Antihypertensive Effects of Artemisia scoparia Waldst in Spontaneously Hypertensive Rats and Identification of Angiotensin I Converting Enzyme Inhibitors

We investigated the antihypertensive effects of Artemisia scoparia (AS) in spontaneously hypertensive rats (SHR). The rats were fed diets containing 2% (w/w) hot water extracts of AS aerial parts for 6 weeks. The AS group had significantly lower systolic and diastolic blood pressure levels than the control group. The AS group also had lower angiotensin I converting enzyme (ACE) activity and angiotensin II content in serum compared to the control group. The AS group showed higher vascular endothelial growth factor and lower ras homolog gene family member A expression levels in kidney compared to the control group. The AS group had significantly lower levels of plasma lipid oxidation and protein carbonyls than the control group. One new and six known compounds were isolated from AS by guided purification. The new compound was determined to be 4′-O-β-d-glucopyranoyl (E)-4-hydroxy-3-methylbut-2-enyl benzoate, based on its nuclear magnetic resonance and electrospray ionization-mass spectroscopy data.


Introduction
High blood pressure is a risk factor for stroke, coronary heart disease and renal vascular disease [1,2]. The control of blood pressure has received considerable attention for public health reasons. Many studies have indicated that foods such as fruits and vegetables have blood pressure-lowering abilities [3][4][5]. Therefore, the clinical importance of fruits and vegetables has received considerable attention.
Halophytes grow widely in the saline environments of coastal sand dunes, salt marshes, mud flats, and inland deserts [6]. They are able to thrive under high salt conditions, although their growth patterns are slightly different depending on the saline concentration. Many studies diet and, thereafter, were significantly lower than that of the control group. At 6 weeks of diet, systolic pressure levels of AS and ASG groups were about 15 mmHg lower than that of the control rats ( Figure 1A). The blood pressure of ASG diet group, which was prepared with the addition of garlic to AS, was similar to AS diet group during the diet. This trend in systolic blood pressure was similar to the results of diastolic blood pressure ( Figure 1B). The renin-angiotensin-aldosterone system (RAAS) plays an important role in controlling blood pressure in the body [30]. In this system, ACE converts angiotensin I to angiotensin II that increases blood pressure by causing the constriction of blood vessels [31,32]. Angiotensin II also promotes oxidative stress, inflammation and fibrosis [33]. The ACE activities and angiotensin II contents in the blood of the rats fed AS and ASG are shown in Figure 2. The AS and ASG groups exhibited significantly lower ACE activities and angiotensin II contents in the blood than the control rats (Figure 2A,B). However, significant differences in the ACE activities and angiotensin II contents between AS and ASG groups were not observed. These results were consistent with the results of systolic and diastolic blood pressure levels. The reduced ACE activity could be due to the reduced expression of ACE protein or due to the reduced catalytic activity by inhibitors. We analyzed ACE protein expression level in the kidney using Molecules 2015, 20 4 immunoblotting. As shown in Figure 2C, there was no significant difference in protein expression levels of ACE among the control, AS, and ASG groups, indicating that the reduced ACE activities of the AS and ASG groups were probably due to the reduced catalytic activity. In vitro high ACE-inhibitory activities in AS and ASG, lower ACE activities and angiotensin II contents in the blood of the rats fed AS and ASG, no significant difference in protein expression levels of ACE among the groups, and lower systolic and diastolic blood pressures of the rats fed AS and ASG lead to a possibility that ACE inhibitors in AS and ASG inhibit ACE activity of rats, which results in the reduction of angiotensin II production and blood pressure of the rats fed AS and ASG.

