J. Biol. Macromol., 14(2), 71- 84 (2014)

Review
Renin Inhibitors in Foodstuffs: Structure-Function Relationship
Saori Takahashi1*, Takeshi Gotoh2, Keishi Hata1, Tetsuo Tokiwano3, Yuko Yoshizawa3,
Kazuyuki Hiwatashi1, Hironobu Ogasawara1, and Kazuyuki Hori1,
1

Akita Research Institute of Food and Brewing (ARIF),
4-26 Sanuki, Arayamachi, Akita 010-1623, Japan2
Department of Applied Chemistry, Akita University,
1-1 Tegata Gakuencho, Akita 010-8502, Japan
3
Fuclty of Bioresource Science, Akita Prefectural University,
241-438 Kaidoubatanishi Nakano, Shimoshinjo, Akita 010-0195, Japan
Received January 22, 2014; Accepted February 10, 2014
Renin, a highly specific aspartic proteinase originated from kidney, is a ratelimiting enzyme in renin-angiotensin system. Recently, we developed effective
expression system for recombinant human (rh) renin using baculovirus-insect cell
expression system, and simple and rapid assay method for rh-renin using internallyquenched fluorogenic (IQF) substrate. Using the purified rh-renin and the IQF
substrate, we constructed rapid and sensitive renin inhibitor screening system. Using
the system, we found renin inhibitors in various foodstuffs. The isolation andcharacterization of renin inhibitors from soybean, rice, and wild vegetables andstructure-function relationship of the inhibitors are also discussed.
Key words: renin, angiotensin, internally quenched fluorogenic substrate, soyasaponin I,
oleic acid, linoleic acid, free fatty acids, kaurenic acid, pimaradienoic acid.
Introduction
Renin-angiotensin system (RAS) is
one of the most important blood pressure*Corresponding
author:Saori
Takahashi, Phone: +81-18-888-2000,
Fax:+81-18-888-2008,
E-mail:saori@arif.pref.akita.jp

control system in mammals. Renin
catalyzes liberation of angiotensin I (AI)from plasma substrate angiotensinogen.
AI is an inactive peptide and activated byangiotensin I converting enzyme (ACE).
ACE cleaves C-terminal dipeptide fromAI and produces active octapeptide

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Renin Inhibitors in Foodstuffs

Fig. 1. Renin-angiotensin system (RAS).
Renin is synthesized mainly in the juxtaglomerular cells in the kidneycortex. The synthesized renin was released into the circulation by several
stimuli. Renin catalyzes the liberation of angiotensin I from plasma
substrate angiotensinogen. Thus, renin is the rate-limiting enzyme in RAS.
angiotensin II (AII) (Fig. 1). AII directly
acts on arterial smooth muscle cells tomaintain blood pressure and increases
release of aldosterone from adrenal cortex
to increase reabsorption of water from thekidney. This also causes high bloodpressure [1]. ACE has been used to screen
inhibitors from various foodstuffs becauseof its simple assay method. Although reninis the most important enzyme in RAS,
screening for renin inhibitors among
foodstuffs has not been well studied due tothe complications of the renin assay. In
this review we describe expression ofrecombinant human (rh) renin in
Spodoptera frugiperda (Sf-9) insect cells,

development of a simple and rapid assay
method for human renin, occurrence ofrenin inhibitors in various foodstuffs,
isolation of renin inhibitors, and structurefunction relationship of them.
Expression of recombinant humanrenin in Sf-9 insect cells
The expression of rh-prorenin was
first demonstrated in Escherichia coli cell[2]. The expressed rh-prorenin formed
inclusion bodies and was difficult to refold.
Recently, we expressed rh-prorenin in E.
coli cells as a fusion protein withthioredoxin and the expressed rh-prorenin
was refolded systematic dialysis andS. Takahashi, et al.

