dithiin class of cyclic molecules^[17]
• The Glutamyl-S-allyl-L-cysteine class of molecules (second initial pool of bioactives) including γ-glutamyl-S-allyl-L-cysteine and γ-glutamyl-S-(trans-1-propenyl)-L-cysteine, two molecules present in garlic in high levels alongside low levels of γ-glutamyl-S-allyl-mercapto-L-cysteine and S-allylcystiene (SAC);^[18] SAC content increases during aging of garlic from 200µg/g to 7,200µg/g^[19] and is seen as the main bioactive of 'aged' garlic^[20]
• The diallyl sulfides, which include; diallyl sulfide, diallyl disulfide (DADS), diallyl trisulfide (DATS or Allitridi), and diallyl tetrasulfide (DATTS),^[21][22][23] which are seen as the main bioactives of garlic oil and main derivatives of allicin;^[22] letting allicin sit for 20 hours results in DADS (66.7%), DATS (14.6%), DAS (13.3%) and diallyl tetrasulfide (5.4%)^[24] with higher polysulfides being of sparse quantities;^[25] diallyl sulfides are seen as the main metabolite(s) of allicin
• A cyclical form of alliin known as cycloalliin^[26] and the fat soluble^[16] cyclical derivatives of allicin^[17] known as vinyldithiins such as 1,2-vinyldithiin;^[27] these derivates are more sparse than the diallyl sulfides^[28]
• Ajoene ((E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide), one of the stable end products of alliin degradation^[29] and made from allicin S-thiolation and 2-propenesulfenic acid addition and again less prominent than diallyl sulfides^[28]
• Allylmercaptane (AM) and allylmethyl sulfide (AMS), which are produced from DADS after oral ingestion^[30] and oxidized into allylmethyl sulfoxide (AMSO) and then allylmethyl sulfone (AMSO₂)^[29]
• S-methylcysteine sulfoxide (Methiin)^[28]
• Dimethyl sulfides as well as allyl methyl sulfides, also present in garlic oil and similar to the diallyl sulfides are derivatives of allicin^[22]
• Thiacremonone (2,4-dihydroxy-2,5-dimethylthiophene-3-one), a cyclic sulfur bearing compound^[31]
• Garlicnins A1, B1-4, C1-3, and D1 (Cyclic sulfoxides);^[32][33][34] thought to be formed sponaneously from allicin
• Allixin, a cyclic non-sulfur component that accumulates in the necrotic areas of garlic bulb^[35][36] that may reach up to 1% of the bulb's dry weight after a year^[35]
• Sodium 2-propenyl thiosulfate^[37]

Finally, the protein fragment of garlic itself contains some bioactives. There is also a 14kDa glycoprotein known to be involved in the induction of natural killer (NK) cells^[38] and thought to be involved in altering T-cell cytokine production, since the extract of garlic where the glycoprotein is found alters T-cells.^[39]

In regards to the sulfur containing compounds, there are two main classes of traditional bioactives, which are pretty much either made from allicin spontaneously reconfiguring itself (this makes diallyl sulfide molecules, Ajoene, and vinyldithiins) or from the Glutamyl-S-allylcysteine being aged (this makes S-allylcysteine mostly). There is a protein fragment involved in immune health, and some cyclical bioactives with unknown origins

With some non-sulfur based bioactives including (note, some variants of garlic will be specified):

• ACE inhibiting dipeptides (Ser-Tyr, Gly-Tyr, Phe-Tyr, Asn-Tyr, Ser-Phe, Gly-Phe, and Asn-Phe)^[40]
• β-Chlorogenin^[26]
• Nitrate at 183mg/kg (34–455mg/kg)^[41]
• The lignans Matairesinol (37.4µg/100g raw weight) and Secoisolariciresinol (26.6µg/100g raw weight)^[42] thus able to produce endogenous enterodiol^[43]
• Quercetin at 47^[44] to 80.6mg/kg^[45] but usually not detectable^[45][46][47]
• Apigenin once reported to be 217mg/kg dry weight^[48] but otherwise not detectable^[46][47]
• Myricetin once reported to be at 693mg/kg^[44] but otherwise not detectable^[47]
• Kaempferol once reported at 1mg/kg^[44] and usually not detectable^[46][45][47]
• Molybdenum at 2.80µg/100g^[49]
• Luteolin at 1.4% of a methanolic extract of the peels^[50] but otherwise at low to undetectable levels in the bulb^[44]
• L-Methionine^[26]
• L-cysteine and related amino acids L-methylcysteine and L-ethylcysteine^[26]
• L-Arginine and related structures such as fructosyl-arginine^[26]
• Glutathione itself (93.5mg/100g^[51])
• Selenium containing molecules including selenate, Dimethylselenide, selenomethionine, selenocysteine, S-methyl selenocysteine and γ-glutamyl-S-methylselenocysteine;^[52] structurally, the selenium replaces the sulfur of some garlic bioactives and of sulfur containing amino acids
• Vitamin C (5.3mg/100g^[51])
• Caffeic acid (2.9mg/kg dry mass^[47]) and ferulic acid (2.6mg/kg dry mass^[47])
• A dietary mannose-containing lectin^[53] that acts on the insulin receptor^[54] and does not agglutinate red blood cells^[55] at 30.3ng/g fresh weight^[54]
• Furostanol saponins Voghieroside (A at 5.7mg/kg, B at 9.1mg/kg, C at 10.6mg/kg, D at 0.4mg/kg, and E at 0.3mg/kg; all isomer pairs) from the variant Voghiera^[6]
• Agigenin (not to be confused with Apigenin) 3-O-trisaccharide from the variant Voghiera^[6]
• Gitogenin 3-O-tetrasaccharide from the variant Voghiera^[6]
• Eugenol diglucoside (0.7mg/kg) and rutinoside (6.1mg/kg) in the variant Voghiera^[6]

The bioactives in garlic that are not the unique sulfur containing ones are not present in very high levels, and it is likely that the things in this list are ingested at much too low of a dose to be bioactive

When looking at overall groups of molecules, an average garlic bulb is about 0.9% γ-glutamylcysteines and up to 1.8% alliin.^[8]

Total flavonoids have ranged from 6.36-9.00mg gallic acid equivalents (GAE) per kilogram dry mass^[56] and elsewhere 6.99–8.70mg,^[48] 19.4mg/kg,^[44] and 0.075–0.12mg/kg.^[46] Garlic skin (the coating of the buds) has a poor content of sulfur containing bioactives.^[57]

One major molecule is Alliin, which is rapidly converted to Allicin (probably not bioactive in the body) and then allicin is further degraded into either Ajoene (which is one major bioactive) or the the vinyldithiin and dithiin molecules (also bioactive), although S-allylcysteine also may play a role

When analyzing commerical garlic products, the allicin content appears to be less than 1ppm (less than 1µg per gram).^[15]

Crushing or cutting a garlic clove to expose it to air (chewing also qualifies for damage, but there is less air exposure) the enzyme known as alliinase is activated and rapidly lyses the cysteine sulfoxides (alliin) to form the alkyl alkane-thiosulfinates of which allicin temporarily forms 70-80% of them.^[8] This rapid conversion of alliin to allicin causes the characteristic garlic odour when initially cut, but due to allicin itself being instable it is then further degraded into ajoene and dithiins.^[29][8]

Despite being the most prominent bioactive in garlic, allicin is highly instable to both heat and oxidation^[29] and is broken down into the more stable vinyldithiins^[17] or ajoene.^[29][58] Even under best case scenarios, allicin in solution can reach a half-life of up to 12 days in 20-50% ethanolic extracts.^[29]

Allicin has poor stability in processed products or in liquid solution, suggesting that products containing allicin may contain the catabolites of allicin instead (due to decomposition)

When looking at S-allylcysteine, it has a melting point of 223.3–223.7°C and is seen as a white crystalline powder (with minor yellowing after prolonged storage);^[59] as the name suggests, it is structurally related to L-cysteine and unlike allicin or its parent compound γ-glutamyl-S-allylcysteine, S-allylcysteine appears to be quite stable and can remain unaltered in garlic for up to two years.^[19] If S-allylcysteine is in an alkaline medium, within six days there may be some detectable catabolites (allylmercaptan and allylsulfide) although these changes do not occur in acidic medium.^[59]

Relative to other compounds in garlic, S-allylcysteine is pretty stable and is commonly seen as an end-point of garlic metabolites; it can still degrade further into some catabolites if placed in an alkaline solution

In general, the oil soluble sulfur components of garlic (arising from the oil fragment) are known to carry the aromatic properties of garlic,^[8] which mainly consist of the main diallyls (sulfide, disulfide, and trisuldie) as well as methyl allyl disulfide (and trisulfide), 2-vinyl-1,3-dithiin, 3-vinyl-1,2-dithiin, and Ajoene.

The most basic variant of garlic supplement is the basic garlic powder, which is simply dehydrating the bulb and crushing it into a powder; it contains some alliin although usually not allicin since whatever allicin was form spontaneously reconfigues into its derivatives.^[28]

Due to the dehydration (and assuming a 65% water content of the garlic cloves), one gram of the basic garlic powder is equivalent to about 2,850mg of raw garlic.

Garlic powder (when 'aging' is not mentioned) is simply a concentrated form of garlic supplement that is pretty much bioequivalent to consuming raw garlic bulbs, assuming the dose is corrected (with one gram of powder being equivalent to 2,850mg of the raw bulb)

Aging garlic for 20 months (at low temperatures and dissolved in 15-20% ethanol) produces 'Aged Garlic Extract', which is usually sold in 10% ethanol.^[19][60][14] The major brand name for aged garlic extract is 'Kyolic', and the process of aging has the benefit of making the garlic supplement odorless.^[19]

The molecular differences in aged garlic extract involves the Glutamyl-S-allylcysteine molecules. During the aging process, the parent molecules (γ-Glutamyl-S-allylcysteine and γ-Glutamyl-S-1-propenylcysteine) passively degrade over time to increase levels of S-Allylmercaptocysteine (SAMC), S-allylcysteine (SAC), and S-1-Propenylcysteine.^[19] Kyolic as a product tends to be standardized to SAC (since it is seen as the major bioactive), and during the aging process SAC can increase from 200µg/g of the dry extract to 7,200µg/g dry extract (associated with full depletion of the parent compounds)^[19] although to be sold on the market as 'Aged Garlic Extract' you only need a minimum of 0.05% (500µg/g) S-allylcysteine. Kyolic was specifically mentioned due to being a product used in many studies (either in humans or in vitro) and while some are partly funded by the producer of the supplement (Wakunaga Pharmaceutical)^[61] there some independent trials that also return positive results.^[20][62]

The aging process does produce some other unique bioactives as well as the above. This includes the tetrahydro-β-carbolines^[63][64] and Nα-(1-deoxy-D-fructos-1-yl)-L-arginine^[65] as well as other fructans.^[66] Some molecules in these categories show direct antioxidant properties^[67] and total fructans may be up to 0.22% of total dry weight.^[66] Due to these unique bioactives and the increased level of Glutamyl-S-Allylcysteine derivatives, aged garlic extract has more antioxidative potential than does standard (fresh) garlic extracts.

Aging garlic tends to encourage the degradation of Glutamyl-S-allylcysteine parent molecules to lose their glutamyl groups, and this increases the levels of its derivatives such as S-allylcysteine. This variant of garlic supplement is also odorless

Garlic oil is a product of garlic manufacturing (usually by steam distillation),^[68][23] which is concentrated for the fat soluble sulfur containing molecules; this mostly refers to diallyl sulfides (end product of ex vivo allicin breakdown) and usualy excludes the glutamyl-S-allylcysteines seen in Aged garlic extract.

Garlic oil tends to contain polysulfides (many sulfur groups) mostly of the diallyl class, and a standard breakdown of bioactives in garlic oil is as follows:^[22]

• Diallyl disulfide (26%)
• Diallyl trisulfide (19%)
• Diallyl tetrasulfide (8%)
• Allyl methyl disulfide (13%)
• Allyl methyl trisulfide (15%)
• Allyl methyl tetrasulfide (6%)
• Dimethyl trisulfide (3%)
• Pentasulfide (4%)
• Hexasulfide (1%)

At times, you can have an oil-macerated garlic oil, which is the same as above but contains Vinyl-Dithiins and Ajoene or an ether-extracted garlic oil, which has a higher content of vinyl dithiins (5.7mg/g) and ajoene (0.4mg/g) relative to garlic, and may have a total allyl sulfide content of 1.4mg/g^[22] The macerated garlic oil contains alliin but not allicin, whereas the garlic oil contains neither.^[28]

Garlic oil is a processed form of garlic catering towards the sulfur donating molecules, particularly diallyl disulfide (DADS). Unlike aged garlic, garlic oil is highly aromatic

Boiling garlic bulbs is known to inactivate the alliinase enzyme which degrades alliin into allicin (which then spontaneously begins producing other bioactives) and due to this boiling garlic bulbs (or, technically speaking, any high level of heat without cutting the garlic bulb first) would cause supplementation of alliin without necessarily forming any of the bioactives made from alliin since alliin does not appear to be metabolized.^[28]

Boiling garlic has been noted to prevent properties of garlic from occurring including vasorelaxation,^[69] anticancer effects,^[70] and the increase in nitric oxide and IFN-α.^[71] Conversely, it has been noted to augment some properties such as memory enhancement (diabetics rats).^[72] Boiling does not modify its antioxidant properties relative to raw garlic when measured in vivo^[73] or when testing the ability to prevent LDL oxidation^[74] and the alterations in triglycerides and cholesterol seen with raw garlic are not affected by boiling.^[73]

The above 'heating' applies to 60s of microwave cooking and 45 minutes of baking in the oven,^[70] but 30s of microwave cooking is fine.^[75] Crushing the garlic and letting it stand for 10 minutes preserves the bioactivity of garlic.^[75]

Boiling garlic, or any heat treatment at high levels without first destroying the structure of the clove, will prevent alliin form converting into its catabolites (diallyl sulfides, vinyl-dithiins, and Ajoene) and prevent their activity in the body. This will prevent the blood flow enhancement and anticancer properties specifically, although the cognitive protective, lipid lowering, and antioxidant effecs seem mostly unaffected

Hydrogen Sulfide (H₂S) is an small gaseous signalling molecule akin to nitric oxide, informally known as a gasotransmitter due to its state^[76] and produced by one of three enzymes known as cystathionine γ-lyase, cystathionine β-synthase, or 3-mercatopyruvate sulfurtransferase.^[77][78] It seems that garlic itself, when incubated in red blood cells, produces H₂S from garlic bioactives^[79] and that H₂S can relax blood vessels.^[80] H₂S is mostly produced in the blood vessels from cystathionine γ-lyase^[81] although it may also be produced nonenzymatically.^[82][79]

The main mechanism of action for garlic, as it pertains to the blood pressure reducing and blood flow enhancing properties, is merely acting as a reservoir of sulfur, which can be used to make the signalling molecule known as hydrogen sulfide (H₂S), which can either directly relax vessels (via ion channels) or indirectly accelerate nitric oxide signaling

There is also some crosstalk with the H₂S signalling pathway and the nitric oxide pathway, since H₂S can degrade the molecule known as S-nitrosoglutathione,^[83] which serves as an intracellular (and to a degree, extracellular^[84]) reservoir of nitric oxide that garlic is known to stimulate.^[85] Due to this, the influence of garlic on blood pressure and blood flow is in part due to nitric oxide signalling per se (the vasorelaxing effect of opening potassium channels to be discussed in the ion channels section^[81]) and in part an influence of the hydrogen sulfide system unto the nitric oxide system.

Hydrogen sulfide production may also underlie the interactions of garlic bioactives and both TRPV1^[86] and TRPA1^[87] as well as the interactions between garlic bioactives and AMPK activation.^[88]

The production of the gasotransmitter hydrogen sulfide (H₂S) seems to underlie pretty much all other mechanisms of action that are attributed to garlic supplementation, which supports the hypothesis that production of hydrogen sulfide is the main mechanism of action of garlic supplementation

Garlic and its bioactives appear to open potassium channels in vitro, and when measuring membrane hyperpolarization and relaxation (maximal efficacy reaching 20% of baseline) it appears that a water extract has been noted to have an EC₅₀ value of 1.15µg/mL and isolated bioactives having EC₅₀ values of 6.2nM (allicin) and 9.9nM (ajoene).^[89] Since garlic is known to spontaneously form H₂S in vitro and H₂S itself is a potent potassium channel opener,^[81] it is thought that the potassium channel opening properties of garlic are secondary to H₂S.

Garlic components appear to be quite potent potassium channel openers in endothelial cells, and this is thought to be due to the production of hydrogen sulfide from these components (with more contribution of sulfur causing more vasorelaxation)

Diallyl sulfide activates TRPA1 (a receptor that responds to noxious cold) with an EC₅₀ of 254μM and reaching maximal stimulation of 90.8%, which is lesser than diallyl disulfide (EC₅₀ of 7.55μM and reaching maximal stimulation of 76.6%) as well as diallyl trisulfide which activates TRPA1 with an EC₅₀ of 0.49μM (490nM) and reaches maximal stimulation of 83.6%;^[21] the efficacy of diallyl trisulfide is greater than the bioactive of mustard oil (allyl isothiocyanate at an EC₅₀ of 1.47μM)^[21] and Allicin itself has a fairly potent EC₅₀ value of 1.32-1.91μM while its precursor (alliin) is ineffective.^[90] This increasing potency correlates with increasing sulfur groups, which suggests that the activation of TRPA1 from hydrogen sulfide^[87] underlies the influence of garlic bioactives on this channel.

Garlic^[91] and mustard oil^[92] also activate TRPV1 and the diallyl sulfides, with EC₅₀ values of 151μM (diallyl sulfide), 36.7μM (diallyl disulfide), and 43.7μM (diallyl trisulfide) all of which are a potency significantly lesser than the reference compound Capsaicin (EC₅₀ of 1.47μM).^[21] Allicin is slightly effective (EC₅₀ of 51.22μM) and its precursor (alliin) is ineffective^[90] and there is a possibility that this is also due to hydrogen sulfide, which is known to activate TRPV1 directly.^[86]

Interestingly, raw garlic has been found to activate TRPA1 and TRPV1 while baking the garlic (400°F for an hour in an oven) has failed to have the same effect^[90] and TRPM8 (a receptor that response to both innocuous cold sensation and menthol from peppermint^[93][94]) has not been activated by either form of garlic.^[90]

Components of garlic appear to stimulate both TRPA1 and TRPV1, and while the efficacy on TRPA1 seems quite potent it is less potent than capsaicin (hot pepper extract) on TRPV1 activation. The activation of these channels is probably secondary to hydrogen sulfide production

Numerous components of garlic have been noted to activate AMPK including Ajoene (LKB1 and SIRT1 dependent signalling, confirmed to not alter ATP concentrations^[95]), S-allylcysteine (SIRT1 and CaMKK dependent signalling^[96]), S-allylmercaptocysteine (LKB1 dependent^[97]), Allicin,^[98] and Thiacremenone.^[99] Noradrenaline secretion is also able to activate AMPK in white (but not brown) adipose tissue^[100] and in this case the molecules of garlic that induce secretion of noradrenaline (S-allylcysteine, allicin, and the Diallyl sulfides^[101]) may indirectly stimulate AMPK.

Seven weeks ingestion of 2-5% of the diet as garlic to rats has been confirmed to increase AMPK in brown (20-80% higher than control) and white (50-100%) adipose tissue as well as the skeletal muscle (10-30%) and liver (10-40%); acting in a dose-dependent manner,^[102] confirming the relevance of this pathway to oral garlic supplementation.

Similar to the other mechanisms of action, there is the possibility that garlic bioactives work secondary to producing hydrogen sulfide as H₂S itself has been noted to activate AMPK^[103] and in the same manner outlined above (dependent on LKB1 and CaMKK^[88]); the connection between garlic per se and AMPK has not yet been confirmed.

