J. Biol. Macromol., 10 (3), 83-91 (2010)

ArticleRenin Inhibitory Activity in Rice and Cereals
Saori Takahashi1,*, Tetsuo Tokiwano2, Nao Suzuki1, Ikuko Kodama3,
Yuko Yoshizawa2, and Takeshi Gotoh4
1

Akita Research Institute of Food and Brewing
4-26 Sanuki, Arayamachi, Akita 010-1623, Japan
2
Faculty of Bioresource Sciences, Akita Prefectural University
241-438, Kaidobata-Nishi, Nakano, Shimoshinjo, Akita 010-0195, Japan
3
Agricultural Experiment Station
Akita Prefectural Agriculture, Forestry and Fisheries Research Center
34-1 Gennpachizawa, Aikawa, Yuwa, Akita 010-1231, Japan
4
Department of Engineering in Applied Chemistry
Graduate School of Engineering and Resources Science, Akita University
1-1 Tegata Gakuencho, Akita 010-8502, Japan
Received October 22, 2010; Accepted November 22, 2010

Our recent study showed the occurrence of renin inhibitory activity in nonglutinous rice. In the present study we screened for renin inhibitory activity in glutinous,
sake, and indica rice, as well as various cereals. Rice extracts tested in this study inhibited renin activity with IC50 values of 88-270 μg/ml. Glutinous rice extracts showed
higher renin inhibitory activity than other rice extracts. On the other hand, extractsof cereals such as buckwheat (soba), rye, and barnyard millet (hie) had renin inhibitory activity, but other cereals had no effects on renin activity. The buckwheat had thehighest renin inhibitory activity in this study. LC/MS analysis of rice and cereal extracts indicated that the major renin inhibitory compounds are oleic acid and linoleic
acid.
Key words: renin, inhibitor, rice, cereals, oleic acid, linoleic acid.
*

To whom correspondence should be addressed. Tel: +81-18-888-2000; Fax:
+81-18-888-2008; E-mail:saori@arif.pref.akita.jp

Introduction
Renin [EC 3.4.15.23] is a key enzyme of the renin-angiotensin system

84

Renin Inhibitor(s) in Rice and Cereals(RAS), the most important blood pressure

purification of recombinant human renin

control system in animals [1]. The enzyme(rh) renin expressed in E. coli and Sf-9 in-

is a highly specific aspartic proteinase

sect cells [13, 14]. Using rh-renin as a tar-

mainly synthesized in juxtaglomerular

get enzyme we screened various foodstuffs

cells in the kidney [2]. Renin catalyzes theand found that soybean and minor legumes

liberation of angiotensin I (AI) from plas-

exhibits renin inhibitory activity [13, 15].

ma substrate angiotensinogen. Angiotensin

The renin inhibitory compound in soybean

converting enzyme (ACE) cleaves AI to

was identified as soyasaponin I [16].

form an active peptide, angiotensin II (AII).

Moreover,On the other hand, chymase, a chymotryp-ture-function relationship of saponins on

sin-like serine proteinase has been reported

renin inhibition and revealed that the 3-O-

[3]. The enzyme is also known to form AII

β-D-glucopyranosiduronic moiety in sa-

from AI in vascular tissues [4-6]. Chymase

ponins (glucuronide saponins) is essential

has no activity in normal tissues and its

for renin inhibition [17]. More recently, we

activitymastfound rh-renin inhibitory activity in non-cell-stimulated tissues. Thus, chymase in-glutinous rice and identified renin inhibi-

hibitors seem to be highly safety, because

tors as oleic acid and linoleic acid [18].

is

only

detectedinthere is no chymase inhibitor target innormal tissues [3].

westudiedthestruc-In the present study, we screened forrenin inhibitory activity in various types of

AII activates the type-1 angiotensin

rice and cereal using rh-renin and re-receptor to induce aldosterone synthesis,

nin-specific substrate, and found renin in-which increases water and salt absorption

hibitory activity in rice and some cereals.

