Pharmacology & Therapeutics 142 (2014) 183–195

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Pharmacology & Therapeutics

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Associate editor: I. Kimura

Potential therapeutic effects of functionally active compounds isolatedfrom garlic

Hyung-Mun Yun a,1, Jung Ok Ban a,1, Kyung-Ran Park a, Chong Kil Lee a, Heon-Sang Jeong b,Sang Bae Han a, Jin Tae Hong a,⁎a College of Pharmacy and Medical Research Center, Chungbuk National University, 48 Gaeshin-dong, Heungduk-gu, Cheongju, Chungbuk 361-763, Republic of Koreab Department of Food Science and Technology, Chungbuk National University, Chungbuk 361-763, Republic of Korea

⁎ Corresponding author. Tel.: +82 43 261 2813; fax: +E-mail address: jinthong@chungbuk.ac.kr (J.T. Hong).

1 These authors contributed equally to this work.

0163-7258/$ – see front matter © 2013 Elsevier Inc. All rihttp://dx.doi.org/10.1016/j.pharmthera.2013.12.005

a b s t r a c t

a r t i c l e i n f o

Available online 11 December 2013

The medicinal properties o

Keywords:GarlicOrganosulfur compoundsCancerCardiovascular disordersNeurological diseasesLiver diseasesAllergyArthritis

f functionally active organosulfur compounds such as allin, diallyl disulfide,S-allylmercaptocysteine, and S-trityl-L-cysteine isolated from garlic have received great attention from a largenumber of investigators who have studied their pharmacological effects for the treatment of various diseases.These organosulfur compounds are able to prevent for development of cancer, cardiovascular, neurological,and liver diseases as well as allergy and arthritis. There have been alsomany reports on toxicities and pharmaco-kinetics of these compounds. The aim of this study is to review a variety of experimental and clinical reports, anddescribe the effectiveness, toxicities and pharmacokinetics, and possible mechanisms of pharmaceutical actionsof functionally active compounds isolated from garlic.

© 2013 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1832. Components of garlic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1843. Pharmaceutical effect of garlic on cancer development . . . . . . . . . . . . . . . . . . . . . . . . . . . 1854. Pharmaceutical effect of garlic on cardiovascular disorders . . . . . . . . . . . . . . . . . . . . . . . . . 1855. Pharmaceutical effect of garlic on neurological diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . 1866. Other pharmaceutical effects of garlic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1887. Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1888. Toxicity and safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1889. Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

188189189190191191192192

191191

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189192

1. Introduction

Historically, garlic has been revered as part of a healthful diet. Theearliest known references indicate that garlic formed part of the dailydiet of many Egyptians (Block, 1985). It was fed particularly to theworking class involved in heavy labor, as in the building of the pyramids(El-Bayoumy et al., 2006). Indeed, a recurring theme throughout earlyhistory is that garlic was given to the laboring classes, presumably to

82 43 268 2732.

ghts reserved.

maintain and increase their strength, thereby enabling them to workharder and be more productive (Rivlin, 2001). Ancient medical textsfrom Egypt, Greece, Rome, China, and India prescribed garlic for anumber of applications including improving performance, reducinginfections, and protection against toxins (Rivlin, 2006). Thesemedicinalproperties, coupled with its savory characteristics, have made garlica true cultural icon in many parts of the world. Thus, garlic is usedtraditionally as a flavor enhancer and has been recognized as not onlya common food additive but also a potent therapeutic agent.

The whole bulbs of garlic contain alliin, γ-glutamyl-S-allylcysteine,S-methylcysteine sulfoxide, S-trans-1-propenylcysteine sulfoxide,S-2-carboxypropylglutathione and S-allylcysteine (Amagase, 2006;

184 H.-M. Yun et al. / Pharmacology & Therapeutics 142 (2014) 183–195

Kimbaris et al., 2006). Recently, a novel organosulfur compound,thiacremonone (2,4-dihydroxy-2,5-dimethyl-thiophene-3-one) wasisolated and identified from heated garlic (Ban et al., 2007). Chemicalcomposition of the preparations obtained by extraction of garlic frac-tions depends on the extraction conditions: temperature, time andsolvent's polarity. The content of organosulfur compounds in garlicbulbs changes during cultivation and storage (Lawson et al., 1991). Itsbiological activities depend onmany factors, including country of originand various processing methods of garlic to isolate new organosulfurcompounds and to decompose organosulfur compounds. Therefore,the development of methods for determination of organosulfurcompounds in garlic and selecting garlic is apparently great importancefor evaluating the biological quality of garlic and garlic products.

Organosulfur compounds have been attributed to the medicinalproperties and health benefits of garlic. Several recent studies haveshown that these organosulfur compounds have anti-cancer, anti-cardiovascular disease, anti-neurological disease, and anti-liver disease

Table 1Chemical structure and quantity of some volatile organosulfur compounds present in garlic.

Sulfur components Contents (mg/g) Origin

Alliin

(MW: 62.28)

25–30 Korea6.7 Korea16.7–21.4 Germ5.3–9.4 Germ22.1 Japan

Allicin

(MW: 162.27)

2.3–4.6 Korea7.7 Austra6.1–7.7 USA g2.4–3.5 Switze5.1–6.6 Chine5.0–5.3 Japan

Diallyl sulfide(DAS)

(MW: 114.2)

0.01–0.02 Greek0.00–0.02 Greek0.00–0.02 Greek0.02–0.23 Korea

Diallyl disulfide (DADS)

(MW: 146.28)

0.08–0.28 Greek0.06–0.231 Greek0.07–0.26 Greek0.57–0.89 Korea

Diallyl trisulfide (DATS)

(MW: 178.34)

0.00–0.20 Greek0.01–0.22 Greek0.01–0.18 Greek0.11–0.39 Korea

E-Ajoene

Z-Ajoene

(MW: 234.4)

0.17 Japan0.17 Japan0.12 Japan0.47 Japan

S-allyl cysteine (SAC)

(MW: 161.22)

0.45 USA g0.36–0.60 Korea

Thiacremonone(MW: 160.19)

0.7 (μg/g) Korea

effects, as well as effects for prevention of allergy and arthritis(Amagase et al., 2001; Amagase, 2006). While these effects are well-known, the exact mechanisms of action have not yet been established.In the present paper, the aim of this study is to review data from a vari-ety of experimental and clinical reports and describe the effectivenessand possible mechanisms of action on how garlic showed the medicalproperties.

2. Components of garlic

Among the several functional compounds of garlic (Table 1), alliin isthe most abundant organosulfur compound in whole garlic. It is a deriv-ative of the amino acid cysteine. Lawson (1998) found that fresh garliccontains alliin (6–14 mg/g). 25.65–30.03 mg/g alliin was detected inKorean garlic cloves and 6.7 mg/g in Korean garlic bulbs (Yoo et al.,2010). 16.7–21.4 mg/g alliin was also detected in dried garlic clovesand 5.3–9.4 mg/g in fresh Germany garlic cloves (Allium sativum L. var.

of garlic Isolation solution References

n garlic cloves Water Yoo et al., 2010n garlic bulbs Water Yoo et al., 2010any dried garlic Water Bloem et al., 2011any fresh garlic Water Bloem et al., 2011ese garlic Water Shimpo et al., 2002

n garlic Water Yoo et al., 2010lian garlic Water Sterling & Eagling, 2001arlic Water Koch & Lawson, 1996rland garlic Water Ziegler & Sticher, 1989se garlic Water Lawson et al., 1991ese garlic Water Ueda et al., 1991garlic Diethyl ether Kimbaris et al., 2006garlic Ethyl acetate Kimbaris et al., 2006garlic Hexane Kimbaris et al., 2006n garlic Dichloromethane Lee et al., 2003

garlic Diethyl ether Kimbaris et al., 2006garlic Ethyl acetate Kimbaris et al., 2006garlic Hexane Kimbaris et al., 2006n garlic Dichloromethane Lee et al., 2003

garlic Diethyl ether Kimbaris et al., 2006garlic Ethyl acetate Kimbaris et al., 2006garlic Hexane Kimbaris et al., 2006n garlic Dichloromethane Lee et al., 2003

ese garlic Soybean oil Naznin et al., 2008ese garlic Rice oil Naznin et al., 2008ese garlic Soybean oil Naznin et al., 2008ese garlic Rice oil Naznin et al., 2008

arlic Water Amagase & Milner, 1993n garlic Water Yoo et al., 2010

n garlic Ethyl acetate Hwang et al., 2007

185H.-M. Yun et al. / Pharmacology & Therapeutics 142 (2014) 183–195

Thermidrome) (Bloem et al., 2011). Shimpo et al. (2002) found22.1 mg/g alliin in water extracts of heated Japanese garlic powder.