Almost all major sulfur-containing bioactives in garlic are implicated in increasing AMPK activation either directly (usually via a LKB1/SIRT1 dependent mean like resveratrol) possibly related to hydrogen sulfide, or indirectly in white adipose tissue via increasing noradrenaline

AMPK activation is thought to underlie the antihepatosteatotic effects of garlic (seen with 10-30mg/kg ajoene daily in mice)^[95] and the antiadipogenic effects of Thiacremonone seem in vitro.^[99]

The pathways related to a reduction in lipid synthesis (fatty liver and obesity) are usually tied back into the AMPK pathway

NF-kB is an inflammatory signalling molecule which is a dimer (pair) of proteins that, when they interact, cross the nucleus to cause a genomic inflammatory response; the most common variant is a heterodimer (different pair) comprised of the p50 and p65 protein^[104] while p50:p50 and p65:p65 homodimers (same pair) have also been reported^[105] where the p50 pairings are thought to be more relevant to inflammatory responses^[106][105] and p65 pairings more relevant to cancer metabolism.^[107] Sulfur bearing compounds tend to interact with the cysteine residues on NF-kB^[108][109] which seems to apply to garlic bioactives.

Thiacremenone (2.5-10μg/mL in macrophages with an IC₅₀ of 8μM) appears to directly interact with NF-kB resulting in its inhibition. This is secondary to oxidizing the sulfhydryl residue on the p50 subunit^[31] which is critical for interaction between p50 subunits and is inhibited by coincubation of reducing agents.^[31] Elsewhere in cancer cells, p65 is thought to be the target of thiacremenone^[107] and the reason for this differential response is not known but likely due to the different cells used.

Thiacremenone appears to directly interact with the protein subunits of nF-kb (either p50 or p65) resulting in its inhibition which is thought to be since it is a sulfur bearing molecule, and sulfur bearing molecules tend to interact with the cysteine residue on these proteins that is needed for their activity

Diallyl disulfide and allylmercaptane have both failed to inhibit TNF-α induced NF-kB activation in isolated endothelial cells at concentrations up to 100µM.^[110]

Histone deacetylases (HDACs) are enzymes that remove acetyl group from histone proteins, and said deacetylation will modify the function of these histones and genomic transcription. As a general statement, deacetylation silences some epigenetic genese in cancer cells and their reacetylation is met with reactivation.^[111][112] Thus, HDAC inhibition appears to be useful for chemotherapy yet limited natural products have this ability (garlic organosulfurs, sulforaphane, and butryic acid from colonic fermentation^[113] as well as allyl isothiocyanates from wasabi and onion^[114]).

Histone deacetylation modifies the genes of a cancer cell to repress or 'silence' a subset of genes, and reactivating these genes by acetylating them is associated with anticancer properties; inhibiting the HDAC enzymes is the main mechanism by which histone acetylation occurs

Diallyl Disulfide (DADS) at 200µM^[115] and allyl mercaptan (AM) at the same concentration^[116] both inhibit HDAC resulting in an enhancement of Sp3 binding to the p21/WAF1 promoter, although AM (92%) is much more potent than DADS (29%)^[115] and when compared to another HDAC inhibitor from garlic (S-allylmercaptocysteine or SAMC) AM remains more potent;^[117] since AM is known to be a direct competitive inhibitor of HDAC^[115][116] and a metabolite of both DADS^[30] and SAMC^[117] it is thought that DADS and SAMC are acting as prodrugs for AM. When comparing the potency of AM against other HDAC inhibitors, it appears to be less potent than the refrence drug Trichostatin A (TCA).^[115]

Of these, oral ingestion of diallyl sulfides (single dose of 200mg/kg; 80% DADS) has been confirmed to inhibit HDAC in rat colonic cancer cells within six hours (3-fold for histone H4 and between 1.4 and 2.5-fold for histone H3) but no longer persisting after 17 hours.^[118] Unpublished research (mentioned here^[113]) suggest this occurs in the liver of mice as well.

Two bioactives in garlic from both the alliin derivatives (DADS) and from the glutamyl-S-allylcysteine derivatives (SAMC) can be seen as molecules that donate an allyl mercaptan (AM) molecule to a cell; also known as allyl mercaptan prodrugs. Allyl mercaptan itself appears to be a relatively potent HDAC inhibitor

Oral ingestion of 500mg of aged garlic extract (Kyolic brand) has been noted to increase plasma S-allylcysteine concentrations 10μg/L (100ng/mL) or more in most (78%) subjects^[119] and elsewhere 2,560mg of this product increased blood levels of S-allylcysteine to 20μg/L (200ng/mL);^[120] both studies have noted a basal S-allylcysteine concentration in the control groups, suggesting that it is an endogenous compound.

Oral ingestion of aged garlic extracts has been confirmed to increase circulating S-allylcysteine (SAC) concentrations to the low nanomolar range

Garlic bioactives (demonstrated with allicin) are thought to passively cross the cellular membrane (without inducing leakage, fusion, nor aggregation of membranes)^[121] and some studies attempting to block exofacial membrane thiols fail to abolish the effects of garlic;^[79] concluding that garlic can exert its main mechanism of action (production of H₂S via sulfur donation) in any thiol containing cell via passive diffusion.^[79]

It seems that no particular transporter is required for the investigated sulfur bearing bioactives, and that they passively diffuse through the cellular membrane without causing damage. If H₂S is produced extracellularly, it may also passively diffuse (like all gasotransmitters)

Allicin is known to be metabolized into diallyl disulfide in the liver^[122] but is inherently instable even in plasma (37°C) and can spontaneously be fully degraded within five minutes;^[15] and the instability limits the bioactivities of allicin per se.^[123][28] Diallyl disulfide (DADS) and Ajoene are also somewhat unstable,^[123][28] but allyl mercaptane (AM) seems to be stable.^[123]

Diallyl sulfide (DAS) can also be metabolized in liver cells to form allyl mercaptane (AM) and allyl methyl sulfide (AMS) with significantly more production of AM over AMS.^[30] This is confirmed in breath tests where AM is eliminated in high levels initially, and later AMS seems to be the prevalent metabolite (DAS and DADS both in low levels at both times),^[124][125] which is thought to be due to a converion of AM into AMS via a methyl donation from S-adenosyl methionine, and AMS is thought to be the primary metabolic end product of allicin metabolism via the breath.^[28] When measuring the urine, oxidized products of allylmethyl sulfoxide (AMSO) and allylmethyl sulfone (AMSO₂) have been detected in rats.^[126]

An alternate pathway involves DAS being metabolized by the CYP2E1 enzyme to produce diallyl sulfoxide, and then being again metabolized by the same enzyme to produce diallyl sulfone.^[127]

When alliin is ingested (cooking garlic prior to activating the alliinase enzyme), the end products of allicin metabolism are not detected in the breath.^[124]

Allicin is highly unstable, and is spontaneously eliminated in plasma rapidly (so no 'effects of garlic' are likely to be due to allicin). The diallyl sulfides are stable outside of the body, but tend to be metabolized into smaller sulfur bearing molecules such as allylmercaptan (AM) and allylmethyl sulfide (AMS) which are thought to underlie most benefits of alicin metabolites

When looking at the glutamyl-S-allylcysteine metabolites, oral ingestion of S-allylcysteine (SAC) results in a serum level of SAC^[128] and some urinary excretion of SAC although the major urinary metabolites seem to be N-acetyl-S-allylcysteine and N-acetyl-S-(2-carboxypropyl) cysteine.^[129][130] S-allylcysteine (SAC) seems stable, although S-allylmercaptocysteine (SAMC) is a little bit unstable.^[28]

S-allylcysteine (major bioactive of aged garlic extract) seems more stable, and oral ingestion of SAC seems to result in serum levels of SAC

Garlic has been noted to have iron chelating properties in vitro comparable with parsley (yet lower than rosemary, sage, and onions)^[131] A rat study has confirmed increased fecal, but not urinary, excretion of cadmium in rats given raw garlic.^[51]

Supplementation of garlic or isolated allicin is well known to reduce lead accumulation in fish,^[132] sheep,^[133] mice,^[134] and rats^[135] with no significant difference between allicin supplements and an equal dose of allicin via fresh garlic^[134] and most efficacy at a dosage of around 6.7-7% of the rat diet (more potent than 3.35%, and 1.70% was ineffective^[51]) and when it is taken concomitantly alongside heavy metal exposure.^[136] Beyond lead, garlic at these relatively high dietary levels has been noted to reduce cadmium^[136] and mercury^[136][51] bioaccumulation and toxicity with somewhat equal potency.

The studies that measure organ damage note that the reduced mineral accumulation is associated with less organ damage, and the protective effects of 6.7% dietary garlic in rats seems equivalent to N-acetyl-D,L-penacillamine (60mg/kg) and 2,3-dimercaptosuccinic acid (60mg/kg), two reference drugs.^[51] The main bioactive seems to be diallyl disulfide or trisulfide,^[51] although S-allylcysteine has not been tested in vivo (in vitro, it was noted that a heated extract containing S-allylcysteine but not allicin metabolites failed to chelate copper^[137] suggesting it is inactive)

Garlic has been confirmed to, at higher than normal doses, protect the body from the damage associated with heavy metals which is associated with less organ accumulation of these minerals. The sulfur compounds (diallyl disulfide and diallyl trisulfide) seem to be implicated as the active ingredients

When tested in humans, consumption of garlic thrice daily (each dose equivalent to 1,200µg allicin) for four weeks in workers in a car battery plant (excessive exposure to lead) was able to reduce various clinical signs of toxicity including headache, irritability, and the reduced blood pressure and muscle reflexes;^[138] the reference drug of D-penicillamine (250mg thrice daily) failed to reduce symptoms, although garlic and D-penicillamine were equivalent in reducing lead levels in the blood (19% and 24% reduction, respectively).^[138]

Appears to be comparable to D-penicillamine in reducing lead levels in the blood with standard oral doses of garlic, attainable via food consumption

Garlic is thought to interact with various enzymes of the P450 class.^[139][127] Of these, CYP2B1 has been noted to be induced with garlic and its prototypical sulfur containing compounds at high doses (200mg/kg diallyl sulfide).^[140][141] The compounds that inhibit CYP2E1 are also known to cause an increase in CYP1A mRNA levels acutely and protein content with daily administration^[127] and all bioactives tested are known to cause an increase in CYP3A2 levels.^[127]

CYP2B1 may be increased with very high doses of sulfur compounds from garlic

CYP2E1 has been reported to be slightly increased in one study with DADS (14 days of 100μM/kg)^[142] although the majority of studies have noted inhibitory effects; inhibition of CYP2E1 has underlied hepatoprotective properties from acetominophen^[143] due to diallyl sulfide and its metabolite diallyl sulfone^[144][145] and later both S-allylmercaptocysteine (AMS)^[146][147] and diallyl disulfide (DADS).^[127] S-allylcysteine has failed to interact with CYP2E1, and propyl sulfides are similarly ineffective.^[127]

CYP2E1 is inhibited by garlic bioactives (diallyl sulfide, diallyl disulfide, and AMS), which may underlie some health effects but also predisposes garlic to some potential drug-drug interactions. These components are found in the garlic oil, so this effect may not be seen with water extracts or aged garlic supplements

Studies assessing CYP2C9 activity have faile to find an influence of garlic (2,000mg of fresh bulb conferring 3.71 mg allicin twice daily for two weeks prior to warfarin).^[148]

No known interaction with CYP2C9

Glutathione S-Transferase (GST) is an enzyme that uses NADPH and a proton to convert glutathione disulfide (GSSG or 'reduced glutathione') into active glutathoine, and serves as a glutathione recycling enzyme.

Injections of DATS (10µM/kg) and DADS (100µM/kg) for two weeks have increased GST activity in rats by 43-54% while DAS is ineffective.^[142]

DAS has been noted to increase GST activity at higher doses (100-500mg/kg) with a potency nonsignificantly greater than the reference drug N-acetylcysteine (500mg/kg) yet was more effective at improving the GSH:GSSG ratio at the higher dose.^[149]

200μM of E-Ajoene has been noted to partially (50%) interfere with GST's reduction of GSSG while enhancing the oxidation of NADPH, resulting in prooxidation.^[150]

Glutathione S-Transferase (GST) is normally benefitted by supplementation of garlic, although concentrations of garlic bioactives that are in the toxic level have the opposite effect

Quinone Reductase (QR) refers to enzymes that use NADPH and a proton to convert a quinone into a semiquinone via the process of reduction, and they are antioxidant enzymes known to reduce toxicity associated with the carcinogenic benzo{a}pyrene.^[151] An induction of QR is thought to be protective against any quinone based carcinogen, or other carcinogens that happen to be inactivated by QR. A relatively important QR is NADPH quinone oxidoreductase 1, or referred to as NQO1.

QR is noted to be increased with DADS ingestion (more potent than DAS and DATS although both are effective^[151]), and the increase is relatively larger than that seen with GST.^[152] It has been noted in most organs (Liver, heart, kidney, spleen, bladder, lungs) at doses as low as 300µg/kg in rats (intestinal tract only) and peaking at 15-30mg/kg in the stomach.^[152] DADS (100μM) induced QR induction to 3.2-fold of control is associated with Nrf2 translocation^[153] and at least when looking at DAS it is thought that Nrf2 is activated secondary to activating MAPKs (ERK/p38, but not JNK).^[149]

Aged garlic extract is also known to induce QR secondary to Nrf2 at 100μg/mL by 2.1-fold (less potency than 100μM DADS at 3.2-fold)^[153] although heated garlic juice at 100mg/kg oral intake to rats for four weeks has failed to increase NOQ1.^[154] Isolated DAS (100-500mg/kg to rats) activates NQO1 to a level equal to 500mg/kg N-acetylcysteine,^[149] and low dose injections of both (10µM/kg) and DADS (100µM/kg) have increased QR activity by 41-91% (while 100µM/kg DAS was inactive).^[142]

Quinone Reductase is an antioxidative enzymes that reduces quinone structures (and plays a role in cancer prevention), and it appears that QR is increased following oral intake of garlic extracts. Several bioactives are known to increase it, with DADS and DATS being active in the lowest doses and likely relevant to garlic ingestion

Diallyl sulfide (DAS) is known to induce transcription of SULT1E1 to 250-fold of baseline secondary to causing nuclear accumulation of CAR, although no perturbations of basal estrogen sulfation were noted in mice treated orally with DAS at 800mg/kg.^[155]

DAS is associated with a relatively potent induction of SULT1E1 of unknown practical significance

Garlic has failed to show inhibitory efects on UGT1A1 using an 80% methanolic extract (3.25% alliin or more) up to 500µg/mL, while EGCG from green tea catechins was most potent (IC₅₀ 7.6+/-0.7µg/mL).^[156] When looking at other enzymes of glucuronidation, this same extract failed to inhibit UGT1A4, UGT1A6, and UGT1A9^[157] while elsewhere isolated diallyl sulfides (1mmol/kg oral ingestion) to rats noted that all tested compounds increased the activity of UDP-glucosyltransferase in the liver and kidney while only DADS increased it in the intestines and lung (and was more effective in the liver and kidneys).^[158]

It is possible that oral ingestion of the diallyl sulfides can increase the activity of UGP-glucosyltransferase in rats, and there is no current human evidence

Garlic (2,000mg of fresh bulb conferring 3.71 mg allicin) twice daily for two weeks before a dose of Warfarin has failed to alter its pharmacokinetics or blood thinning abilities overall,^[148] although in subjects with a wild type VKORC1 gene there was an increase in the S-warfarin EC₅₀ (22%) with garlic.^[148] While this evidence suggests no adverse interaction and low doses of garlic (4.2g of the raw cloves) do not necessarily interact with platelets^[159] there have been reported case studies of high garlic intakes causing cerebral hemhorraging when coingested with Warfarin.^[160]

Garlic does not appear to alter the blood thinning abilities of Warfarin supplements when taken in normal doses, although there is some limited evidence of adverse interactions with higher doses. Coingestion is possible but must be approached cautiously with the oversight of a medical professional

It appears that garlic can reduce overall exposure (AUC) of the drug saquinavir (a protease inhibitor used in HIV therapy) by 51% associated with intake of 8g raw garlic^[161] which is mostly due to a rapid reduction in C_max (54%) and is mostly normalized after 10 days of washout.^[162] Another antiviral known as ritonavir has its efflux hindered in vitro with garlic due to P-glycoprotein inhibition^[163] but this does not seem to occur in humans following consumption of 10mg of a garlic concentration (1g raw garlic equivalent).^[164]

Garlic ingestion at levels of which it is concievable to eat in the diet (two cloves weight 8g) is able to half the bodily exposure to the anit-HIV drug saquinavir

Garlic appears to be touted for, amongst other things, longevity promotion^[165] which appears to be a traditional usage of garlic as well.^[8][9] It seems to currently be thought that this 'antiaging' property is due to an increase in its cardiovascular and cerebral benefits improving vitality in older age, although other benefits of garlic are known to treat 'comorbidities' of aging.^[166]

There appears to be traditional usage of garlic for the purpose of 'longevity' and 'anti-aging', and the best evidence at this point in time (mostly reviews) tend to suggest that this is an increase in functionality and vitality during the aging process causing the perception of youth

When assessing isolated human fibroblast cells via the Hayflick system (seeing how many times a cell can divide until it cannot anymore, and an increased Hayflick limit being thought to demonstrate increased cellular antiaging) has noted some potential with garlic extract.^[167]

There has been one study investigating a dietary lectin from garlic (as well as bananas) that physically interacted with the pro-insulin receptor and secondary to siganlling through this receptor and ERK it reduced oxidative stress in stem cells;^[54] this was observed in mice following weekly ingestion of 10ng of the lectin (330mg of raw garlic bulb equivalent) for 6-8 weeks, suggesting it is relevant to oral ingestion.^[54]

Diallyl trisulfate (DATS) appears to promote longevity in C. elegans^[168] via activating transcription of skn-1, which is the worm homologue of the human gene Nrf2.^[169] Concentrations of 5-10μM (20μM appeared less effective and higher doses toxic) increased lifespan by 11.7-12.6%, although this was reduced to 6.8-9% when the possible ability of DATS reducing bacterial contamination was accounted for.^[168] The daf-2 and daf-16 pathways were not involved, and the worm model for caloric restriction (eat-2) did not experience benefits.^[168]

DATS appears to have very minor longevity promoting benefits in worms (C. Elegans) that may not benefit those undergoing a calorically restricted diet

There are a few studies in senescence accelerated mice (SAMP8) as a model for aging which note improvements in cognitive performance which is usually impaired (and thought to reflect the cognitive impairment of aging)^{[170][171][172]} and garlic is associated with a reduction in brain atrophy associated with aging.^[172][173] S-allylcysteine itself has been noted to improve cognition associated with aging in SAMP10 mice at 40mg/kg of the diet,^[174] which was similar to the aforementioned studies using 2g of aged garlic extract per 100g of food.