in kidney and increases blood pressure.
Thus, control of RAS is the major target ofMaterials and Methods

cardiovascular disease therapies. ACE in-

Materials

hibitory peptides in various foodstuffs have

KWK2136),

been demonstrated to control RAS in vitro

PEE3753), and rutin (Lot No. EPP5152)

and in vivo [7-10]. Although renin is thewere obtained from Wako Pure Chemical

most important enzyme in RAS, substan-

Industries Ltd. (Osaka, Japan). Four im-tial screening for renin inhibitors amongportant rice cultivars in Akita Prefecture,

foodstuffs has not been undertaken because

gluteinous rice strains (Tatsukokomochiof the complexity of the renin assay [11,and Kinunohada) and rice strains for sake12]. Recently, we developed a method of

brewing (Miyamanishiki and Akitasake-

Oleic acid (Lot No.
linoleic

acid

(Lot

No.

S. Takahashi et al.

komachi), grown in 2008 were obtained85

Preparation of Rice and Cereal Ex-

from Agricultural Experimental Station,

tractsSample powder (25 g) was

Akita Prefectural Agriculture, Forestry, andsoaked in 200 ml of methanol at room

Fisheries Research Center (Yuwa, Akita,

temperature for 1 h then centrifuged at

Japan). Indica rice was obtained from Ki-

10,000 x g for 30 min. The supernatanttokushiryo Co. Ltd. (Tokyo, Japan). Rye,

was evaporated to dryness, and the dry

foxtail millet (awa), and Chinese millet

matter was dissolved in 100% methanol for(kibi) were obtained from Ikegaya Co. Ltd.

renin inhibition assay.

(Shizuoka, Japan). Flour for bread, bar-Renin Activity

The rh-renin ex-

nyard millet (hie), and amaranthus werepressed in Sf-9 cells was prepared by theobtained from Yamamoto Koujisyouten Co.

method of Takahashi et al. [14]. The inter-Ltd. (Hyougo, Japan). Buckwheat (soba)

nally quenched fluorogenic (IQF) substrate

was obtained from Totachiya Co. Ltd.

for human renin,(Tokyo, Japan). Weak flour was obtained(Nma)-Ile-His-Pro-Phe-His-Leu*Val-Ile-T

from Nisshin Seifun Co. Ltd. (Tokyo, Ja-

hr-His-Lys-2,4pan).

-dinitrophenyl(Dnp)-D-Arg-D-Arg-NH2 (*,N-methylanthranyl

scissile peptide bond) and areferencecompoundcustom-synthesized

at

were
the

Peptide Institute (Osaka, Japan). Hydrolysis of the IQF
substrate at the Leu-Val bondwas

spectrophotometricallydetermined.

The

reaction

mixture contained 39 µl of 50
mM sodium phosphate buffer,
pH 6.5, 0.1 M NaCl, 0.02%
Fig. 1 Effects of Rice Extracts on rh-Renin Activity
rh-Renin was incubated with the indicatedamounts of Tatsukomochi (●), Kinunohada (◆),
Miyamanishiki (○), Akitasakekomachi (◇), and Indica rice (▲) extracts. Each result is the mean value
for triple determinations.

NaN3, 0.02% Tween 20, 1 µlof 1 mM IQF substrate solution in DMSO, 5 µl of inhibitor solution, and 5 µl of
rh-renin solution (5 µg/ml) ina total volume of 50 µl. The

86

Renin Inhibitor(s) in Rice and Cereals

reaction mixture was incubated at 37°C foracid as eluent (0.4 ml/min, a linear gradient

30 min and the reaction was terminated byfrom 70% to 80% acetonitrile for 10 min).

adding 0.2 ml of 0.1 M triethanolamine, pH

Five microliters of a 10 ppm solution of

9.5. The increase in fluorescence intensity

fatty acid samples in MeOH was injected.

was measured at an emission wavelength

Linoleic acid (m/z 279) and oleic acid (m/zof 440 nm upon excitation at 340 nm. The

281) were detected at tR 3.4 and tR 5.1 min,sample concentration required to inhibit

respectively.

