J. Biol. Macromol., 17 (1), 3-13 (2017)

ArticleInhibition of renin-angiotensin system related enzymes (renin, angiotensin
converting enzyme, chymase, and angiotensin converting enzyme 2) by water
shield extracts.
Saori Takahashi,*1 Ai Satou,1 Hiroshi Shimoda,2 and Keishi Hata1
1

Akita Research Institute of Food and Brewing, 4-26 Sanuki, Arayamachi, Akita
010-1623, Japan
2
Oryza Oil & Fat Chemical, 1 Numata, Kitagatacho, Ichinomiya, Aichi 493-8001, Japan
Received January 25, 2017; Accepted February 21, 2017
We screened for the inhibitory activities of 19 wild vegetable and fruit extracts forrenin-angiotensin system (RAS)-related enzymes, including renin, angiotensinconverting enzyme (ACE), chymase, and angiotensin-converting enzyme 2 (ACE2).
Among them, a hot water extract of Brasenia schreberi (Water shield, Junsai)
strongly inhibited renin and chymase activities and significantly inhibited ACE
and ACE2 activities. We also tested the RAS-related enzyme inhibitory activities of
13 polyphenols isolated from Brasenia schreberi. The polyphenols mostly had little
effect on ACE and ACE2 activities. Among them, hypolaetin 7-O-glucoside showed
the strongest chymase inhibitory activity with an IC50 value of 3.8 μM. Some otherpolyphenols also inhibited renin and chymase activities.
Key words: Brasenia schreberi, renin-angiotensin system, inhibition, chymase.
Introduction
The renin-angiotensin system (RAS) is

The enzyme catalyzes the liberation ofangiotensin I (AI) from the plasma

one of the most important blood pressure
control systems in mammals (Fig. 1).
Renin is a highly specific aspartic
proteinase that is mainly synthesized injuxtaglomerular cells in the kidney.

substrate angiotensinogen. The produced
prohormone AI is further processed to thepressor peptide angiotensin II (AII) by
angiotensin-converting enzyme (ACE) or
chymase. Chymase is a chymotrypsinlike serine proteinase that cleaves
C-terminal aromatic amino acids [1-3].
Using angiotensin I as the substrate,
human chymase cleaves the Phe8-His9*To whom correspondence should be
addressed:
E-mail: saori@arif.pref.akita.jp
Fax: (+81)18-888-2008

4

Chymase inhibitor from water shieldbond of AI to produce AII. The produced
AII directly acts on arterial smoothmuscle cells to maintain blood pressure
and increases the release of aldosterone
from the adrenal cortex to increase
sodium retention in the kidney. Recently,
the ACE homologous carboxypeptidase
angiotensin-converting enzyme 2 wasmeasurement of renin, ACE, and ACE2
activities using internally quenched (IQF)
substrates [9, 10]. In the present study, wedeveloped a novel assay method forchymase using an IQF substrate forhuman renin. Using these assay systems,
we screened for RAS-related enzyme
inhibitory activities in various foodstuffs

discovered [4, 5]. The enzyme hydrolyzes
AI and AII to produce angiotensin 1-9(Ang 1-9) and angiotensin 1-7 (Ang 1-7),
respectively. Ang 1-9 is also processed toAng 1-7 by ACE. Ang 1-7 evokes
vasodilation involving interaction withMas [6-8] (Fig. 1).
Recently, we developed a rapid andsensitive assay method for theand found strong enzyme inhibitory
activities in a hot water extract ofBrasenia schreberi (Junsai). We also
tested the RAS-related enzyme inhibitory
activities of polyphenols isolated from
Brasenia schreberi (Junsai) and foundhypolaetin
7-O-glucosidestrongly
inhibited chymase activity.

Angiotensinogen (Liver)

Renin
Angiotensin I (AI)
(D-R-V-Y-I-H-P-F-H-L)

ACE

ACE2
Ang (1-9)(D-R-V-Y-I-H-P-F-H)

ChymaseACE

ACE2
Angiotensin II (AII)(D-R-V-Y-I-H-P-F)

AT1 receptor
Vasoconstriction
Cell proliferation
Hypertrophy

Ang (1-7)(D-R-V-Y-I-H-P)

AT2 receptor

AT1-7 receptor(Mas receptor)

ACE
Ang (1-5) (D-R-V-Y-I)

Vasodilatation
Antiproliferation

Fig. 1 Renin-angiotensin system
Renin is a rate-limiting enzyme of RAS. Renin produces AI from angiotensinogen. AI
further processed to AII by ACE or chymase.