When looking at rodent studies, in senescence accelerated mice (SAMP8) given aged garlic extract at 2% of the diet (0.1% total dietary S-allylcysteine) after two months of life it was noted their lifespan was normalized to senescence resistant mice (SAMR1)^[170] and elsewhere SAMR1 mice have failed to experience an increase in lifespan with aged garlic extract at 2% of the diet.^[171]

The prolongation of lifespan seen in SAMP8 is not seen in another strain of senescence accelerated mice (SAMP10)^[172] but the attenuation of brain atrophy seen during aging also applies to not only SAMP10 but also the senescence resistant SAMR1 mice.^[173]

There may be a longevity promoting effect of garlic ingestion, but this seems to only occur in the mice which are prone to accelerated aging. The mice who are genetically resistant to aging recieve no apparent extension of lifespan with garlic, but all groups seem to have improved cognitive performance during aging associated with reduced brain atrophy

Under physiological conditions, the concentration of hydrogen sulfide in the brain has been reported to be in the range of 50-160μM which is thought to be due to the enzyme cystathionine β-synthase, since its inhibition lowers brain concentrations of H₂S.^[175]

The inhibition of adenosine deaminase (the enzyme that degrades the adenosine neurotransmitter into inosine and ammonia^[176]) by methylmercury is inhibited by garlic extract at 100µg/mL^[177] which is simply thought to be due to the interaction between thiol groups (known to sequester toxic minerals) and mercury, since it was also seen with glutathione (50µM).^[177]

Garlic ingestion at 25-100mg/kg (20% ethanolic extract) to mice over two weeks has been noted to decrease levels of both MAOA and MAOB in the brain to a level comparable to 15mg/kg Imipramine.^[178]

The antidepressant effects of garlic have been noted to be partially blocked by coadministration with prazosin, the α1-adrenergic receptor blocker.^[178]

25-100mg/kg of a 20% ethanolic extract of garlic to mice for two weeks prior to antidepressant testing (forced swim and tail suspension tests) in a manner that is partially blocked by baclofen, a GABA_B agonist.^[178]

Garlic ingestion at 25-100mg/kg (20% ethanolic extract) to mice over two weeks has been noted to decrease levels of both MAOA and MAOB in the brain to a level comparable to 15mg/kg Imipramine.^[178]

The antidepressant effects of garlic appear to be partially inhibited by coadministration of p-CPA (Fenclonine), a serotonin synthesis inhibitor.^[178]

Injections of 300mg/kg S-allylcysteine to mice over eight weeks appears to increase then levels of the 5-HT_1A receptor in the mouse dentate gyrus to 223.4% of control.^[179]

One study in adult rats given 250mg/kg of fresh garlic homogenate for 21 days, which say improvements in cognition, noted increases in plasma and brain concentrations of free tryptophan as well as similar increases in serotonin and the metabolite 5-HIAA.;^[180] alongside the increase in free tryptophan in plasma was a decrease in total tryptophan,^[180] which suggests that the total tryptophan (90% in bound form to proteins in serum) is being freed up which is the main determinant of increased serotonin synthesis.^[181][182]

The antidepressant effects of garlic appear to be partially inhibited by blockade of the D₂ dopamine receptor by sulpiride.^[178]

In NGF-treated PC12 cells, aged garlic extract (1-2.5mg/mL) and isolated S-Allylmercaptocysteine (5-25µg/mL, no concentration dependence) augmented the neuronal growth induced by NGF; there was no effect on growth without NGF and S-allylcysteine was ineffective up to 250µg/mL.^[183] High doses (300mg/kg) of injected S-allylcysteine have been noted induce neurogenesis in mice^[179] which were correlated with an increase in 5-HT_1A receptor expression (known to induce neurogenesis in the dentate gyrus^[184][185]).

The components of aged garlic extract have been noted to increase neurogenesis, and the mechanisms of this are not well understood. There are no practical studies on garlic for this purpose so it isn't known how relevant the above information is for supplementation

Oral administration of 10mg/kg diallyl disulfide (DADS), but not 1mg/kg, to rat pups is able to suppress neuronal growth without inducing inflammation.^[186] This is thought to be due to suppressing neuronal proliferation (with no influence on cell viability) which was seen to a mild degree at 10µM DADS (0.1-1µM ineffective) while both S-allylcysteine and S-allylmercaptocysteine were inactive,^[186] and it was thought that DADS reduced BDNF concentrations and thus reduced ERK phosphorylation.^[186]

The components of garlic oil appear to suppress neurogenesis in young rats, with no evidence in adult rats. Although a higher dose was used it was still reasonable, and due to this it may be prudent to avoid giving youth higher than normal doses of garlic oil supplements

A dose of diallyl disulfide to rat pups that is able to reduce neurogenesis (10mg/kg for six weeks) seems to also cause impaired performance in learning as assessed by cross-over latency.^[186]

When diallyl disulfide impairs neurogenesis, it can result in less than optimal memory performance in young rats

S-allylcysteine, at a dosage that attenuates cognitive deficits associated with Alzheimer's has failed to outperform control in otherwise healthy rats.^[187]

250mg/kg of fresh garlic homogenate fed to otherwise healthy adult rats over 21 days is able to improve cognitive performance as assessed by a step-through passive avoidance test (near doubling in latency)^[180] which was thought to be related to the increase in serotonin synthesis seen as serotonin itself has cognitive promoting effects.^[188] Raw garlic has been used elsewhere, where 1,000mg/kg (but not 2,000mg/kg) to rats improved performance in physical memory tests (rope climbing and rotarod treadmill test) but was not significantly effective in a learning maze test.^[189]

A larger dose of 1,000mg/kg garlic extract (fresh, aged, and boiled) to diabetic rats for four weeks noted that while cognitive function could be preserved relative to control, it only occurred with boiled garlic.^[72]

Mixed evidence as to whether garlic supplements can improve cognition in otherwise healthy rodents, with the only hint being that lower doses of raw garlic appear to be more effective than higher doses (250mg/kg in rats is about 40mg/kg for humans and 1,000mg/kg is 160mg/kg)

25-100mg/kg of a 20% ethanolic extract of garlic to mice for two weeks prior to antidepressant testing (forced swim and tail suspension tests) appeared to exert antidepressant effects in a dose-dependent manner, with 100mg/kg being as potent as the reference drugs (imipramine at 15mg/kg and fluoxetine at 20mg/kg)^[178] although garlics benefits were partially blocked by all tested antagonists (sulpiride, p-CPA, prazosin, and baclofen).^[178]

Garlic is being investigated for Alzhiemer's disease in part because S-allylcysteine does show promise itself^[190] and there were a lot of studies conducted on senescence accelerated mice (SAMP8) which showed improved cognition during the aging process^{[170][171][172]} which were used initially to explain antiaging properties of garlic, but SAMP8 is also the mouse model for Alzheimer's disease.^[191]

The mouse model of aging that has been used is also the mouse model for Alzheimer's, so with the success (somewhat, see the longevity section for clarification) in this mouse line previously the research was expanded into assessing Alzheimer's disease

Garlic is known to suppress amyloidogenesis (production of amyloid proteins) in vitro which is seen with fresh garlic but not boiled.^[192] This is thought to be due to the compounds mostly in the aged garlic extract, since thiacremonone has been found to inhibit LPS induced amyloidogenesis in vitro (2-5µg/mL being as potent as 2µg/mL thiacremenone) and in vivo (21 days of 1-10mg/kg oral intake)^[193] and with isolated S-allylcysteine.^[194] S-allylcysteine has also been demonstrated to induce destabilization of amyloid in vitro^[194] and when tested in transgenic mice who develop Alzheimer's disease-like pathology, oral ingestion of 2% aged garlic extract in the diet appeared to attenuate accumulation of both water soluble amyloids (sAβ40 by 39.6% and sAβ42 by 33.3%) and detergent resistant amyloids (fAβ40 by 46.5% and fAβ42 by 39.3%) in both Tg2576 and TgCRND8 strains of mice.^[195]

Beyond reductions in amyloid proteins directly, S-allylcysteine is known to reduce apoptosis in neurons (PC12) caused by said amyloid proteins^[196] and also H₂O₂ and reactive oxygen species (ROS).^[197] A protein known as SNAP25 is also known to be reduced in Alzheimer's rodent models^[197][198] which, due to its importance in building synaptic connections,^[199] is thought to contribute to the pathology of the condition (since Alzheimer's is associated with poor synaptic connections^[200]); aged garlic extract (2% of the mouse diet) or S-allylcysteine in vitro can preserve up to 70% of SNAP25 levels^[197] which is thought to be secondary to the antioxidative effect. Diallyl disulfide was confirmed to not be active.^[197]

Components in garlic appear to exert a reducing effect on amyloid protein content (reducing its synthesis and inducing its degradation) as well as protecting cells from the toxic effects of amyloid signalling. This is noted with the bioactives of aged garlic extract mostly, and appears to occur following oral ingestion of the standard aged garlic extract dose

The learning deficits that occur alongside LPS injections (which are associated with amyloid production) are inhibited by thiacremonone when it inhibits amyloid production.^[193] S-allylcysteine, known to be active in isolation in SAMP8 mice in attenuating learning deficits^[174] has been shown to also attenuate learning deficits in the streptozotocin model of Alzheimer's disease^[201] associated with partially reversing oxidative and genomic damage as well as apoptosis of cells.^[187]

As mentioned previously, supplementation of 2% of the diet as aged garlic extract (usually 0.1% S-allylcysteine) is known to improve cognition in SAMP8 mice when taken as a daily supplement throughout their lifetimes^{[170][171][172]} and other types of mice (TgCRND8) also note improvements in cognitive function in later life with this same dose.^[195]

In animals that have Alzheimer's (various models of Alzheimer's) and are treated with garlic, there are improvements in cognition relative to the Alzheimer's control. This improvement is significant, but not to the level of normalizing cognition relative to nondiseased control

In rats supplemented with garlic, the increase in cardiac size seen with hypertension when given at 0.5% of the diet.^[202]

The increase in diabetes induce cardiac hypertrophy is reduced in rats with oral intake of 10-100mg/kg of garlic oil (every other day)^[203] and 100mg/kg of the water extract of garlic for eight weeks (intraperitoneal administration to diabetic rats) was able to attenute the increase in contractility seen with diabetes^[204] which is a benefit seen in the periphery as well as the heart.^[205]

When fed to rats in instances where the heart tissue would normally enlarge, standard doses of garlic appear to attenuate the increase somewhat

In cardiomyocytes, while garlic extract alone did not alter cardiomyocytes the influence of noradrenaline (hypertrophy) is prevented in a manner that is partially prevented by inhibiting either nitric oxide signalling or hydrogen sulfide signalling.^[57] The reduction in oxidative stress seen with garlic was also partially inhibited by preventing the actions of the gasotransmitters^[57] and this has been seen in rodents given 2-4mL/kg of the juice or 2-4mg/kg of the oil before being treated with isoprenaline (cardiotoxin via the same receptors as adrenaline).^[206]

Inhibiting MAPK activation (p38, ERK, JNK) has also been implicated in rats fed 10-100mg/kg of garlic oil, as has suppression or ERK5/MEK5.^[203] Both the three standard MAPKs^[207][208] as well as ERK5/MEK5^[209] are known to be involved in cardiac hypertrophy, suggesting that this inhibition is relevant to the actions of garlic on heart tissue.

Protective effects are, at least in part, mediated by the two gasotransmitters nitric oxide and hydrogen sulfide although other mechanisms are involved. The protection from noradrenaline is interesting, since garlic itself increases noradrenaline and may circumvent its own harm

As demonstrated by allicin in vitro, the sulfur containing compounds are thought to be able to passively diffuse into red blood cells without disrupting membrane function.^[121]

Red blood cells are capable of reducing sulfur into H₂S at a steady rate when given substrate and in a manner not inhibited by antioxidants (ie. reducing agents);^[82] it can be stimulated with glucose^[82] and red blood cells incubated with garlic the production of H₂S is accelerated.^[79] Red blood cells seem account for 75% of garlic derived H₂S,^[79] whereas the rest is due to spontaneous formation of H₂S extracellularly from any thiol component (including N-acetylcysteine and homocysteine).^[79]

Red blood cells are able to accept garlic bioactives in a passive manner (not requiring a membrane receptor) and when inside the cell it facilitates conversion of garlic bioactives into hydrogen sulfide

It has been noted that garlic, at 4% of the mouse diet over 22 weeks, stimulated red blood cell (RBC) production and turnoever without increasing plasma erythropoietin nor spenic Hif1α.^[210] There was actually a mild reduction in plasma erythropoietin, but no mouse was anemic.^[210]

The authors detected increase bilirubin and iron (breakdown products of heme via the enzyme heme oxygenase 1 or HO-1^[211][212]) within 5-10 days, and since carbon monoxide (CO) is a gasotransmitter produced by this reaction^[212] they investigated the pathway and noted that the CO signalling pathway (MAPK p38β activation, resultin in Hsp70 upregulation and Hsp110 stabilization, which then stabilizes the major transcription factor Gata-1^[213][214]) was upregulated and replicated with carbon monoxide exposure.^[210]

The authors suspected that an induction of heme oxygenase 1 (HO-1) to 2.5-fold of control accelerated CO production within the cell and stimulated the above pathway; there was no influence on HO-2.^[210] CO is known to stimulate RBC production independent of erhythropoitein^[215] as it is the third major gasotransmitter.^[216]

Garlic appears to also stimulate the third major gasotransmitter, carbon monoxide, which underlies a possible increase in red blood cell production and turnover seen in mice given the standard doses of garlic

The deposition of calcium in the arterial wall (a process known as arterial calcification, and commonly referred to as 'arterial stiffening') is an early predictor of atherosclerosis^[217] and is highly correlated with overall plaque volume post mortem^[218][219] and risk of death from cardiovascular disease;^[220] similar to Vitamin K supplementation, garlic is said to reduce calcification^[221] and persons who routinely consume garlic supplementation^[222] or fighterfighters who take garlic (alongside CoQ10^[223] seem to have less biomarkers of stiffness as assessed by PWV, a measure of aortic stiffness.^[224]

One study with garlic (1,200mg aged garlic extract) in persons at high risk for cardiovascular disease who were already on aspirin and statin therapy but further given garlic for one year noted that the 22.2% increase in coronary calcium seen in placebo was attenuated to 7.5%.^[225]

White adipose tissue appears to be highly correlated to coronary artery calcium levels^[226] and an aged garlic extract (250mg) alongside some other confounds (100μg of B12, 300μg of folic acid, 12.5mg of B6, and 100mg Arginine) in humans for one year appears to prevent the progression of coronary calcium buildup, associated with a relative (to placebo) reduction in homocysteine and less white adipose generation.^[226]

Mechanisms that underlie the interactions between garlic and white/brown adipose tissue may also underlie coronary calcification, a phenomena that contributes to arterial stiffness (and therapeutic effect of Vitamin K)

In endothelial cells NF-kB activation is implicated in promoting immune cell recruitment and proliferating smooth muscle cells, all of which contribute to atherogenesis and inhibiting its activation preventing plaque formation to a degree.^[227][228] NF-kB can be activated in these cells by oxidants such as H₂O₂,^[229] oxidized LDL cholesterol (oLDL; via macrophage activation into foam cells),^[230] or inflammatory factors such as TNF-α, IL-1, and IFN-γ.^[231]

S-allylcysteine can reduce the activation of NF-kB in response to oxidized LDL cholesterol, TNF-α, and H₂O₂ at concentrations of 2.5mM or higher^[232] and elsewhere both aged garlic extract (1-5mg/mL) and S-allylcysteine (1-20mM) reduced activation in a concentration dependent manner associated with buffering intracellular glutathione.^[233] When looking at lower concentrations, 100µM has twice failed to significantly reduce NF-kB activation in response to these stressors.^[232][233]

One study has noted that equivalent levels of aged garlic extract (2mg/mL) and S-allylcysteine favored the former in protecting the cells viability (assessed by LDH leakage)^[234] which implies other bioactives. That being said, diallyl disulfide (DADS) and allylmercaptane (AM) have failed to inhibit TNF-α induced NF-kB activation in endothelial cells (1-100µM).^[110]

When looking at the level of the endothelium (vessel wall exposed to the blood) S-allylcysteine and aged garlic extract appear to be effective in reducing inflammatory activation, which is one of the first events in atherosclerotic pathology. However, this happens at higher than normal doses and may not be too relevant following supplementation

When looking at LDL oxidation rates, S-allylcysteine has weak protective effects at 100µM and stronger effects at 1-10mM in vitro in response to copper induced oxidation;^[232][235] and due to S-allylcysteine not being a metabolite of allicin heat treatment does not affect this capacity.^[137] The mechanism of action for S-allylcysteine in these models does not appear to be related to chelating copper ions,^[137] but may be more related to direct antioxidant effects.

Diallyl sulfide and disulfide have been noted to inhibit LDL oxidation in vitro,^[236][237] which is currently thought to be related to their copper chelating properties.^[137] In fact, one study in mice given 9mg/kg allicin only noted that, when their LDL was extracted, it exhibited reduced binding to copper stimulation^[238] and a water garlic extract has shown inhibition of calcium binding to proteoheparin sulfate,^[239] both of which form a complex with LDL to initiate plaque formation.^[239][240]

In human studies, 6 months supplementation of 7,200mg of aged garlic extract has shown a trend to reduce oLDL in hypercholesterolemic men that failed to reach significance, but TBARS (a biomarker of lipid peroxidation) was reduced.^[241] Another study in this population (900mg garlic powder for 12 weeks) has failed to find an influence on LDL oxidation.^[242]

Two weeks supplementation of 600mg garlic powder to otherwise healthy persons resulted in a 34% reduction in LDL oxidation^[243] and a review mentioning a small (unpublished) pilot study noted that 3,600mg aged garlic extract in older individuals (no mention of hypercholesterolemia) attenuated the rate of LDL oxidation after six weeks.^[244]

There appears to be mild protective effects against LDL oxidation associated with the antioxidants in garlic, but the potency is not to a remarkable magnitude (significantly weaker than olive leaf extract, the supplemental reference for this category) and human studies fail to find an effect in persons who are at risk for cardiovascular disease

Oxidized LDL cholesterol (oLDL) is known to activate immune cells via the oLDL receptor, a property not seen with normal LDL cholesterol.^[245] Activation of the oLDL receptor on macrophages accelerates their conversion to foam cells which are a major determinate of atherosclerosis,^[244][246] and interventions that could reduce this process include immunosuppressive agents (prevent the already made foam cell from adhereing to the artery), reducing oxidized LDL so less can activate the receptor, or reducing receptor expression itself (less activation independent of oLDL concentrations).

In vitro, activation of immune cells is known to increase CD36 receptor expression inherently (contributes to atherosclerosis^[247]) and this process is increased in the precense of homocysteine (risk factor for atherosclerosis^[248][249]) yet coincubation with aged garlic extract at 5mg/mL is able to suppress the increased expression by 48.6-61.8% (with and without homocysteine, respectively).^[250][251] This appears to be related to the PPARγ pathway (known to increase CD36 expression^[252]) since it blocked the increase by a PPARγ agonist.^[253]

Uptake of oxidized LDL into THP-1 macrophages has been noted to be reduced at 5mg/mL aged garlic extract by 85.6+/-2.8%^[250] and replicated in this cell line at 61.8+/-7.4%;^[253] primary macrophages also note inhibition at the degree of 50.5+/-7.1%.^[253] One study in mice given 9mg/kg allicin that confirmed a reduction in atherosclerotic plaque noted less oLDL uptake from macrophages,^[238] suggesting biological relevance.

The expression of the LDL receptor on leukocytes has been noted to not be modified by 900mg garlic powder supplementation over 12 weeks in hypercholesterolemic men;^[242] this study also failed to find reductions in cholesterol overall.

Garlic extract does appear to, in vitro, reduce the ability of macrophages to take up already oxidized LDL cholesterol secondary to suppressing the signalling in the PPARγ pathway. That being said, the in vitro studies use a high dose and while this is thought to apply to supplementation of garlic in humans it has not been confirmed

One study in adults (aged 45-75) lasting for 48 months noted that supplementation of garlic daily (900mg of the powder) was associated with a stasis of plaque in the main carotid arteries whereas placebo experienced an increase in plaque.^[254] This study later was statistically evaluated, and while it was elaborated that the increase of plaque seen in placebo (74.9% increase over four years relative to their own baseline) was more than garlic (14.2%) overall, but it was only statistically significant in women but not men.^[255] Later, this was replicated with aged garlic extract (250mg) in persons at intermediate risk for cardiovascular disease where arterial calcium progression was significantly attenuated; this study did have nutrient confounds though.^[256]

Limited studies have measured plaque levels in humans or their progression over time, and while the protective effect seems moderate and shows gender differences (more protective in women) there does appear to be a mild protective effect associated with daily garlic ingestion

Garlic intake in the diet has been noted to actually be an independent predictor of endothelial function, with a higher intake being associated with better flow mediated vasodilation (FMD; indicative of blood flow).^[257]

The enzyme cystathionine γ-lyase has been detected directly in the endothelial tissue of rats, although the other H₂S producing enzyme cystathionine β-synthase is not present.^[81] It is thought that the former enzyme (cystathionine γ-lyase) mostly underlies the fasting levels of hydrogen sulfide, which were detected at 46μM in rat serum.^[81]

The enzymes that produce hydrogen sulfide are present in the endothelium

Incubation of blood vessels with 50-500μg/mL garlic is able to reduce blood vessels in a concentration dependent manner that correlates with the production of H₂S, and the molecules that confer more H₂S (diallyl disulfide and trisulfide) seem to induce relaxation to a larger degree.^[79] H₂S can directly induce vasorelaxation via acting as a potent potassium channel opener (independent of cGMP production)^[81][258] or acts indirectly through nitric oxide signalling and cGMP.^[258] Studies that cause vessel relaxation with hydrogen sulfide note that the benefits are partially prevented by both blocking potassium channels (blocking the effects of H₂S) or blocking cGMP (blocking nitric oxide signalling), supporting the two independent mechanisms.^[258]

The hypertension induced by blocking H₂S production (via abolishing the cystathionine γ-lyase enzyme) is circumvented by providing donation of H₂S,^[259] supporting the involvement of H₂S directly rather than another property of the enzyme.