Figure 2.
Serum ACE activity (A); serum angiotensin II content (B); ACE expression level of kidney (C); and plasma nitric oxide content (D) in rats fed diets of AS and ASG for 6 weeks. Values are expressed as mean ± SD (n = 8). * p < 0.05 and ** p < 0.01, compared with control (Tukey's test).
Endogenous nitric oxide (NO), which is released by endothelial cells of blood vessel through NO synthesis, is a major vasodilator, possibly resulting in lowered blood pressure [34]. No significant difference for the plasma NO concentration among the groups was observed ( Figure 2D), although there was a trend that plasma NO concentrations in the rats fed AS and ASG appeared to be slightly higher than that of the control rats. Vascular endothelial growth factor (VEGF) increases NO production by stimulation of endothelial NO synthase (eNOS) and leads to vasodilation [35,36]. In addition, NO suppresses the activation of ras homolog gene family member A (RhoA) that plays a role in regulating vasoconstriction The renin-angiotensin-aldosterone system (RAAS) plays an important role in controlling blood pressure in the body [30]. In this system, ACE converts angiotensin I to angiotensin II that increases blood pressure by causing the constriction of blood vessels [31,32]. Angiotensin II also promotes oxidative stress, inflammation and fibrosis [33]. The ACE activities and angiotensin II contents in the blood of the rats fed AS and ASG are shown in Figure 2. The AS and ASG groups exhibited significantly lower ACE activities and angiotensin II contents in the blood than the control rats (Figure 2A,B). However, significant differences in the ACE activities and angiotensin II contents between AS and ASG groups were not observed. These results were consistent with the results of systolic and diastolic blood pressure levels. The reduced ACE activity could be due to the reduced expression of ACE protein or due to the reduced catalytic activity by inhibitors. We analyzed ACE protein expression level in the kidney using immunoblotting. As shown in Figure 2C, there was no significant difference in protein expression levels of ACE among the control, AS, and ASG groups, indicating that the reduced ACE activities of the AS and ASG groups were probably due to the reduced catalytic activity. In vitro high ACE-inhibitory activities in AS and ASG, lower ACE activities and angiotensin II contents in the blood of the rats fed AS and ASG, no significant difference in protein expression levels of ACE among the groups, and lower systolic and diastolic blood pressures of the rats fed AS and ASG lead to a possibility that ACE inhibitors in AS and ASG inhibit ACE activity of rats, which results in the reduction of angiotensin II production and blood pressure of the rats fed AS and ASG.
Molecules 2015, 20 5 via inhibition of dephosphorylation by myosin phosphatase [37]. Angiotensin II promotes VEGF secretion and the activation of RhoA [38,39]. However, in case of VEGF, relation between VEGF expression and angiotensin II is somehow complicated because ACE inhibition was also reported to induce the expression of VEGF via bradykinin that is increased by ACE inhibition [40,41]. The rats fed AS and ASG had higher levels of VEGF expression and lower levels of RhoA expression in the kidney and skeletal muscle compared to the control rats, although their expression levels were slightly different in each tissue ( Figure 3). The AS more efficiently enhanced VEGF expression and suppressed RhoA expression in the kidney. In contrast, the ASG was more effective in the skeletal muscle of SHR. However, there were no statistically significant differences among the AS and ASG groups. These results indicate that AS and ASG reduce blood pressure in SHR via the enhancement of VEGF expression and the suppression of RhoA expression. In this study, we demonstrated that AS and ASG inhibited ACE activity, but did not increase NO generation. However, no significant difference in NO content and inhibition of ACE activity between AS and ASG was observed, indicating that additional amount of garlic in AS could not synergistically Endogenous nitric oxide (NO), which is released by endothelial cells of blood vessel through NO synthesis, is a major vasodilator, possibly resulting in lowered blood pressure [34]. No significant difference for the plasma NO concentration among the groups was observed ( Figure 2D), although there was a trend that plasma NO concentrations in the rats fed AS and ASG appeared to be slightly higher than that of the control rats. Vascular endothelial growth factor (VEGF) increases NO production by stimulation of endothelial NO synthase (eNOS) and leads to vasodilation [35,36].
In addition, NO suppresses the activation of ras homolog gene family member A (RhoA) that plays a role in regulating vasoconstriction via inhibition of dephosphorylation by myosin phosphatase [37]. Angiotensin II promotes VEGF secretion and the activation of RhoA [38,39]. However, in case of VEGF, relation between VEGF expression and angiotensin II is somehow complicated because ACE inhibition was also reported to induce the expression of VEGF via bradykinin that is increased by ACE inhibition [40,41]. The rats fed AS and ASG had higher levels of VEGF expression and lower levels of RhoA expression in the kidney and skeletal muscle compared to the control rats, although their expression levels were slightly different in each tissue ( Figure 3). The AS more efficiently enhanced VEGF expression and suppressed RhoA expression in the kidney. In contrast, the ASG was more effective in the skeletal muscle of SHR. However, there were no statistically significant differences among the AS and ASG groups. These results indicate that AS and ASG reduce blood pressure in SHR via the enhancement of VEGF expression and the suppression of RhoA expression.
In this study, we demonstrated that AS and ASG inhibited ACE activity, but did not increase NO generation. However, no significant difference in NO content and inhibition of ACE activity between AS and ASG was observed, indicating that additional amount of garlic in AS could not synergistically induce an antihypertensive effect. Reduction of blood pressure by AS and ASG could be due to ACE inhibition, decreased oxidative stress, enhanced endothelial function, or direct action on the vascular smooth muscle. Our study demonstrated a possibility that ACE inhibitors in AS played a role in reduction of blood pressure as intake of AS reduced ACE activity and angiotensin II content, but did not suppress ACE expression. There could be another possible mechanism that ACE inhibitors reduce blood pressure. It has been reported that endothelial dysfunction is an early feature of angiotensin II-mediated hypertension [41]. Therefore, it may be possible that reduced angiotensin II content by ACE inhibitors of AS could improve endothelial function and played a role in reduction of blood pressure.