Allicin features the thiosulfinate functional group, R-S(O)-S-R(Table 1). The compound is not present in garlic unless tissue damageoccurs (Block, 1985), and is formed by the action of the enzyme alliinaseon alliin (Block, 1985). It is considered that 1 mg alliin is equivalentto 0.45 mg allicin (European Scientific Cooperative of Phytotherapy.Monographs on the medicinal uses of plant drugs: fascicules 1 and 2(1996); Barnes et al., 2007). The amount of allicin which was known asa major bioactive compound of garlic and garlic preparations is about2.3–4.6 mg/g in Korean garlic (Yoo et al., 2010). About 7–9 mg/g allicinwas detected in Australian garlic. Sterling and Eagling (2001) reportedthat allicin was detected about 6.1–7.7 mg/g in the USA garlic, 2.4–3.5 mg/g in Switzerland garlic (Ziegler & Sticher, 1989), 5.1–6.6 mg/gin Chinese garlic and 5.1–6.6 mg/g in Japanese garlic (Ueda et al.,1991). Commercial garlic preparations are often standardized on thecontent of sulfur-containing constituents, particularly to alliin, or onthe allicin yield (Barnes et al., 2007).

Diallyl sulfide (DAS) is easily transformed from allicin (Senguptaet al., 2004) (Table 1). Kimbaris et al. (2006) found that garlic oil con-tains DAS (23 μg/g) by simultaneous distillation extraction, analyzedby FT-Raman spectroscopy. Kimbaris et al. (2006) found that diethylether, ethyl acetate or hexane extract or extracts of garlic were com-posed of DAS about 10–20, 0–20 or 0–20 μg/g, respectively. Lee et al.(2003) found that dichloromethane extracts of Korean garlic werecomposed of DAS about 0.02–0.23 mg/g.

Diallyl trisulfide (DATS) begins its biochemical synthesis withγ-glutamyl-S-alk(en)yl-L-cysteine, which is hydrolyzed and oxidizedto produce alliin (Block, 1985) (Table 1). Alliin is the odorless precursorof DATS. Processing of garlic (cutting or chewing) generates a vacuolarenzyme (allinase), which acts upon alliin to give rise to allicin andother alkyl alkane-thiosulfinates (Block, 1985). Allicin and relatedthiosulfinates are decomposed to yield various organosulfur com-pounds including DATS. Kimbaris et al. found that diethyl ether, ethylacetate or hexane extracts of garlic were composed of DATS about0–200, 10–220 or 1–180 μg/g, respectively (Kimbaris et al., 2006). Leeet al. found that dichloromethane extracts of Korean garlic were com-posed of DATS about 0.11–0.39 mg/g (Lee et al., 2003).

Ajoene (4,5,9-trithiadodeca-1,6,11-triene-9-oxide) is the degrada-tion product of allicin (Table 1). Incubation temperature is a very impor-tant factor for ajoene formation. When incubation temperature was at40, 60 and 80 °C, the amount of ajoene concentration also increasedgradually, with the highest amount of E-ajoene (172.0 μg/g of garlic)and Z-ajoene (476.3 μg/g of garlic) found in Japanese garlic withrice oil at 80 °C, indicating its optimal temperature (Naznin et al.,

Table 2Epidemiological studies showing anticarcinogenic properties of garlic.

OSCs Type of cancer Country Cases/cont

Garlic Gastric Iran 217/394Garlic Garstric Korea 136/136Garlic Garstric Uruguay 160/320Garlic Garstric Venezuela 292/485Garlic and onion Garstric Serbia 102/204Garlic and onion Garstric Italy 126/561Garlic Stomach Lithuania 379/1137Garlic Stomach China 750/750Garlic Stomach China 98/196Garlic Stomach China 187/333Garlic Stomach China 201/201Garlic and onion Stomach 10 Europe 330/521,4Garlic Colon Argentina 110/220Garlic Colon USA 1192/1192Garlic Colon Switzerland 223/491Garlic Colon USA 212/3500Garlic Colon Netherlands 443/3123

OR, odds ratios.

2008). Soybean oil was composed of E-ajoene (172.0 μg/g of garlic)and Z-ajoene (120 μg/g of garlic).

S-allyl cysteine (SAC), a major transformed product from γ-glutamyl-S-allyl-L-cysteine, is the water-soluble organosulfur com-pounds and its concentration increases through a long-term extractionin an aqueous medium (Table 1). SAC is detected in the blood, and itsblood concentration and pharmacokinetic parameters are well-associated with doses of orally administered SAC in animal studies(Amagase, 2006). Amagase and Milner found that aged garlic containsSAC about 0.45 mg/g (Amagase & Milner, 1993). Yoo et al. (2010)found that water extracts of Korean garlic were composed of SACabout 0.36–0.60 mg/g.

Diallyl disulfide (DADS) is an organosulfur compound found inplants of the genus Allium (Table 1). Along DATS and diallyl tetrasulfide,it is one of the principal components of the distilled oil of garlic. Itis yellowish water in soluble liquid and has a strong garlic odor. It isproduced during the decomposition of allicin, which is released duringincision of garlic and other plants of the Alliaceae family (Amagaseet al., 2001). DADS has many health benefits from garlic, but it is alsoan allergen causing garlic allergy. Highly diluted, it is used as a flavoringin the food. Kimbaris et al. found that diethyl ether, ethyl acetate orhexane extracts of garlic were composed of DADS 80–280, 60–231 or70–260 μg/g, respectively (Kimbaris et al., 2006). In addition, Lee et al.found that dichloromethane extracts of Korean garlic were composedof DADS about 0.57–0.89 mg/g (Lee et al., 2003).

Thiacremonone (2,4-dihydroxy-2,5-dimethyl-thiophene-3-one)was isolated and identified as a novel and major organosulfur com-pound (0.3%) in High-Temperature-High-Pressure (HTHP)-treated gar-lic (Hwang et al., 2006) (Table 1). The heated samples were juiced andthen filtered on a Buchner funnel under a vacuum. Heated garlic juicewas partitioned consecutively in a separating funnel using ethyl acetate.Isolation of the compounds from the ethyl acetate layer of heated garlicjuice was subjected to column chromatography on silica gel. This frac-tion was purified by preparative RP-HPLC on a Younglin SP930D Instru-ment. It was recently found that heated Korean garlic containsthiacremonone about 0.7 μg/g (Hwang et al., 2006).

3. Pharmaceutical effect of garlic on cancer development

3.1. Anti-cancer effect of garlic in epidemiological studies

Over the last 30 years, there have been many literature reports ofepidemiological studies indicating that a garlic-rich diet decreases riskof some cancers such as gastric, stomach, and colon as shown inTable 2. Those who consumed garlic more than 3 times/week have

rols OR OR (95% CI) References

0.35 0.35 (0.13–0.95) Pourfarzi et al., 20090.5 0.50 (0.27–0.91) Kim et al., 20020.67 0.67 (0.38–1.18) De Stefani et al., 20010.5 0.5 (0.3–0.8) Munoz et al., 20010.11 0.11 (0.02–0.6) Lazarevic et al., 20100.53 0.53 (0.25–0.82) Palli et al., 20010.5 0.50 (0.23–1.08) Zickute et al., 20050.68 0.68 (0.37–1.26) Setiawan et al., 20050.5 0.50 (0.19–0.81) Gao et al., 20020.66 0.66 (0.37–1.17) Takezaki et al., 20010.21 0.21 (0.09–0.50) Setiawan et al., 2005

57 0.77 0.77 (0.50–1.20) Gonzalez et al., 20060.22 0.22 Iscovich et al., 19920.7 0.7 Le Marchand et al., 19970.39 0.39 Levi et al., 19990.68 0.68 Steinmetz et al., 19941.36 1.36 Dorant et al., 1996

Table3

Invivo

labo

ratory

stud

iessh

owingan

ticarcinog

enicprop

erties

ofga

rlic

anditsco

nstituen

ts.