50% of renin activity under the assay condition was taken as the IC50 value. One unitof renin inhibitory activity results in 50%
inhibition of 25 ng of rh-renin.

Inhibition of rh-Renin by Rice Extracts

Identification of Oleic Acid and Linolenic Acid by LC-ESI-MS

Results and Discussion

Nonglutinous rice is the mostimportant agricultural product in Japan,

LC-ESI-MS

especially in rice-producing counties in theanalysis was performed with an Agilent

Tohoku area. Recently, we found rh-renin

1100 Series HPLC system and API-2000

inhibitory activity in nonglutinous rice ex-mass spectrometer (Applied Biosystems)

tracts and identified the active compounds

in the negative mode. HPLC was carried

from Akitakomachi, the most importantout on a TSKgel ODS-100V (Tosoh, 3 µm,

rice cultivar in Akita Prefecture, as oleic

φ2.0 x 50 mm) column used at 40 °C withacid and linoleic acid. Moreover, theacetonitrile–H2O containing 0.1% formic

structure-function relationship of fatty ac-

S. Takahashi et al.

ids on rh-renin activity was also investi-

87

bitory activity as nonglutinous rice.

gated [18]. In the present study, we tested

The amount of rice extracts, IC50

the effects of glutinous, sake, and Indica

values, inhibitor units, and oleic acid andrice extracts on rh-renin activity to under-

linoleic acid contents are summarized instand the existence of renin inhibitors in

Table 1. The glutinous rice, Tatsukomochi

rice universally. As shown in Fig. 1, all ofand Kimunohada, gave large amounts of

the rice extracts tested in this study inhi-

extract with 10.0 and 10.1 mg/g powder,

bited rh-renin activity in a dose-dependent

respectively. The yields of sake rice andmanner. Among them, Tatsukomochi ex-

nonglutinous rice extract were 5.7 to 8.8

tract, the most important glutinous rice in

mg/g powder. Indica rice extract showed

Akita Prefecture, had the highest renin in-

the lowest yield of 3.7 mg/mg powder. Our

hibitory activity with an IC50 value of 88.0

previous study showed that oleic acid andµg/ml. Another glutinous rice extract, Ki-

linoleic acid are the major rice rh-renin in-

nunohada, also showed high renin inhibi-

hibitors [18]. Thus, we measured the con-

tory activity with an IC50 value of 125.0

centration of these free fatty acids in rice

µg/ml. On the other hand, sake and Indica

extracts. The IC50 values of rice extracts

rice extracts showed nearly the same inhi-

are correlated with the contents of oleic
acid and linoleic acid. To evaluate the rh-renin inhibitory activity per gram weight, we calculated renin inhibitor unit (IU)
per gram powder. Nonglutinous
rice had 4.04 to 9.47 U/mg of
powder. Glutinous rice, Tatsukomochi

andKinunohada,

showed 22.73 and 16.03 IU,
respectively. Glutinous rice had
two- to three-fold higher renin
Fig. 2 Effects of Cereal Extracts on rh-Renin
Activity
rh-Renin was incubated with the indicatedamounts of buckwheat (●), rye (▲), and barnyard millet (■) extracts. Each result is the meanvalue for triple determinations.

inhibitory activity than the otherstrains. Indica rice showed thelowest value of 2.74 IU. These
results showed that glutinous
rice extracts had higher renin

88

Renin Inhibitor(s) in Rice and Cereals

inhibitory activity than the other rice ex-values of buckwheat, barnyard millet, andtracts.

rye extracts were determined to be 173,
265, and 490 µg/ml, respectively. No other

