J. Biol. Macromol. 11(3), 83-89 (2011)

ArticleIsolation and Identification of Human Renin Inhibitor from Aralia cordata (Udo)
Saori Takahashi,1,* Tetsuo Tokiwano,2 Keishi Hata,1 Mika Hokari,1
Yuko Yoshizawa,2 and Takeshi Gotoh3
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, Japan3
Department of Applied Chemistry, Akita University, 1-1 Tegata Gakuen-cho, Akita 010-8502, Japan
Received August 08, 2011: Accepted October 22, 2011
We screened for human renin inhibitory activity in wild vegetables and found the activity inmethanol extract of Aralia cordata. Physico-chemical data on the isolated inhibitors were identical tothose of (–)-kaur-16-en-19-oic acid (kaurenic acid, KA) and pimaradienoic acids (PDA, 8:2 mixture ofpimara-8(14), 15-dien-19-oic acid: pimara-9(11) 15-dien-19-oic acid). KA and PDA also inhibited porcine
renin but had nearly no effect on other proteinases. This is the first demonstration of renin inhibitors inwild vegetables.
Key words: renin, inhibitor, Aralia cordata, kaurenic acid, pimaradienoic acid.

Introduction
Renin (EC 3. 4. 23. 15) is a highly specific aspartic
proteinase mainly synthesized by juxtaglomerularcells in the kidney cortex. The enzyme catalyzes
the liberation of decapeptide angiotensin I (AI)from plasma substrate angiotensinogen. This is thefirst step of the renin-angiotensin (RAS) system, awell-characterized physiological modulator ofblood pressure and electrolyte balance [1]. The
produced AI is an inactive peptide and activatedby angiotensin converting enzyme (ACE, EC 3. 4.
15. 1). ACE removes a dipeptide His-Leu from the
C-terminus of AI. The produced octapeptide
giotensin II (AII) directly acts on arterial*Corresponding author: Saori Takahashi.
Phone: +81-18-888-2000
Fax: +81-18-888-2008
E-mail: saori@arif.pref.akita.jp

smooth muscle cells to maintain blood pressure.
AII also accelerates the release of aldosterone
from the adrenal cortex to induce sodium retention
in the kidney. This also induces high bloodpressure. Thus, control of RAS is the major targetof cardiovascular disease therapies [2, 3].
ACE inhibitory peptides in various
foodstuffs have been demonstrated to control RAS[4-6]. Renin is one of the most important enzymes
in RAS. However, the screening for renin
inhibitors among foodstuffs has not been wellstudied because of the need for preparation ofhuman renin and the complication of the reninassay. Recently, we screened for renin inhibitory
compounds in various foodstuffs using
recombinant human (rh)-renin as a target enzyme[6-10] and found that fermented soybean paste
miso contains a renin inhibitory compound,soyasaponin I, which is derived from soybean[10].
More recently, we found renin inhibitory
activity in rice and cereals [11, 12]. The purified
rice inhibitor preparation contained two
unsaturated fatty acids, oleic acid and linoleic acid[11]. In the present study, we screened the extractsof 34 wild vegetables from Akita prefecture, Japan,
for renin inhibitory activities. The screening
showed that some wild-vegetable extracts had
renin inhibitor activities. Methanol extract ofAralia cordata showed the highest renin inhibitory
activity. We isolated active compounds from A.
cordata and the chemical structures of the purified
renin inhibitors were identified as kaurenic acidand pimaradienoic acids.

N-methylanthranyl-(Nma)-Ile-His-Pro-Phe-HisLeu*Val-Ile-Thr-His-Nε-2, 4-dinitrophenyl-Lys(Lys(Dnp))-D-Arg(r)-r-HN2 (*, scissile peptide
bonds) [14]. The reaction mixture contained 40 μlof 50 mM sodium phosphate buffer, pH 6.5,
containing 0.1 M NaCl, 25 μM IQF substrate,
0.02% Tween 20, 5 μl of renin solution (5 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 M triethanolamine, pH 9.5.
The increase of fluorescence intensity wasmeasured at an emission wavelength of 440 nm
upon excitation at 340 nm. The sample
concentration required to inhibit 50% of renin
activity under the assay conditions was taken as
the IC50 value.

Materials and Methods
Preparation of inhibitor solution andphysico-chemical analysis Freeze-dried wild
vegetables were extracted with methanol andevaporated to dryness. The evaporated sample wasdissolved in methanol at a final concentration of
10 mg/ml. The sample was used for measuringrenin inhibitory activity. Isolated compounds wereanalyzed by ESI-HR-MS, 1H (400 MHz) and 13C(100 MHz) NMR, and [α]D measurements.

Materials Porcine pepsin (Lot SDK5232),
bromeline (Lot DPG0653), bovine trypsin (Lot
TSQ4876) were obtained from Wako Pure
Chemical (Osaka, Japan). Subtilisin (Lot
20K1585), and rabbit lung ACE (Lot 056K74251)
were from Sigma (St. Louis, MO, USA). Papain(Lot DL739221) was obtained from MP
Biomedicals (Illkirch, France). Aminopeptidase M(Lot DL739221) was from Pierce (Rockford, IL,
USA). Fluorogenic, internally quenched
fluorogenic (IQF), and peptidyl-pNA substrates
were obtained from Peptide Institute (Osaka,
Japan). Porcine kidney renin was purified by apreviously reported method [13]. Recombinanthuman (rh)-renin expressed in Sf-9 insect cells
was prepared by the method of Takahashi et al[14].