Preventive Effects of AS on Oxidative Stress in SHR
Oxidative stress is increased in high blood pressure in response to vessel stimulation [42]. Breakdown of redox balance produces excessive reactive oxygen species and lead to oxidize cellular components such as DNA, RNA, proteins, and lipids. Malondialdehyde (MDA) and protein carbonyls (PCO) are important markers of lipid peroxidation and protein oxidation, respectively [43,44]. In addition, the increase of oxidative stress induces inflammation by activating NF-κB via tumor necrosis factor alpha (TNF-α) [45]. In this study, we found that AS and ASG more efficiently suppressed the increasing levels of oxidative stress-and inflammation-related biomarkers. That is, the rats fed with AS had lower levels of MDA in the plasma and PCO in the liver ( Figure 4A,B) than the control rats. In addition, the rats fed with the AS had higher total glutathione content in the liver than the control rats ( Figure 4C,D). The rats fed with the AS had lower levels of TNF-α in plasma and NF-κB expression in kidney than the control rats ( Figure 4E,F). These results indicate that AS and ASG reduce oxidative stress and inflammation in SHR. Garlic has been reported to inhibit hypertension by suppressing NF-κB expression and lowering ROS concentration [46]. In particular, reduction of oxidative stress and inflammation of ASG was more effective than those of AS, although they were no statistically significant differences between the AS and ASG groups. Therefore, it seemed apparent that reduced oxidative stress by AS and ASG at least partially played a role in lowering of blood pressure.
We found that AS and ASG reduce blood pressure in SHR via the inhibition of plasma ACE activity, the enhancement of VEGF expression, and the suppression of RhoA expression. However, antihypertensive effects between AS and ASG had no significant difference, suggesting that mainly AS has contributed to the antihypertensive effect of ASG. In particular, AS may reduce blood pressure via an inhibition of ACE, one of the important factors in regulation of blood pressure. In addition, ACE inhibitors have been reported to show various preventive effects for heart failure, myocardial infarction, and diabetic nephropathy [37][38][39][40][41][42][43][44][45][46][47][48][49]. Therefore, we attempted the isolation and identification of the ACE inhibitors from AS aerial parts with guided purification of ACE inhibitory assay. The hot water extracts of the AS aerial part were solvent-fractionated to obtain n-hexane, chloroform (CHCl 3 ), ethyl acetate (EtOAc), water-saturated n-butanol (BuOH), and H 2 O fractions. The ACE inhibitory activities of these fractions were evaluated by comparing their activity against ACE extracts of rabbit lung. The CHCl 3 and EtOAc fractions showed higher ACE inhibitory activity than the other fractions. The n-hexane and BuOH fractions showed very low ACE inhibitory activity, similar to the H 2 O fraction. Therefore, we pursued purification of ACE inhibitors from the CHCl 3 and EtOAc fractions.
Seven ACE inhibitors 1-7 were isolated from the CHCl3 and EtOAc fractions of AS aerial parts by various column chromatographic procedures with the guided assay of ACE inhibition. The structures of the isolated compounds were determined by NMR and ESI-MS analyses. Among them, six known compounds previously isolated from AS aerial parts were identified as [50], salicylic acid (4) [51], 3,4-dihydroxybenzoic acid (5) [52], caffeic acid (6) [52], and isofraxidin 7-O-β-D-glucopyranoside (7) ( Figure 5). Compounds 4-6 were identified via comparison of their NMR and MS spectroscopic data with values previously reported in the relevant literature. Compounds 1 and 7 were identified by 1Dand 2D-(HSQC, HMBC, 1 H-1 H COSY, and NOE) NMR and MS spectroscopic data. These compounds have been identified from Michelia hedyosperma [53] and Apium graveolens [54], respectively. Seven ACE inhibitors 1-7 were isolated from the CHCl 3 and EtOAc fractions of AS aerial parts by various column chromatographic procedures with the guided assay of ACE inhibition. The structures of the isolated compounds were determined by NMR and ESI-MS analyses. Among them, six known compounds previously isolated from AS aerial parts were identified as eugenol 2 , salicylic acid (4) [51], 3,4-dihydroxybenzoic acid (5) [52], caffeic acid (6) [52], and isofraxidin 7-O-β-D-glucopyranoside (7) ( Figure 5). Compounds 4-6 were identified via comparison of their NMR and MS spectroscopic data with values previously reported in the relevant literature. Compounds 1 and 7 were identified by 1D-and 2D-(HSQC, HMBC, 1 H-1 H COSY, and NOE) NMR and MS spectroscopic data. These compounds have been identified from Michelia hedyosperma [53] and Apium graveolens [54], respectively.