Garlic is known to produce hydrogen sulfide, and hydrogen sulfide is known to reduce blood pressure secondary to positively influencing the nitric oxide signalling system (to be discussed); prior to that, however, hydrogen sulfide has some direct properties in reducing blood pressure via opening potassium channels

Garlic has been noted to increase the activity of nitric oxide synthase (NOS) enzymes in vitro^[260] and oral intake of 2.86g/kg aged garlic extract in rats caused an increase in nitric oxide (30-40%) within 15-60 minutes after oral ingestion in a manner that was blocked by inhibiting constitutive NOS enzymes (eNOS and nNOS)^[261] suggesting its relevance in vivo.

Homocysteine is known to inhibit NOS enzymes via oxidative modification of a part of the NOS enzyme called tetrahydrobiopterin.^[249] While aged garlic extract does not appear to alter homocysteine concentrations in vitro but prevents homocysteine from suppressing NOS enzymes^[262] and the ability of superoxide (O₂^-) to inhibit NOS via tetrahydrobiopterin^[263] may also be negatively affected since superoxide levels in hypertensive perosns supplementing garlic (250mg of garlic pearls) has been noted.^[264] Diallyl disulfide and trisulfide have also been noted to reduce the inhibitory effects of oxidized LDL on NOS activity,^[265] which was thought to be secondary to Akt signalling.^[265]

There are a few compounds that are elevated in instances of cardiovascular disease which then suppress the nitric oxide synthase (NOS) enzyme and reduce nitric oxide levels; garlic appears to reduce the inhibitory effects of many of these stressors to indirectly preserve nitric oxide signalling

S-Nitrosoglutathione is one of a few molecules in the S-nitrosothiol class (alongside S-nitrosocysteine and S-nitrosocysteinylglycine) that is produced when nitric oxide reacts with the thiol, and serves as an intracellular reservoir of nitric oxide. The nitric oxide is either released enzymatically^[266] or decomposition is induced by hydrogen disulfide (H₂S),^[83] and despite mostly being intracellular it can be found in plasma in the low nanomolar range.^[84]

Despite being a reservoir of nitric oxide and nitric oxide relaxing blood vells, the blood vessel relaxing properties of S-nitrosothiols is not mediated by nitric oxide at the physiological level.^[267][268] Garlic (in vitro at 45µg/mL) has been noted to stimulate the release of nitric oxide from S-nitrosothiols (physiological relevance uncertain) but to also prolong the relaxation associated with S-nitrosoglutathione.^[85]

In isolated HUVEC cells, S-allylcysteine increased cGMP production in a concentration dependent manner (20-40μM) which does imply nitric oxide signalling, but blocking the NOS enzymes have failed to abolish the effects.^[269]

The S-nitrosothiol class of molecules reduce blood pressure and serve as a donor of nitric oxide, but oddly at a physiological level the blood pressure reduction is not related to nitric oxide; garlic appears to stimulate the release of nitric oxide and prolong relaxation from S-nitrosoglutathione though, suggesting a possible benefit to blood pressure by increasing the activity of a normally redundant pathway (in regards to blood pressure)

Finally, one study has noted that a plasma increase in nitric oxide (by 224%) in humans correlated with an increase in interferon alpha (IFN-α) by 386% following ingestion of 2g of raw garlic, and this increase was noted for at least seven days (length of the study).^[71] The authors suggested that the increase in nitric oxide preceding the increase in IFN-α although IFN is known to also directly stimulate NOS activity.^[270][271]

Although more research is needed to figure of what is doing what and how, it seems that an increase in interferon alpha (IFN-α) is associated with the increase in nitric oxide seen with raw garlic

Mechanistically, phenolic rich garlic extracts are able to inhibit the ACE enzyme in the 2-10μg/mL,^[272] with the 'bound' phenolics (ie. glycosides) having an IC₅₀ of 3.48μg/mL and the 'free' phenolics (aglycone) less potent at an IC₅₀ of 14.81μg/mL.^[272] Elsewhere, there has been a noted ACE inhibitory activity of leaf extracts of garlic (thought to be related to ajoenes and thiosulfates)^[273] and dipeptides in the water soluble fraction of garlic which are less potent than captopril.^[40]

One study in rats using fresh garlic juice (100mg/g intraperitoneal injections) noted that, after eight weeks of treatment, that there was a highly significant reduction in serum ACE activity that extended to both diabetic rats (the ones being investigated) as well as control rats.^[274] This parallels previous research in hypertensive rats (oral ingestion of a basic extract)^[275] and in rats undergoing ischemia/reperfusion given a different species of garlic with similar bioactives (Allium ursinum),^[4] and oddly the magnitude of reduction was less in the diabetic group (of which it was normalized to nondiabetic control) than it was in nondiabetic rats (nearly abolished the activity).^[274]

There are several compounds in garlics that demonstrate the ability to directly reduce ACE activity which should theoretically reduce blood pressure, and it appears that prolonged intake of garlic is able to reduce ACE in rats. This has been confirmed in both diseased models (diabetes, hypertension) and at least one study noted a potent reduction in otherwise healthy control rats

1,200mg of aged garlic supplementation for 12 weeks in persons with metabolic syndrome (nonhypertensive) failed to significantly reduce blood pressure or blood flow relative to placebo^[61] and one study with 250mg of garlic pearls for two months in normotensive controls failed to find a reduction in blood pressure.^[264] One study, however, found a small reduction of 2.4-4.2% blood pressure in hypercholesterolemic persons who were also normotensive due ot 900mg garlic supplementation thrice daily^[276] and 7.2g of aged garlic has shown a similar effect (5.5%).^[277]

In otherwise normotensive persons, it seems that garlic is quite unreliable in its effects and is either wholly ineffective or causes a mild reduction in blood pressure

Supplementation of garlic (240-960mg conferring 0.6-2.4mg S-allylcysteine) for twelve weeks in hypertensive persons noted that only the middle dose (480mg of garlic conferring 1.2mg S-allylcysteine) was able to reduce systolic blood pressure significantly (11.8+/-5.4mmHg) as the higher dose trended to reduce blood pressure by 7.4+/-4.1mmHg.^[20] Reductions in blood pressure have been noted elsewhere in hypertensives to the tune of 6% or 8mmHg (systolic) and 10% or 9mmHg (diastolic)^[264] and in persons with high cholesterol (appeared to be hypertensive) by 4.7% systolic and 5.7% diastolic (1,200mg garlic for 12 weeks).^[278]

When looking at persons who are being treated for their elevated blood pressure yet are still hypertensive (140mmHg or greater), supplementation of 900mg aged garlic extract (2.4mg S-allylcysteine) daily for 12 weeks is associated with a 10.2+/-4.3mmHg reduction in systolic blood pressure relative to placebo with no influence on diastolic.^[279] In this same population (hypertensives), garlic as aged garlic extract at doses of 600-1,500mg in divided doses was able to reduce blood pressure in the range of 3-6.3% (4.3-7.6mmHg) systolic and 3.7-5.4% (4-5mmHg) diastolic; performing equally to the reference drug atenolol after 24 weeks, although only 900-1,500mg was effective at 12 weeks and 300mg was not as effective as the reference drug (2% reduction after 24 weeks).^[280]

One study has noted large variability, with one subject noting a 40mmHg reduction yet 33.8% of the subjects reporting less than 5mmHg changes.^[20]

Although the magnitude of reduction is somewhat variable in hypertensive persons, it seems that a reduction at around 5-10% is almost always seen with supplementation of garlic suggesting an antihypertensive effect. In hypertensive persons, this has once performed equally to the reference drug atenolol

A meta-analysis of hypertensive patients in nine double-blind trials with either placebo or another active treatment ranging from 3 to 26 weeks in length found a significant reduction in both systolic (-9.1mmHg, 95% CI −12.7 to −5.4mmHg) and diastolic blood pressure (-3.8mmHg, 95% CI −6.7 to −1mmHg) with a high heterogenity between trials; restricting the analysis to high-quality trials yielded a significant lowering in only systolic blood pressure (–5mmHg, 95% CI –8.7 to –1.2mmHg), with no significant change in diastolic levels.^[281]

A meta-analysis found that garlic significantly reduces systolic blood pressure and possibly diastolic blood pressure (depending on the quality of the trials included). However, trials past 26 weeks have not been conducted to see if garlic can affect blood pressure in the long-term.

When looking at components that influence platelet function, numerous compounds have been noted to exert inhibitory effects on aggregation including ajoene (50-80% inhibition at 300-600µM),^[16] sodium 2-propenyl thiosulfate,^[37] most cysteine related amino acids,^[26]

Components that seem to work in the physiological range include ajoene, which interacts with and inhibits the fibrinogen receptor (IC₅₀ of 800nM)^[282] which is a particular mechanism noted with mixed garlic extracts^[283] and some other isolated compounds such as sodium 2-propenyl thiosulfate.^[37] Some human evidence does note that following ingestion of garlic supplements (7,200mg of the aged garlic extract) there is a reduction of fibrinogen binding to platelets (by around 30%)^[241] and inhibition has been noted at a lower dose of 2,400mg aged garlic extract as well;^[128] other ligands, such as collagen or von Willebrand factor, are inhibited only at a higher dose of 7,200mg.^[128]

S-Ethylcysteine and S-1-Proponyl-L-cysteine have been noted to have inhibitory effects on aggregation at 780nM.^[26]

Most bioactives in garlic appear to exert inhibitory effects on platelet aggregation in vitro, but at higher concentrations than are likely to occur with oral supplements. When looking at compounds that are likely to be active, Ajoene seems to be a likely candidate and the main mechanism is preventing fibrinogen from binding to its receptor

When investigating platelet function in humans, 7,200mg of aged garlic extract for 10 months in hypercholesterolemic men appears to reduce platelet aggregation induced by ephedrine and collagen (but not ADP)^[241] although reduced rates ADP aggregation has been noted elsewhere (5mL of juice in otherwise healthy persons over 13 weeks) independent of maximal aggregatory potential.^[284] One study has noted that 2,400-7,200mg of aged garlic extract could reduce ephedrine and collagen aggregation at all doses yet only the highest dose tested inhibited ADP,^[128] so it is possible that particular pro-aggregatory agent is more resistant to the effects of garlic.

Dietary levels of garlic (4.2g of raw garlic cloves) over the course of one week in otherwise healthy persons does not modify platelet function,^[159] suggesting that despite the forementioned supplemental studies on garlic that moderate food ingestion has no effect.

In a comparative study, 80mg of ginkgo biloba (EGb-761) was more effective than 250mg garlic oil (21.5% diallyl disulfide) over 180 days both reduced blood viscosity to the same level, but ginkgo was deemed more effective due to a higher baseline level and larger statistical significance.^[285]

Supplements of aged garlic extract appear to have an ability to inhibit platelet aggregation when consumed by both healthy persons and those with cardiovascular complications. Moderate consumption of garlic in the diet may not be associated with such as effect

In obese rats with metabolic syndrome given aged garlic extract (2.86g/kg bodyweight; 0.1% of the diet being S-allylcysteine) with or without physical exercise for four weeks, supplementation appeared to further the weight and fat loss seen with exercise and was additive in reducing triglycerides (but no interaction was seen with cholesterol nor inflammatory markers).^[286]

Rodent research suggests that the reduction in triglycerides is additive with exercise

One study in otherwise healthy persons given 5mL of a Kyolic garlic juice product has been noted to trend to reduce triglycerides by 13%, although the effect was not statistically significant after thirteen weeks^[284] and other studies in otherwise healthy persons without high triglycerides have failed to find a reducing effect of garlic (100mg/kg raw bulb in elderly).^[287]

When selectively looking at studies of people without any problems in triglyceride levels (ie. normal levels), it seems that there are unpredictable but minor alterations in triglycerides

1,200mg of aged garlic supplementation for 12 weeks in persons with metabolic syndrome failed to reduce triglycerides more than placebo,^[61] and a failure to significantly reduce triglycerides has also been noted in hypertensives^[264] and hypercholesterolemics.^[277]

400mg of garlic (1mg allicin) twice daily for six weeks in persons with high cholesterol has been noted to reduce triglycerides by 6.3%.^[288]

In otherwise healthy controls, there is no significant reduction in triglycerides with 250mg of garlic supplementation over two months,^[264] 600mg of twice daily garlic supplementation,^[278] 900mg garlic in three divided doses for 12 weeks,^[276]

A 1994 meta-analysis on the effects of garlic supplementation on triglycerides, which noted that many of the preliminary trials had a bit of methodological problems, noted that garlic was associated with a statistically significant reduction of triglycerides by 0.31mmol/L (95% CI of 0.14-0.49)^[289] although a later meta-analysis looking at a larger pool of evidence noted a trend to reduce triglycerides that failed to reach statistical significance with a 5.45mg/dL reduction yet a 95% CI between a 14.18mg/dL reduction and a 3.27mg/dL increase.^[290]

Mechanistically, allylmercaptan (5-125µg/mL) appears to reduce cholesterol synthesis with an IC₅₀ value of around 25µg/mL (450µM)^[291][292] and causing 20% inhibition at 5µg/mL.^[291] Despite this potency, it tends to be the weakest of the tested sulfur compounds.^[293] Diallyl disulfide (DADS, a more potent compound) has an IC₅₀ of 64+/-7µM in inhibiting cholesterol synthesis^[292] and S-allylcysteine (SAC) seems active as well.^[294]

Inhibition of HMG-CoA has been reported with the water soluble bioactives Allicin (IC₅₀ 17+/-2μM), Ajoene, S-allylcysteine, S-ethylcysteine, and S-prpoylcysteine although Aliin is inactive^{[294][295][296]} and components in the oil fragment such as diallyl disulfide (inhibitory at 5μM).^[293]

The components of garlic appear to be inhibitors of HMG-CoA (molecular target of statin drugs) and secondary to that can inhibit cholesterol synthesis. This mechanism appears to occur potently enough and at a low enough concentration with some of the stable garlic products that it likely explains the observed human effects

The first meta-analysis on garlic and cholesterol (1993) noted that low doses of supplemental garlic, correlating to approximately one half to a full clove of garlic day, are associated with reductions in cholesterol in persons with high cholesterol by 0.59mmol/L (around 9%);^[297] subsequently, a meta-analysis on the influence of garlic on cholesterol noted that there was a statistically significant reduction in total cholesterol by 0.77mmol/L (95% CI of 0.65-0.89) which was a 12% reduction relative to control.^[289] Further meta-analyses have been conducted on the topic as well, concluding a 0.41 mmol/L reduction ^[298] and an 8% reduction^[290] which were similar in magnitude to the first two meta-analyses although they had differeing conclusions; one meta-analysis stating that the study quality was of questionable clinical relevance^[298] and the other directly stating clinical relevance and an alternative to statin therapy (in some populations).^[290]

These meta-analyses tended to assess studies that used a lower dose garlic supplementation of around 700-1,200mg, and tended to last lengths of over 8 weeks (with some studies lasting up to 24 weeks). The subjects were those with hypercholesterolemia and a total cholesterol level exceeding 200g/dL.

When looking at all meta-analyses conducted, they all report a reduction in circulating cholesterol associated with garlic supplementation and this reduction seems to be of a similar magnitude in all analyses at around 10% for persons with high cholesterol (over 200mg/dL)

While the above meta-analyses tended to only assess total cholesterol, LDL and HDL have both been directly investigated.

In regards to LDL, there are mostly reductions associated with 7,200mg of the extract (4.6%),^[277] 900mg over twelve weeks (14.2%),^[276] 600mg of garlic extract known as Allicor over 12 weeks (13.8%),^[278] 400mg over 12 weeks (17.3%),^[288] whereas 250mg of the pearls^[264] and 1,200mg of the extract in persons with metabolic syndrome over 12 weeks^[61] have failed.

Improvements (increases) in HDL-C have been noted with 600mg of garlic (Allicor) over 12 weeks (11.5%),^[278] 400mg over 12 weeks (15.7%),^[288] while nonsignificant reductions have been seen with 7,200mg of garlic^[277] and 900mg over 12 weeks,^[276] and no significant influence with 250mg of garlic pearls^[264] or 1,200mg over 12 weeks in persons with metabolic syndrome.^[61]

Garlic seems to reduce LDL-C in a fairly reliable manner (although some negative evidence does exist) whereas it is a bit less reliable on HDL-C yet still seems to have a beneficial effect there. The magnitude of change for LDL and HDL appears to be in the range of 10-15% when looking at persons with high cholesterol

An in vitro assessment of garlic extract has noted that, against the α-glucoside enzyme, that garlic has an inhibitory effect with an IC₅₀ of 136.3µg/mL^[299] although elsewhere garlic extracts have failed to have any inhibitory potential on both α-glucoside and α-amylase when 100µL (1mg dry plant weight) was tested.^[131] It was also noted that inhibitory actions of spices on the enzymes of carbohydrate metabolism correlated well with the flavonoid content^[131] which garlic is known to be pretty low in.

There is not a lot of evidence on this particular topic overall, but the one study that suggested garlic may have a potential inhibitory effect on glucose absorption is directly contradicted by another; practical significance of garlic and carbohydrate absorption not yet clear but doesn't look promising

In vitro, S-allylcysteine sulfoxide appears to stimulate insulin secretion from pancreatic β-cells isolated from normal rats.^[300]

Supplementation of 1,200mg aged garlic in persons with metabolic syndrome for three months has failed to reduce fasting insulin or to improve insulin sensitivity.^[61]

In healthy as well as diabetic rabbits, 250-350mg/kg of garlic extract appeared to have a dose-dependent reduction in blood glucose after four hours with a potency comparable to 250-500mg/kg Metformin.^[301] Acute effects like this have been noted with isolated components such as S-allylcysteine sulfoxide^[302] and with the garlic extract except with an oral glucose tolerance test.^[303]

Supplementation of 1,200mg of aged garlic for 12 weeks in persons with metabolic syndrome has failed to significantly reduce blood glucose.^[61]

Advanced glycation end products (AGEs) are small molecules developed from the process of glycation, and their production in states of hyperglycemia are thought to mediate damage to tissues and the genome in diabetes.^[304][305] AGEs include compounds like carboxymethyllysine, which is the major AGE formed from oxidative breakdown^[306] and is at two-fold higher concentrations in diabetics relative to normoglycemic persons^[307] and can induce inflammatory damage via NF-kB.^[308]

Advanced glycation end products (AGEs) are formed in the blood when a combination of high blood glucose and high oxidation are seen for long periods of time, and the production of AGEs is mostly relevant to diabetes and mediates a fair bit of organ damage seen in diabetics (the -pathy comorbidities)

Aged garlic extract at 28-84mg/mL range has been shown to inhibit over 50% glycation in vitro, which was seen with 30-80mM of isolated S-allylcysteine^[309] and S-allylcysteine was noted to have Amadorin like activity and inhibited carboxymethyllysine formation at the lowest tested concentration of 10mM;^[309] sulfur containing compounds (DAS, DADS) are less effective than cysteine containing compounds in attenuating glycation (LDL)^[236] although it may be beneficial when oxidation is also a stressor^[237] and while S-ethylcysteine has once been reported to be most effective^[237] but seems comparable to S-allylcysteine and S-propylcysteine in the 5-15µM range.^[236]

Amadorin-like compounds are those that ultimately inhibit AGE production, but inhibits the previous step of an amadori compound being oxidized into an AGE (of which the reference drug for this mechanism is Pyridorin).^[310] Garlic extract also has also been noted to normalize the activity of both enzymes of polyol synthesis (aldose reductase and sorbitol dehydrogenase) after oral ingestion in diabetic rats,^[311] so it is possible multiple pathways are involved.