Compo

nents

Canc

ertype

Doses

and

conc

entrations

Adm

inistration

Duration

Mecha

nism

sinhibition

Animal

mod

elRe

ferenc

es

DAS

DMBA

-ind

uced

skin

carcinog

enesis

10mg/kg

Topical

3times/w

for28

wApo

ptosis,p

53an

dp2

1;Bcl-2an

dsurvivin

–Sw

issalbino

mice

Kalra

etal.,20

06DAS

NNK-ind

uced

lung

canc

er20

0mg/kg

p.o.

Daily

for3d

NNK-metab

olizingen

zymeactiv

ity62

%A/Jmice

Yang

etal.,20

01DADS

HL-60

xeno

graft

42mg/kg

Peritone

alcavity

3times/w

for21

wG1ph

asearrest

andp2

1WAF1

63.40%

SCID

mice

Zhao

etal.,20

06DATS

Mou

seco

loncarcinom

aCT

26allograft

50mg/kg

p.o.

Every5dfor32

dApo

ptosisan

dsub-G1arrest

50%

BALB

/cmice

Wuet

al.,20

11DATS

Tran

sgen

icad

enocarcino

maof

mou

seprostate

(TRA

MP)

≑80

mg/kg

Gav

age

3times/w

for13

wApo

ptosis

STAT3

,Bcl-2,B

cl-xL

andSu

rvivin

41%

TRAMPmice

Chan

dra-Ku

ntal

&Sing

h,20

10DATS

Hum

anprostate

canc

erPC

-3xe

nograft

(ortho

topic)

40mg/kg

Oral

5times/w

for4w

Growth

andTR

AIL

;DR4

,DR5

andcasp

ase-8

50%

BALB

/cnu

demice

Shan

karet

al.,20

08

DATS

TRAMP

≑40

–80

mg/kg

Oral

Thrice/w

for13

wProlife

ration

;Cyclin

B125

–46

%TR

AMPmice

Sing

het

al.,20

08DATS

DMNAan

dTP

Aindu

cedtw

o-stageskin

papillo

mamod

el0.2–

0.9(μg/mou

setissue

)To

pical

Twice/w

for20

wAP-1,

COX-2,JNKan

dAkt

75–99

%ICRmice

Shrotriyaet

al.,20

10

Ajoen

eSk

intumor

250(μg)

Topical

Twice/w

for20

d–

95%

ICRmice

Nishika

waet

al.,20

02SA

MC

Hum

ancoloncanc

erSW

620xe

nograft

300mg/kg

Oral

Daily

for16

dApo

ptosis,caspa

se3an

dPA

RP1

N90

%BA

LB/c

athy

micnu

demice

Lian

get

al.,20

11SA

CHum

anprostate

canc

erCW

R22R

xeno

graft

1000

mg/kg

i.p.

Daily

for7w

Apo

ptosisan

dcasp

ase3

;Bcl-2

62.40%

BALB

/cathy

micnu

demice

Chuet

al.,20

07SA

CHum

anprostate

canc

erPC

-3xe

nograft

300mg/kg

Oroga

stricfeed

ing

Daily

for28

dMetea

stasis

;E-cad

herin

71%

CB-17SC

ID/SCIDmice

How

ardet

al.,20

07SA

CHum

anNSC

LCA-549

xeno

graft

240mg/kg

Oral

Daily

for8w

Prolife

ration

;PI3K/Akt,N

F-κB

andCo

x-2

42%

BALB

/CAnN

-Fox

n1nu

demice

Tang

etal.,20

10

SAC

Hum

anNSC

LCA-549

xeno

graft

480mg/kg

Oral

Daily

for8w

Prolife

ration

;PI3K/Akt,N

F-κB

andCO

X-2

64%

BALB

/CAnN

-Fox

n1nu

demice

Tang

etal.,20

10

Garlic

etha

nol

extract

DMA-ind

uced

coloncanc

er1%

Diet

Daily

for8w

Cellgrow

th,A

P-1an

db-actenin

;p21

67%

F344

rat

Kim

etal.,20

07

DAS,diallylsulfide

;DADS,diallyld

isulfide

;DATS

,dially

ltrisu

lfide

;S-ally

lmercaptoc

ysteine;

SAC,

S-allylycysteine

;,D

ecrease;

,Inc

rease.

186 H.-M. Yun et al. / Pharmacology & Therapeutics 142 (2014) 183–195

lower risk of gastric cancer than those who never or infrequentlyconsumed it (OR 0.42; P for trend b 0.01) (Pourfarzi et al., 2009).A significant decrease in the risk of gastric cancer was observed withincreased intake of garlic (OR = 0.50 [CI = 0.27–0.91]) (Kim et al.,2002). Garlic consumption was strongly associated with a reduced riskof stomach cancer (OR 0.67, 95% CI 0.38–1.18) (De Stefani et al.,2001). Frequent consumption of garlic was negatively associated withgastric cancer (OR 0.5, 95% CI 0.3–0.8) (Munoz et al., 2001). Allium veg-etables (e.g. onion, garlic, leek) decreased the risk of stomach cancer(OR 0.11; 95% CI 0.02–0.60) (Lazarevic et al., 2010). For gastric tumors,a significant protective effect was associated with frequent consump-tion of garlic (Palli et al., 2001). Stomach cancer riskwas inversely asso-ciated with consumption of raw vegetables such garlic (OR = 0.59, 95%CI = 0.37–0.96; 1–6 times/week vs. almost never) (Zickute et al.,2005). Intakes of garlic were inversely associated with risk of coloncancer (Iscovich et al., 1992; Le Marchand et al., 1997; Levi et al., 1999).

3.2. Anti-cancer effect of garlic in in vivo and in vitro studies

Preclinical studies have provided convincing evidence that severalnatural organosulfur compound analogues are highly effective inaffording protection against cancer induced by different carcinogens indifferent experimental animals listed in Table 3. DAS, DADS, DATS, ajoene,SAC, and garlic extracts are documented to inhibit lung, liver, prostate andskin tumor development (Shankar et al., 2008; Chandra-Kuntal & Singh,2010; Wu et al., 2011). DAS topical treatment (10 mg/kg, 3 times/weekfor 28 weeks) induced apoptosis and inhibited tumor growth throughmodulation of p53 and Ras-induced signaling pathways including p21/waf1, survivin, bcl-2, PI3K/Akt, andMAPKs in DMBA induced skin tumorsin Swiss albino mice (Kalra et al., 2006). DADS (42 mg/kg, 3 times/weekfor 21 weeks)was protective against cancers of thehumanpromyelocyticleukemia HL-60 via G1 phase arrest and induction of p21/WAF1 expres-sion (Zhao et al., 2006). DATS treatment (about 80 mg/kg, 3 times/weekfor 13 weeks) suppresses prostate cancer development in transgenic ad-enocarcinoma of mouse prostate mice via a visible decrease in the levelsof pSTAT3 (Chandra-Kuntal & Singh, 2010). SAC treatment (240 mg/kgand 480 mg/kg) in an vivo model of NSCLC A549-implanted nude miceshowed an anti-tumor effect, a decrease of 42 and 64% in tumor growthby inhibition of proliferation of cancer cells via reduction of PI3K/Akt,NF-κB, and COX-2 expression (Tang et al., 2010). Garlic ethanol extractinhibited DMH-induced overexpression of Activator Protein-1 (AP-1)and β-catenin genes related to cell proliferation that also upregulatedthe expression of p21Waf1/Cip1 mRNA and a cell cycle-regulating gene(Kim et al., 2007). Naturally occurring organosulfur compound analoguescan also suppress proliferation of cancer cells in culture listed in Table 4including human colon, lung, prostate, liver, breast, keratinocytes,leukemia, and gastric cancer cells.