ACE Inhibitory Activity of the Isolated Compounds
The ACE inhibitory activities of the identified compounds 1-7 at the same concentration (1 mM) were evaluated using ACE crude extract from rabbit lung. Quercetin (Q), which is widely distributed in nature and has ACE inhibitory activity [55,56], was used as a positive control. The ACE inhibitory activities of the identified compounds 1-7 are shown in Figure 6. As expected, these compounds exhibited ACE inhibitory activities (28.3%˘1.46%-55.8%˘1.54%), although their activities were very low in comparison to that (49.3%˘2.6%) of captopril (1 nM), which is used as a drug. However, the ACE inhibitory activities of the isolated compounds 1-7 were higher and similar when compared to that of Q. Compounds 1, 6, and 7 had higher ACE inhibitory activity than other compounds. In addition, the new compound 2 had relatively high ACE inhibitory activity at the same concentration (1 mM) under the reaction conditions used. Several phenolic compounds 5-7 identified in this study have been reported to show ACE inhibitory activity [54,[56][57][58]. These observations indicated that the phenolic compounds identified have in vitro ACE inhibitory activities. In addition, phenolic compounds, which are widely distributed in various plants, have been reported to show anti-oxidative and anti-inflammatory activities [59,60]. Therefore, the phenolic compounds identified in this study may be responsible for anti-oxidative activity of AS.
to show ACE inhibitory activity [54,[56][57][58]. These observations indicated that the phenolic compounds identified have in vitro ACE inhibitory activities. In addition, phenolic compounds, which are widely distributed in various plants, have been reported to show anti-oxidative and anti-inflammatory activities [59,60]. Therefore, the phenolic compounds identified in this study may be responsible for anti-oxidative activity of AS.

Materials
The aerial parts of AS were collected May, 2013 in Shinan County, South Korea. They were washed with water, immediately steamed for 2 min, and dried at 45˝C for 2 days. The dried samples were stored at´20˝C until used in the experiments. Garlic was collected June, 2013 in Shinan County, South Korea. Voucher samples (AS, MNU AS 20130601; garlic, MNU G 20130602) were deposited in the herbarium of the laboratory. The dried AS aerial parts (4 kg) were extracted with distilled water (40 L) at 121˝C for 30 min. After filtration with No. 2 filter paper (Whatman, Maidenstone, UK), the residues were repeatedly extracted with distilled water (20 L), as described above, and then filtered (No. 2 filter paper) again. The extracted solutions were combined and were freeze-dried to obtain AS water extract powder. Additionally, to obtain the water extract powder of AS and garlic mixture, the dried AS aerial part and fresh garlic (ASG) were mixed in the proportion of 3.6:0.4 (w/w). Extraction, filtration, and freeze-drying of the ASG mixture were performed by the same extraction procedures as described in the preparation of AS powder.

Animal Studies
Male SHR rats (80˘10 g, 4 week-old) were obtained from Central Lab. Animal Ltd. (Seoul, Korea). All rats were housed in room temperature (25˘1˝C) with humidity (55%˘5%) and light cycle (12 h: 6:00 to 18:00). Food and water were available ad libitum. All rats were fed the standard rodent diet (Harlan Rodent diet, 2018S) for 3 weeks. All experimental procedures were approved by the Ethics Committee of Mokpo National University (no. MNU IACUC-2014-011). The rats used in animal studies were randomly divided into three groups: control group that was fed AIN-93G diet; AS group that was fed the diet containing 2% (w/w) AS water extract; ASG group that was fed the diet containing 2% (w/w) ASG water extract. The AS and ASG diets were prepared from 2% (w/w) AS and ASG water extracts, respectively, based on manufacture protocol of AIN-93G purified diet for rodents. The rats were fed these experimental diets for 6 weeks. Food and water intake were recorded during the AS and ASG diets.