Inhibition of rh-Renin Activity by Cereal Extracts

cereal extracts had rh-renin inhibitory ac-

For cereals, growth istivity. This is the first demonstration of re-possible in sterile land. Therefore, cold re-nin inhibitory activity in cereals. Table 2

gions and highlands can be used for indis-

shows a summary of the total extracts, IC50

pensable crops. Moreover, cereals are avalues, IU, and concentrations of oleic acidstaple food in many countries around theand linoleic acid. Buckwheat showed theworld, including Japan. In this study, we

highest renin inhibitory units among theused buckwheat (soba), rye, foxtail millet

renin inhibitory cereals. Renin inhibitory(awa), barnyard millet (hie), Chinese millet

cereals contain relatively high concentra-(kibi), and amaranthus as cereals. Flour fortions of oleic acid and linoleic acid. On thebread and weak flour were also used as aother hand, concentrations of these unsa-

control for rye. Among methanol extracts

turated fatty acids in non-renin inhibitoryof the different cereals studied, buckwheat

cereals are lower. The concentrations of

extract had the highest rh-renin inhibitory

oleic acid and linoleic acid seem to be cor-

activity followed by barnyard millet andrelated with rh-renin inhibitory activity.

rye extracts, as shown in Fig. 2. The IC50

Buckwheat contains several functional

S. Takahashi et al.

89

compounds such as hyperin, quercetin,

nical assistance. This study was supported

isoquercetin, or rutin [19]. Among these,in part by a Grant-in-Aid for Scientificrutin is a well-known functional compound

Research (No. 20380081) from the Japan

in buckwheat. This compound inhibits al-Society for the Promotion of Science.

pha-glucosidase

activity

[20].

Ru-

tin-containing foodstuffs are expected to be

References

a good source for the development of po-

1. Persson, P. B., (2003) Renin: origin, se-werful anti-diabetes functional foods [20].

cretion and synthesis. J. Physiol., 552,

Thus, we tested the effects of rutin on

667-671.

rh-renin activity. Rutin had no effects of

2. Khayat, A., Gonda, S., Sen, S., andrh-renin activity up to 1.0 mg/ml (data not

Smeby, R. R. (1981) Responses ofshown). As shown in Table 2, oleic acidjuxtaglomerular cell suspension toand linoleic acid are major renin inhibitory

variouscompounds in buckwheat. However, the157-167.

stimuli.

Hypertension,

3,

existence of other unknown rh-renin inhi-

3. Takai, S., Jin, D., and Miyazaki, M.

bitory compound(s) in buckwheat extract(2010) New approach to blockade ofcannot be ruled out. More detailed studies

the renin-angiotensin-aldosterone sys-

are necessary for the identification of renintem: Chymase as an important target

inhibitory compound(s) in cereals.

to prevent organ damage. J. Pharmacol. Sci., 113, 301-309.

Conclusion

4. Takai, S., Shiota, N., Yamamoto, D.,

In the present study, we investigated the

Okunishi, H., and Miyazaki, M.

effects of rice and cereal extracts on(1996) Purification and characteriza-

rh-renin activity and found that glutinous

tion of angiotensin II-generating chy-

rice exhibits higher renin inhibitory activi-

mase from hamster cheek pouch. Lifety than other rice extracts. Moreover,Sci., 58, 591-597.

buckwheat contains the highest renin inhi-

5. Takai, S., Shita, N., Sakaguchi, M., Mu-

bitory activity among the tested cereals.

raguchi, H., Matsumura, E., andThis is the first demonstration of renin in-

Miyazaki, M. (1997) Characterization

hibitory activity in cereals.

of chymase from vascular tissues. Clin.
Chim. Acta, 265, 13-20.