Multiple sulfur bearing components in garlic are known to reduce the rate of glycation of proteins (hemoglobin and LDL mostly) and it isn't clear which ones are most potent in a living system. Beyond that aforementioned glycation (and the AGE pathway) there also seems to be some interaction with the polyol pathway which ultimately results in similar organ damage

In streptozotocin induced diabetic rats, there is a reduction in glycation of the kidneys and red blood cells seen with 500mg/kg of fresh garlic extract over eight weeks, which occurred alongside a reduction in blood glucose (57%) and slight restoration of insulin; glycated hemoglobin (HbA1c) was reduced 43%^[312] and elsewhere in rats a normalization of blood glucose (250-500mg/kg garlic extract) after 30 days was associated with a normalization of the enzymes in polyol synthesis.^[311]

Some studies that note a failure to reduce blood glucose also note no changes in HbA1c, although it seems that organ protection from diabetes may still occur when neither of the aforementioned two do.^[313]

When blood glucose is reduced there seems to also be a reduction in HbA1c in rodents, but blood glucose reductions with garlic are not necessarily commonplace. Even if blood glucose is not reduced though, there seem to be some protective organ effects

Garlic appears to be quite a popular alternative medicine for diabetes, being one of the most popular reported alternative medicine (2001 data)^[314] and being used in the Malaysian region as well (13.3% of those who reported using complementary medicine, less than both Momordica Charantia and Java).^[315]

Garlic appears to be frequently used as an alterative medicine for the treatment of diabetes

Numerous rat studies that use doses of garlic that are able to exert some protective effects in the state of diabetes fail to actually find reductions in blood glucose in streptozotocin induced diabetes^{[203][204][205][274][313]} or have a mild and clinically irrelevant reduction;^[72] there is limited evidence that notes that 500mg/kg of the fresh bulb can eventually work after seven to eight weeks of supplementation^[312][316] but most evidence, as assessed by meta-analysis, suggests no significant effect.^[317]

When looking at alloxan induced diabetes, garlic at 10mL/kg ingestion to rats after diabetes has stabilized appears to normalize blood glucose relative to undiabetic control, which was as effective as onion juice at the same dose;^[318] both garlic sulfoxides (S-allylcysteine sulfoxide) and onion sulfoxides (S-methylcysteine sulfoxide) are effective individually^[319] (as well as DMSO actually, or dimethyl sulfoxide^[320]) suggesting the sulfoxide group is active in this model of diabetes.

Although both toxins cause pancreatic β-cell toxicity and necrosis, streptozotocin works via directly alkylating the DNA and alloxan causes an intracellular oxidative stress.^[321] It is not sure if these differing mechanisms matter due to the beneficial effects seen after diabetes has stabilized (rather than ingestion alongside the toxin).

Animal models of the interactions of garlic and diabetes are very divergent, with the alloxan models of diabetes showing great promise in normalizing blood glucose yet the streptozotocin models showing little to no effect

In a single blind pilot study on type II diabetics, 24 weeks of garlic supplementation at 900mg (300mg thrice daily with an 0.6% allicin content) given alongside Metformin therapy noted that while there was a modest improvement in blood glucose associated with garlic (The 1.78% reduction with metformin was increased to 3.12% with the combination) there were significant improvements on lipid profiles of the patients; most notably the 6.72% increase in HDL-C.^[322] This study of study was replicated with 250mg of garlic extract two to three times daily alongside Metformin over twelve weeks, the addition of garlic was able to increase the 16% reduction in fasting blood glucose seen with Metformin to 23% and also showed a most additive benefit on postprandial blood glucose as well as HbA1c;^[323] this study also noted hypolipidemic effects of garlic not seen with Metformin alone.^[323]

Human studies in diabetics given garlic note very modest improvements in blood glucose concentrations when paired with Metformin, suggesting it may be an effective add-on to Metformin.

Garlic powder at 0.8% of the rat diet (the powder of which was 1.5-5% diallylsulfides) appears to stimulate plasma levels of noradrenaline and metabolic rate,^[324][101] which are thought to be due to diallyldisulfide (able to stimulate noradrenaline in isolation in a dose-dependent manner)^[324] although other sulfur bearing compounds including alliin, S-allyl-L-cysteine, and diallyl trisulfide (most potent) also stimulated noradrenaline secretion whereas diallyl sulfide was barely effective.^[101] Adrenaline itself has once been noted to not be increased^[101] but may also be stimulated with all sulfur components tested.^[325]

Diallyldisulfide has been shown to increase the thermogenic rate in a manner which is blocked by β-adrenergic receptor blockers,^[101] and since noradrenaline itself stimulates thermogenesis secondary to acting upon brown adipose tissue via the β-adrenergic receptors^{[326][327][328]} it is hypothesized that garlic works vicariously through an increase in noradrenaline, a mechanism similar to ephedrine.^[329][330]

Garlic components are able to stimulate the secretion of noradrenaline, and this increased secretion of noradrenaline works on the β-adrenergic receptors of brown adipose tissue to increase thermogenesis (heat production)

Protein uncoupling refers to proteins (UCPs) that enable proton dissipation across the mitochondrial wall, which induces bioenergetic inefficiency and 'uses' cellular energy for heat production without necessarily increasing ATP levels in a cell.^[331] While UCP1 is known to mediate the ability of brown adipose tissue to increase heat production^[331] UCP2 is more generally widespread in tissues^[332] and UCP3 highly expressed in muscle tissue.^[333]

It appears that 2-5% garlic in the diet of rats for seven weeks is able to increase uncoupling protein mRNA in both white (220-700% for UCP2 only) and brown (200-230% for UCP1, 170-210% for UCP2, 10% for UCP3) adipose tissue as well as the liver (30% for UCP2) and skeletal muscle (70-90% for UCP2 and 90-240% for UCP3).^[102] Elsewhere, an increase in brown adipose tissue UCP has been replicated but to a larger degree in rodents on a high fat diet over 28 days^[325] yet there were no differences in noradrenaline secretion.^[325]

This is associated with an increase in AMPK activation in all tissues, which is known to regulate UCPs and itself may be regulated by noradrenaline (at least in brown adipose);^[334] and at least with thiacremonone the increase in UCP2 is prevented when AMPK is blocked.^[99]

The influence of noradrenaline on tissues may increase the activity of AMPK and the rate of protein uncoupling, resulting in an increased energy expenditure via passive heat diffusion. There appear to be some interaction with diet and garlic (being more effective on a high fat diet) which are not related to noradrenaline

AMPK activation is known to negatively regulate adipogenic factors mainly by inactivating acetyl CoA carboxylase 1 (ACC-1) which, secondary to producing less Malonyl-CoA (rate limiting substrate), suppresses adipogenesis^[335] and alleviate any suppressive effects on fat oxidation such as CPT-1 downregulation.^[336]

Thiacremonone, secondary to AMPK activation, can exert antiadipogenic effects in vitro (300-600μM)^[99] and 100μM of 1,2-vinyldithiin (1,2-DT) has also shown antiadipogenic effects not yet tied back to AMPK.^[27]

In vitro, garlic components appear to suppress lipogenesis (cellular fat accumulation) and reduce the proliferation of fat cells. This is thought to just be secondary to AMPK activation

Oral ingestion of 2-5% garlic in the diet of high fat fed rats is able to attenuate the increase in adipogenic gene expression in white adipose tissue of which includes SREBP-1c, PPARγ, and C/EBPα.^[102]

Antiadipogenic effects have been noted in rats following oral ingestion of the highest nontoxic doses of garlic

In research animals, garlic supplementation at 0.8% of the diet is known to stimulate the production of brown fat over the course of 28 days^[324] although one study separating rats into a high carb or high fat diet only noted such an increase in the high fat group;^[325] previous studies noting an overall increase have used high fat models.^[324]

Brown fat tissue in rodents given garlic extracts (0.8% of the diet) tend to have higher mitochondrial content than do controls irrespective of diet.^[325]

A supplement containing aged garlic extract (250mg) alongside some other confounds (100μg of B12, 300μg of folic acid, 12.5mg of B6, and 100mg Arginine) in humans for one year appears to promote the production of brown adipose tissue relative to placebo.^[226]

There appears to be an increase in the overall quantity of brown fat in rats that are fed garlic, and preliminary evidence suggests that this may also apply to human consumption of garlic (study is, however, confounded with other components in the diet)

Secretion of IL-6 and CRP from isolated adipocytes stimulated by macrophages appears to be attenuated with incubation of 100μM of 1,2-vinyldithiin;^[27] as these cytokines are secreted from visceral fat and exacerbate metabolic syndrome^[337][338] garlic has been tested in persons with metabolic syndrome, but 1,200mg of an aged garlic extract for 12 weeks failed to influence serum C-reactive protein or IL-6.^[61]

Technically may reduce the inflammatory response from adipose tissue, but oral supplementation of garlic does not appear to be significantly effective

Adiponectin is known as an antiinflammatory adipokine that also has both antidiabetic and cardioprotective roles,^[339][340] and its increase is thought to be therapeutic for metabolic syndrome.

1,200mg of aged garlic supplementation daily (Kyolic brand) for twelve weeks in persons with metabolic syndrome is able to significantly increase plasma adiponectin by increasing it by 313.79+/-179.44ng/mL rather than the reduction of 271.88+/-187.18 seen in placebo.^[61]

Oral supplementation of garlic may be able to increase adiponectin concentrations in the serum of persons with metabolic syndrome

1,200mg of aged garlic daily for 12 weeks in persons with metabolic syndrome does not influence circulating leptin.^[61]

Animal studies investigating the effects of garlic on the body tend to measure weight even if it is not the primary end point, and there have been some instances where garlic administration to a control group over a prolonged period of time (shortest being 8 weeks) has resulted in weight loss relative to a control which recieved saline or placebo chow.^[204]

Garlic appears to have traditional usage (Egypt and Rome) in being given to labourors to reduce fatigue (and entice more work to be done) while reducing infection risk.^[341] It is also reported to have been given to Roman athletes to bolster endurance and strength.^[341]

Improvements in physical performance have been seen in mice subject to a treadmill endurance test 30 minutes after garlic ingestion where, although both effective, aged garlic extract was more reliable than raw garlic bulbs.^[342] Subsequently aged garlic extract (2.86g/kg of the diet, conferring 0.1% of total dietary S-allylcysteine) was noted to improve submaximal treadmill performance associated with improvements in nitric oxide levels but no alterations in lactic acid^[343] and improved swimming performance in rats prior to and after inducing cardiac damage (isoprenaline).^[206] 1g of raw garlic (but not 2g) to rats has improvements on a rotarod treadmill test relative to control^[189] and the higher doses of raw garlic circumventing the benefits of lower doses has been noted previously.^[342]

100mg/kg of the garlic bulb has been reported to improve pulmonary hypertension in periods of hypoxia^[344] and garlic has been noted to increase nitric oxide by 30-40% within 15-60 minutes^[343] which both suggest a peripheral mechanism of action.

Rodent research suggests that garlic may be beneficial for the purpose of oxygenation during exercise secondary to interactions with nitric oxide signalling. It seems to benefits rats during submaximal exercise or in rats that have some manner of impairment to exercise function (ie. cardiac damage)

Some human studies cited in a review but unavailable online^[341] have noted reductions in subjective perceptions of fatigue, and it seemed most positive results were associated with instances of sickness or fatigue that were being attenuated. This is seen with a production known as KYOLEOPIN that has additional B vitamins (B3, B5, B6) and liver extract showing anti-fatigue effects in humans.^[345]

In nine competitive male cyclists given 4,650mg of garlic bulb extract (46.5mg allicin) in three divided doses for one week prior to a hypoxic exercise test to exhaustion, there was no significant effect on oxygen consumption nor heart rate and the overall time to exhaustion was similarly unaltered.^[346]

In persons with coronary artery diease (CAD) on stable drug therapy who were then subject to six weeks of garlic oil supplementation (equivalent to 1g of raw garlic) reduced peak heart rate (12%) and work load on the heart during exercise without effect on resting heart rate, which resulted in improved exercise capacity.^[347]

The human evidence is (mostly unavailable) mixed, and it seems that garlic may have a potential role in improving physical performance during instances of cardiac damage. The one study looking at maximal performance in elite athletes has failed to show any benefit, and animal research suggests garlic just helps with submaximal exercise

When surveying complementary medicines used for the treatment of osteoarthritis (OA), it appears that garlic is one of the more popular options (36% of persons who reported using herbal products) comparable to ginger (30%)^[348] although still less popular than fish oil or vitamin supplements (overall category).^[349]

When looking at the overall prevalence of OA (hip) in the population, it seems that there were two categories of food that were significantly associated with a reduced risk of developing OA; allium vegetables (of which garlic and onions are the most popular) as well as noncitrus fruits.^[350]

Garlic appears to frequently be used for the treatment of osteoarthritis in persons who venture to complementary medicine, and there appears to be a reduced occurrence of OA development in those who eat allium vegetables

In mice given adjuvant induced arthritis, 1-2mg/kg injections of thiacremenone appears reduce paw volume (indicative of edema) in a dose dependent manner with a potency lesser than 10mg/kg indomethacin.^[31] This was associated with a suppression of NF-kB activity.^[31]

Animal studies suggest that some of the bioactives have general antiinflammatory effects against research chemicals that induce arthritis

Garlic extract appears to have antibacterial properties against common dental strains of bacteria (Streptococcus mutans, Streptococcus sanguis, Streptococcus salivarius, Pseudomonas aeruginosa, and lactobacillus^{[351][352][353]}) with similar efficacy between 5-100% concentration^[352] and possibly effective against drug resistant variants (Streptococcus mutans^[353]); these benefits are thought to be related to allicin and its derivates as well as thiosulfates^[354] and the potency seems to be less than that of 0.2% chlorhexidine gluconate^[352] and minimum inhibitory concentration (MIC) values have been reported to be in the 4-32µg/mL range.^[353]

Garlic does appear to have relatively effective anti-bacterial properties with efficacy against oral bacteria, although the active compounds may be the sulfur containing compounds (and thus usage as an oral paste limited by the taste properties and 'breath' side-effect)

Garlic oil (100mg/kg) has been noted to attenuate bone loss and improve tensile strength over a month when given to a rat model of osteoporosis over the course of a month^[355] and this has been replicated elsewhere using the same dose and time frame in the same rat model^{[356][357][358]} where garlic oil seems to be of similar potency to lovastatin (900µg/kg) but less effective than the reference drug of 10µg/kg 17β-estradiol.^[357] Garlic oil likely works via increasing estrogen in the body^[355] which has been measured at 44% preservation relative to control^[358] although it is not clear if this is a direct induction of estrogen or merely a preserving effect.

This protective effect is associated an increase calcium absorption from the intestines^[355] and reduction in urinary calcium and phosphorus excretion,^[356][357] both mechanisms that occur from increased estrogen signalling. Furthermore it seems that the increase in IL-6 and TNF-α seen during estrogen deficiency from macrophages^[359] which is thought to be due to increased intracellular oxidative stress; garlic oil can attenuate both the cytokine release and the oxidative stress in these cells.^[358] A decrease of TNF-α (38.7%) but not IL-6 has been noted in menopausal women consuming 400mg of a dry garlic extract thrice a day (1,200mg, said to be equivalent to 2g raw garlic) over one month^[360] which suggests that this dose of garlic is active in humans. The above mechanisms may also be related to estrogen, since macrophages express an estrogen receptor^[361] which can be activated to regulate oxidative functions.^[362]

Garlic oil appears to be able to preserve bone loss in animal models of menopause and is thought to just work through increasing estrogen in the body, and one biomarker seen in these studies (TNF-α) is affected in menopausal women consuming raw garlic products which suggests that they also affect humans. No human studies have measured bone loss per se yet

Garlic has been noted to increase both IFN-α^[71] following oral ingestion of garlic raw garlic cloves (weighing 2g with the blood measured within an hour).

An increase in interferon alpha (IFN-α) has been noted in humans given raw garlic supplementation, and appears to be fairly rapid

IFN-γ has its expression in macrophages increased with aged garlic extract (IC₅₀ 37mg/mL) in macrophages infected with Leishmania but not uninfected cells^[363]

Garlic has been noted to increase IFN-γ in various animal models given the aged garlic extract^[39] and in humans given 2,650g of Aged Garlic Extract.^[120] This is thought to underlie the macrophage activation in response to bacterial infections and, when cooperating with Th1 cells, an increase in immunity. Elsewhere, garlic oil has been noted to increase IFN-γ when given to rats at 100mg/kg orally while doubling the dose suppressed IFN-γ relative to control.^[364]

Garlic can increase the production and secretion of IFN-γ in macrophages, and an increased serum level of IFN-γ has been confirmed in humans given the aged garlic extract

Garlic appears to suppress LPS-induced macrophage activation in a concentration dependent manner^[269] which was noted to also occur with isolated S-allylcysteine (20-80µM) and thought to be due to NF-kB inhibition^[269] although HO-1 induction (a downstream effect of Nrf2 signalling) has also been implicated with the extracts.^[365]

When looking at components of the oil, DAS appears to suppress the LPS-mediated induction of PGE₂ and COX2 at 1-10μM reaching 20-67% inhibition (DADS and allyl mercaptan inactive) while all compounds very mildly reduce nitrite accumulation in the concnetration range of 100nM to 2μM (DADS being more potent); this appeared to be due to directly interacting with the nitric oxide.^[366]

2.5-10μg/mL of thiacremenone in isolated RAW 264.7 macrophages (as well as THP-1 cells) appears to reduce LPS-stimulated nitric oxide production with an IC₅₀ of 8μM attributed to inhibiting NF-kB^[31] which is thought to be due to thiacremenon by oxidizing the sulfhydryl residue of p50; a mechanism that was inhibited by the reducing agents dithiothreitol and glutathione.^[31]

Thiacremenone appears to directly interact with the p50 subunit of NF-kB, and secondary to oxidizing p50 is may be able to suppress overall NF-kB activation at a fairly low concentration

Macrophages with this particular infection also secrete more IFN-γ and expression iNOS to a larger degree when they are incubated with aged garlic extract (37mg/mL), although this does not occur in uninfected macrophages^[363] and similar effects are seen with the IL-12 cytokine^[367] IFN-γ and IL-12 (assuming cooperation with NK cells, CD4+ and CD8+ T-cells, and dendritic cells) are major determining factors in treating infection from Leishmania,^[367] and since these changes in cytokines have been noted in mice infected with Leishmania treated with an extract containing the glycoprotein (14kDa) it supports garlic's biological relevance.^[39]

Macrophages have also noted increased killing potential and phagocytosis in response to Leishmania infection,^[368] which may be related to the released IL-12 stimulating Th1 cells to release more IFN-γ, which activates this process in macrophages.

