The Antihypertensive Effect of Black Garlic (Allium Sativum) in Spontaneously Hypertensive Rats via

Research in Health and Nutrition (RHN) Volume 2, 2014

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The Antihypertensive Effect of Black Garlic (Allium Sativum) in Spontaneously Hypertensive Rats via Scavenging of Free Radicals Yelian Miao*1, Jieyu Chen2, Guangyong Zhou3, Xiaobian Xu4, Qimei Zhang5, Jining Wang6 College of Food Science and Light Industrial Engineering, Nanjing University of Technology, Jiangsu 211816, China 2 Faculty of Bioresource Science, Akita Prefectural University, Akita 010-0195, Japan 6 School of Economics and Management, Nanjing University of Technology, Jiangsu 211816, China *1,3,4,5

ylmiao@njut.edu.cn

*1

Received 17 Sep, 2013; Accepted 10 Nov, 2013; Published 10 Jan, 2014 © 2014 Science and Engineering Publishing Company

Abstract Black garlic (Allium sativum) is a new garlic product with high free-radical-scavenging ability. In the present study, the antihypertensive effect of black garlic was investigated in vivo using spontaneously hypertensive rats (SHRs, 185±12 mm Hg) as the test animals. Total antioxidant capacity (TAOC) and malondialdehyde (MDA) content in both plasma and hypothalamic paraventricular nucleus (PVN) of the rats were measured to explore underlying biochemical mechanism of the antihypertensive effect.

The administration of black garlic for 14 days significantly lowered the blood pressure of SHRs to 121±10 mm Hg (with a decline rate of 34.6% in average), while it did not affect the normal blood pressure of Wistar rats. The black garlic had more antihypertensive effect on SHRs than fresh garlic. In the plasma of SHRs receiving the black garlic, the T-AOC increased from 4.2±1.0 U/mL to 5.4±1.1 U/mL (with an increase rate of 28.6% in average), and the MDA content decreased correspondingly from 10.2±2.2 nmol/mL to 7.9±0.7 nmol/mL (with a decrease rate of 22.5% in average). In the PVN of SHRs receiving the black garlic, the T-AOC increased from 4.4±0.7 U/mg-protein to 7.2±1.6 U/mg-protein (with an increase rate of 63.6% in average), and the MDA content decreased correspondingly from 8.2±1.5 nmol/mgprotein to 3.9±1.2 nmol/mg-protein (with a decrease rate of 52.4% in average). The findings indicated that the black garlic exerts a potential antihypertensive effect through scavenging excessive oxygen free radicals (OFRs) in the plasma and PVN of SHRs. Keywords Black Garli, Antioxidant Activit, Antihypertensive Effect, Oxygen Free Radical, Hypertension

Abbreviations CSAR, cardiac sympathetic afferent reflex FRAP, ferric reducing ability of plasma MDA, malondialdehyde OFRs, oxygen free radicals PVN, paraventricular nucleus SHRs, spontaneously hypertensive rats T-AOC, total antioxidant capacity TBA, thiobarbituric acid

Introduction Hypertension is a commonly occurring cardiovascular disease in the world. It is estimated that by 2025, the incidence of hypertension will increase to 24% in developed countries and to 80% in developing countries (Messerli et al., 2007; Quiñones et al., 2013).

Hypertension may be caused by oxygen free radicals (OFRs) in the human body (Russo et al., 1998; Bogdan et al., 2000). Antioxidant therapy helps to scavenge excessive OFRs, and to prevent the pathogenesis of hypertension and its complications (Vaziri et al., 2000; Ortiz et al., 2001). Recent research also indicated that excessive OFRs in hypothalamic paraventricular nucleus (PVN) contribute to the potentiation of vasoconstrictor angiotensin II (Ang II) and cardiac sympathetic afferent reflex (CSAR), which may induce

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hypertension (Han et al., 2007; Grassi et al., 1999). In contrast, microinjection of tempol (i.e. a superoxide anion scavenger) into the PVN of hypertensive rats can abolish the potentiation of Ang II and CSAR, and lower the high blood pressure (Han et al., 2011; Koga et al., 2008).