Acknowledgments
We thank Ms. Mika Hokari for tech-

6. Takai, S., Jin, D., Sakaguchi, M., andMiyazaki,M.

(1999)

Chy-

90

Renin Inhibitor(s) in Rice and Cereals

mase-dependent angiotensin II forma-

12. Murakami, K., Takahashi, S., Suzuki F.,tion in human vascular tissues. Circu-

Hirose, S., and Inagami, T. (1980) In-

lation, 100, 654-658.

termediate molecular weight renin and7. Okamoto, A., Hanagata, H., Matsumoto,renin binding protein(s) in the hog

E., Kawamura, Y., Koizumi, Y., andkidney. Biomed. Res., 1, 392-399.

Yanagida, Y. (1995) Angiotensin I

13. Takahashi, S., Ogasawara, H., Wata-

converting enzyme inhibitory activi-nabe, T., Kumagai, M., Inoue, H., andties of various fermented foods. Biosci.

Hori, K. (2006) Refolding and activa-

Biotechnol. Biochem., 59, 1147-1149.

tion of human prorenin expressed in

8. Vercruysse, L., Camp, J. V., andEscherichia coli: Application of re-

Smagghe G. (2005) ACE Inhibitory

combinant human renin for inhibitor

peptides derived from enzymatic hy-

screening. Biosci. Biotechnol. Bio-

drolysates of animal muscle protein: A

chem., 70, 2913-2918.

review. J. Agric. Food Chem., 53,
8106-8115.

14. Takahashi, S., Hata, K., Kikuchi, K-I.,
and Gotoh, T. (2007) High-level ex-

9. Wu, J., Aluko, R. E., and Nakai, S.

pression of recombinant human renin(2006) Structural requirements of an-

in Sf-9 cells: Rapid purification andgiotensin-I- converting enzyme inhi-characterization. Biosci. Biotechnol.

bitory peptides: Quantitative struc-

Biochem., 71, 2610-2613.

ture-activity relationship study of di-

15. Takahashi, S., Hori, K., Kumagai, M.,

and tripeptides. J. Agric. Food Chem.,and Wakabayashi, S. (2007) Human54, 732-738.

renin inhibitory activity in legumes. J.

10. Guang, C. and Phillips, R. D. (2009)

Biol. Macromol. 7, 49-54.

Plant food-derived angiotensin I con-

16. Takahashi, S., Hori, K., Shinbo, M.,verting enzyme inhibitory peptides. J.

Hiwatashi, K., Gotoh, T., and Yamada,

Agric. Food Chem., 57, 5113-5120.

S. (2008) Isolation of human renin in-

11. Haber, E., Koerner, T., Gage, L. B.,

hibitor from soybean: Soyasaponin I is

Kliman, B., and Purnode, A. (1969)

the novel human renin inhibitor from

Application of a radioimuuunosaasy

soybean. Biosci. Biotechnol. Biochem.,

for angiotensin I to the physiologic

72, 3232-3236.

measurements of plasma renin activity

17. Takahashi, S., Hori, K., Hokari, M.,

in normal human subjects. J. Clin.

Gotoh, T., and Sugiyama, T. (2010)

Endocrinol. Metab., 29, 1349-1355.

Inhibition of human renin activity by

S. Takahashi et al.

91

saponins. Biomed. Res. 31, 155-159.

pounds from buckwheat (Fagopyrum

18. Takahashi, S., Tokiwano, T., Hata, K.,

esculentum Möench) hull. J. Agric.

Kodama, I., Hokari, M., Suzuki, N.,

Food Chem., 45, 1039-1044.

Yoshizawa, Y., and Gotoh, T. (2010)

20. Li, Y. Q., Zhou, F. C., Gao, F., Bian, J.

The occurrence of renin inhibitor inS., and Shan, F. (2009) Comparative

Rice: Isolation, identification, andevaluation of quercetin and rutin as

structure-function relationship. Biosci.

inhibitor of α-glucosidase. J. Agric.

Biotechnol. Biochem. 74, 1713-1715.

Food Chem., 57, 11463-11468.

19. Watanabe, M., Ohshita, Y., and Tsushida T. (1997) Antioxidant com-Communicated by Ueno Hiroshi