73

Fig. 2. Expression of rh-renin in Sf-9 insect cells (A) and SDS-PAGEof purified rh-renin.
A, Western blotting of culture medium using anti-human renin antiserum. M, molecular weight marker (Kaleidoscope molecular weightmarker, Bio-Rad), A 10 μl of culture mediums were used for theelectrophoresis. B, SDS-PAGE of rh-renin. After the electrophoresis,
proteins were stained with Coomassie brilliant blue R-250. M,
molecular weight marker (Low molecular weight marker, GE-HealthCare) ; lane 1, culture medium (10 μl); lane 2, pepstatin column eluate(1.3 μg of protein); lane 3, purified rh-renin (1.0 μg of protein).
activated by trypsin [3]. The active rhrenin was used for the screening of renin
inhibitors and found that commerciallyavailable fermented soybean, miso
contained renin inhibitory activity [3]. On
the other hand, expression of rh-proreninor rh-renin in mammalian and insect cells
has been reported [4-9]. In these cases,
major expressed protein was inactive
prorenin. Trypsin treatment was essential
for the activation of prorenin. We also
used a recombinant baculovirus, vhpR,
carrying human preprorenin cDNA in thepolyhedrin
locusofAutorgapha

californica multiple nuclear polyhedrosis
virus (AcMNPV) [10]. On an infection
culture at a MOI of 1 pfu/cell using SF900II serum-free medium on an orbital
shaker at 100 rpm at 28°C. Cell grewcontinuously until day 3, but total cellnumbers and viability decreased at day 4
and 5 of culture. Renin activity was notdetected until day 3, but a small amount ofrenin activity was detected at day 4 andappeared dramatically at day 5. Western
blotting using anti-human renin antiserum[11], 43 kDa prorenin was detected at days
3 and 4 cultures. On the other hand, only a

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Renin Inhibitors in Foodstuffs

40 kDa mature renin was detected in the
day 5 culture (Fig. 2A). These results
clearly show that the expressed prorenin
was activated by proteinase appearing atlate stage of culture. Our recent study also
showed that 32 kDa cysteine proteasederived from AcMNPV gene is theprorenin processing enzyme [12]. Using
the day 5 culture, we purified rh-reninusing
pepstatin
affinitycolumnchromatography and Mono Q FPLC. The
purified rh-renin showed single proteinband on SDS-PAGE with the molecularweight of 40 kDa [10] (Fig. 2 B).
Development of internally quenched
fluorogenic substrate for human renin
In 1969 Haber et al. developed
sensitive renin assay method using
angiotensin I radioimmunoassay [13].
This method is highly sensitive, but wehave to prepare various reagents including
125

I-labeled AI, angiotensinogen, and antiAI anti-serum. In 1980, Murakami et al.
developed fluorogenic substrate for
porcine renin, succinyl (Suc)-Arg-ProPhe-His-Leu*Leu-Val-Tyr-4methylcoumaryl-7-amide (MCA) [14].
Porcine renin cleaves the substrate at LeuLeu bonds, releasing Leu-Val-Tyr-MCA.
This peptide was then cleaved byaminopeptidase to release free 7-amino-4methyl- coumarin. This method isrelatively simple, but the sensitivity forhuman renin is very low. Recently, wedeveloped novel internally quenched

fluorogenic (IQF) substrates for porcine[15] and human renin [10, 16]. The
structures of both substrates are as
follows: 2-(methylamino) benzoyl (Nma)His-Pro-Phe-His-Leu*Leu-Val-Tyr-[N ξ (2,4- dinitro phenyl)-Lys] [Lys(Dnp)]-DArg (r)-r-NH2 and Nma-Ile-His-Pro-PheHis-Leu*Val-Ile- His-Thr-Lys(Dnp)-r-rNH2 (*, scissile peptide bond). Hydrolysis
of Leu-Leu bond of porcine renin
substrate or Leu-Val bond of human renin
substrate was spectrophotometricallydetermined. In the case of human reninassay, the reaction mixture contained 1 μlof 1 mM IQF substrate in DMSO, 44 μl of
sodium phosphate buffer, pH 6.5,
containing 0.1 M NaCl, 0.02% Tween 20,
and 0.02% NaN3, and 5μl of rh-renin
solution in a total volume of 50 μl. The
reaction mixture was incubated at 37℃
for 30 min and the reaction was terminatedby adding 0.1 M triethanolamine, pH 10.5.
The increase in the fluorescence intensity
was measured at an emission wavelengthof 440 nm upon excitation wavelength at
340 nm. The Km and kcat values of rh-renin
for the synthetic substrate at the above
conditions were 35.7μM-1 and 833s-1
respectively. The assay system is suitable
for the measurement of rh-renin activity
and the screening of rh-renin inhibitorsfrom various foodstuffs and natural
products.
Renin inhibitor in soybean
Using the rh-renin as target enzyme,