Black garlic (Allium sativum) is a new garlic product processed by the fermentation of fresh garlic in a temperature- and humidity–controlled room for a month. It is black in color with a fruit-like sweetness and non-irritating order. In particular, black garlic has outstanding antioxidative activity since it is rich in bioactive compounds such as ajoene, S-allyl-L-cysteine and polyphenols (Zhou et al., 2010; Wang et al., 2010; Kim et al., 2012). Experimentation on rats showed that black garlic has a higher ability to increase antioxidase activity, and to decrease malondialdehyde (MDA) content in the blood and the liver tissue, in comparison with fresh garlic (Apitz-Castro et al., 1992; Zhu et al., 2008). The heat-extracts of black garlic enforces anti-tumor activity with a 50% cure rate of BALB/c mouse fibrosarcoma since it enhances the cellular immunity by raising the activity of natural killer cells (Sasaki et al., 2007). Black garlic also has favourable hepatoprotective, nephroprotective, hypolipidemic, and antiobesity effects, but no hypoglycemic effects (Jung et al., 2011). A black garlic formulation containing 10% black garlic extract is effective in protecting skin from UVB photodamage (Kim et al., 2012).

In the present study, black garlic with high freeradical-scavenging ability was prepared, and its antihypertensive effect was investigated in vivo using spontaneously hypertensive rats (SHRs) as the test animals. Total antioxidant capacity (T-AOC) and MDA content in both plasma and PVN of the rats were measured to explore underlying biochemical mechanism of the antihypertensive effect.

Materials and Methods Garlics Fresh garlic (Allium sativum) with purple skin, produced in Henan province, was obtained from a local market in Nanjing, China. Black garlic was prepared by maintaining the fresh garlic in an incubator with a temperature of 65℃ and a relative humidity of 70% for 30 days (Miao, 2006).

The content of main compounds and the free-radicalscavenging ability of fresh and black garlics are shown

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Research in Health and Nutrition (RHN) Volume 2, 2014

in Table 1. Moisture was measured by drying a 5 g garlic sample at 105℃ for 24 h. Alliin and S-allyl-Lcrysteine were measured using a high performance liquid chromatograph (HPLC) system (Ichikawa et al., 2006; Kodera et al., 2002). Total polyphenols were measured with the Folin–Ciocalteu method (Li et al., 2009; Kim et al., 2012). Ajoene was measured using a LC-MS system and its amount was expressed by the peak area of LC chromatogram (Iberl et al., 1990; Fu, 2006). The free-radical-scavenging ability was determined with the ABTS method (Re et al., 1999; Zulueta et al., 2008). The content of main compounds and the free-radical-scavenging ability were expressed on the basis of dry garlic. The black garlic contained only little alliin and other volatile organic sulfides, so that it did not have unacceptable odor and sharp taste of fresh garlic. Moreover, the black garlic had a freeradical-scavenging ability of 63.4 g-Trolox/kg, which was about 9 times higher than that of fresh garlic. The black garlic’s high ability of free-radical-scavenging resulted from remarkable increases in the content of bioactive compounds, i.e. total polyphenols, ajoene and S-allyl-L-cysteine.

TABLE 1 THE CONTENT OF MAIN COMPOUNDS AND THE FREE-RADICALSCAVENGING ABILITY OF GARLICS

Garlic

Fresh garlic

Black garlic

Moisture (%,w.b.)

66.2

50.3

Alliin (%,d.b.)

3.8

0.7

Total Poly- phenols (g/kg)

0.8

Ajoene (mAU•s/ kg)

0.45×10

S-allyl-L- cysteine (mg/kg) Free-radical- scavenging ability (g-Trolox/kg)

21.0

106.1

7.1

63.4

4.7 6

4.44×106

Preparation of Animal Model Normotensive Wistar rats (male: n=18, female: n=18) and spontaneously hypertensive rats (SHRs, male: n=18, female: n=18), weighing approximately 150-260 g (20-week-old), were supplied by Shanghai Laboratory Animal Center of Chinese Academy of Sciences. The male Wistar rats, female Wistar rats, male SHRs and female SHRs were housed separately in polycarbonate boxes, and had free access to food and tap water. The animal room was maintained at approximately 22°C and 50% humidity with a 12-h light/dark cycle. All of animal experimental design was approved by the Institutional Animal Care and Ethical Committee of Nanjing Medical University.