DAS andDATS suppressed the proliferation and induced apoptosis ofhuman colon cancer cells through oxidative modification of β-tubulin(Hosono et al., 2005). DAS decreased the cell growth of HEK 293Tkeratinocytes through inhibition of cyclooxygenase-2 (Elango et al.,2004). DAS and garlic extract increased cell death through regulationof the expression of Bcl-2, Bax, and p53 in NCI-H460 and NCI-H1299non small cell lung cancer cell lines (Hong et al., 2000). DADS increasedapoptosis through induction of the G2/M cell cycle arrest and regulationof the expression of apoptosis regulatory proteins in human A549, NCIH460 and NCI1299 lung cancer cells (Hong et al., 2000; Wu et al.,2005). In addition, DADS inhibited proliferation via induction of theexpression of p21/waf1 and the G2/M cell cycle arrest in human coloncancer cells (Robert et al., 2001; Filomeni et al., 2003). Similarly DATSwas shown to induce apoptosis and G1 phase arrest in human coloncancer cells (Hosono et al., 2005; Wu et al., 2005). DATS induced apo-ptosis through modulation of Bcl-2 expression and phosphorylation ofJNK and ERK in human prostate PC-3 cells (Xiao et al., 2004). This is anovel aspect in the biological profile of this garlic compound givingnew insights into the understanding of the molecular mechanisms of

Table 4In vitro laboratory studies showing anticarcinogenic properties of garlic and its constituents.

OSCs Cancer cell type IC50 (mM) Treatmenttime

Mechanisms References

DAS HCT-15 and DLD-1 human colon tumor cells N100 24 h Apoptosis and G1 phase arrest ; Tubulin polymerizationApoptosis and G1 phase arrest ; Tubulin polymerizationApoptosis and G1 phase arrest ; Tubulin polymerization

Hosono et al., 2005

HEK 293T keratinocytes 30 72 h Proliferation and Cox-2 Elango et al., 2004NCI-H460 and NCI-H1299 non small cell lungcancer cell line

20–25 48 h Apoptosis, p53 and Bax Bcl-2 Hong et al., 2000

DADS Human A549 lung cancer 25–50 48 h Apoptosis, intracellular ROS and G2/M cell cycle arrestApoptosis, intracellular ROS and G2/M cell cycle arrestApoptosis, intracellular ROS and G2/M cell cycle arrest

Wu et al., 2005

HCT-15 and DLD-1 human colon tumor cells N100 24 h Apoptosis, G1 phase arrest ; Tubulin polymerizationApoptosis, G1 phase arrest ; Tubulin polymerizationApoptosis, G1 phase arrest ; Tubulin polymerization

Hosono et al., 2005

PC3 prostate cancer 35 24 h Apoptosis ; Bcl-2 Xiao et al., 2004HEK 293 T keratinocytes 16.1 72 h Proliferation and Cox-2 Elango et al., 2004Caco-2 and HT29 human colon cancer cells b200 24 h Proliferation and histone deacetylase activity ; p21/waf1 Filomeni et al., 2003HT29 human colon adenocarcinoma 100 24 h G2/M phase arrest and DNA fragmentation G2/M phase

arrest and DNA fragmentation G2/M phase arrest andDNA fragmentation

Robert et al., 2001

NCI H460 and NCI1299 Non small cell lung cancer b5 48 h Apoptosis, p53 and Bax ; Bcl-2 Hong et al., 2000DATS Mouse CT26 colon cancer cells 20 24 h Apoptosis and sub-G1 arrest Apoptosis and sub-G1

arrest Apoptosis and sub-G1 arrestWu et al., 2011

DU145 LNCaP human prostate cancer 20–40 24 h Apoptosis ; STAT3 and Bcl-2 Chandra-Kuntal &Singh, 2010

Human BGC823 gastric cancer 25–50 72 h Apoptosis Li et al., 2006HCT-15 and DLD-1 human colon tumor cells 10–15 24 h Apoptosis and G1 phase arrest Tubulin polymerization

Apoptosis and G1 phase arrest Tubulin polymerizationApoptosis and G1 phase arrest Tubulin polymerization

Hosono et al., 2005

Human HEK 293 T keratinocytes 3 72 h Proliferation COX-2 Elango et al., 2004DU145 and PC3 prostate cancer 22 24 h Apoptosis Bcl-2 Xiao et al., 2004J5 human liver tumor 50–100 24 h Cell growth ; G2/M arrest Cell growth ; G2/M arrest Wu et al., 2005HepG2 human hepatoma 100–1000 72 h Cell growth ; B(a)P-induced phase-1 Chun et al., 2001

SAMC SW480, SW620, Caco-2 human colorectal cancercellsSW480, SW620, Caco-2 human colorectalcancer cells

400–800 72 h Apoptosis, Caspase 3 and PARP1 Liang et al., 2011

MCF-7 breast cancer N61 8 d Anchorage-dependent cell growth Pinto & Rivlin, 2001MCF-7ras breast cancerMCF-7ras breast cancer b61 8 d Anchorage-dependent cell growth Pinto & Rivlin, 2001

SAC NSCLC A-549 human lung cancer 5–10 (mM) 72 h Proliferation and Akt/mTOR/NF-kB Tang et al., 2010MCF-7 breast cancer N62 8 d Anchorage-dependent cell growth Pinto & Rivlin, 2001MCF-7ras breast cancerMCF-7ras breast cancer b62 8 d Anchorage-dependent cell growth Pinto & Rivlin, 2001

Ajoene HL-60 human leukemia 20 12 h Proliferation ; G2/M arrest and proteasomeProliferation ; G2/M arrest and proteasome

Xu et al., 2004

Thiacremonone SW620 and HCT116 human colon cancer cells 583–722 24 h Apoptosis, NF-kB and Caspase-3 Ban et al., 2007AGE MCF-7 breast cancer N10 (μg/ml) 8 d Anchorage-dependent cell growth Pinto & Rivlin, 2001

MCF-7ras breast cancerMCF-7ras breast cancer N10 (μg/ml) 8 d Anchorage-dependent cell growth Pinto & Rivlin, 2001Garlic extract NCI H460 and NCI1299 Non small cell lung cancer N200 48 h Apoptosis, p53 and Bax ; Bcl-2 Hong et al., 2000Garlic oil HL-60 human leukemia 25–50 (μg/ml) 96 h Proliferation Seki et al., 2000

DAS, diallyl sulfide; DADS, diallyl disulfide; DATS, diallyl trisulfide; S-allyl mercaptocysteine; SAC, S-allylycysteine; AGE, aged garlic extract.Decrease; , Increase.

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its potential antitumor action (Xu et al., 2004). As a possiblemechanismof this inhibition, it has been suggested that allyl sulfides may act toincrease activity of glutathione S-transferase (GST), which catalyzesconjugation of electrophilic compounds with GSH (Sparnins et al.,1986; Sparnins et al., 1988). The effect of these volatile substanceson (CYP1A1, 1A2 and 1B1)-mediated bioactivation of B(a)Pwas investi-gated using a human hepatoma cell model (HepG2). The cell viability,an indicator of the capacity to inhibit B(a)P bioactivation, was increasedby treatments of 100–1000 μMDADS and 10–100 μMDATS. In addition,DATS induced apoptosis of human BGC823 gastric cancer cells (Li et al.,2006) and human HEK 293T keratinocytes (Elango et al., 2004). SAMC(S-allylmercaptocysteine), the garlic-derived compound DADS inducedapoptosis through induction of active caspase-3 and PARP1 in humanSW480, SW620 and Caco-2 colorectal cancer cells (Liang et al., 2011).DAS and DADS inhibited arylamine N-acetyltransferase activityand 2-aminofluorene (2-AF)-DNA adduct formation in HL-60 cellline. This was the first report of garlic components affecting humanleukemia cell NAT activity and 2-AAF-DNA adduct formation (Linet al., 2002). Thiacremonone, a novel recently identified organosulfurcompound from garlic, induced apoptotic cell death and increased

susceptibility of cancer cells with the combination of chemotherapeuticagents. Treatment of thiacremonone inhibited colon cancer cellgrowth, followed by induction of apoptosis, as well as suppression ofNF-κB target anti-apoptotic genes (Bcl-2, cIAP1/2, and XIAP) and in-flammatory genes (iNOS and COX-2), whereas it induced apoptoticgene (Bax, cleaved caspse-3, and cleaved PARP) expression (Ban et al.,2007; Ban et al., 2009a). Moreover, oral treatment of mice withthiacremonone (1 mg/kg) by administering it in drinking water for4 weeks significantly augmented docetaxel (1 mg/kg, i.p., four times)-induced decrease of tumor growth accompanied with regulationof NF-κB activity and NF-κB target genes. In the molecular dockingexperiment, NF-κB and thiacremonone exhibited binding affinity of−5.8 kcal/mol, and the binding pocket for thiacremonone was formedby two beta-sheet peptide segments (Arg356, and Val412, Leu437–Leu440) and a loop (Gly361–Gly365) in close proximity to the DNAbinding site of NF-κB. Essentially, thiacremonone was shown to formtwo and three hydrogen bonds with the surrounding amino acids.Among these, one hydrogen bond interaction (between C1_O andNH of Val412) was conserved (unpublished data). These results suggestthat induction of apoptosis and inhibition of NF-κB by thiacremonone

Table 5Cholesterol lowering effect of garlic in human.