S. Takahashi, et al.

we screened the inhibitory activity ofvarious foodstuffs and found that miso
extract contains rh-renin inhibitory
activity originated from soybean. Thus,
we try to isolate renin inhibitor fromsoybean.
Before isolation of rh-renin
inhibitor from soybean, we investigatedthe localization of renin inhibitor insoybean. Soybean was separated into twoparts, embryo and cotyledon. Both parts
were extracted with hot water and theextracts were concentrated by Sep-Pak
ODS column (Millipore, USA) andevaluated for renin inhibitory activity.
Embryo extract contained about 3-foldhigher inhibitory activity than cotyledon
extract, hence we used soybean embryo
for isolation of renin inhibitor.
Approximately 70 mg of soybean renin
inhibitor (SRI) was obtained from 750 g ofsoybean embryo. The isolated SRI gavesoyasapogenol B moiety and sugar chain
unit as rhamnopyranosyl (1 → 2)
galactopyranosyl
(1
→
2)
1
13
glucopyranosiduronic acid for H and C
NMR spectra [17-19]. Finally, the SRI
was identified as soyasaponin I by directcomparison with standard compound for[α]D, mixed melting point, 1H NMR, andIR spectra (Fig. 3), [16].
Soyasaponin I inhibited rh-renin
activity in a dose dependent manner with
IC50 value of 30 μg/ml. Kinetic studies
with soyasaponin I indicated partial
noncompetitive inhibition with Ki value of

75

Fig. 3. Structures of soyasaponins.
Soyasaponin I and II inhibited rh-renin
activity. On the other hand, soyasapogenol
B had no effect on rh-renin.
37.5μM. The inhibitory spectra ofsoyasaponin I are shown in Table I.
Soyasaponin I also inhibited porcine renin
activity with an IC50 value of 30 μg/ml.
Soyasaponin I weakly inhibited porcine
pepsin or cysteine proteases, papain andbromeline, and had no effect on serine
proteases or metallo proteinases. These
results clearly indicate that soyasaponin I
is a renin-specific inhibitor in soybean.
The effects of soyasaponin on bloodpressure of spontaneously hypertensive
rats (SHR) were also investigated [20].
Oral administration of commerciallyavailable soybean saponin (Wako Pure
Chemical Industries, Osaka) at 80 mg/kg
of body weight per day to SHR/Izm strain
for eight weeks significantly decreased the

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Renin Inhibitors in Foodstuffs

systolic blood pressure. These results
show that soybean saponin inhibited
human and porcine renin activity in vitro,and improved hypertension in SHR.
The structure
andfunction
relationship of saponins on renin
inhibition was also investigated using
several saponins and sapogenols [21].

Soyasaponin I and II (from soybean) (Fig.
3), chikusetusaponin IV (from Panax
japonicas Rhizome), glycyrrhizin (formlicoriceroot),
monoglucuronyl
glycyeehetic acid (form licorice root), andKichiascoparia
fruitsaponins(momordins) inhibited rh-renin activity.
On the other hand, sapogenols(soyasapogenol B from soybean (Fig. 3)
and glycyrrhetic acid from licorice root),saikosaponins b2 and c (from bupleurumroot), and ginsenoside Rb1 (from ginsengroot) had no effects on rh-renin activity.
These results clearly indicate that the 3-Oβ-D-glucopyranosiduronic acid moiety insaponins is essential for renin inhibition.
Renin inhibitor from rice
We found rh-renin inhibitory
activity in cooked rice after screening