Blood Pressure Measurement
The systolic and diastolic blood pressure levels of rats during the special diets were measured by tail-cuff method using Heater Scanner LE 5650/6 and Storage Pressure Meter LE 5002 (Panlab, S.L., Barcelona, Spain). The rats were placed on a chamber preheated at 37˝C. A clamp type transducers and cuff were set up on the tails of rats. Systolic and diastolic blood pressures of the rats were measured at 37˝C for 10 min. The rats were trained in an acrylic restrainer every day for one week before measuring blood pressure. Systolic and diastolic blood pressures were measured when rats were still unperturbed in the chamber during the inflation-deflation cycles.

Determination of Plasma ACE Activity
ACE activity in serum was measured by using HHL as a substrate [61]. The serum samples (50 µL) were mixed with 0.1 M sodium borate buffer (pH 8.3, 100 µL) containing 300 mM NaCl and 5 mM HHL (50 µL). The mixture was incubated at 37˝C for 45 min, and then the enzymatic reaction was stopped with the addition of 2 N HCl solution (200 µL). The reaction mixture was separated with ethyl acetate (2.0 mL). The upper layer (ethyl acetate layer, 1.5 mL) was concentrated and dissolved with 1 M NaCl solution (1 mL). The absorbance was measured at 228 nm and the hippuric acid content in the serum was calculated using calibration curve of hippuric acid standard.

Determination of Serum Angiotensin II content
Angiotensin II content in serum was determined by using Rat angiotensin II EIA kit (RayBiotech, Inc., Norcross, GA, USA).

Determination of NO Content in Plasma
The NO content in plasma was measured according to Griess assay [62]. The plasma samples (0.1 mL) were mixed with zinc sulfate (50 µL, final concentration 15 mg/mL) and then centrifuged at 1000ˆg for 10 min. The supernatant (100 µL) was mixed with vanadium (III) chloride (100 µL, final concentration 8 mg/mL) and added to 2% sulfanilamide in 5% H 3 PO 4 solution (50 µL) and 0.1% N-(1-naphthyl)ethylenediamine (50 µL). After incubation at 37˝C for 30 min, the absorbance was measured at 540 nm. The NO content in the plasma was calculated using standard curve of sodium nitrite.

Western Blotting
The tissues (0.2 g) were mixed with a lysis buffer containing 1% Triton X-100, 50 mM HEPES (pH 7.4), 100 mM sodium pyrophosphate, 10 mM NF, 10 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, pepstatin (1 µg/mL), leupeptin (1 µg/mL), 10 mM Na 3 VO 4 , and aprotinin (0.1 mg/mL). After protein extraction at 4˝C for 2 h, the homogenate was centrifuged (15,000ˆg, 4˝C) for 40 min. The protein content in the supernatant was determined by using Bradford protein assay [63]. The protein solution (0.1 mL) combined with 0.1 mL of tricine buffer (BioRad, Hercules, CA, USA) was denatured for 5 min at 95˝C. The denatured proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The proteins were transferred electrophoretically to polyvinylidene fluoride membranes (Pall Corporation, Pensacola, FL, USA). After blocked with a blocking buffer containing 3% bovine serum albumin (BSA) for 1 h, the membrane was incubated with primary antibodies (VEGF, RhoA, NF-κB p65) overnight at room temperature. After washing, the membrane was incubated with HRP-conjugated goat anti-rabbit immunoglobin G for 1 h and then the bands were visualized by chemiluminescence (ECL) reagent. Visualized protein bands were photographed and their intensities were analyzed by ImageJ software (NIH, Bethesda, MD, USA).

Determination of Thiobarbituric Acid Reactive Substances (TBARS)
The plasma (0.1 mL) was mixed with 0.6 M HCl solution (0.5 mL) containing 20% TCA and 1 M NaOH solution (0.3 mL) containing 0.67% TBA. The mixture was boiled for 20 min, cooled, and then separated with water-saturated n-butanol (800 µL). The absorbance of the upper layer (n-butanol layer) was measured at 532 nm and TBARS levels in the plasma were calculated using standard curve of MDA [64].