Rat Grouping and Garlic Administration The Wistar rats, as well as SHRs, were randomly

Research in Health and Nutrition (RHN) Volume 2 Issue 1, January 2014

divided into three groups (6 male and 6 female rats in each group), i.e. (1) Group VI: rats receiving the administration of deionized water; (2) Group FG: rats receiving the administration of fresh garlic; (3) Group BG: rats receiving the administration of black garlic. In addition, the data obtained from Wistar rats and SHRs before the treatment was used as the control values, respectively.

The flesh portions of fresh and black garlics were mashed to puree in a mortar. 100 mL of garlic homogenate with a concentration of 16% (w/v) were prepared by mixing 47.4 g of the fresh garlic puree (with a moisture content of 66.2%,w.b.) or 32.2 g of the black garlic puree (with a moisture content of 50.3%,w.b.) with deionized water, and stored at -10°C. The frozen garlic homogenates were thawed by tap water before being used.

The deionized water, fresh garlic homogenate and black garlic homogenate were administrated to rats once a day at 10:00 AM for 14 days, using an injector equipped with an intragastric administration needle.

The administration volume was 2 mL each time for a rat weighing 200-260 g, and 1.5 mL each time for a rat weighing 150-200 g. The average dose of garlic in dry matter was 1.38 g/kg rat. It corresponded to that an adult weighing 70 kg receives 10 g of dry garlic, based on the assumption that the dose for adults is one tenth of that for rats (Chen, 2006).

Measurement of the Body Weight, Blood Pressure and Heart Rate of Rats Body weight was measured every three days using an electronic balance. Systolic blood pressure and heart rate were measured for the awake rats at 24 h before the first administration and 24 h after the last administration, with the caudal artery method (Li, 2002). The testing apparatus consisted of a tail sleeve, a pressure sensor, a bridge amplifier (QUAD bridge, AD Instruments, Australia) and a PowerLab data analytical processing system (8SP, AD Instruments, Australia). The measurement for each rat was in triplicate, and their average was used.

Biochemical Determinations for the Plasma and Hypothalamic PVN of Rats The T-AOC and MDA content in plasma and PVN of rats were determined. In order to determine T-AOC and MDA content in plasma, 0.2 mL of blood was collected from each rat by tail venipuncture under the anesthetized condition with intraperitoneale (i. p.)

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injection of chloral hydrate (400 mg/kg), at 24 h before the first administration and 24 h after the last administration. Then, the blood samples were centrifuged at 3000 r/min and 4°C for 10 min. The plasma (supernatant) was separated and stored at 80°C until being assayed. In order to determine TAOC and MDA content in PVN, all rats were sacrificed under the anesthetized condition with chloral hydrate (400 mg/kg, i. p.) at 24 h after the last administration. The brains were taken out quickly, and coronally cut in 500 μm thick slices using a cryostat. PVN (0.84 mm anterior and 0.60 mm posterior to the bregma) regions in the frozen slices were micro-dissected using a 26-gauge stainless steel tubing, and then stored at -80°C until being assayed.

Before the assay, the frozen plasma was homogenized and thawed in ice water. The frozen PVN was homogenized by sonication with a pH 7.5 homogenization buffer (containing 140 mM NaCl, 5 mM KCl, 0.8 mM MgC12, l.8 mM CaC12, l mM Na2HPO4, 25 mM HEPES, l% glucose) in ice water, followed by centrifugation at 3000 r/min and 4°C for 10 min to attain the supernatant for the assay.