IL-10 is usually a negative regulator of both Th1 cells^[369] and macrophage stimulation,^[370] but it seems unaffected in macrophages.^[367]

Garlic appears to increase the activation of macrophages (phagocytosis refers to consuming a bacterial invader, killing potential refers to actually digesting it), and this seems to be due to increasing how much IL-12 the macrophage releases when it detects a bacteria; allowing Th1 cells (T cells) to increase macrophage activity via IFN-γ. This is thought to be relevant following garlic consumption since we have detected increases in serum IFN-γ

Neutrophils are the immune cells that are seen as the first-line defense against bacterial invasions, and are known to mediate their immune response through rapid production of superoxide (O₂^-) which effectively destroys bacteria via oxidative damage;^[371] this oxidative damage can, if overactivated, damage human tissue via the same mechanism.^[372]

When incubated in neutrophils that are stimulated with PMA, aged garlic extract (3mg/mL) appears to slightly attenuate superoxide production^[67] which may be due to some direct superoxide scavengers in aged garlic extract^[67] and raw garlic also appears effective (chloroform extract most effective).^[373]

Neutrophils mediate oxidative damage to ward off bacterial infections, and excessive activation is known to cause tissue damage associated with 'highly inflammatory states'. Aged garlic extract seems to have an antioxidant effect in activated neutrophils

Neutrophils are attracted to sites of infection or injury by chemoattractants (chemicals that attract), with chemoattractants such as CINC-1 (mouse equivalent of IL-8),^[374] TNF-α, or IL-1β^[375] causing an increase in the neutrophil's CD11b/CD18 receptor which binds to the endothelium via the intercellular adhesion molecule-1 (iCAM-1) on the blood vessel.^[376][377]

Garlic oil has been noted to reduce the response of neutrophils to IL-8^[378] and has reduced levels of the adhesion factors such as iCAM-1^[379] and reduced receptor (CD11b) expression on neutrophils^[379] which are thought to be due to the diallyl sulfides (DAS, DADS, and DATS) although each molecule seemed to have differential mechanisms.^[380] Expression of iCAM-1 and CD11b was not affected unless there was an inflammatory stimuli that would otherwise increase them.^[380]

This is thought to underlie the ability of garlic oil or raw garlic to suppress neutrophil infiltration of tissue, both intestinal tissue in response to stressors^[379][380] and the endothelium.^[381][382] This has been noted with oral ingestion of 10-50mg/kg of garlic oil in rats in a dose dependent manner (although 100mg/kg worsened neutrophil infiltration)^[379] and replicated with isolated diallyl sulfides in the dosage range of 0.025-0.125mM/kg (equivalent to the 10-50mg/kg garlic oil dose).^[380] Interestingly, since 100mg/kg of the oil was ineffective (and actually augmented recruitment)^[379] and 50mg/kg of DATS was less effective than DAS and DAS^[380] it implicates hydrogen sulfide since H₂S has been noted to suppress neutrophil migration in instances of activate neutrophils^[383] yet augments it otherwise.^[384]

Garlic oil (and the diallyl sulfides) appear to have immunosuppressive and antiinflammatory effects on neutrophils when they are activated. This has been confirmed in rodents with oral ingestion of low doses of garlic oil (higher doses seem to have the opposite effect). Although it needs to be confirmed, this is likely due to an immunomodulatory effect of hydrogen sulfide

Garlic is investigated for its interactions with natural killer cells due to its reported immunomodulatory health and improved resistance to infections, where along with cytotoxic T cells the natural killer cells play a major role in both innate and adaptive immunity.^[385] Natural killer (NK) cells are known to produce interferon gamma (IFN-γ^[385]) and tumor necrosis factor alpha (TNF-α^[385]) amongst other cytokines, both of the aforementioned beening elevated in humans given garlic.^[120] NK cells themselves are primed by various factors including IL-15 from either dendritic cells^[386] or macrophages,^[387] IL-12,^[388] and IL-18^[389] which increase their activity.^[385]

A glycoprotein of 14kDa has been isolated from the R10 fraction of fresh garlic,^[38] which is the same fraction that causes a shift from Th2 cytokines to Th1 cytokines (suggesting the same bioactive).^[39] This glycoprotein, at 20mg/kg (IP injection) appears to itself increase natural killer cell activity against tumors^[38] and since the sulfur donating compounds (in this case, diallyl trisulfide and disulfide) do not appear to influence NK cells^[390][391] it seems this may be the main bioactive.

In accordance with its 'boosting immunity and resistance' claims, garlic is investigated for its interactions with natural killer cells which are involved in both adaptive (in response to an antigen) and innate immunity as well as tumor killing. This may be related to a glycoprotein in the garlic rather than the sulfur bearing molecules

Natural killer cells are known to expression pattern recognition receptors (PRRs)^[392] which respond to pathogen-associated molecular patterns (PAMPs) similar to macrophages and one subset of T cell (γδ-T cell); this contributes to antibacterial recognition and targeting^[393] and is underlies an ability of NK cells to respond to bacterial infections such as the common cold.

In otherwise healthy humans using 2.56g of aged garlic extract daily for 45 days, there was a doubling of NK cell activity in serum relative to placebo^[120] which, alongside an induction in γδ-T cell population, were thought to underlie the reduction in infection rate of the common cold seen with garlic.^[120]

Natural killer cell increases have been noted in otherwise healthy humans, and this is thought to in part contribute to the nonspecific immunity and reductions in the common cold occurrence seen with garlic

In male mice incubated with Sarcoma-180 tumors, 10mL/kg of raw garlic juice (0.162% allicin), heated juice (0.266% alliin), garlic powder (0.462% alliin) or aged garlic extract (0.003% allicin) every other day for three weeks noted minor reductions in tumor size but increased NK cell cytotoxicity against YAC-1 cells in the range of a 485-742% increase, with a potency similar to polysaccharide K (from Trametes Versicolor) except from the dehydrated powder which outperformed.^[394] The glycoprotein by itself (20mg/kg) has augmented NK cell activity against K562 tumor cells when injected into mice.^[38]

When looking at human evidence a study in persons with cancer of the colon, liver, or pancreas given aged garlic extract (500mg) for twelve weeks alongside standard chemotherapy failed to note any changes in quality of life yet an increase in natural killer cells (34%) preceded the increase in overall activity; activity per cell was unaffected.^[119] This was thought to be secondary to attenuating a reduction in NK cell content, since there were numerous reductions in placebo on a case per case basis while none occurred in the garlic group.^[119] This potential 'immunoprotective' effect has been observed in mice exposed to stress with or without aged garlic extract, where the ability of their NK cells to attack tumor cells (YAC-1) is fully preserved with garlic relative to control.^[395]

In both humans and rodents with cancer, an increase in natural killer cells is seen relative to control when garlic is ingested. The human study did not investigated whether there was any cytotoxicity to the tumor (and they were not seen to a large degree in the animal study either)

Natural Killer cells (as well as cytotoxic T-cells) are known to contribute to acetominophen induced toxicity secondary to inflammatory signalling^[396][397] and it appears their infiltration into liver (Kuppfer) cells are reduced with Thiacremonone (10-50mg/kg in mice),^[398] the antiinflammatory molecule of garlic extracts.

Natural killer cells can be involved in pathological inflammatory responses that cause organ damage; unrelated to the stimulatory effect of garlic on natural killer cells per se, but when garlic reduces the damage to the liver associated with acetominophen natural killer cells no longer infiltrate the liver

There is an atypical variant of T-cell known as a γδ-T cell, which respond to pathogen-associated molecular patterns (PAMPs)^[399][400] rather than the typical protein antigens.^[401][402] Priming γδ-T cells has been noted in tea drinkers^[403] thought to be due to the metabolite of L-theanine known as ethylamine (since this molecular structure primes γδ-T cells^[404]) and associated with some medicinal products such as apple polyphenols.^[405]

Garlic supplementation (2.56g of aged garlic) daily for 45 days resulted in somewhat of an unreliable increase in magnitude when looking at a case by case basis, but overall an 8-fold increase was noted and this was seen alongside a significant reduction in cold frequency.^[120] The activity of this receptor (expression of the γδ-TCR) was not necessarily increased at basal conditions.^[120]

A particular T cell known as a γδ-T cell appears to be involved in innate immunity and is thought to be relevant to reducing infection risk when it is elevated. Garlic appears to increase levels of this T-cell about 8-fold following ingestion of a high dose of aged garlic extract

A extraction of garlic (thought to contain a 14kDa glycoprotein as the bioactive^[38]) given to mice at 20mg/kg injections appeared to reduce lesions size from Leishmania major to a level more than the reference drug (60mg/kg glucantime) but also additive with it over the course of 20 days.^[39] This was associated with a chance from a Th2 cytokine profile (less IL-4 and IL-10) to a Th1 cytokine profile (increased IFN-γ and IL-2).^[39] Other studies in Leishmania major have replicated increases in IFN-γ^[363] and IL-12 (macrophages)^[367] suggesting it can useful for this infection.

Garlic appears to be able to promote an increase in IFN-γ and IL-2, reflective of a Th2 cytokine profile in T-cells which tends to be more associated with anti-tumor and anti-infective properties of T cells

250-750mg/kg garlic extract (water extract of fresh bulbs) to rats appears to increase total white blood cell and CD4+ T-cell count in a manner that is antagonistic to coingestion of onions.^[406]

S-allylcysteine appears to reduce the NF-kB activation in T cells that occurs due to H₂O₂ or TNF-α.^[407]

The common cold is a collection of infectious diseases which cause similar symptoms of nasal congestion and discharge, sneezing, sore throat and cough (possibly also malaise, lethargy, and headaches).^[408] Garlic appears to be quite a popular supplement for the treatment of the common cold (30% of garlic users in Australia, 2007, reported garlic for this purpose^[13]) in part due to marketing and in part due to traditional usage to 'bolster immunity'.

A meta-analysis has been conducted on the topic of garlic and the common cold,^[409] and due to its inclusion criteria it accepted a lone study^[410] which used a raw garlic powder supplement conferring 180mg allicin daily for twelve weeks; cumulative sick days was redued from 366 in placebo down to 111 in garlic (70% reduction) while the average length of symptoms was reduced from 5.01 in placebo to 1.52 in the garlic group (70% reduction).^[410] Other studies found (and excluded) by the meta-analysis include those using supplements confounded with other ingredients (such as nigella sativa, echinacea, and panax ginseng),^[411][412] had complications with controls,^[413][414] or one case also included influenza (whereas the meta-analysis only looked at the common cold) but overall the supplement (600mg garlic powder) was associated with a 1.7-fold reduction in acute respiratory disease in children treated with garlic relative to placebo.^[415]

Following up this meta-analysis was a new study which used aged garlic extract at a relatively higher dose of 2.56g daily, and it was noted that after 90 days of supplementation there was a reduced number of collective sick days with garlic relative to placebo (61%) as well as reported sickness incident (58%) and symptom severity (21%).^[120] The symptom frequency, assuming the subject was sick, was not significantly influenced.^[120]

There is not a large body of evidence investigating the link between garlic and sickness from either the common cold or the flu, but garlic appears to be quite effective in the limited evidence that does exist. It is most effective in preventing sickness from occurring, mildly effective in reducing severity of symptoms but doesn't seem effective in reducing the frequency of symptoms assuming you are already sick

In otherwise healthy mice given an aged garlic extract (0.1% S-allylcysteine; total oral dose of 10mL/kg) noted that the stress-induced reduction in spleen size and cell count were reduced 75% and 55% with garlic relative to stressed control.^[395] The reduction in natural killer cell activity and immunity against an antigen were also both significnatly ablated with garlic, suggesting an immunoprotective effect against stress^[395] which has also been noted in alloxan-induced diabetic rats given either garlic or nigella sativa (where total lymphocytes were preserved).^[416]

One study on human cancer patients investigating the alterations in natural killer (NK) cells in response to garlic ingestion (500mg of aged garlic extract) noted that there was a relative increase in NK cell content in the experimental group relative to placebo; this was thought to be mostly due to attenuating a decrease in NK cell content that was observable in placebo.^[119]

One study (not an intervention, but a longitudinal study nested within the Women's Interagency HIV Study) noted that usage of garlic supplements, supposedly popular amongst persons with HIV, was not associated with any beneficial nor adverse changes in HIV viral load or HAART therapy adherence; CD4+ cells seemed similar between those using garlic and those not as well.^[417]

When looking for phytoestrogenic properties, garlic extract (50% methanolic extract of the outer flakes) injected at 30mg/kg failed to increase uterine weight; the authors suggested this was due to the low phenolic content.^[50]

Limited studies looking for direct phytoestrogens (things from plants that directly activate the estrogen receptor) have failed to find anything

Diallyl sulfide (DAS) is known to cause a very large (250-fold) induction in the mRNA of SULT1E1 (via causing nuclear translocation of CAR)^[155] and SULT1E1 produces Phase II estrogen sulfotransferases that sulfate estrogen specifically;^[418][419] since these sulfated variants actiate the estrogen receptors less it is seen as a temporary anti-estrogenic mechanism of action, until estrogen sulfatases reverse the process.^[419]

Despite how ablating the gene causes a drastic induction of estrogen^[420] the alterations in its activity seen with DAS were very minor as there was a small spike in estrogen and no changes in sulfated estrogen;^[155] that being said, exogenous estrogen (injections) were rapidly cleared.^[155]

Diallyl sulfide greatly increases the enzyme that sulfates estrogens, but at resting conditions in normal rats this does not alter estrogen metabolism too much; high levels of exogenous estrogen are, however, rapidly excreted

In a rat model of menopause, oral ingestion of 100mg/kg garlic oil for a month is able to preserve approximately one third^[355] to 44%^[358] of the estrogen lost during removal of the uterus.

In menopausal rats, there is an increase (or preservation) in circulating estrogen by a bit less than half associated with the garlic oil bioactives

In some studies noting testicular toxicity associated with high doses of garlic, reductions in serum testosterone have been noted with 7.5% of the rat diet as spring garlic (9-21% reduction, more from the alcoholic than aqueous extract)^[421] and while 5% of this garlic in the diet doesn't significantly reduce testosterone, higher doses (10-30% of the diet) dose-dependently reduce testosterone to 10% of baseline at the highest dose.^[7]

Lower doses of garlic are known to be protective of the testicles against prooxidative toxins (due to their antioxidative properties), and in this scenario garlic can be met with a relative increase in testosterone.^[422] One study has also noted an increase in intratesticular testosterone to a near doubling in rats fed garlic (0.8%) on a high protein diet of 25-40% protein yet not with 10% protein,^[423] the mechanisms underlying this particular testosterone boost are not known.

The doses that impair spermatogenesis in the testicles also reduce the activity of enzymes in testosterone synthesis (Star, Cyp11a, Hsd3β5 and Hsd17β)^[424] while the context in which testicles are protected is met with a relative increase of these enzymes.^[422]

Garlic is associated with both testicular protective effects and testicular toxic effects, and the influence on testosterone follows the effects on the testicles (protection is met with an increase, toxicity met with a decrease). There are no human studies at this point in time, and moderate garlic consumption is unlikely to influence testosterone in most persons

In isolated keratinocytes (genetically modified as a model for growth hormone secretion on a cellular level^[425]), 500-2,000µg/mL of aged garlic extract and 125-250µg/mL of S-allylcysteine seem to induce growth hormone secretion in a dose and time dependent manner;^[426] 0.25-1µg/mL Pycnogenol was minimally effective but worked at lower concentrations and not in a dose dependent manner.^[426]

Preclinical evidence suggests that aged garlic extract may cause growth hormone secretion, but there are currently no studies in living models to assess the practical relevance

In anaethized rats, injections of diallyl disulfide (20-30mM) appears to increase LH secretion similar to how injections of noradrenaline increased LH secretion;^[423] as diallyl disulfide has been noted to increase plasma noradrenaline previously^[325] the authors thought that it may work vicariously through an increase in plasma noradrenaline.

In rats fed 0.8% garlic in the diet, it appears that after 28 days there is a reduction in circulating corticosterone by approximately half.^[423]

In hyperthyroidic rats, garlic (500mg/kg of the bulb extract) appears to reduce excessive circulating concentrations of T3 and T4 with a potency comparable to Fenugreek (220mg/kg of the 20% ethanolic seed extract);^[427] oddly, the two are antagonistic when coingested.^[427]

In normal rats given 500mg/kg of garlic, there was a nonsignificant trend to reduce T3 levels in serum.^[427]

Nrf2 (Nuclear factor erythroid like-2) is a nuclear protein that, when activated, influences the genome (specifically, the 'antioxidant response element' or 'ARE') in such a way that it orchetrates an antioxidant response in a cell; it is commonly activated by pro-oxidative stressors,^[428][429] and as such is seen as a protein that mediates the 'antioxidant response to oxidizing stressors' (ie. hormesis).

Some components of garlic have been noted to interact with Nrf2/ARE signalling, including S-allylcysteine^[19] and diallyl disulfide.^[430] It has been noted that activation of Nrf2/ARE signalling is abrogated by inhibition of MAPK (ERK, p38, and JNK).^[430] and activation of this pathway (as assessed by induction of HO-1, increased by Nrf2^[431]) occurs at 100µM of diallyl disulfide.

One study assessing diallyl trisulfide and its effects in ischemia failed to find an interaction with Nrf2.^[432]

Components of garlic appear to activate Nrf2 siganlling downstream of activating MAPKs (JNK, p38, and ERK all implicated) and due to activating Nrf2/ARE signalling it orchestrates an antioxidant response in cells

Oral ingestion of garlic supplements have been confirmed to increase intracellular glutathione concentrations in white blood cells (PMBCs) following ingestion of 2,650mg of aged garlic supplementation^[120] and in red blood cells both glutathione peroxidase (12%) and SOD (24%) have been noted to be increased following consumption of 100mg of raw garlic per kilogram bodyweight, with no significant influence on catalase nor xanthine oxidase.^[287]

Higher doses of garlic supplementation have been noted to increase antioxidant enzymes in red and white blood cells in humans

Aged garlic extract appears to be able to directly sequester superoxide radicals, reaching 54% sequestering at 3,000µg/mL (HPX-XOD ex vivo system) with an EC₅₀ of 800µg/mL^[67] and despite aged garlic extract being more effective than the bulb in sequestering superoxide^[433] it seens S-allylcysteine and S-allylmercaptocysteine are not highly active but fructosyl-arginine and a carboline (MTCdiC) are active.^[67]

Ex vivo, aged garlic extract seems to directly sequester free radicals which seems to be associated with some of the unique (and not sulfur containing) bioactives present

High concentrations of H₂S are known to stimulate superoxide production^[434]

Superoxide (O₂^-) in the serum of hypertensives appears to be significantly reduced with oral supplementation of 250mg garlic pearls over two months, with no significant changes in healthy controls (who did not have excessive levels of superoxide).^[264]

Excessive levels of H₂S are known to stimulate H₂O₂ production.^[434]

S-allylcysteine is known to reduce oxidative damage to cells induced by H₂O₂.^[407]

Peroxynitrate (ONOO^-) can be scavenged in vitro by garlic extracts^[435] and while heat treatment (before or after cutting the garlic) does not modify this effect both pickling as well as microwaving can.^[436]

S-allylcysteine and S-allylmercaptocysteine appear to reduce TBARS in vitro.^[433]

In hypertensive persons, lipid peroxidation appears to be significantly reduced with oral supplementation of 250mg garlic supplementation for 8 weeks.^[264]

In a mouse model of DNA damage (cyclophosphamide induced genotoxicity), it seems that garlic at 250mg/kg is nonsignificantly more protective than 10mg/kg curcumin with both being significantly more protective than 100mg/kg saffron^[437] and when investigating pairings all three agents were additively protective.^[437]

When looking at rodent studies, garlic appears to reduce genotoxicity (DNA damage) from various stressors, and relative to other tested DNA protective agents garlic seems to be of respectable and comparable potency

250mg of garlic pearls (2.5% garlic oil) daily for two months in persons with essential hypertension (a state associated with increased oxidation and subsequent DNA damage^[438][439]) is able to significantly reduce DNA damage as assessed by urinary 8-OHdG, with healthy controls experiencing a nonsignificant decrease.^[264]

A reduction of DNA damage has been noted with low dose garlic ingestion in hypertensive persons

Helicobacter pylori infection is a gastric bacterial infection known to cause ulceration and is associated with a variety of gastric disease states such as gastritis and gastric carcinoma^[440] while eradicating the infection increases remission from these diseases.^[441] Although the standard therapy (a PPI paired with two of the following; metronidazole, amoxicillin, or clarithromycin^[442]) is highly effective, bacterial resistance sometimes occurs; garlic does not appear to be associated with said resistance^[443] and due to possible antibacterial properties it is investigated as a possible alternative for resistant patients.