Determination of Protein Carbonyl Content
The protein extract (2.0 mg) obtained from liver tissue was mixed with 0.1 mL of 2.0 M HCl solution containing 10 mM DNPH. The mixture was incubated for 1 h in a dark place at room temperature and heavily shaken at 15 min intervals. The reaction mixture was added to 1.0 mL of a cooled 10% (w/v) trichloroacetic acid solution and centrifuged at 3000ˆg for 10 min. The protein pellet was washed three times with 2.0 mL of ethanol/ethyl acetate = 1:1 (v/v). The pellets were then dissolved in 1.5 mL of 6 M guanidine hydrochloride (pH 2.3). After incubation for 10 min at 37˝C, the absorbance was measured at 370 nm. The PCO content was calculated in nmol of carbonyls groups per mg of protein, using the extinction coefficient of 2.2ˆ10 4 /M¨mL [65].

Determination of TNF-α Level in Plasma
The levels of TNF-α in the plasma was measured using an ELISA kit (Koma Biotech Inc., Seoul, Korea) according to manufacturer's instructions. Briefly, the plasma (0.1 mL) was added in an ELISA kit and incubated for 2 h. After washing with phosphate saline buffer containing 0.0005% Tween-20 (PBST, 4 times), the plate was added to anti-Rat TNF-α (100 µL), incubated for 4 h, and washed with PBST (4 times). The plate was incubated with the addition of streptavidin-HRP conjugate (100 µL) for 30 min followed by treatment of TMB substrate solution (100 µL) for 10 min. After stopping the reaction with 2 M H 2 SO 4 solution (100 µL), the absorbance of each plate well was measured at 450 nm by ELISA reader (µQuant, Bio-Tek Instruments, Inc., Winooski, VT, USA). The levels of TNF-α in the plasma were quantified from a calibration curve of the standard (47-3000 pg/mL).

Assay for ACE Inhibitory Activity
The ACE inhibitory activities of the fractions and the isolated compounds were measured according to the methods described by Cushman et al. [48], with slight modification. The samples (20 µL) were mixed with 0.1 M sodium borate buffer (pH 8.3, 130 µL) containing 300 mM NaCl and 5 mM HHL (50 µL). The mixture was then preincubated at 37˝C for 10 min. After preincubation, the mixture was added to 100 µL of ACE extract from lung acetone powder of rabbit. The mixture was incubated at 37˝C for 90 min and then the enzymatic reaction was stopped by the addition of 2 N HCl solution (200 µL). The reaction mixture was separated with ethyl acetate (2 mL). The upper layer (ethyl acetate layer, 1.5 mL) was concentrated and the hippuric acid fraction was dissolved with 1 M NaCl solution (1 mL). The absorbance (A) was measured at 228 nm. Q was used as a positive control. The absorbance (B) of Q and the samples was measured by the same procedure as described above except for ACE extract. The ACE inhibitory activities of the samples were determined as the percentage decrease in absorbance compared to that of a control test. The dose-response curve of each compound at the concentration of ď1 mM was a linear for ACE inhibitory activity. The isolated compounds were assayed at the concentration of 1 mM. ACE inhibitory activity p%q " rpcontrol´pA´Bq{controlsˆ100

Statistical Analysis
Data are expressed as mean˘standard deviation (SD) using the SPSS, (IBM, Armonk, NY, USA) 19.0 package program. Statistical differences were measured by one-way analysis of variance followed by Tukey's multiple test. A p < 0.05 was considered significant.

Conclusions
AS attenuated an increment of blood pressure in SHR via the enhancement of VEGF expression and the suppression of RhoA expression by reduced ACE activity and angiotensin II production. However, in this study, we could not see significant synergistic effects of garlic with AS in lowering blood pressure. Possible explanations are as follows: (1) not enough garlic was added to AS; (2) sulfur compounds having antihypertensive activity are volatile and lipophilic. They might not be extracted when garlic and AS were extracted with water in autoclave; (3) garlic may not be synergistic with AS although we do not understand the mechanism. Further studies are required. Reduced ACE activity and angiotensin II content in the plasma of the rats fed AS must have been probably or at least partially due to the ACE inhibitors present in AS. These results led to isolation and identification of ACE inhibitors present in AS for the utilization of this plant as a food and medicinal herb. Seven ACE inhibitors, including one new phenolic glycoside, were isolated and identified from the aerial parts of AS with guided purification of ACE inhibitory assay. Further studies on how much these ACE inhibitors contribute to the antihypertensive effects of AS and how they are absorbed in the body remain to be investigated.