For the plasma and PVN, the T-AOC was determined with the FRAP (ferric reducing ability of plasma) method (Benzie and Strain, 1996), and the MDA content was determined with the TBA (thiobarbituric acid) method (Placer et al., 1966). The soluble protein contained in PVN was measured with the Bradford method (Bradford, 1976). Commercial assay kits (Nanjing Jiancheng Bioengineer Institute, China) were used for the biochemical determination. T-AOC and MDA content in the plasma was expressed as U/mL and nmol/mL, while T-AOC and MDA content in the PVN was expressed as U/mg-protein and nmol/mgprotein, respectively. Data were obtained from each group of twelve rats throughout the experiments.

Statistical Analysis The group data were expressed as mean ± SD (standard deviation), and analyzed by one-way analysis of variance (ANOVA) using State 9.2 software (STATA Corporation, USA). Differences between the means were considered to be significant when p<0.05.

Results and Discussion Body Weight and Heart Rate of Rats Both the Wistar rats and the SHRs originally had a body weight of 217±30 g. The body weight did not

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Research in Health and Nutrition (RHN) Volume 2, 2014

change significantly during the administration of deionized water, fresh and black garlics (Table 2).

The heart rate of rats before and after the administration is shown in Fig. 1. It was 352±26 times/min in the Wistar rats and 393±33 times/min in the SHRs before the administration. The heart rate in both the Wistar rats and the SHRs did not change significantly after the administration. In general, the heart rate of adult Wistar rats ranges from 250 times/min to 400 times/min (Zhang and Sannajust, 2000), while that of adult SHRs ranges from 350 times/min to 400 times/min (Li, 2002). It was confirmed that both the Wistar rats and the SHRs used in the present study had a normal heart rate during the administration.

blood pressure of 115±11 mm Hg and 185±12 mm Hg, respectively. After the administration, the blood pressures in Groups VI, FG and BG of Wistar rats, and in Group VI of SHRs were almost the same as those before the administration, respectively. However, the blood pressure of SHRs declined significantly to 164±11 mm Hg (with a decline rate of 11.4% in average) in Group FG, and to 121±10 mm Hg (with a decline rate of 34.6% in average) in Group BG. The difference between the blood pressures in Group BG and Group FG of SHRs was significant (p<0.05). It should be noted that the high blood pressure of SHRs was lowered to that of Wistar rat by administrating of the black garlic.

The black garlic had more antihypertensive effect on SHRs than the fresh garlic.

TABLE 2 BODY WEIGHT (MEAN±SD) OF RATS DURING THE

300

Wistar rats

Time (d)

SHRs

VI

FG

BG

VI

FG

BG

0

221±11

217±18

213±24

208±31

229±44

214±54

1

220±10

217±16

214±26

208±36

230±43

214±58

4

219±11

214±18

211±25

204±27

225±46

215±48

7

217±12

211±22

207±23

206±23

229±48

218±47

11

221±12

211±21

209±26

212±25

228±43

219±48

14

221±14

214±17

210±24

211±23

227±43

219±49

VI, FG and BG: rats receiving the administration of deionized water, fresh garlic and black garlic, respectively.

Heart rate（times/min）

550

450

350

250 VI

FG

BG

CR

VI

FG

BG

150 Wistar rats

SHRs

FIG. 1 HEART RATE (MEAN±SD) OF RATS BEFORE AND AFTER THE ADMINISTRATION CR: control before the administration; VI: after the administration of deionized water; FG: after the administration of fresh garlic; BG: after the administration of black garlic; ++: p<0.05 vs. CR of Wistar rats

Lowering of High Blood Pressure by Black Garlic The blood pressure of rats before and after the administration is shown in Fig. 2. Before the administration, the Wistar rats and the SHRs had a

8

250 ££ $$ ££ $$ &&

++

200 150 100 CR

VI FG

BG

CR

VI

FG

BG

50 Wistar rats

SHRs

FIG. 2 BLOOD PRESSURE (MEAN±SD) OF RATS BEFORE AND AFTER THE ADMINISTRATION CR: control before the administration; VI: after the administration of deionized water; FG: after the administration of fresh garlic; BG: after the administration of black garlic; ++: P<0.05 vs. CR of Wistar rats; ££: P<0.05 vs. CR of SHRs; $$: P<0.05 vs. VI of SHRs; &&: P<0.05 vs. FG of SHRs.