In vitro, garlic appears to be able to suppress the growth of helicobacter pylori in a concentration dependent manner and is synergistic with omeprazole (a PPI).^[444] The garlic powder has an MIC of 250-500μg/mL^[25] which is thought to be due to either allicin (3-6μg/mL, but unstable) or the diallyl sulfides, since garlic oil had an MIC of 8-32μg/mL;^[445][25] DADS has an MIC of 100-200μg/mL although DATTS has a potent 3-6μg/mL MIC^[25] yet DATS was highly effective at 1μg/mL.^[446] In simulated gastric environments the mucus that may protect helicobacter pylori is antagonized by garlic oil^[445] and while additive with dextin, garlic oil itself is antagonized by rapeseed oil and peptone (protein digest of pepsin).^[445]

In looking at human evidence, a pilot study using 4mg of garlic oil four times daily (with meals) over fourteen days failed to eradicate or suppression helicobacter pylori;^[447] this relative ineffective has been noted elsewhere with the oil^[448] and a study using the food product up to 10 sliced cloves daily has failed to find a protective effect.^[449] It was mentioned in a letter to the editor (Helicobacter Journal)^[450] that the above trials were of short duration and small sample size, and in drawing data from a larger intervetion on gastric ulceration^[62] where subjects were given 400mg aged garlic extract with 2mg garlic oil (each twice daily) over 7.3 years, and ended up confirmed a lack of clinically relevant effects seen with garlic supplementation.^[450]

Garlic oil components appear to be highly effective in suppressing the growth of helicobacter pylori when tested outside of a living system, but studies using garlic as a food product or as the supplemental oil have pretty much failed to replicate this potency

When looking at studies assessing the interaction of garlic and alcohol, protective effects have been noted with aged black garlic extract (100mg/kg in rats^[451]), garlic oil (50-200mg/kg in mice^[452][68]), or the bulbs themselves (250mg/kg in rats^[453]). Garlic oil appears to be gram per gram as effective as Vitamin E in reducing liver damage from alcohol^[454] and has once been used alongside Metadoxine (pharmaceutical treatment for alcohol damage to the liver and ADHD) and successfully prevented all damage in mice.^[455]

Other protective effects on the liver have been noted in response to acetominophen with isolated Thiacremonone (IP injections of 10-50mg/kg),^[398] Ajoene (20-100mg/kg),^[456] S-allylmercaptocysteine (200mg/kg),^[147] garlic extract^[457] and oil (200mg/kg).^[458] Garlic components appear to have a potency comparable to N-acetylcysteine when directly compared^[457][458] and coingestion of the two appears to be additive.^[459]

Oral ingestion of garlic appears to be protective against liver damage induced by alcohol in rodent models when preloaded daily for about a week prior to acute ethanol toxicity

CYP2E1 is thought to mostly underlie the protective effects of garlic on the liver, and it is thought that secondary to preventing the activation of CYP2E1 that there is a preservation of glutathione (noted numerous times^{[453][451][452]}) resulting in less oxidative damage. CYP2E1 is increased in the liver in response to alcohol and mediates its toxicity^[460][461] (being so relevant that mice who lack the enzyme do not experience alcohol damage^[462]), and CYP2E1 has been confirmed to be reduced relative to alcohol control in one study (55% inhibition in response to 100mg/kg aged black garlic extract^[451]) and the main CYP2E1 inhibiting compound in garlic (diallyl disulfide^[127]) has alone been noted to protective livers from alcohol damage associated with CYP2E1 inhibition.^[463]

Susbequently, one study noted that DADS protected liver cells from damage by activating MAPKs (p38, JNK, and ERK) which then activated Nrf2/ARE signalling and produced HO-1;^[430] a known protective agent in the liver^[464][465] which has been directly linke to CYP2E1 mediated toxicity before.^[466] Protective effects have been noted at 25-100mg/kg oral intake for one week prior to alcohol ingestion, and in vitro effects seem to be maximized (2.5-fold HO-1 protein levels) at 100µM.^[430]

Other bioactives that have been noted to inhibit CYP2E1 and thus confer protective effects include thiacremonone.^[398]

CYP2E1 inhibition is thought to be the main protective effect of garlic in regards to alcoholic liver damage, although the induction of heme-oxygenase 1 (HO-1) appears to also be vital. The exact signalling pathway(s) need to be further elucidated

Activation of AMPK (S-allylcysteine) in liver cells appears to reduce lipogenesis.^[96]

Isolated ajoene (10-30mg/kg in mice) appears to reduce liver fat buildup from a high fat diet secondary to AMPK activation.^[95] AMPK activation per se suppresses a protein known as S6K1 which would normally promote genomic activity of LXRa-SREBP-1c;^[467] and preventing SREBP-1c activity suppresses an increase in lipogenic proteins.^[468]

In rats with fatty liver, S-allylmercaptocysteine (confirmed to be hepatoprotective at 200mg/kg in rats^[469]) has circumvented the NAFLD induced suppression of AMPK/LKB1 activation by directly activating it;^[97] this activation of AMPK was thought to underlie the reduction in apoptosis and increase in autophagy.^[97]

The components of garlic that can activate AMPK (Ajoene, S-allylmercaptocysteine) appear to have hepatoprotective and lipolytic properties in animal models of steatohepatitis (liver fat)

When tested in humans with hepatopulmonary syndrome (an instance of portal hypertension and increased intrapulmonary vasodilation with lesser oxygen delivery to tissue^[470]) given 125mg of garlic oil in two divided doses each day over 9-18 months, supplementation was associated with an increase in arterial oxygen levels (24.66% more than baseline) and a decrease in alveolar-arterial oxygen gradient (28.35% lower than baseline), both of which were more significant than placebo;^[471] there was reversal of hepatopulmonary syndrome in two thirds of patients on garlic (rather than one in placebo) and during followup the instances of death were much greater in placebo (7 persons out of 20) than with garlic (2 persons out of 21).^[471]

Preliminary evidence suggests that garlic oil is highly therapeutic for hepatopulmonary syndrome

Mechanistically, diabetic kidneys are known to increase expression of VEGF^[472] which increases ERK-1;^[473] when garlic protects the kidneys from damage these increases are no longer seen.^[313] Kidney levels of δ-aminolevulinic acid (δ-ALA; a substrate of heme synthesis) are usually suppressed high levels of oxidation as is seen with diabetes^[474][475] yet they are normalized with S-allylcysteine at 150mg/kg for 45 days.^[476]

Although the exact mechanism of kidney protection is not known, it appears to be associated with a reduction in the expression of proteins that cause renal damage and seems to be associated with less oxidation

500mg/kg of a water extract of fresh garlic to diabetic rats was able to reduce the abnormal urination (despite not reducing blood glucose) associated with effectively normalizing serum proteins; histological examination of the kidney showed no abnormalities in the diabetic rats given garlic, despite diabetes causing toxic effects.^[313] This study is duplicated in Medline.^[477]

High levels of the water extract appear to be protective of the kidneys in diabetic animals

It appears that garlic (water extracts) possess spermicidal activity in ejaculated human semen, with complete immobilization at 500mg/mL concentrations in an irreversible manner^[478] which is thought to be due to Diallyl trisulfide (Allitridum) since it fully immobilized sperm at 7.5mg/mL.^[479]

Diallyl trisulfide appears to be a spermicide capable of fully immobilizing sperm, although the concentration at which this occurs seems too high for standard oral supplementation of garlic

Spring garlic (a pink variant of allium sativum) at 7.5% of the rat diet did not alter testiclar weight yet exerted an apparently toxic effect as assesed by DNA fragmentation and abnormal histology of the seminiferous tubules.^[421] Elsewhere, 10-30% of the diet as spring garlic for one month in has impaired spermatogenesis and reduced the weight of the testes (while 5% was ineffective)^[7] which is replicated a few times.^{[424][480][481]} As 5% seems to be the NOAEL on this parameter, its estimated human dose (assuming 15-30g of food intake for a 250-300g rat) is around 2,500-6,000mg/kg for a rat and 400-960mg/kg in humans.

Studies that measure apoptosis of germ cells and spermatids do note an increase in apoptosis^[421][424] and when measuring hormones that may influence testicular function there is a dose and time-dependent increase in luteinizing hormone (LH)^[7] and decreases in both testosterone^{[480][421][7][424]} and follicle-stimulating hormone (FSH).^[424][480]

Oral doses of spring garlic at 7.5% of the rat diet or higher appear to be dose-dependent testicular toxins associated with causing cellular death. Oddly, all studies have used the spring garlic variant and not standard white garlic and despite the above toxicity it seems to only occur at moderately high oral doses of garlic (not possible with supplements, possible with food)

Garlic is known to exert protective effects on the testicles, as injections of garlic (correlating to 300mg/kg daily) to rats undergoing lindane toxicity normalized the altered testicular function (lipid peroxidation)^[482] and the toxic pro-oxidative effects of TCDD have also been attenuated with 5-40mg/kg oral ingestion of garlic extract (70% ethanolic extract of the bulb).^[483]

When in the presence of testicular toxins that happen to be prooxidative in nature, garlic appears to be protective of testicular function secondary to its antioxidant effects

Hydrogen sulfide (H₂S) is a proerectile gasotransmitter^[484][485] similar to nitric oxide,^[486] but while nitric oxide works via the cGMP system (which is also the target of PDE5 inhibitors like Viagra^[487]) H₂S is known to directly open potassium channels^[81] and can cause relaxation in penile tissue.^[488] H₂S is synthesized directly in penile tissue as blocking the enzyme of synthesis (mostly cystathionine γ-lyase) prevent L-cysteine from making H₂S^[488][489] and glibenclamide (potassium channel blocker) can prevents most of the proerectile effects of H₂S.^[488] There are some other mechanisms associated with H₂S rather than potassium channels, but blocking cAMP and cGMP signalling (two proerectile signalling molecules) only blocks 22.5% and 4.7% of the relaxing effects respectively^[489] and thus it is thought that potassium channels mediate the last 72.8%.^[484]

The enzymes of synthesis are expressed on the dorsal neurons innervating the penis^[484] and in live rats receiving intrapenile injections of this enzyme inhibitor there was a significant reduction in nerve-induced penile tumensence.^[490]

Hydrogen sulfide appears to be a relaxing agent in penile tissue similar in concept to nitric oxide, but works via different mechanisms of opening potassium channels (still results in a relaxation of the tissue and more blood engorgement).

Garlic, perhaps through hydrogen sulfide signalling, has been dubbed vegetable viagra.^[491]

In diabetic rats, who have erectile dysfunction secondary to endothelial dysfunction,^[492] there are proerectile effects from isolated S-allylcysteine.^[493]

Preliminary evidence suggests that garlic may be proerectile, but it is limited

Bacterial prostatitis (characterized by recurrent urinary tract infections and prostatic inflammation^[494]) tends to be treated with antimicrobial agents.^[495]

Garlic at 9mg/kg bodyweight twice daily in rats for three weeks underperformed relative to the reference drug (Ciprofloxacin 2.5mg/kg twice daily) but was mildly additive in suppressing bacterial growth and may have been synergistic in reducing organ damage.^[496]

In regards to bacterial prostate inflammation, garlic may be effective but at a lower potency than the reference drug; they may work well together though

Garlic is thought to be benficial to the prostate in part due to the beneficial effects on prostate cancer and animal evidence, as well as preliminary evidence for the treatment of benign prostatic hyperplasia.^[497]

In 27 subjects with benign prostatic hyperplasia, 200mg/kg raw garlic (via a 1mL/kg concentrated water extract) for the course of one month was effective in reducing prostatic size by around 32% (assessed via transrectal ultrasonography), and symptoms of benign prostatic hyperplasia (urinary frequency and volume, IPSS scoring) were significantly improved relative to their own baseline values.^[498]

Consumption of high levels of garlic, based on preliminary evidence in humans, appears to significantly reduce the size of the prostate and improve symptoms of benign prostatic hyperplasia

In isolated neuroblastoma cells (SH-SY5Y), diallyl disulfide (DADS) at 10-100μM appears to cause cellular accumulation in the G₂/M phase and apoptosis (via a mitochondrial dependent pathway) secondary to oxidative stress in a manner that is blocked by inhibition of c-Jun/JNK signalling;^[499] JNK activation being known to positively influence oxidative stress in many neural cells^[500][501] and DADS appeared to prevent a complexation of GST-π-1 and JNK (GST-π-1 normally suppresses JNK,^[500] so preventing this allows JNK to be activated and cause oxidative stress).^[499]

Diallyl disulfide appears to prevent a negative regulator (GST-π-1) from suppressing JNK in neuroblastoma cells, and the relative increase in JNK signalling causes apoptosis

In glioblastoma cells (T98G and U87MG), 100μM of either diallyl sulfide (DAS) or diallyl sulfide (DADS) and 25μM of diallyl trisulfide (DATS) cause concentration dependent cytotoxicity with DADS being slightly more effective than DAS and DATS requiring less of a concentration to act;^[502] they activated p38 (but not p42/44) to cause apoptosis and JNK1 to cause cell death (via oxidation) in a manner dependent on the mitochondria^[502] and due to the increasing potency correlating with sulfur groups and the known ability for hydrogen sulfide to activate p38 and JNK in cancer cells^[503] it is thought hydrogen sulfide mediates these anticancer effects.

Another target in glioblastoma cells is HDAC inhibition, as DATS (known to inhibit HDAC^[504][114]) injected to mice bearing glioblastoma tumors (U87MG) at 10-10,000μg/kg for seven days (after three weeks of tumor growth) caused dose-dependent reductions in tumor size with the highest dose reducing tumor size to 10% of control and halving HDAC activity in the tumor.^[505] The highest dose of DATS did not appear to be toxic as assessed by liver histology.^[505]

Sulfur containing compounds also appear to be cytotoxic to glioblastoma cells, and this therapeutic benefit has been confirmed in mice recieving injections of DATS

In a study that confirmed that treating heliobacter pylori infections reduced the occurrence of precancerous gastric lesions, the group given garlic supplementation (800mg aged garlic with 4mg distilled garlic oil in two divided doses) for 7.3 years after treatment did not have a further reduction in formation of gastric lesions^[62] although when followed up another 7.4 years later the group treated with garlic seemed to have a nonsignificant reduction in cancer diagnosis and mortality (HR of 0.65 and a 95% CI of 0.35-1.20),^[506] a similar nonsignificant reduction in risk also seen with vitamin therapy (250mg Vitamin C, 100IU Vitamin E, 37.5μg selenium taken twice daily).^[506]

The protective effects of garlic supplementation on the occurrence of stomach cancer appears to be statistically insignificant or nonexistent

S-allylcysteine has been noted to protect research animals from gastric cancer induced by DMBA and increase apoptosis rates in this research model (albeit high doses of 200mg/kg oral intake)^[507][508] and this is also seen with a water extract of garlic (250mg/kg);^[509] this protective effect has been noted against MNNG as well, and a combination of S-allylcysteine (100mg/kg) and lycopene (1.25mg/kg) taken over three weeks alongside said toxin appeared to have additive effects (synergism not demonstrated)^[510] which has been replicated elsewhere.^[511]

S-allylmercapto-L-cysteine (SAMC) at the oral dose of 100-300mg/kg in rats with implanted stomach tumors was able to reduce tumor size by 31.36-37.78% (weak dose dependence) associated with pro-apoptotic changes in Bax and Bcl-2 and an increase apoptosis index of 20.74-30.61%.^[512]

Garlic extract, thought to be due to the S-allylcysteine content, is protective of the stomach against carcinogenic insults; it appears to be additive with lycopene, the main bioactive in tomato products

When looking at risk factors for multiple myeloma, a diet high in garlic (although shallots were pooled together in the analysis) appeared to be associated with a reduced risk for myeloma with an Odds Ratio of 0.60 and a 95% confidence interval of 0.43-0.85;^[513] consumption of soy products and green tea appeared to be associated with even less of a risk, however.

Consumption of garlic and related vegetables appear to be associated with reduced risk for myeloma

Ajoene appears to be investigated for its role in inducing apoptosis of leukemic cancer cells,^[58] the mechanism appears to be prooxidative means (efficacy halved by N-acetylcysteine in vitro), specifically peroxides, which led to activation of NF-kB in a concentration dependent manner between 1-40μM (up to 20% apoptosis)^[514] with no influence on otherwise healthy PBMCs at this concentration range.^[514] This apoptosis is dependent on the mitochondrial pathway^[515] and is associated with a block in the G2/M phase of the cell cycle.^[516]

Ajoene in isolation appears to have cytotoxic effects in leukemic cells. The potency is somewhat moderate, but occurs at a low enough concentration that it may be physiologically relevant

Ajoene has been reported to enhance the cytotoxicity of cytarabine via enhancing its suppression of Bcl-2 (16% of cytarabine alone) and activation of caspase-3 (212% more than cytarabine alone);^[58] Bcl-2 being a protein that enhanced cell survival via facilitating a withdrawal into a quiniscient phase^[517] and suppresses mitochondrial lysing^[518] while capsase-3 is a biomarker for mitochondrial lysing. In CD34+ cells, particularly blast cells (usually more drug resistant associated with the G0 quiescent phase, which reduces how effective drugs are at inducing apoptosis^[519][520]) which are known to overexpression Bcl-2 and P-glycoprotein (drug efflux protein) more often than not,^[521][522] and due to this Ajoene is thought to allow proper apoptosis in drug resistant cells.^[58]

Ajoene from garlic oil appears to be able to reduce drug resistance to cytarabine

In BALB/c mice bearing fibrosarcoma tumors (WEHI-164), intravenous infusions of 100mg/kg of an aged garlic extract appeared to increase survival time (60.6% longer than control) to a degree similar to the reference drug of 500µg/kg Naltrexone (54.5%) although a combination appeared to be synergistic (154.5%).^[523] This appeared to be related to an increase in T-cell cytotoxicity and IFN-γ secretion (although not in count)^[523] and this study is duplicated in Medline.^[524]

When looking at associations of vegetable intake and cancer occurrence in a secondary analysis of data, it seems that garlic ingestion is associated with a reduced risk of prostate cancer with an odds ratio (OR) of 0.77 and a 95% confidence interval (CI) of 0.64-0.91.^[525] This study drew data from a few surveys where garlic itself was either associated with less risk^[526][527] or trended towards it,^[528] and studies where allium vegetables (garlic plus onion) were associated with reduced risk^[529][530] or trended towards it (but failing to be statistially significant).^[531][532]

There appears to be a negative correlation with prostate cancer and garlic intake in the diet, which implies a protective effect; the magnitude of this protective effect is somewhat modest

Diallyl trisulfide (DATS) is known to induce oxidative stress in cancer cells^[499] which extends to prostatic cancer cells;^{[533][534][535]} it seems that DATS reduces the ferritin protein level in a cell and increases the labile iron pool, and said increased free iron causes intracellular damage^[533] and that this increase in iron-mediated oxidative damage was due to JNK1/Itch signalling^[533][535] and dependent on p66Shc phosphorylation;^[534] a molecular target influenced by JNK1/Itch that mediates the release of iron to cause oxidative damage^[536] and may suppress Akt.^[537] This pathway is, in part, regulated by acetylation of the p300/CBP promoter^[538] and HDAC inhibition is hypothesized to underlie the above.^[115]

While this mechanism can occur in noncancerous cells as well, the potency in cancer cells (PC-3) is significantly higher than noncancerous cells (PNT1A), as the suppression of Akt seen in cancer cells^[537][534] does not occur in noncancerous cells^[534] at the cytotoxic concentration (40μM) and the ERK1/2 phosphorylation is lesser; the cytotoxic concentration induces iron release at a significantly higher level in cancer cells than normal cells and this release of iron does not appear to cause oxidative stress in normal cells,^[534] possibly because the expected decrease in iron containing proteins seen in cancer cells (would cause oxidative damange as mentioned earlier^[539]) was not seen, but a relative increase was noted in normal cells (reduces oxidative damage^[540]).

Such cytotoxicity may also be seen with DADS (noted to be cytotoxic to DU145 cells via JNK, a cell line more sensitive than PC3 and LNCap at 200-400μM^[541] but also active at 25-40μM^[542]) and S-allylcysteine has also been implicated although the mechanisms not well elucidated.^[543]

While the induction of oxidative stress is known to be the main cytotoxic effect there are some 'antisurvival' effects in prostate cancer cells, as DATS inhibits STAT3 activation at 20-40µM which prevented the cytokine IL-6 from helping the cancer cells survive^[544] (which IL-6 normally does^[545]) and the histone deacetylase (HDAC) inhibition is thought to be relevant in this tissue as well.