Preparation Doses Duration Effect References

Tablet Garlic (KWAI) 900 mg/d 24 w TC, TG and LDL ; HDL Ashraf et al., 2011Allicor (NAT-Farma, Moscow, Russia) 300 mg/d 12 m TC, TG and LDL Sobenin et al., 2010Allicor (NAT-Farma, Moscow, Russia) 600 mg/d 12 w TC, TG and LDL ; HDL Sobenin et al., 2008Garlic powder tablet 800 mg/d 6 w TC, TG and LDL ; HDL Kojuri et al., 2007Tablet garlic (Garlex-Bosch Pharmaceuticals) 600 mg/d 12 w TC and LDL ; HDL Ashraf et al., 2005Aged ‘Kyolic’ garlic 2.4 g/d 2 w TC and TG ; LDL and HDL Williams et al., 2005Garlic powder tablet 920 mg/d (alliin 43.2 mg/d) 2 w TC and LDL ; TG and HDL Turner et al., 2004Garlic tablet (Garlet) 800 mg/d 3 m TC, TG, LDL and HDL− Ziaei et al., 2001Garlic powder tablet 880 mg/d 12 w TC and LDL ; TG and HDL Kannar et al., 2001AGE capsule 7200 mg/d 5 m TC and LDL ; TG and HDL− Yeh & Liu, 2001

TC, Total Cholesterol; TG, Triglycerides; LDL, Low-density lipoproteins; HDL, High-density lipoproteins.Decrease; , Increase.

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could provide a specific and causative mechanism for the inhibition ofcancer cell growth, as well as provide novel evidences for combinationof chemotherapeutic agents with thiacremonone. Taken together,organosulfur compounds isolated from garlic could exert great benefi-cial effects due to its relatively low toxicity and better anti-cancer activ-ities against different types of cancers with various action mechanisms.Therefore organosulfur compounds could be potentially useful as che-motherapeutic agents for the treatment of cancers.

4. Pharmaceutical effect of garlic on cardiovascular disorders

Numerous studies have confirmed the ability of garlic to reduce pa-rameters associated with cardiovascular disease. A brief description ofsome of these studies is given below.

4.1. Effect of garlic on cholesterol

Experimental evidence indicates that garlic ingestion lowers bloodcholesterol levels and inhibits cholesterol synthesis (Table 5). Zengand his colleagues deduced from 26 studies that hypercholesterolemicpatients treated with garlic (garlic powder and aged garlic extract)had a mean serum TC and TG concentration, that were 0.28 (95%CI, −0.45, −0.11) mmol/l (P = 0.001) and 0.13 (95% CI, −0.20,−0.06) mmol/l (P b 0.001) lower than that of patients treatedwith pla-cebo, respectively (Zeng et al., 2012). Several experimental studies haveindicated that garlic and its constituents inhibit key enzymes involvedin cholesterol and fatty acid synthesis in cultured rat hepatocytes andhuman HepG2 cells (Gebhardt, 1993; Yeh & Yeh, 1994; Liu & Yeh,2001; Yeh & Liu, 2001). Cultured hepatoma cells were treated withaqueous garlic extract and radiolabeled cholesterol was quantified.

Table 6Blood pressure effect of garlic in human.

Preparation Doses

AGE 960 mg/d (2.4 mgAllicor (coated tablets containing 150 mg garlic powder,INAT-Farma, Moscow, Russia)

600 mg/d

Garlicin tablet (produced by Xinjiang Tianshan Pharmaceutical Factory) 24 mg/dAGE Kyolic 3 g/d (S-allylcysteClove of garlic 1 g/dDried garlic powder tablets (Futura Hvidløg Fortew; Dansk Droge,Ishøj, Denmark)

920 mg/d (alliin 1

Garlic pearls (GP; Ranbaxy Laboratories Ltd., Mumbai, India). 250 mg/dAqueous garlic extract (20%, w/v) (Kastamonu garlic). ∼10 g/dGarlic tablet 1 g/d (1.5 mg allicGarlic oil 12.3 mg/dGarlic tablet Garlet 800 mg/d (1 mg aGarlic powder 1200 mg/d (1 mgGarlic tablet (Garlet) 800 mg/dGarlic 134 g/mKwai garlic tablets 900 mg/d

SBP, systolic blood pressure; DBP, diastolic blood pressure; , Decrease; , Increase.

Garlic extract reduced cholesterol synthesis by up to 75% withoutevidence of cellular toxicity. These results indicate that compounds con-taining an allyl-disulfide or allyl-sulfhydryl group are most likely re-sponsible for the inhibition of cholesterol synthesis by garlic and thatthis inhibition is likely mediated at sterol 4 alpha-methyl oxidase. Ithas also been shown that the more water-soluble compounds like SACpresent in aged garlic extract are less cytotoxic and more efficient ininhibiting cholesterol biosynthesis than the lipid-soluble organosulfurcompounds such as DAS (Yeh & Liu, 2001).

4.2. Effect of garlic on blood pressure

In patientswith uncontrolled hypertension (SBP N/= 140 mm Hgatbaseline), systolic blood pressure was on average 10.2 +/− 4.3 mm Hg(P = 0.03) lower in the garlic group who consumed four capsules ofaged garlic extract (960 mg containing 2.4 mg SAC) daily for 12 weekscompared with controls over the 12-week treatment period (Riedet al., 2010). Garlic reduces blood pressure (BP) in two-kidney, one-clip (2K-1C) rats through enhancement of nitric oxide (NO) synthesisin in vivo and in vitro as well as in human (Sobenin et al., 2008;Qidwai et al., 2000; Ried et al., 2010) (Table 6). Garlic appears to exertthis effect by modulating the activity of several mechanisms that arevital in BPhomeostasis in favor of hypotension.Onepossiblemechanismby which garlic might induce its hypotensive effect could be throughthe direct and indirect vasodilatory actions of NO. The drinkingwater was replaced with an L-Arg solution (10 mg/ml; average intakeof 300 mg/day) from 7 to 14 days. A day after surgery, there was a sig-nificant reduction of mean blood pressure in the L-Arg-treated groupcompared to control (129 ± 7 vs 168 ± 6 mm Hg) (Kim-Park & Ku,2000; Gouvea et al., 2003). Morihara et al. (2002) also reported that

Duration effect References

S-allylcysteine) 12 w SBP ; DBP − Ried et al., 201012 w SBP ; DBP Sobenin et al., 2008

4 w SBP ; DBP Cheng et al., 2006ine 14 mg) 20 m SBP ; DBP Macan et al., 2006

2 m SBP ; DBP Jabbari et al., 200508 mg) 12 w No significance Turner et al., 2004

8 w SBP ; DBP Dhawan & Jain, 20044 m Hypertention Durak et al., 2004

in) 12 w No significance Gardner et al., 200116 w SBP Zhang et al., 2001

llicin, ajoene) 8 w SBP and DBP −; Hypertention Ziaei et al., 2001Allicin) 4 w SBP ; DBP Mortazavi et al., 2001

3 m Hypertention Ziaei et al., 20011 m SBP ; DBP Qidwai et al., 200012 w No significance Superko & Krauss, 2000

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extract of garlic (2.86 g/kg) increasedNOproduction by 30–40% from15to 60 min after administration (Morihara et al., 2002). Elevating evi-dence indicates that hydrogen sulfide (H2S) plays a cell signaling rolesimilar to NO and CO. In vivo and in vitro cardiovascular effects of H2Sinclude decreased blood pressure and cardioprotection against ischemicdamage and vasorelaxation (Zhao et al., 2001; Sivarajah et al., 2006;Koenitzer et al., 2007). A recent study by Benavides et al. suggests thatH2S may underlie the beneficial effects that garlic exerts on the cardio-vascular system (Benavides et al., 2007). Garlic and garlic-derived or-ganic polysulfides, including DATS and DADS induced H2S productionin a thiol-dependent manner (Calvert et al., 2010). Garlic-mediatedand H2S-mediated vascular smooth muscle relaxation indicate thatboth were based on NO signaling pathways (Benavides et al., 2007).