++

CR

Blood pressure（mmHg）

ADMINISTRATION (G)

It has been reported that adult normotensive Wistar rats have a blood pressure of about 120 mm Hg, while adult SHRs have a blood pressure higher than 170 mm Hg (Shi, 1989). An agent is considered to be antihypertensive when the high blood pressure of hypertensive rats declines to a normal blood pressure level or by an amount of more than 20 mm Hg after its administration (Chen, 2006).

Change of Antioxidative Activity in Plasma Figure 3 shows the plasma T-AOC and MDA content of rats before and after the administration. Before the administration, the SHRs had a lower plasma T-AOC of 4.0±1.0 U/mL and a higher plasma MDA content of 10.2±2.2 nmol/mL, in comparison with those of Wistar rats (plasma T-AOC: 4.9±0.7 U/mL, plasma MDA content: 6.0±0.8 nmol/mL), respectively (p<0.05).

Research in Health and Nutrition (RHN) Volume 2 Issue 1, January 2014

After the administration, the plasma T-AOC in Group BG of SHRs increased to 5.4±1.1 U/mL (with an increase rate of 28.6% in average) (Fig. 3A).

Correspondingly, the plasma MDA content in Group BG of SHRs decreased to 7.9±0.7 nmol/mL (with a decrease rate of 22.5% in average) (Fig. 3B). In contrast to Group BG of SHRs, other groups did not have significant change in the levels of plasma T-AOC and plasma MDA content. It was indicated that the antioxidative activity of plasma in SHRs was increased by the administration of black garlic.

10

A

££ $$ &&

6

++

4

The PVN MDA content levels were roughly the same at 3.7±0.6 nmol/mg-protein in Groups VI, FG and BG of Wistar rats. Group VI of SHRs had a PVN MDA content of 8.2±1.5 nmol/mg-protein, which was much higher than that in Group VI of Wistar rats (p<0.05).

The high PVN MDA content in Group VI of SHRs decreased to 6.0±1.0 nmol/mg-protein (with a decrease rate of 26.8% in average) in Group FG, and to 3.9±1.2 nmol/mg-protein (with a decrease rate of 52.4% in average) in Group BG (Fig. 4B). The difference between PVN MDA content levels in Group BG and Group FG of SHRs was significant (p<0.05). It should be noted that the PVN MDA content of SHRs was decreased to that of Wistar rats by the administration of black garlic.

12

2 VI

CR

FG

BG

CR

VI

FG

BG

0 Wistar rats

SHRs

18

B MDA（nmol/mL）

4A). The differences between PVN T-AOC levels in Group BG of SHRs and other groups were significant (p<0.05).

++

££ $$ &&

12

A T-AOC（U/mg-protein）

T-AOC（U/mL）

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$$ &&

8

4 VI

FG

BG

VI

FG

BG

0 Wistar rats

SHRs

6 12 VI

FG

BG

CR

VI

FG

BG

0 Wistar rats

SHRs

FIG. 3 PLASMA T-SOD (A) AND MDA CONTENT (B) (MEAN±SD) OF RATS BEFORE AND AFTER THE ADMINISTRATION CR: control before the administration; VI: after the administration of deionized water; FG: after the administration of fresh garlic; BG: after the administration of black garlic; ++: P<0.05 vs. CR of Wistar rats; ££: P<0.05 vs. CR of SHRs; $$: P<0.05 vs. VI of SHRs; &&: P<0.05 vs. FG of SHRs.

Change of Antioxidative Activity in PVN Figure 4 shows the PVN T-AOC and MDA content of rats after the administration. The PVN T-AOC was almost the same at 4.4±0.7 U/mg-protein in Groups VI, FG and BG of Wistar rats, and Group VI of SHRs. It increased to 4.8±1.0 U/mg-protein in Group FG of SHRs, and to 7.2±1.6 U/mg-protein (with an increase rate of 63.6% in average) in Group BG of SHRs (Fig.