Takahashi et al.

5

Materials and Methods
Materials
Recombinant human renin expressed in
Sf-9 insect cells was prepared by themethod of Takahashi et al. [11].
Recombinant human chymase expressedin Pichia pastoris was obtained from
Sigma-Aldrich (St. Louis, MO, USA).

10 mg/ml.

Recombinant human ACE and ACE2
were from R&D Systems (Minneapolis,
MN,
USA).
Internallyquenched
fluorogenic substrates were from Peptide
Institute, Inc. (Saito, Osaka, Japan).
Polyphenols isolated from Brasenia
schreberi (Junsai) (13 kinds ofcompounds) were obtained from Oryza
Oil & Fat Chemical (Aichi, Japan) [12]:renin:[2-(N-Methylamino)benzoyl](Nma)-Ile-His-Pro-Phe*His-Leu-Val-IleThr-His-[Nε-(2,4-dinitrophenyl)-Lys][Lys(Dnp)]-D-Arg-D-Arg-NH2 (*, scissile
peptide bond). The cleavage site of the
enzyme was determined by LC-MS
analysis (data not shown). The reaction
mixture contained 40 μl of 0.1 M
Glycine-NaOH, pH 9.0, 0.1 M NaCl, 25

compound 1, ethyl gallate; compound 2,
caffeoyl glucose; compound 3, hypolaetin
7-O-glucoside; compound 4, quercetin
3-O-(6”-galloyl) glucoside; compound 5,
quercetin 3-O-glucoside; compound 6,
kaempferol 3-O-(6”-galloyl) glucoside;
compound 7, gossypetin; compound 8,
kaempferol 3-O-glucoside; compound 9,
junsainoside A; compound 10, quercetin;μM IQF substrate, 0.02% NaN3, 0.02%
Triton X-100, 5 μl of chymase solution(0.4 μg/ml), and 5 μl of inhibitor solution
in a total volume of 50 μl. The reaction
mixture was incubated at 37°C for 30 min,and the reaction was terminated byadding 200 μl of 0.1 sodium borate buffer,
pH 10.5. The increase of fluorescenceintensity was measured at an emission

compound 11, tiliroside; compound 12,
kaempferol; compound 13, methyl caffeic
acid.

wavelength of 440 nm upon an excitation
wavelength at 340 nm.

Preparation of hot water extracts offoodstuffs
Lyophilized sample powder was added towater and heated at 105°C for 30 min.
After centrifugation, the supernatant waslyophilized. The sample was dissolved in
distilled water at a final concentration of

Measurement of chymase activity
As the recombinant human chymase
cleaves Phe8-His9 of angiotensin I toyield angiotensin II, we tested various
IQF substrates for ACE and renin andfound that the enzyme efficiently cleaves
the following IQF substrate for human

Measurement of renin, ACE, andACE2 activities
Renin activity was measured using(Nma)-Ile-His-Pro-Phe-His-Leu-Val-IleThr-His-Lys(Dnp)-D-Arg-D-Arg-NH2 as
the substrate [11]. ACE and ACE2
activities
were
measuredusing
Nma-Phe-His-Lys(Dnp) and Nma-His-

6

Chymase inhibitor from water shield

Pro-Lys(Dnp) as substrates, respectively[9, 10].
Inhibition of renin, ACE, chymase, andACE2 activities by polyphenols
The 13 kinds of polyphenols isolated
from Brasenia schreberi [12] were used
for enzyme inhibition assays at a final
concentration of 10 μM (Table 1). Data

deviation (n = 3). The significance ofdifferences was analyzed using a
one-way ANOVA with Dunnett’s multiple
comparison
tests.
The
sampleconcentrations required to inhibit 50% of
enzyme activity under the assay
conditions was taken as the IC50 value(Table 2).

are expressed as the mean ± standard

Table 1 Inhibition of renin, ACE, chymase and ACE2 by Junsai polyphenols____________________________________________________________________________
Relative activity (%)____________________________________________________________________________
Renin

ACE

ChymaseACE2

Means ± SD

Means ± SD

Means ± SD

Means ± SD____________________________________________________________________________
Control