4.3. Effects of garlic on platelet aggregation

Like cholesterol and blood pressure lowering effects, garlic also has abeneficial effect on platelet aggregation in human as well as in animal(Table 7). Platelet aggregation and subsequent thrombus formationare significantly reduced by garlic and its constituents. All constituentsdisplayed a biphasic pattern of inhibition that was only significant forSEC at 0.78 μmol/l, SMC at 3.125 and 6.25 μmol/l, SPC at 0.78, 3.125and 6.25 μmol/l, and BC at 0.78, 3.125 and 25 μmol/l. Aqueous garlic ex-tracts (final concentration, 1 mg/ml) inhibited human platelet aggrega-tion (Pierre et al., 2005). DATS inhibited platelet aggregation andCa(2+) mobilization in a concentration-dependent manner without in-creasing intracellular cyclic AMP and cyclic GMP. DT at high concentra-tions partially blocked the binding of IP(3) to its receptor (Qi et al.,2000). The mechanism of inhibition of platelet aggregation by garlic'sconstituents has also been addressed, and it is thought to work via theinhibition of calcium mobilization (Qi et al., 2000). A recent study hasalso found another garlic component, sodium 2-propenyl thiosulfate,tomodulate cyclooxygenase activity in canine platelets, thus preventingtheir aggregation (Chang et al., 2005). Overall, there are sufficient evi-dences in terms of reducing the risk of cardiovascular diseases and itsuggests that organosulfur compounds could be potentially developedinto a health product for the cardiovascular system.

Table 7Anti-platelet aggregatory effect of garlic in human.

Preparation Doses andconcentrations

Duration Effect References

Garlic tablet 225 mg/d 5 y & 9 m PA Manoharanet al., 2006

Garlic oil 9.9 g/d 4 h Nosignificance

Wojcikowskiet al., 2007

AGE 7.2 g/d 6 w PA Steiner & Li, 2001AGE 5 ml/d 13 w PA Rahman &

Billington, 2000SEC (In vitro) 0.78 μM 5 min PA Allison et al.,

2006aSMC (In vitro) 3.125–6.25 μM 5 min PA Allison et al.,

2006aSPC (In vitro) 0.78–6.25 μM 5 min PA Allison et al.,

2006aBC (In vitro) 0.78–25 μM 5 min PA Allison et al.,

2006aAGE (In vitro) 80 mg/ml 10 min PA Allison et al.,

2006bAqueous garlicextracts

(In vitro)1 mg/ml 15 min PA Pierre et al.,2005

Diallyltrisulfide

(In vitro) 10 μg/ml 5 min PA Qi et al., 2000

SEC, S-ethylcysteine; SMC, methyl-L-cysteine; SPC, S-1-proponyl-L-cysteine; BC,β-chlorogenin.PA, Platelet aggregation, : Decrease

5. Pharmaceutical effect of garlic on neurological diseases

Recent studies have demonstrated beneficial effects of AGE and oneof its active ingredients SAC in Alzheimer disease models (Chauhan,2006; Ray et al., 2011a,b) (Table 8). The therapeutic effects of SACwere also assessed in various models of neurodegenerative diseases in-cluding stroke (Kim et al., 2006; Atif et al., 2009), ischemia/reperfusion(Ashafaq et al., 2012), Alzheimer's disease (Perez-Severiano et al., 2004;Javed et al., 2011), and Parkinson's disease (Garcia et al., 2010). Themo-lecular mechanisms of these effects may include protecting neuronsagainst oxidative/nitrosative (reactive oxygen/nitrogen species) stress,mitochondrial damage, and subsequent cell death. SAC also reducesedema formation in the ischemic rat brain through the inhibitionof LPO (Numagami et al., 1996) and produces neuroprotective effectson the Aβ-induced oxidative damage, and learning deficits (Perez-Severiano et al., 2004).

Thiacremonone also inhibited LPS-inducedmemory impairment, glialactivation, pro-inflammatory mediator expression, and amyloidogenesisin in vitro and in vivo via the inactivation of NF-κB via blocking ofphosphorylation of IκBα in mice brain as well as cultured astrocytesand microglial BV-2 cells (Lin et al., 2012). These results indicated thatthiacremonone inhibited neuroinflammation and amyloidogenesisthrough inhibition of NF-κB activity, which controls the genes in-volved in not only inflammation but also amyloidogenesis. Therefore,organosulfur compound in garlic could be applied for intervention ofinflammation-related neurodegenerative disease including Alzheimer'sdisease, stroke, and Parkinson's disease.

6. Other pharmaceutical effects of garlic

6.1. Pharmaceutical effect of garlic on liver diseases

Recent human study suggests that garlic offers protection againstoxidative stress and antioxidant activities in alcoholic liver disease pa-tients (Table 9). Preclinical studies have shown that garlic amelioratesalcohol-induced oxidative stress (Zeng et al., 2008; Nencini et al.,2010), inhibits induction of cytochrome P450 (CYP) (Kishimoto et al.,1999) and prevents fatty liver and liver cirrhosis (Zeng et al., 2008).Garlic is also shown to reduce the ethanol-induced increase in thelipid peroxidation and to increase the levels of antioxidants suchas glutathione (GSH), ascorbic acid, catatase (CAT), and glutathionereductase (GR) in the rat liver (Nencini et al., 2010). Garlic and itsorganosulfur components have been shown to increase the activity ofGSH-related antioxidant system in rat liver (Wu et al., 2001), andhave been demonstrated to provide protective effects against oxidativestress by chemicals (Yang et al., 2001; Khosla et al., 2004; Park et al.,2005; Agarwal et al., 2007). Treatment of SAMC attenuated non-alcoholic fatty liver disease (NAFLD)-induced liver injury, fat accumu-lation, collagen formation and free fatty acids through reductionof MAP kinase pathways and the NF-κB and AP-1 activity (Xiaoet al., 2013).

One of the major protective functions of garlic is to decrease the ox-idative damage in liver (Gedik et al., 2005). Indeed, garlic prevents theinfection induced loss of GSH and decrease of activities of catalase andSOD. These components are normally lowered during oxidative damageinduced by infection as it has been also described by Georgieva et al.(2006) who showed an increase in malondialdehyde (MDA) levels ofEimeria tenella-induced coccidiosis. In accordance, Kiruthiga et al.(2007) have found that garlic significantly decreased lipid peroxidationand increased GSH, catalase and SOD. The antioxidative property of gar-lic has been previously ascribed mainly to its four major chemical com-ponents, i.e. allinin, SAC, DADS, and allicin (Chung, 2006). In addition,Raso et al. (2001) have viewed that the oxidative stress is mainly dueto NO produced by the stimulated iNOS. The inhibitory effect of garlicis mainly attributed to the impaired action of iNOS (Gu et al., 2003;Chang & Chen, 2005). Our data also showed that thiacremonone

Table 8Effect on neurological diseases of garlic.

Components Diseases Doses Duration Effects References

DADS Alzheimer's diseases 20 mg/kg 4 months Anti-amyloidogenic, anti-inflammatory and anti-tangle effects Chauhan, 2006SAC Alzheimer's diseases 20 mg/kg 4 months Anti-amyloidogenic, anti-inflammatory and anti-tangle effects Chauhan, 2006SAC Alzheimer's diseases 30 mg/kg 15 days Cognitive and neurobehavioral improvement Javed et al., 2011Thiacremonone Alzheimer's diseases 1, 3, and 10 mg/kg 1 month Memory improvement, glial inactivation, and anti-amyloidogenesis Lin et al., 2012SAC Ischemia 300 mg/kg 30 min before MCAO Reduction of infarct size Kim et al., 2006DAS Ischemia 100, 150, and 200 mg/kg 7 days before MCAO Increase in Bcl-2 and decrease in caspase-3 Lin et al., 2012AGE Ischemia 1.2 ml/kg 30 min before MCAO Reduction of SOD, GPX, and EC-SOD activities Aguilera et al., 2010SAC Parkinson's diseases 120 mg/kg 5 days Reduction of TNF-α, iNOS, and GFAP expression Garcia et al., 2010

MCAO, Middle cerebral artery occlusion; GPX, Glutathione peroxidase; SOD, Superoxide dismutase; EC-SOD, Extracellular superoxide dismutase.

