J. Biol. Macromol., 7 (3), 49-54 (2007)

ArticleHuman Renin Inhibitory Activity in Legumes
Saori Takahashi*, Kazuyuki Hori, Masanori Kumagai, and Saburou Wakabayashi
Institute for Food and Brewing, Akita Prefectural Agricultural, Forestry, and Fisheries ResearchCenter, 4-26 Sanuki, Arayamachi, Akita 010-1623, Japan
Received August 6, 2007; Accepted August 17, 2007
The effects of minor legume (legumes except soybeans and peanuts) extracts on humanrenin activity were investigated.
this study.

All minor legume extracts showed renin inhibitory activity in

Minor legumes can be divided into two groups, high renin inhibitory (Group 1) andlow renin inhibitory (Group 2) groups.

Group 1 minor legumes include Vigna angularis, V.

anguiculata subsp. sesquipendalis, and Phascolus vulgaris.

Group 2 minor legumes include P.

coccineus var. albus, P. coccineus, Lablab purpureus, Canavalia gladiata, Vicia faba, and Pisum
sativum.

This is the first demonstration of renin inhibitory activity in minor legumes.

Key words: angiotensin; bean; inhibitor; legume; renin
controlled by several stimuli [1].

Introduction
The renin-angiotensin-aldosterone system

Renin

activity is also regulated by renin-binding(RAS) plays an essential role in blood pressure

protein (RnBP), an endogenous renin inhibitor

control in animals.

first isolated from porcine kidney [2-7].

Renin catalyzes theTheliberation of angiotensin I from plasma substrate

expression and characterization of recombinantangiotensinogen.

RnBPs has been reported [8-12].

The produced decapeptide

RnBP

angiotensin I is an inactive peptide activated byinhibits renin activity by forming a complex ofangiotensin converting enzyme (ACE).

renin, called high molecular weight renin [2,ACE cleaves C-terminal dipeptide fromangiotensin I.

The produced angiotensin II

13-15].
ACE has been used as a target enzyme inraises blood pressure by vasoconstriction as well

RAS for screening inhibitors because of its

as stimulation of the synthesis and release of

simple assay method; however, renin is a

aldosterone.

rate-limiting enzyme in RAS, so it was not used

Thus, renin is a key enzyme of RAS.
secretion of renin into the circulation is_________________________________*Corresponding author (Tel:+81-18-888-2000; Fax: +81-18-888-2008;
E-mail: saori@arif. pref.akita.jp).

The_________________________________
The abbreviation used are: RAS, reninangiotensin-aldosterone system; ACE,
angiotensin converting enzyme; rh,
recombinant human; RnBP, renin binding
protein; Nma, N-methylanthranyl; Dnp, 2, 4
dinitrophenol.

50

Renin Inhibitor in Legumes

because the measurement is very complicated.

been equilibrated with distilled water.

Recently, we expressed recombinant (rh) humancolumn had been washed with distilled water,

renin in Escherichia coli cells, refolded andand then the adsorbed materials were eluted withactivated by trypsin [16].

methanol.

Using rh-renin as a

The

The eluate was evaporated totarget enzyme, we screened the renin inhibitory

dryness, and the dry matter was dissolved in

activity in fermented soybean paste (miso) anddistilled water to evaluate the renin inhibitory

found that soybean, the major ingredient of miso,

activity.

had renin inhibitory activity [16].

In theThe hydrolysis of the fluorogenic substratepresent study, we screened renin inhibitory

at the Leu-Val bond was spectrophotometrically

activity in minor legumes (legumes except

determined. The reaction mixture contained 38

soybean and peanut) and found that they hadµl of 50 mM sodium phosphate buffer, pH 6.5,

renin inhibitory activity.

0.1 M NaCl, 0.02% NaN3, 0.02% Tween 20, 2 µl

Minor legumes areclassified into two groups, high renin inhibitoryof 1mM Nma-Ile-His-Pro-Phe- His-Leu-Val-Ile-(Group 1) and low renin inhibitory (Group 2)

Thr-His-Lys-DnpNH2 in DMSO, 5 µl of humangroups.

renin solution (5 µg/ml of 20 mM sodium
phosphate buffer, pH 6.5, 1 mM EDTA, 10 µM

Materials and Methods

leupeptin, 0.1% heat-inactivated bovine serum

Minor legumes were obtained from
Fukutane Co. Ltd. (Fukui, Japan).