S. Takahashi, et al.

several foodstuffs. Therefore, we tested
the effects of local cultivar rice extracts on
rh-renin activity. Table II shows the effectsof methanol extracts of various
nonglutinous rice strains cultivated in
Akita Prefecture on rh-renin activity [22].
All of the rice extracts tested inhibited rhrenin activity with IC50 values of 150280μg/ml. Next we tried to isolate ricerenin inhibitor (RRI) from Akitakomachi
because it is the most important rice strain
in Akita Prefecture. Akitakomachi powder(300 g) was soaked in methanol (2.4
litters) at room temperature for 1 h andthen centrifuged at 10,000 x g for 30 min.
The supernatant was evaporated todryness. The dry matter was used forfurther purification. After several column
chromatography steps, we isolate RRI
preparation (59.6 mg). The preparation
was used for the identification of RRI. The
1

H NMR spectrum of the isolated RRI
showed signals arising from a mixture ofunsaturated fatty acids, and the negative
ESI-MS gave m/z 279 and 281 peaks.
These data indicate that the RRI was a
2.5:1 mixture of linoleic acid and oleic
acid. The isolated RRI preparation
inhibited rh-renin activity with an IC50
value of 9.9μg/ml. To clarify the direct
effects of oleic acid and linoleic acid on
rh-renin activity, we used commerciallyavailable extra pure reagents for rh-renin
inhibitory activity. Oleic acid and linoleic
acid inhibited rh-renin activity in a dosedependent manner with IC50 values 8.0

77

and 10.5μg/ml, respectively.
We further investigate the structurefunction relationship of fatty acids on rhrenin activity; we tested the effects ofseveral saturated and unsaturated fatty
acids on it. Figure 4 shows the structuresand IC50 values of various free fatty acids
on rh-renin activity. In the case of C18
unsaturated fatty acids, mono- to triunsaturated fatty acids, cis-vaccenic acid,oleic acid, linoleic acid, and linolenic acidinhibited rh-renin activity. On the other
hand, saturated fatty, stearic acid had noeffect on activity. A C16 saturated fatty
acid, palmitic acid, also had no effect on
the activity. Other polyunsaturated fatty
acids, arachidonic acid, eicosapentanoic
acid (EPA), and docosahexanoic acid
(DHA), also inhibited rh-renin activity.
These results indicate that free unsaturated
fatty acids inhibited rh-renin activity.
In connection with these results, theeffects of circulating natural lipids onhuman plasma renin activity have beenreported [23, 24]. Kotchen et al. found thatacetone soluble lipid extracts from humanplasma inhibit the in vitro renin activity[23]. Furthermore, they isolated a renin
inhibitor from human plasma by affinity
chromatography
using
mouse
submaxillary grand renin as a ligand andconcluded that circulating linoleic acidinhibits renin activity [24]. The effects of
n-3 polyunsaturated fatty acids (PUFAs)
on a model of human renin hypertension
had also been reported [25]. Fischer et al.

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Renin Inhibitors in Foodstuffs

compared the effects of PUFAs with direct
renin inhibition in electrophysiologicalremodeling. They treated doubletransgenic rats expressing the human renin
and angiotensinogen genes weeks 4 to 7
with n-3 PUFAs ethyl-esters. SpragueDawley rats were used as control. Dietaryn-3 PUFAs increased the cardiac contentsof EPA and DHA. At week 7, mortality inthe hypertension model rats was 31%,
whereas none of the n-3 PUFAs or
aliskiren-treated hypertension model rats
died. Aliskiren is the first orally active
inhibitor of human renin to be approvedfor clinical use as an antihypertensive
agent [26]. Aliskiren-treated hypertension
model rats and Sprague-Dawley rats were
normotensive. These results indicate thatn-3 PUFAs and aliskiren improved highblood pressure and the development ofcardiac hypertrophy.
Renin inhibitor from cereals
We further screened for renin
inhibitory activity in various types of riceand cereals and found renin inhibitory
activity in rice and some cereals [27]. At
the beginning, we tested the effects of