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reduced acute hepatic injury through inactivation of NF-κB and STAT3by inhibition of pro-inflammatory cytokine production (unpublisheddata). Taken together, all these observations indicate the usefulness ofgarlic in the prevention of liver diseases.

6.2. Pharmaceutical effect of garlic on allergy

Several studies suggested anti-allergic properties for garlic extract(Table 9). Kyo et al. (2001) reported that in their rodent basophilecell line model, addition of garlic extract reduced histamine release(anti-histamine effect). They showed suppression of IgE-mediatedantigen-specific skin reaction in murine model too and concludedthat garlic extract could beneficially balance, or modify the functionof mast cells, basophile, and activated T lymphocyte factors, whichall play a leading role in allergic cascade reactions and inflammation.Three-time intra peritoneal injections of the aged garlic extract causeda significant decrease in the hallmark criteria of allergic airway in-flammation levels which included eosinophil percentage in lavage,peribronchial lung eosinophils, IgG1 level in lavage and serum, mu-cous producing goblet cell grade and peribronchial and perivascularinflammation (Zare et al., 2008). Aged red garlic extract reduces ciga-rette smoke extract-induced cell death by increasing GSH content andreducing ROS generation in human bronchial smooth muscle cells(Jeong et al., 2012).

6.3. Pharmaceutical effect of garlic on arthritis

Several studies have also shown that garlic extract is helpful atpreventing arthritis (Table 9). Consumption of alliums (garlic, leeks,and onions) (P = 0.029) showed the strongest protective associationwith hip osteoarthritis (OA) (Williams et al., 2010). They suggestedthat diallyl disulfide, a compound found in garlic and other alliums, re-presses the expression of matrix-degrading proteases in chondrocyte-like cells, providing a potential mechanism of action (Williams et al.,2010). DAS (48.9 mg/kg) significantly inhibited the increase of IL-1βgeneration and COX-2 gene expression through inactivation of NF-κBinduced by monosodium urate crystals in a rat model and in synovial

Table 9Other pharmaceutical effects of garlic.

Components Diseases Doses and concentrations Duration Effec

Raw garlic cloves Liver diseases 2 cloves/body (2.4 g) 45 days OxidGarlic extracts Liver diseases 250 mg/kg 5 days OxidGarlic oil Liver diseases 200 mg/kg 15 days DecrSAMC Liver diseases 200 mg/kg Three times/week ReduAqueous garlic extract Liver diseases 250 mg/kg 28 days ReduAged garlic extract Allergy 250 mg/kg 24 h DecrGarlic extract Allergy 20 mg/kg 48 h DecrDADS Arthritis 2.5–10 μM 6 h ProtDAS Arthritis 48.9 mg/kg 24 h DecrThiacremonone Arthritis 10 mg/kg 20 days Inhi

GR, Glutathione reductase; GSH, Glutathione; CAT, Catalase; SOD, Superoxide dismutase.

cells and chondrocytes (Lee et al., 2009). A 1–10 mg/kg dose ofthiacremonone also suppressed the carrageenan and mycobacteriumbutyricum-induced inflammatory and arthritic responses as well as ex-pression of iNOS and COX-2, in addition to NF-κB DNA-binding activity(Ban et al., 2009a).

7. Pharmacokinetics

There have beenmany reports on the pharmacokinetics of garlic andits constituents. The oil-soluble organosulfur compounds in garlic,including allicin, ajoene, and vinyldithiins are not found in blood orurine, even after consumption of a large amount of garlic (Lawsonet al., 1992). Therefore, they are likely not the active compounds. Allicin,perfused into isolated rat livers, showed a remarkable first-pass effectand is metabolized to DADS and allyl mercaptan, whereas ajoenes andvinyldithiins were recovered in the effluent (Egen-Schwind et al.,1992a,b). Allicin disappeared very rapidly when incubated with liverhomogenate (Egen-Schwind et al., 1992a,b). No allicin was detected ineither serum or urine from 1 to 24 h after ingestion of 25 g of raw garlic(∼90 mg allicin) (Lawson et al., 1992). DADS is a metabolite of allicin(Egen-Schwind et al., 1992a,b). The maximum concentration ofradiolabeled DADS in the liver of mice occurred 90 min after intraperi-toneal administration (Pushpendran et al., 1980). Seventy percent ofthe radioactivity, which was no longer DADS, was distributed in theliver cytosol, of which 80%wasmetabolized to sulfate. DADS, like allicin,was not detected in human blood or urine from 1 to 24 h after oralingestion of 25 g of crushed raw garlic (Lawson et al., 1992). The insta-bility and/or metabolism of such compounds likely contributes to theinconsistent results found in the clinical cholesterol studies using garlicoil (Berthold et al., 1998) and garlic powder products (Simons et al.,1995; Neil et al., 1996; Breithaupt-Grogler et al., 1997; Berthold et al.,1998; Isaacsohn et al., 1998; McCrindle et al., 1998). Metabolitesof garlic constituents, such as N-acetyl-S-(2-carboxypropyl)-cysteine,N-acetylcysteine, and hexahydrohippuric acid have been detected inhuman urine after ingestion of garlic (Jandke & Spiteller, 1987). Afterconsumption of garlic, N-acetyl-S-allyl-cysteine is found in humanurine. At present, SAC is the only reliable human compliance marker

ts References

ative stress and antioxidant activities Mirunalini et al., 2010ative stress, GR and CAT activities Nencini et al., 2010ease in oxidation process El-Khayat et al., 2010ction of MAPK, NF-κB, AP-1, and cytochrome P450 2E1 activity Xiao et al., 2013ction of GSH, CAT, and SOD activities Gedik et al., 2005eased 25–45% of the ear swelling Kyo et al., 2001eased allergic airway inflammation levels Zare et al., 2008ective effects in hip osteoarthritis Williams et al., 2010ease in IL-1 and COX-2 Lee et al., 2009bition of the carrageenan paw edema Ban et al., 2009b

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used for studies involving garlic consumption because it is detectableand increases quantitatively in the blood after oral intake of garliccapsules (Steiner & Li, 2001). The pharmacokinetic studies of SAC dem-onstrated rapid absorption and almost 100% bioavailability after oral ad-ministration. The absorbed SAC seems to be metabolized to N-acetyl-SAC in urine of rats, dogs, and humans by N-acetyltransferase, whichis mainly found in liver and kidney. However, it has been shown that,when SAC is almost completely eliminated from the liver, it is readilyretained at a comparatively high concentration in the kidney. Thus, itcan be speculated that SAC may be transformed into N-acetyl-SAC byN-acetyltransferase in the liver, and then, a portion of N-acetyl-SACmay be deacylated to SAC by acylase in kidney, followed by its reabsorp-tion. In addition, since both the safety and effectiveness of SAC havebeen reported, this compound appears to play an important role ingarlic's medicinal effects. Though individual compounds such as SAChave shown activity in studies and are absorbed by the body, it is likelythat a synergism of various compounds provides the benefits of garlic.Thus, the activity of various sulfur compounds could not alone beresponsible for the benefits of garlic, and fixation on a single group ofcomponents can lead to mistakes and wrong conclusions. Overall, theactive principles in garlic have not been fully characterized. However,it is assumed that the bioavailability of these sulfur-containing com-pounds will play an important role in determining the biological re-sponse to various garlic preparations.