X-linked inhibitor of apoptosis (XIAP; an independent predictor for prostate cancer recurrence^[546]) has also been noted to be inhibited by DATS at the active concentration range (20-40µM) and oral dose (1-2mg/kg in mice),^[547] and this appears to play a role since overexpressing XIAP (to prevent its inhibition by DATS) prevents apoptosis via DATS.^[547]

DATS appears to be cytotoxic towards prostatic cancer cells by causing a release of iron within the cell, and the iron causes oxidative damage; it is antisurvial via STAT3 and HDAC inhibition (and XIAP seems to play a role). All these mechanisms appear relevant to oral supplementation of garlic, and all of them suggest selectivity towards prostatic cancer cell death rather than healthy prostatic cell death

Increased expression of the IGF-1 receptor has been noted in prostatic cancer cells^[548] and is independently associated with increased prostate cancer risk^[549] thought to be through Akt signalling, which can increase cell survival^[550] downstream of NF-kB;^[551] a negative regulator of Akt, PTEN, is also reduced in prostate cancer^[552] and correlated with poor prognosis.^[553] Garlic, via the DADS component (20-40μM in PC-3 cells), appears to suppress the mRNA for the IGF-1 receptor while increasing levels of the IGF binding protein (IGFBP3);^[554] alongside the apoptosis induced was a suppressing of both NF-kB and Akt signalling.^[554] DADS showed direct docking potential on the IGF1 receptor.^[554]

Similar to the IGF-1 receptor, the androgen receptor is increased in prostate cancer cells and a main determinant of early prostate cancer.^[555] DATS has been confirmed to suppress androgen receptor signalling in prostatic cancer cells (40µM in both LNCaP and TRAMPc1 cells) and was able to inhibit androgen-induced cell growth; while there was less suppressive effects with DAS and DADS (some seen with allyl mercaptan) and a decrease in promoter activity was noted.^[556] In TRAMP mice given 1-2mg of DATS thrice weekly via oral gavage (where carcinoma occurrence was reduced 34-41%), the androgen receptor protein content was reduced by 71% (2mg) relative to controls.^[556] There appeared to be a suppressive effect on the androgen receptor in normal prostates, but to a much lesser degree (appoximately 15%).^[556]

The diallyl sulfides tested, either directly or indirectly, appear to suppress the signalling of two prosurvival hormones that cause poor prognosis for prostate cancer; IGF-1 and androgens (such as DHT). At least the androgen receptor has been noted to be suppressed in vivo

When looking at rodent studies, oral gavage of 1-2mg DATS has been confirmed to reduce STAT3 signalling in TRAMP mice bearing prostate tumors^[544] associated with inhibition of XIAP^[547] and reduced androgen receptor protein content;^[556] this dose elsewhere reduced prostatic proliferation by 34–41% in TRAMP mice^[391][556] and has reduced tumor size in mice bearing PC-3 tumors.^[557]

Oral ingestion of very low doses of DATS appear to suppress prostate tumor growth and proliferation in mice bearing varying types of prostate tumors

A pilot study of nine men with prostate cancer have noted that 200mg/kg of raw garlic daily for the course of a month was able to reduce circulating levels of prostate specific antigen (PSA).^[498] Free PSA was reduced from 1.92+/-1.04 to 0.75+/-0.31 (61%) and total PSA was reduced from 8.87+/-2.5 to 3.59+/-0.78 (60%) relative to baseline values.^[498]

Preliminary human evidence has noted that high dose garlic consumption is associated with reduced PSA levels in serum, with the magnitude of reduction being surprisingly high but likely an overestimate (based on the trial not having a placebo control and being the first of its kind)

One study assessing the risk of urothelial cell carcinoma of the bladder associated with dietary supplements noted that no tested study, including garlic supplements, was associated with a reduced risk.^[558]

No noticeable risk reduction is seen with garlic supplements for bladder carcinomas

Interferon Alpha (IFN-α), particularly INF-α_2b, is known to not only stimulate the immune system to cause indirect tumor cytotoxicity but may directly stimulate bladder cancer cells.^[559][560] It tends to be weaker than the reference drug (Bacillus Calmette-Guerin or BCG) when injected^[561] but is sometimes used as an adjuvant.^[562] Since garlic is said to hold potential against bladder cancer secondary to stimulating macrophage, T cell, and NK cell activity^[563] and is confirmed to increase serum IFN-α concentrations in humans (384% of baseline)^[71] it is thought that garlic may play a potential immunomodualtory role against bladder cancer pending more research.^[564][565]

There are possible indirect anti-tumor properties of garlic secondary to increase interferon alpha, but this is a preliminary topic and not directly linking garlic and bladder cancer yet

In C3H/He mice bearing MBT-2 bladder cancer cells, injections of 25mg of garlic extract have shown antitumor effects although demonstrating some toxicity.^[566][567]

In vitro, both DAS and DADS have been noted to inhibit arylamine N-acetyltransferase activity in a concentration dependent manner (10-100µM),^[568] a mechanism also seen in colon cancer cells.^[569] Decreases in the cells in the S and G0/G1 phases have been noted alongside an increase in the G2/M phase.^[570][571]

DADS (5-75µM) in T24 human bladder cancer cells has been noted induce concentration dependent (mitochondrial dependent) apoptosis associated with increasing intracellular H₂O₂ concentrations.^[570] This same cancer cell had antiproliferative effects with DATS with an IC₅₀ value of 20μM and 13μM (36 and 72 hours) and induced apoptosis via a mitochondrial dependent pathway, this was associated with suppressing activation of Akt.^[571]

Finally, S-allylmercaptocysteine (SAMC) appears to also have apoptotic effects.^[572] The inhibitor of differentiation (ID-1) is an antiapoptotic factor (preserves cells) associated with drug resistance^[573][574] and SAMC has been noted to suppress ID-1 activity (but it itself is subject to resistance by ID-1, and blocking ID-1 enhanced the effects of SAMC).^[572]

Diallyl sulfides have the typical cytotoxic effects in bladder cancer cells, and while injections of garlic have been confirmed to reduce bladder tumors in size there are no well controlled oral studies in rodents. Some promise (due to direct toxicity and the immunostimulation) but highly preliminary evidence

Dietary intake of garlic is known to be negatively correlated with the risk of developing colonic adenocarcinoma^[575][576] with an odds ratio of 0.87 (95% CI of 0.77-0.99) at the highest quintile of intake (1.6 servings daily).^[575] Elsewhere, using data from the Women's Health study it was noted that garlic consumption was associated with a 48% reduced risk of colon cancer (RR 0.52, 95% CI 0.30–0.93) when comparing one or more servings a day against none.^[577]

Oral ingestion of garlic appears to be associated with a reduced risk of developing colorectal cancer

The mechanism of HDAC inhibition appears to be relevant for cancer cells, as mRNA and protein content of p21^WAF1/CIP1 (increases with HDAC inhibition^[578] due to being regulated by acetylation of the CDKN1A gene^[579]) are both increased with DADS at 200µM, although more potently with allyl mercaptan (both less potent than the reference drug Trichostatin A).^[115] Greater acetylation of the CDKN1A promotor has been confirmed at this concentration of DADS,^[580] and histone H3 and H4 are acetylated in Caco-2 cells yet only H3 in HT-29 cells.^[580]

In the presence of DADS, colon cancer cells release calcium into the cytosol (SW480; dependent on calcium channels but not the mitochondria^[581]) and induce oxidative cell death via a mitochondrial dependent pathway (HT-29 cells).^[582]

colon cancer cells accumulate in the G2 phase of the cell cycle^{[115][580][582]} in a manner which is additive with the other HDAC inhibitor butyrate (short chain fatty acid produced from intestinal microflora) in HT-29 cells.^[582]

Diallyl disulfide is able to induce apoptosis in colonic cancer cells, which is thought to be due to HDAC inhibition and its mechanisms of apoptosis paralleling that seen in prostatic cancer cells

HDAC inhibition has been confirmed in rat colon cells following ingestion of 200mg/kg garlic oil (80% pure for DADS) within six hours (3-fold for histone H4 and between 1.4 and 2.5-fold for histone H3) but no longer persisting after 17 hours.^[118] This time course (inhibition for less than 24 hours) has also been noted in vitro with 200µM DADS.^[580]

Diallyl sulfide (DAS) in the diet at 100-300ppm (100-300mg/kg) for 10 weeks in mice in a genetic model of familial adenomatous polyposis (prone to colon cancer^[583][584]) dose-dependently reduced incidence of precancerous intestinal polyps by 19-32%.^[585]

Relatively large doses of garlic oil appear to inhibit HDAC following oral ingestion to rats, but this inhibition is fairly short lived

In patients with basal cell carcinoma (BCC; a nonmelanoma type of skin tumor that is usually seen as benign since it rarely metastasizes) of either nodular or superficial origin given a topical cream containing 0.4% Ajoene to put on and surrounding the BCCs for six months, Ajoene was able to reduce tumor size in 21 out of 24 patients (median reduction of 42% and 55% for nodular and superficial) associated with reducing Bcl-2 positive cells (39% and 50%, respectively).^[586]

Topical application of Ajoene to BCC tumors has been noted to half their size and Bcl-2 expression after six months

Although allium vegetables (garlic, onion, spring onion, and leeks) are associated with a lower risk for spontaneous preterm delivery overall (odds ratio of 0.81 with a 95% CI of 0.72-0.94) it appeared to be slightly stronger with garlic in particular; having an odds ratio of 0.74 and a 95% confidence interval of 0.56-0.97.^[587]

There appears to be an association with more dietary garlic intake and a reduced risk for spontaneous birth

One meta-analysis on the interactions of garlic and PAD has been conducted, but it was inable to find more than a single study^[588] and said study^[589] failed to find a significant protective effect, since the improvement seen in subjects treated with 800mg garlic powder (28%) was statistically similar to placebo (18%) over the course of twelve weeks.

The lone study did note that benefits seen were highly associated with the antithrombotic effects of garlic, ^[589] but since the dose tested tends to be lower than that normally used to thin blood it may have been too low to provide clinical benefit.

Current studies have failed to find an improvement in peripheral arterial disease, but the lone study and the meta-analysis based on the single study could merely be investigating too low a dose of garlic

S-adenosyl methionine (SAMe) is a supplement catered to reduce depression and improve joint health, and despite its traditionally high price it appears to be effective at both claims.

It appears that SAMe can activate the enzyme that produces H₂S, cystathionine β-synthase, and even without any particular coincubation of substrate it can produce H₂S in vitro at 2mM.^[175]

S-adenosylmethionine is known to produce hydrogen sulfide, and while this may not account for all mechanisms of action (since SAMe is also a methyl donor) production of H₂S may underlie the benefits of SAMe and thus there would be similar benefits from garlic and SAMe supplementation

Garlic (300mg thrice daily) has been tested alongside fish oil (4g fish oil containing 720mg EPA and 480mg DHA thrice daily) in hypercholesterolemic men, and the cholesterol reducing properties of garlic were neither augmented nor hindered with the combination of fish oil and the triglyceride reducing properties of fish oil were similarly unaffected; the net result on LDL-C (known to increase with fish oil) was a lesser reduction with the combination than with garlic alone.^[276]

The two do not appear to interact with each other following standard oral doses, but the LDL lowering effect of garlic may complement the adverse effect of fish oil in increasing LDL

Magnolia Bark is derived from magnolia officinalis, and is sometimes used in a chewing gum format for its dental health properties, and chewing gum per se is known to reduce volatile sulfur components (VSCs) in the oral muscoa (ie. garlic breath).^[590] A chewing gum containing both magnolia (0.15%) and zinc (acetate, effective on its own^[591]) at 0.012% appears to significantly reduce the levels of VSCs in the mouth following a meal,^[592] suggesting that it may treat garlic breath.

Magnolia bark and/or zinc acetate, included in chewing gum, may be able to reduce garlic breath more than placebo gum

Grape Seed Extract is a source of procyanidins and marketed as a blood health promoting antioxidant.

In high levels (that which are deemed 'toxic') garlic can induce organ damage secondary to prooxidative effects,^[593] which is sometimes seen when antioxidants are superloaded (ie. Vitamin C); Grape seed extract appears to negative these effects when coincubated.^[594]

One study using one week of pretreatment with garlic (20mg/kg) alongside milk thistle (50mg/kg) noted that, in response to toxicity from N-nitrosodiethylamine (a liver carcinogen), that while both agents were equally effective in normalizing liver enzymes and oxidative stress while the combination was synergistic at increasing apoptosis rates.^[595] The increase in apoptotis rates in response to a carcinogen suggest synergism in regards to liver cancer, but possibly not general liver damage (as oxidation and liver enzymes were not additive and of similar potency).^[595]

May be synergistic in reducing liver cancer development, although this synergism may not extend to general liver protection against oxidation and damage

Nicotine is a cognitive enhancing alkaloid found in cigarettes where it contributes to their addictive potential, and in excessive levels nicotine can induce oxidative damage to organs.^[596][597]

Injections of a garlic extract (125mg/kg) alongside a dose of nicotine sufficient to induce oxidative damage for 21 days fully prevented alterations in glutathione and lipid peroxidation seen with nicotine as well as markers of kidney damage (BUN and creatinine) in all tested organs except the heart (where it attenuated the damage).^[598] This is likely due to the antioxidative properties, as a study replicating this protective with 100mg/kg garlic extract or isolated S-allylcysteine also saw the same protection with 100mg/kg vitamin E.^[599][600] Garlic oil also seems effective, secondary to antioxidant enzymes^[601] and has been confirmed to reduce nicotine-mediated DNA damage (albeit less effective than curcumin and green tea catechins).^[602]

Garlic extract, secondary to its antioxidative properties, can reduce the endothelial and tissue damage seen in rats given nicotine

In a rat model of menopause, the inclusion of garlic oil (100mg/kg for a month) is able to increase (albeit not normalize) intestinal calcium absorption.^[355] This was associated with an increase in serum estrogen^[355] and since a reduction in calcium absorption is known to both be caused by estrogen deficiency and treated with estrogen^[603] it is though that garlic works via estrogen.

Both ginger and garlic appear to individually have antibacterial properties,^[604][605] and ginger has previously been noted to be synergistic with Tazma honey from the stingless bee (Apis mellipodae);^[606][607] a synergism that also extends to garlic in regards to the strains of Salmonella (NCTC 8385), Staphylococcus aureus (ATCC 25923), Listeria moncytogenes (ATCC 19116) and Streptococcus pneumonia (ATCC 63).^[608]

Honey's antibacterial properties are known to be synergistic with other agents, and garlic appears to be one of those agents that honey is synergistic with

Tender cluster beans (Cyamopsis tetragonoloba) is a food product with a high fiber content that is mostly guar gum, which is known to reduce triglycerides and cholesterol^[609][610] including in humans;^[611] the addition of garlic (1% of the diet) to these beans (15% of the diet) exerted additive benefits in reducing total cholesterol, LDL-C, and triglycerides and increasing HDL-C.^[612] The benefits of the combination extended to the liver's lipid profile (improving phospholipids and reducing cholesterol and triglycerdes) but did not augment the bile flow rate which was increased with the dietary fiber.^[612]

Garlic appears to be additive with dietary fibers in improving the cholesterol profile of subjects, but the amount that the two work together does not appear to be synergistic

Captopril is an anti-hypertensive drug known as an ACE inhibitor, and a related drug known as S-allylmercaptocaptopril has been made which is essentiall a conjugate of allicin with captorpil;^[613] it appears slightly more effective than captopril^[614][615] possibly due to the beneficial cardiovascular effects of H₂S signalling.

In rats given 125-250mg/kg of fresh garlic homogenate (or 0.11-0.22mg/kg S-allylcysteine) during three weeks of fructose induced hypertension either by itself or alongside 30mg/kg captopril noted that all groups were able to reduce blood pressure to similar levels, although there were additive benefits with any combination therapy.^[616] This dosage range appears to also be beneficial for cardiac damage, although 500mg/kg garlic was deemed excessive.^[617]

Low doses of garlic, possibly secondary to hydrogen sulfide signalling, appear to be additively beneficial with captopril

The TRP receptors are known to interact with one another, and the main TRP that garlic components act upon (TRPA1) is sometimes seen as antagonistic to the main TRP that capsaicin acts upon (TRPV1).^[618] Interactions with the TRPs are not unique to the aforementioned two supplements by any means, and have been noted in the same plant family of allium (including onions, which are allium cepa^[91]) and other compounds that confer sulfur (such as ferula asafoetida^[619]), amongst others.

This antagonism between TRPA1 activation and TRPV1 activation is thought to be relevant as there has been antagonism between compounds that act on these receptors noted in mice (such as onions and garlic^[406] or fenugreek and garlic^[427]), and one case study of a capsaicin insensitivity (low TRPV1 levels on their tongue) noted a hypersensitivity to garlic despite normal receptor density.^[620]

Despite the ultimate effects of TRPA1 and TRPV1 sometimes being similar, activation of one will suppress activation of the other which results in antagonism when agonists of both are taken

When looking at either garlic oil or its main bioactives (the diallyl sulfides), there is possible toxicity associated with these compounds.

500mg/kg DAS oral intake seems well tolerated in rats over seven days.^[149]

No toxic effects are noted at 90mg/kg oral intake in rats of DADS.^[152]

135mg/kg diallyl disulfide has been noted to cause decreases in red blood cell packed volume (hemolytic anemia)^[152] which has been noted elsewhere.^[621]

Oral ingestion of isolated diallyl disulfide at 180mg/kg to rats has been noted to not cause clinical signs of toxicity, but a discoloration and thickening of the stomach wall with some lesions was noted.^[152] Relative organ weights are also increased at this dosage, although hearts were increased in size at 225mg/kg.^[152]

Garlic oil has been noted to be lethal at a dose of 100mg/kg (rats) when given after a day long fast due to acute pulmonary oedema with severe congestion, although it was well tolerated in a fed state.^[622]

Garlic oil appears to be able to exert toxicity, and while this is more than the the supplementation dose and higher than that readily eaten via garlic it is still low enough that overdosing on garlic oil can cause adverse effects

S-allylcysteine in particular appears to have a high safety threshold (sometimes said to be 30-fold safer than other sulfur bearing garlic components), and its LD₅₀ in female mice (9.39 g/kg) or male mice (8.89 g/kg) is much higher than that of allicin (female 0.363 g/kg and male: 0.309 g/kg) and diallyl disulfide (female: 1.3 g/kg and male: 0.145 g/kg).^[8][19] The LD₅₀ of S-allylcyteine via injections is actual comparable to other dietary amino acids such as L-methionine (via injections).^[59]

S-allylcystine appears to be capable to being toxic, but it is comparable with many dietary amino acids and (practicall speaking) it is pretty much nontoxic

At times, subjects taking garlic supplements reported smelling of garlic as a side effect. This has been noted to occur at a frequency of 21% (9% in placebo)^[623] but rarely is to a level that causes dropouts^[623] (although it has been noted in longer trials^[254]). Overconsumption of garlic has been reported to exacerbate the smell (possibly causing it to a problematic degree), and apparently a traditional recommendation to attenuate the scent of garlic is to coingest the garlic with milk as washing the skin is ineffective.^[165] Milk itself has been confirmed to reduce bad breath as a side effect of garlic.^[624]

Heartburn is sometimes reported as a side-effect that causes dropout, usually at the rate of a single persons in studies of around 40 participants.^[360][322]

As a general statement, the side-effects of garlic supplementation or ingestion of garlic as a food product (beyond the taste) would be infrequent heartburn and a scent of garlic. While the scent of garlic does not appear to be bad enough for people to drop out of a study, the heartburn has been noted to cause dropouts

A case report of a women with chronic recurring menorrhagia who received a hysterectomy (surgical removal of the uterus) noted that, after the surgery, there was a significant reduction in blood pressure requiring clinical intervention;^[625] this was thought to be related to her consumption of garlic (reported to be higher than average but exact amount not disclosed) and its anti-platelet effects.^[625] Such cases have been reported elsewhere in ophthalmic surgery,^[626] plastic surgery,^[627][628] and dermatological surgeries^[629] where said bleeding problems are associated with garlic consumption (either the raw vegetable or aged garlic supplements) and a case of a spinal epidural hematoma is also associated with garlic ingestion.^[630]

Garlic, dosage not specified aside from being reported as being 'large dietary amounts', has been reported to cause bleeding disorders associated with surgery. It would be highly prudent to stop garlic usage prior to surgery due to these interactions

Although rare, it appears to be possible to be allergic to garlic^[631][632] which may result in eosinophilic esophagitis,^[633] anaphylaxis,^[634] headaches,^[635] and urticaria;^[632][631] allergies may be either following ingestion or topical contact of garlic.

Allergies to garlic do not always appear to be associated with allergies to other allium vegetables such as onions,^[632][631] (although at times there is cross sensitivity^[635]) and at times when raw garlic turns out positive in a subject cooked garlic does not induce an immune response^[632][634] (again, it does not occur with 100% reliability^[631]).

Alliin lyase has been determined to be an allergin and underlied cross-reactivity with leek, shallot, and onion.^[636]

Garlic is a very uncommon allergin, but said allergies to garlic do exist. Being allergic to garlic as a food product most likely will result in an allergy to supplements, and heating the garlic may have some beneficial effects in reducing the allergenicity of the garlic