S. Takahashi, et al.

79

Fig. 4. Structures of free fatty acids and IC50 values.
Saturated fatty acids, palmitic acid and stearic acids, had noeffect on rh-renin activity. Unsaturated fatty acids inhibited rhrenin activity in a dose-dependent manner. The IC50 values areindicated in the parenthesis. N. I., no inhibition.
glutinous, sake, and Indica rice extracts
on rh-renin activity to understand theexistence of renin inhibitor in rice
universally. Table 3 shows the summary of
the amount of rice extracts, IC50 values,
inhibitor units (IU), and oleic acid andlinoleic acid contents of various rice
extracts. Rice extracts tested in this study
inhibited rh-renin activity with IC50 valuesof 88-270 μg/ml. In the previous study, we
found that oleic acid and linoleic acid are
the major rice rh-renin inhibitors [24]. The
IC50 values of rice extracts are correlatedwith the contents of oleic acid and linoleic
acid. To evaluate the renin inhibitory
activity per gram weight, we calculated

the renin inhibitor per gram extracts.
Nonglutinous rice, Akitakomachi andother strain had 4.04 to 9.47 IU of extract(Table 3) [22]. Glutinous rice,
Tatsukomochi and Kimunohada showed
22.73 and 16.03 IU, respectively. Indica
rice showed the lowest value of 2.74 IU.
These results clearly showed thatglutinous rice extracts had higher renin
inhibitory activity than other rice extracts.
Cereals are staple food in many
countries around the world including
Japan. Table 4 shows effects of methanol
extracts of various cereals on rh-renin
activity. Flour for bread and weak flour
were also used as a control. Among

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Renin Inhibitors in Foodstuffs

different cereal extracts, buckwheat
extract showed highest rh-renin inhibitory
activity followed by barnyard millet andrye extracts. On the other hand, control
flours, foxtail millet, Chinese millet, andamaranth extracts had no effects on rhrenin activity. The concentration of oleic
acid and linoleic acids are also shown in
Table 4. Renin inhibitory cereals containrelatively high concentration of theseunsaturated free fatty acids. The
concentration of oleic acid and linoleic
acid seem to be correlated with rh-renin
inhibitory activity. However the existenceof other unknown renin inhibitory
compound in cereals cannot be ruled out.
Renin inhibitor from wild vegetables
We tested renin inhibitory activityof methanol extracts from 34 wild

vegetables grew in Akita prefecture [28].
Among them, Aralia cordata (Udo)extract showed highest renin inhibitory
activity. Thus, we tried to identify renin

S. Takahashi, et al.

81

PDA inhibited rh-renin activity dosedependent manner with IC50 values of 46.3
and 51.2 μM, respectively. The effects ofKA and PDA on several proteases werealso investigated. Table 5 shows summaryof inhibition of proteases by KA and PDA.
KA and PDA strongly inhibited rh-renin
and porcine renin activities. Bothcompounds also weakly inhibitedbromeline. But they had no effect on otherproteases. These results indicate that KA
and PDA are renin-specific inhibitor from
A. cordata.

Fig. 5. Structures of (-)-pimera8(14), 15-dien-19-oic acid (A)and pimera-9(11), 15-dien-19oic acid (B), and (-)-kaur-16-en19-oic acid (C).
inhibitor from A. cordata. Fromlyophilized powder, we finally obtained apolar mixture of two isomers and a lesspolar compound by preparative TLC.
These compound were identified as an 8:2
mixture of (-)-pimera-8(14),15-dien-19oic acid and pimera-9(11),15-dien-19-oic
acid (PDAs, Fig 5 A and B), and (-)-kaur16-en-19-oic acid (KA, Fig 5C). KA andConclusion
In this study we isolated several
renin inhibitory compounds fromfoodstuffs. The isolated compounds aresoyasaponin I from soybean, oleic acidand linoleic acid from rice, and KA andPDA from A. cordata. As for renin
inhibitors from foodstuffs, safety is
guaranteed. These compounds will bebeneficial ingredients for a therapeutic
treatment against hypertension and relateddiseases.
Acknowledgement
This study was supported in part by
Grant-in-Aid for Scientific Research (no.
18580134 and no. 2038081) from theJapan Society for the Promotion of
Science and by a Grant for City Area
Program from the Ministry of Education,
Culture, Sports, Science, and Technology(MEXT) of Japan.

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Renin Inhibitors in Foodstuffs

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Communicated by Ueno Hiroshi