The

albumin), and 5 µl of bean extracts in a total
volume of 50 µl. The reaction mixture wasfluorogenic substrate for human reninincubated at 37 °C for 120 min and the reaction

N-methylanthranyl (Nma)-Ile-His- Pro-Phe-

was terminated by adding 0.2 ml of distilled

His-Leu-Val-Ile-Thr-His-Lys-2, 4 dinitrophenyl

water.

(Dnp)NH2 was custom-synthesized at thewas measured at an emission wavelength of 440

Peptide Institute (Osaka, Japan).

nm upon excitation at 340 nm.

Recombinant

The increase of fluorescence intensity

The sample

human renin was prepared by the method ofconcentration required to inhibit 50% of renin

Takahashi et al. [16] or obtained from Cayman

activity under the assay condition was taken as(LA, USA).

the IC50 value.

Twenty-five grams of legumes were soaked

SDS-PAGE was performed by the method

in 250 ml of distilled water at room temperatureof Laemmli [17] using 5-20% polyacrylamide

overnight, autoclaved at 115 °C for 30 min andgel (E-T520L, ATTO, Tokyo, Japan).

then cooled to room temperature.

electrophoresis, the proteins were stained with

The sample

was homogenized in a food processor

After

Coomassie brilliant blue R-250.

(MX-T10G, TOSHIBA, Tokyo, Japan) andcentrifuged at 10,000 x g for 30min.

The

Results and Discussion

supernatant was applied to a Sep-Pac Vac C18

Fig. 1 shows the classification of minor

35 cc (Waters, Massachusetts, USA) that had

legumes. Minor legumes used in this study wereS. Takahashi, et al.

51(Family)(Genus)(Species)(Japanese name)

FabaceaeVigna

angularis

1. Tanba dainagon adzuki
2. Natsu wase adzuki

unguiculata subsp. sesquipendalis

3. Midori sasage
4. Akadane sanjaku naga sasage
5. Honkintoki namame
6. Toramaru uzuranamame
7. Naga uzuranamame

vulgaris

Group 1

Group 2

Phascolus

coccineus var. albus

8. Oosirohana namame

9. Oobenihana namame

coccineus

10. Shirohana wase fujimame

Lablab

purpureus

Canavalia

gladiata

Vicia

faba

12. Wase soramame
13. Ninntoku issun soramame

Pisum

sativum

14. Usui endou
15. Tsurunasi fusanari endou

11. Shiro natamame

Fig. 1. Classification of Minor Legumes. Minor legumes classified into six groups.
classified as 6 genera, Vigna, Phascolus, Lablab,recombinant human renin, we evaluate the renin

Canavalia, Vicia, and Pisum. In the present

inhibitory activity of the legume extracts.

study, we used 15 minor legumes including 6

interestingly, all minor legume extracts showed

genera for renin inhibitory activity.

human renin inhibitory activity, as shown in Fig.

The renin inhibitory activities of 15 legume

2.

Very

Partitional clustering analysis [18] of theextracts were tested using rh-renin as the target

inhibitory data clearly showed that minor

enzyme and newly developed human renin

legumes could be classified into two groups

substrate.

according to their renin inhibitory activity, aIn our previous study, we used hog

angiotensinogen as a substrate and angiotensin I

high renin inhibitory group (Group 1) and low

radioimmunoassay to evaluate human renin

renin inhibitory activity group (Group 2). Group

inhibitory activity.

The assay method was very

1 legumes included Vigna angularis (Tanba

sensitive but we need a relatively long time of 2

dainagon adzuki and Natsu wase adzuki), V.

days to measure renin activity.

anguiculata subsp. sesquipendalis (Midori

With thepresent assay method using a synthetic substrate,sasage and Akadane sanjaku naga sasage), andwe can measure human renin activity within a

Phascolus vulgaris (Honkinntoki namame,

few hours.

Toramaru uzuranamame, and Naga

Using a synthetic substrate and52

Renin Inhibitor in Legumes

protein pattern.

Group 2

60

40

20

Mucna prurienus

Molecular weight marker
1. Tanba dainagin adzuki
2. Natsu wase adzuki
3. Midori sasage
4. Akadane sanjaku naga sasage
5. Honkinntoki namame
6. Toramaru uzura namame
7. Naga uzura namame
8. Ooshirohana namame
9. Oobenihana namame
10. Shirohana wase fujimame
11. Shiro natamame

Relat ive renin a ctiv ity (%)

Group 180

Molecular weight marker
11. Shiro natamame
12. Wase soramame
13. Ninntoku issun soramame
14. Usui endou
15. Tsurunashi fusanari endou

100

(kDa)
)

2)
,9

(0

5)

0)