MDA（nmol/mg-protein）

B CR

##

8

$$ $$ &&

4 VI

FG

BG

VI

FG

BG

0 Wistar rats

SHRs

FIG. 4 PVN T-SOD (A) AND MDA CONTENT (B) (MEAN±SD) OF RATS AFTER THE ADMINISTRATION VI: administration of deionized water; FG: administration of fresh garlic; BG: administration of black garlic; ##: P<0.05 vs. VI of Wistar rats; $$: P<0.05 vs. VI of SHRs; &&: P<0.05 vs. FG of SHRs.

Mechanisms for the Antihypertensive Effect of Black Garlic OFRs are metabolites of oxygen in the human body,

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including superoxide free radicals (O2-), superoxide anions (O2-), hydroxyl free radicals (OH), hydroxyl anions (OH-), hydrogen peroxide (H2O2), singlet oxygen (1O2), lipidic superoxide free radicals (·ROO) and alcoxyl free radicals (·RO). In addition, brain is an organ with the highest metabolism rate in the body.

Most of the energy required for brain activity is provided by aerobic metabolism. Although the mass of brain tissue is only 2% of the body, the brain consumes 20% of total oxygen for the body. Because 50% of the consumed oxygen is reduced to OFRs, the brain tissue is subjected to more attack of OFRs than the other organs (Bosnjak et al., 2003).

Based on the experimental results of present study and the medical evidences reported previously (Han et al., 2007; Grassi et al., 1999; Han et al., 2011; Koga et al., 2008), it may be presumed that the existence of excessive OFRs in the plasma and PVN of SHRs is the main reason for high blood pressure. When the black garlic is given to SHRs, its bioactive compounds, such as polyphenols, ajoene and S-allyl-L-crysteine, pass through the blood brain barrier into PVN to scavenge the excessive OFRs, which results in the decline of high blood pressure via abolishing the potentiation of Ang II and CSAR. Therefore, the black garlic has great potential to prevent the pathogenesis of hypertension, as a health-oriented food product.

In addition to the scavenging of excessive OFRs, inhibition of plasma angiotensin-converting enzyme (ACE) may also contribute to the antihypertensive effect of black garlic (Quiñones et al., 2013). ACE is an important factor controlling vascular tone by producing extremely potent Ang II. Pomegranate juice and several polyphenols such as procyanidins have been demonstrated to inhibit ACE activity (Aviram and Dornfeld, 2001; Actis-Goretta et al., 2003; Kivimäki et al., 2013). Further investigations are needed.

Conclusions (1) The administration of black garlic for 14 days significantly lowered the blood pressure of SHRs to 121±10 mm Hg (with a decline rate of 34.6% in average), while it did not affect the normal blood pressure of Wistar rats. The black garlic had more significant antihypertensive effect on SHRs than fresh garlic.

(2) In the plasma of SHRs receiving the black garlic, the T-AOC increased from 4.2±1.0 U/mL to 5.4±1.1

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Research in Health and Nutrition (RHN) Volume 2, 2014

U/mL (with an increase rate of 28.6% in average), and the MDA content decreased correspondingly from 10.2±2.2 nmol/mL to 7.9±0.7 nmol/mL (with a decrease rate of 22.5 % in average).

(3) In the PVN of SHRs receiving the black garlic, the T-AOC increased from 4.4±0.7 U/mg-protein to 7.2±1.6 U/mg-protein (with an increase rate of 63.6% in average), and the MDA content decreased correspondingly from 8.2±1.5 nmol/mg-protein to 3.9±1.2 nmol/mg-protein (with a decrease rate of 52.4% in average).

(4) It was indicated that the black garlic exerted a potential antihypertensive effect via scavenging excessive OFRs in the plasma and PVN of SHRs. ACKNOWLEDGEMENT

The present study was financially supported by the National Basic Research Program of China (973 Program, 2009CB724700), and the National Nature Science Fund of China (71173103/G0310). The authors thank professor Ling Chen of the School of Basic Medical Science, Nanjing Medical University, China for giving us advice and chance to use laboratory for the animal experimentation. The authors declare no conflict of interest.

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