8. Toxicity and safety

Although garlic has been used safely in cooking as a popular condi-ment or flavoring and used traditionally for medicinal purposes, it iscommonly known that excessive consumption of garlic can cause prob-lems. Adverse effects that have been documented in humans include aburning sensation in the mouth and gastrointestinal tract, nausea, diar-rhea, vomiting (Fulder, 1989; Barnes et al., 2007) and body odor. Theallergenic potential of garlic is well recognized, and the allergens havebeen identified as DADS, allylpropyl sulfide and allicin (Papageorgiouet al., 1983; Barnes et al., 2007). Since 1932, several reports haverevealed the following adverse effects associated with raw garlic andgarlic powder: 1) stomach disorders and diarrhea (Nakagawa et al.,1980; Caporaso et al., 1983; Desai et al., 1990); 2) decrease of serumprotein and calcium (Miyamoto, 1938; Shashikanth et al., 1986); 3) ane-mia (Katsunuma, 1932; Kuzutani, 1934; Nakagawa et al., 1980);4) bronchial asthma (Lybarger et al., 1982; von Kirsten & Meister,1985); 5) contact dermatitis (Mitchell, 1980; Parish et al., 1987;Lembo et al., 1991; McFadden et al., 1992; Garty, 1993; Burden et al.,1994); and 6) inhibition of spermatogenesis (Dixit & Joshi, 1982; Qianet al., 1986). Allicin can be an oxidizing agent that not only impedes bac-terial growth (Shashikanth et al., 1986) but also can damage the intesti-nal lining and the stomach (Kodera, 1997). Raw garlic juice (0.5 ml)caused significant damage to the epithelial mucosal membrane after

Table 10Toxicological properties of garlic extracts.

Tests Preparation Doses andconcentrations

Administration Duration effect

Acute toxicity Garlic extract 30 mg/kg P.P., I.P., S.C. 7 days No efChronic toxicity Garlic extract 60–2000 mg/kg Oral 6 months No w

decreMutagenicity Garlic extract

vs raw garlic100–2500 mg/kg Oral 18 days Increa

Cytotoxicity Garlic extractvs raw garlic

0.012–1.2 mg/ml In vitro 7 days Growchang

General toxicity Garlic extractvs raw garlic

5 ml/kg Oral 5 days Stomvariou

Teratogenicity Garlic extract 275.5 mg/kg Oral 5 days/week Preveterato

Clinical study Garlic extract 7.2 g/day Diet 6 months Reduc

MNC, Micronucleated cell; PC, Polychromatocytes; CHE, Chinese hamster embryo.

2 h in rats. Enteric-coated garlic products are designed to deliver allicin(1–5 mg, depending on the product label claim) directly into the intes-tinal tract. After 24 h of exposure, ulcers, shrinkage, and bleeding in theepithelial mucosa were detected. When three kinds of commerciallyavailable enteric-coated garlic preparations, i.e., Garlicin, Garlique, andGarlinase 4000, were used at dosages of 133, 108 and 60.5 mg/rat, re-spectively, each caused severe damage to the duodenal mucosa after2 h of exposure (Amagase et al., 2001). According to their study,enteric-coated garlic powder products, which are designed to generateallicin in the delicate intestine, may be hazardous to the intestinal tract(Amagase et al., 2001).

On the other hand, a number of toxicological and clinical studies ofgarlic extracts have been performed with no adverse effects. The safetyof garlic extracts has been well established by the following studies(Table 10): 1) acute and subacute toxicity tests (Nakagawa et al.,1984a,b); 2) chronic toxicity test (Sumiyoshi et al., 1984); 3) mutage-nicity tests (Yoshida et al., 1984); 4) general toxicity tests (Nakagawaet al., 1980; Kanezawa et al., 1984); 5) teratogenicity tests (SegmentsI, II, and III) (Assayed et al., 2010); 6) toxicity test conducted by theU.S. Food and Drug Administration; and 7) clinical studies conductedon N1000 subjects (Hasegawa et al., 1983; Kawashima et al., 1989;Steiner et al., 1996). The United States National Cancer Institute testedtoxicity of typical garlic compounds. In humans, daily doses of up to60 g of fresh garlic and 120 mg of essential oil of garlic, over a periodof three months, did not result in any serious disorders (Anonymous,1997). Recent clinical trials report garlic extracts to be safe as a comple-mentary medicinewith warfarin (Macan et al., 2006). Such characteris-ticsmay come from theAGEprocessingmethod and clearly differentiatethe extract from other preparations. SAC is a safe compound and itsbiological effects are well studied. The U.S. National Cancer Institute(NCI) investigated the toxicity of SAC when compared with other garliccompounds and found that SAC has less toxicity than allicin and DADS.The oral 50% lethal dose in mice is as follows for allicin: 309 mg/kgin males and 363 mg/kg in females; for DADS: 145 mg/kg in malesand 130 mg/kg in females; and for SAC: 8890 mg/kg in males and9390 mg/kg in females (Amagase, 2006).

9. Perspective

In vitro and in vivo as well as clinical trials have demonstrated thatgarlic may be an important traditional medicine to treat a variety ofconditions such as cancer, cardiovascular disease, neurological disease,and liver disease as well as allergy and arthritis. Garlic contains at leastalliin, γ-glutamyl-S-allylcysteine, S-methylcysteine sulfoxide, S-trans-1-propenylcysteine sulfoxide, S-2-carboxypropylglutathione, SAC, andthiacremonone, which have a wide variety of pharmaceutical proper-ties. Recent study demonstrated that garlic (Allicin) had selectivityon the sulfonylurea receptors (SURs) (Harikesh et al., 2012). SURsare membrane proteins which are the molecular targets of the

s Model References

fect (LD50 N 30 mg/kg) Wistar rat, dYY mouse Nakagawa et al., 1984beight gain and toxic sign, slightlyased food consumption

Wistar rat Sumiyoshi et al., 1984

se of MNC and PC Mouse, Chinese hamster Yoshida et al., 1984

th inhibition and morphologicale

CHE cell, HEp-2 cell line Yoshida et al., 1984

ach injury, weight loss, swelling ofs organs

Wistar rat Kanezawa et al., 1984

ntion of cypermethrin-inducedgenic effects.

Wistar rat Assayed et al., 2010

tion in cholesterol, blood pressure Men (32–68 age group) Steiner et al., 1996

192 H.-M. Yun et al. / Pharmacology & Therapeutics 142 (2014) 183–195

sulfonylurea class of antidiabetic drugs whose mechanism of action isto promote insulin release from pancreatic beta cells. More specifically,SUR proteins are subunits of the inward-rectifier potassium ion chan-nels Kir6.x (Campbell et al., 2003). Four Kir6.x and four SUR subunitscompose an ion conducting channel referred to as the KATP channel(Aguilar-Bryan et al., 1998). Garlic extract exhibited anti-diabetic andanti-hypertensive effects as a vasodilator by hyperpolarizing the mem-brane of normal vascular smooth muscle cells. The hyperpolarizationin vascular smooth muscle was induced due to K+ channel opener ac-tivity. Anti-hypertensive activity of garlic extract was suppressed byglibenclamide (nonselective SUR blocker) whereas nateglinide (selec-tive SUR1 blocker) exhibited antihypertensive effect with garlic extract(Harikesh et al., 2012). These studies validated that garlic will be effec-tive in the treatment of hypertensive diabetes. Garlic also showedantihypertensive activity by selective opening of SUR2. Under cerebralischemic conditions SUR1 (the regulatory subunit of the KATP- and theNCCa-ATP-channels) is expressed in neurons, astrocytes, and microglia(Simard et al., 2012). SUR1 is involved in animal stroke throughpreventing brain swelling and enhancing neuroprotection (Ortegaet al., 2012). Garlic-derived organic polysulfides also induced H2S pro-duction (Benavides et al., 2007). H2S is the first identified endogenousgaseous opener of ATP-sensitive K+ channels in vascular smooth mus-cle cells (Zhao et al., 2001). H2S lowers blood pressure, protects theheart from ischemia and reperfusion injury, inhibits insulin secretionin pancreatic beta cells, and exerts anti-inflammatory, anti-nociceptiveand anti-apoptotic effects through the activation of ATP-sensitive K+

channels (Tang et al., 2010).Thus, functionally active components isolated from garlic have

beneficial effects in a variety of diseases and will have a wide range ofapplications.We suggest that this reviewwill shed light on a foundationfor further studies to investigate mechanisms underlying the effectsof functionally active components isolated from garlic, and clinicalapplications of these components.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by the National Research Foundation ofKorea (NRF) grant funded by the Korean Government (MISP) (No.MRC, 2008-0062275), by a grant (A101836) from the Korean HealthTechnology R&DProject,Ministry for Health,Welfare and Family Affairs,Republic of Korea.

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