.7

15
n

(1

14
n

B

B

ea

13
n

ea

12
n

ea

ea
B

11

ea
B

B

n

(1

.0

8)

5)

.7

(1

(0

.3

7)

0)
.9

(0

0)
.4

.6
10

(1

(0
9

n
ea
B

ea

n

7)

2)
.4

(0

B

8

ea
B

n

7)

7
n

.2
(0

6

.2
(0

5
ea

ea
B

ea

ea

B

ea
B

B

B

n

6)

6)
.3

(0
n

2)

4
n

.4
(0

(0
3

n

.3

7)
2

.3
B

(0
1

n
ea

ea

B

C

n

on

tr

ol

0

97 →
66 →
45 →

Fig. 2. Inhibition of Human Renin Activity
by Minor Legume Extracts.
Legume extracts eluted from the
Sep-Pak Vac C18 35 cc column were used forrenin inhibition assay. The final
concentrations of the extracts were 0.2
mg/ml. Renin activity without legumeextracts was used as a control. Beans 1 to 15
are the same as bean names in Fig. 1. Beans 1
to 15 extracts (n = 5) showed significant
difference compared to control at p<0.05 by
student's t-test. IC50 values of bean extracts(mg/ml) were indicated in the parentheses.

30 →20.1 →
14.4 →

Group 1

Group 2

Fig. 3. SDS-PAGE of Minor Legume
Extracts. Water extracts (10µl) oflegumes were loaded on the gel. Afterelectrophoresis, gels were stained with
Coomassie Brilliant Blue R-250.

The grouping of minor legumes correlated
uzuranamame).

Group 2 legumes included P.

very well with their protein pattern, shown incoccineus (Ooshirohana namame), P. coccineus

Fig. 3. IC50 values of bean extracts werevar. albus (Oobenihan namame), Lablab

determined to be 0.27-0.42 mg/ml for Group 1

purpureus (Shirohana wase fujimame),and 0.67-1.75 mg/ml for Group 2, respectively.

Canavalia gladiata (Shiro natamame), Vicia

Our previous results showed that fermented

faba (Wase soramame and Ninntoku issun

soybean, miso, had renin inhibitory activity

soramame), and Pisum sativum (Usui endou andderived from soybean.

Tsurunasi fusanari endou) (Fig. 2).

soybean renin inhibitor was estimated to be

The minor

The IC50 value of thelegume extracts showed several protein bands on

1.6-1.9 mg/ml [16].

SDS-PAGE (Fig. 3).

comparable to those of minor legumes.

Samples 1 to 7 showed

These values are
To ourmajor 30 kDa protein bands and minor proteinsknowledge, this is the first demonstration ofnearly the same mobility, but samples 8 to 15

renin inhibitory activity in minor legumes.

showed various proteins with different motilities.

preliminary results showed that the molecular

These results suggest that beans could beweights of renin inhibitory compound(s) in

divided into two groups according to theirminor legumes were 500-1,500.

The

Our

S. Takahashi, et al.

identification of legume renin inhibitor may

53

3. Inoue, H., Fukui, K., Takahashi, S., andhave scope to develop functional foods for

Miyake, Y., Molecular cloning and sequence

regulating blood pressure.

analysis of a cDNA encoding a porcine
kidney renin-binding protein. J. Biol. Chem.,

Conclusion

265, 6556-6561 (1990).

Minor legumes, especially adzuki, have

4. Inoue, H., Takahashi, S., Fukui, K., andbeen used for traditional sweets for several

Miyake, Y., Genetic and molecular

centuries in Japan. In the present study, we

properties of human and rat renin binding

discovered renin inhibitory activity in minor

protein with reference to the function of thelegume extracts.

leucine zipper motif. J. Biochem., 110,

Among them, adzuki had

relatively high renin inhibitory activity.

The

identification of active compound(s) will493-500 (1991).
5. Inoue, H., Takahashi, S., and Miyake Y.,

provide a possibility to develop antihypertensive

Modulation of active renin secretion by reninfunctional foods using legumes.

binding protein (RnBP) in mouse pituitary
AtT-20 cells transfected with

Acknowledgments

human renin and RnBP cDNAs. J. Biochem.,

We thank Ms. M. Sugawara and A. Omiya
for technical assistance.

The screening of renin

inhibitory activity in legumes was examined in
the course of research for the Japan Bean Fund
Association.

This study was supported in partby Grants-in-Aid for Scientific Research (No.
18580134) from the Japan Society for the
Promotion of Sciences.
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Communicated by Ueno Hiroshi