100.0 ± 8.0

100.0 ± 2.8

100.0 ± 5.8

100.0 ± 3.3

Compound 1

102.7 ± 2.7

99.8 ± 0.8

56.7 ± 2.7**

101.0 ± 3.4

Compound 2

103.4 ± 3.0

99.2 ± 5.6

97.8 ± 4.0

94.9 ± 5.8

Compound 3

95.4 ± 1.3

100.8 ± 1.6

10.0 ± 2.4**

104.9 ± 1.5

Compound 4

87.7 ± 0.6**

100.7 ± 2.3

42.1 ± 0.9**

97.5 ± 6.7

Compound 5

100.1 ± 1.5

93.0 ± 4.3

68.1 ± 0.4**

95.8 ± 4.7

Compound 6

91.0 ± 2.1*

107.9 ± 5.1

49.4 ± 2.0**

95.3 ± 1.4

Compound 7

97.2 ± 1.2

99.2 ± 6.0

45.2 ± 1.2**

97.4 ± 7.8

Compound 8

101.0 ± 2.9

107.2 ± 6.6

79.1 ± 2.3**

92.6 ± 11.5

Compound 974.0 ± 1.9**

98.2 ± 0.3

39.1 ± 1.9**

86.9 ± 0.5

Compound 10

80.8 ± 1.9**

102.3 ± 5.3

33.6 ± 0.4**

89.2 ± 3.5

Compound 11

75.3 ± 2.6**

99.4 ± 5.2

84.5 ± 1.7**

92.1 ± 4.5

Compound 12

73.7 ± 1.0**

90.5 ± 2.9

62.4 ± 1.3**

81.0 ± 1.3*

Compound 13

106.6 ± 0.9

104.1 ± 5.8

96.3 ± 1.5

97.2 ± 6.0______________________________________________________________________________*, p<0.01; **, p<0.001

Takahashi et al.

7

Results and Discussion
Chymase activity
We determined the kinetic parameters ofrecombinant human chymase for the IQF
substrate. The Km, kcat, and kcat/Km valuesof chymase for (Nma)-Ile-His-ProPhe-His-Leu-Val-Ile-Thr-His-Lys(Dnp)-D

activity in foodstuffs, although there areseveral report of chymase inhibitors frommicroorganisms [13] and synthetic
compounds [14, 15].

-Arg-D-Arg-NH2 were 7.69 μM, 1,280 s-1,
and 166 μM-1 s-1, respectively. In aprevious study, endogenous chymase
substrates and AI from various animals
were used for the chymase assay [1].
Among them, human AI was one of themost suitable substrates for humanchymase, with Km, kcat, and kcat/Km valuesof 60 μM, 198 s-1, and 3.3 μM-1 s-1,

occurrence of renin inhibitory activities
in ethanol extracts of various foodstuffs[16-18]. Moreover, we have identified therenin inhibitors as soyasaponin I fromsoybean [19], oleic acid and linoleic acidfrom rice [20], and (-)-kaur-16-en-19-oic
acid, pimara-8(14), 15-dien-19-oic acid,and pimara-9(11), 15-dien-19-oic acidfrom Aralia cordata (Udo) [21]. In therespectively. The kcat/Km value of the IQF
substrate is about 50-fold higher than thatof human AI, indicating that (Nma)Ile-His-Pro-Phe-His-Leu-Val-Ile-ThrHis-Lys(Dnp)-D-Arg-D-Arg-NH2 is an
excellent substrate for human chymase.
Chymase inhibitory activities offoodstuff extracts
We tested the chymase inhibitory activity

present study, we also tested the renin
inhibitory activity of hot water extracts
from 19 wild vegetables and fruits (Fig. 2,
closed bars). As already mentioned,
Aralia cordata (Udo) and Glycine max(Soy) extracts inhibited renin activity.
Moreover, Brasenia schreberi (Junsai)
extracts strongly inhibited renin activity,
andMatteuccia
struthiopterisof hot water extracts from 19 wild
vegetables and fruits (Fig. 2, open bars).
Among them, the Brasenia schreberi(Junsai),
Araliacordata(Udo),
Matteuccia struthiopteris (Kusasotetsu),
and Glycine max (Soy) extracts strongly
inhibited chymase activity. The IC50
value of the Brasenia schreberi (Junsai)
extract was 12.5 μg/ml. This is the first
demonstration of chymase inhibitory(Kusasotetsu), Corchorus olitorius (Naltajute), Oenanthe stolonifera (Seri), andBassiascoparia(Tonburi),
Chrysanthemum
morifolium(Chrysanthemum) extracts effectively
inhibited renin activity. The identificationof renin inhibitors from these vegetables
is necessary to understand thestructure-function relationship of renin
inhibitors from foodstuffs.

Renin inhibitory activity of foodstuff
extracts
Our previous studies demonstrated the8Chymase inhibitor from water shield

0

20

40

60

80

100

120

Relative activity (%)

Fig. 2 Inhibition of renin and chymase activities by extracts of fruits andvegetables.
The reaction was carried out at 37°C for 30 min with 500 ng/ml of renin or 40 ng/ml ofchymase and hot-water extracts of fruits and vegetables at a final concentration of 0.1
mg/ml. Each result is the mean value of triplicate determination. Open bars indicate
chymase activities, and filled bars indicate renin activities.

Takahashi et al.

0

20

40

9

60

80

100

120

Relative activity (%)
Fig. 3 Inhibition of ACE and ACE2 activities by extracts of fruits and vegetables
The reaction was carried out at 37°C for 30 min with 20 ng/ml of ACE or 10 ng/ml of
ACE2 and hot-water extracts of fruits and vegetables at a final concentration of 0.1
mg/ml. Each result is the mean value of triplicate determination. Open bars indicate
ACE2 activities, and filled bars indicate ACE activities.

10

Chymase inhibitor from water shield

ACE and ACE2 inhibitory activity offoodstuff extracts
Our recent study demonstrated theoccurrence of an ACE2 inhibitor insoybean, and the inhibitor was identified
as nicotianamine [10]. In the present
study, we screened the ACE and ACE2
inhibitory activities of hot water extracts

sativus (Cucumber), Glycine max (Soy),
Abelmoschus
esculentus(Okura),Chrysanthemum
morifolium(Chrysanthemum),andSolanum
lycopersicum (Tomato) equally inhibited
ACE and ACE2 activities. These results
suggest that both enzymes are inhibitedwith the same inhibitor compound. Inof 19 wild vegetables and fruits (Fig. 3,
open bars for ACE2 and closed bars for
ACE). The extracts of Brasenia schreberi(Junsai),
Araliacordata(Udo),
Asparagus
officinalis(Asparagus),
Corchorus
olitorius(Naltajute),
Oenanthe stolonifera (Seri), Cucumis

connection with this matter, the ACE2
inhibitor
isolatedfromsoybean,nicotianamine, inhibited both ACE andACE2 activities [11]. The isolation of an
ACE and/or ACE2 inhibitor in these
extracts is necessary to clarify the
problems.

Table 2 IC50 values (μM) of Junsai polyphenols on renin and chymase______________________________________________________
Renin
Chymase______________________________________________________
Compound 1
N.D.
11.6
Compound 2
N.D.
N.D.
Compound 3
N.D.
3.8
Compound 4
58.0
5.4
Compound 5
N.D.
18.5
Compound 6
74.0
6.1
Compound 7
N.D.
7.7
Compound 8
N.D.
>50.0
Compound 9
23.0
5.8
Compound 10
64.0
5.4
Compound 11
>100
>50.0
Compound 12
38.0
16.2
Compound 13
N.D.
N.D.
______________________________________________________
N.D., not determinedTakahashi et al.

11

RAS-related
enzymeinhibitory
activities of polyphenols from Brasenia
schreberi (Junsai)
Recently, various kinds of polyphenols
were isolated from Brasenia schreberi
produced in Akita Prefecture, Japan [12].
We tested the renin, ACE, chymase, andACE2 inhibitory activities of 13 kinds ofactivity of various vegetables forantihyperlipidemic activities and foundthat Brasenia schreberi extracts hadstrong inhibitory activities against
triglyceride and cholesterol secretions
from human HepG2 hepatoma cells [22].
Moreover, Brasenia schreberi extracts
inhibited human immunodeficiency virus

compounds as described in “Materials
and Methods”. Table 1 shows the resultsof the inhibition studies. None of thecompounds inhibited ACE and ACE2
activities
exceptcompound
12.
Compound 12 inhibited ACE2 activity
weakly, with an IC50 value of about 120
μM. On the other hand, compounds 4, 6,
9, 10, 11, and 12 significantly inhibited

type 1 (HIV-1) reverse transcriptase
activity and HIV-1 replication [23, 24].
The present study showed a novel
physiological function of Brasenia
schreberi extracts. Brasenia schreberi
will
be
a
good
sourcefor
anti-hypertensive functional foods.

renin activity. The IC50 values ofcompounds 4, 6, 9, 10, and 12 ranged
from 23.0 μM to 74.0 μM (Table 2). The
IC50 value of compound 11 was estimatedto be >100 μM. Moreover, compounds 1
and 3 to 12 significantly inhibitedchymase activity (Table 1). The IC50
values are also summarized in Table 2.
Among these compounds, compound 3

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Communicated by Kiyoshi Yasukawa