Results and Discussion
Screening of renin inhibiton We tested
renin inhibitory activity of methanol extracts from34 wild vegetables (Fig. 1). Among them, Pilea
hamanoi (Mizu), Souchus asper (Oninogeshi), A.
elata (Taranoki), and A. cordata (Udo) extracts(final concentration of 0.1 mg/ml) inhibited renin
activity by more than 20% compared with thecontrol and A. cordata extract had the highest
renin inhibitory activity. Thus, we tried to purify
renin inhibitory compounds from A. cordata.

Renin activity The rh-renin activity wasmeasured using internally quenched fluorogenic(IQF) substrate:2

Co n tro l
An e mo n e flac c ida (Nirin so u )Alliu m grayi (No biru )S ambu c u s sie bo ldian a (Niwato ko )He me ro c allis lo n gitu ba (No kan zo u )S milax o ldh ami (S h io de )Ro dge rsia po do ph ylla (Yagu ru maso u )Ho sta lan c ifo lia (K o bagibo u sh i)
S c e ptro c n ide mac ro stac h ya (Miyamairaku sa)
Eryth ro n iu m japo n ic u m (K ataku ri)
P u e raria th u n be rgian a (K u zu )O smu n da japo n ic a (Ze n mai)
Ho sta sie bo ldian a (O o bagibo u sh i)

Wild ve ge table s

De n an th e sto lo n ife ra (S e ri)
De n taria marc ro ph ylla (K o n ro n so u )An ge lic a e du lis (Maru bae zo n yu u )Cirsiu m japo n ic u m (No azami)
Cirsiu m pu rpu ratu m (Fu jiazami)
Matte u c c ia stru th io pte ris (K o go mi)
Cac alia h astata (Ho n n a)
Vio la vagin ata (S u mire saish in )Ru me x japo n ic u s (Gish igish i)
P e tasite s japo n ic u s (Fu ki)
P te ridiu m aqu ilin u m (Warabi)
Wasabi japo n ic a (Wasabi)
Co n do n o psis lan c e o lata (Tsu ru n in jin )Astilbe o do n to ph ylla (To riash ish yo u ma)
P o lygo n u m c u spidatu m (I tado ri)
An th risc u s n e mo ro sa (K o sh yaku )Cac alia de lph in iifo lia (Mo mijigasa)
P ile a h aman o i (Mizu )S o n c h u s aspe r (O n in o ge sh i)
Aralia e lata (Taran o ki)
Aralia c o rdata (Udo )

0

20

40

60

80

100

Relative renin activity (%)

Fig. 1. Inhibition of rh-renin activity by wild-vegetable extracts.
Crude methanol extracts of wild vegetables were used for rh-renin inhibition assay. The final
concentration of the extracts was 0.1 mg/ml. Each result is the mean value of triplicate determinations.

3

Purification and identification of renin
inhibitors from A. cordata (Udo)Freeze-dried A. cordata powder (75 g) was
soaked in 1.2 liters of methanol at room
temperature for 1 h then centrifuged at 10,000 x g
for 30 min. The supernatant was evaporated todryness, and the dry matter (8.1 g) was dissolvedin 240 ml of 100% methanol. Insoluble materials
were removed by centrifugation and thesupernatant was evaporated to dryness (5.7 g). A
part of the sample (1 g) was dissolved in 10%
methanol and applied to Sep-Pak Vac C18 35cc
(Waters, Bedford, USA) that had been equilibrated
with distilled water. The column was washed with
distilled water and 50% methanol. The adsorbed
materials were eluted with 100% methanol. The
eluate was evaporated to dryness, and the dry
matter (0.116 g) was dissolved in 10 ml of 100%
methanol. The sample was put on a Sephadex
LH-20 column (2.0 x 60 cm) equilibrated with
100% methanol. The column was eluted with thesame solvent and fractions containing rh-renin
inhibitory activity were pooled. The active
fractions were evaporated to dryness (21.6 mg).
The dry matter was purified by silica gel
chromatography (hexane–EtOAc) to afford a 4:4:1
mixture of three compounds (31.1 mg), whichwere separated by PTLC (Merck RP-18, 100%
acetonitrile) into a polar mixture of two isomers
and a less polar compound. These compounds
were identified as an 8:2 mixture of(–)-pimara-8(14), 15-dien-19-oic andpimara-9(11), 15-dien-19-oic acids (PDAs, Fig. 2
B and C), and (–)-kaur-16-en-19-oic acid (KA, Fig.

A

B

C

Fig. 2. Chemical structures of(–)-kaur-16-en-19-oic acid (KA) (A),pimara-8(14), 15-dien-19-oic acid (B), andpimara-9(11), 15-dien-19-oic acid (C).

2A), on the basis of HR-MS, NMR, and []D data.
The 1H and 13C NMR shifts were identical to thosereported in the literature [15, 16] (Fig. 2).
Fig. 3. Effects of KA and PDA on rh-renin activity.
The rh-renin was incubated with the indicatedamount of KA (-○-) and PDA (-●-). Each result isthe mean value for triplicate determinations.

4

Table 1. Effects of KA and PDA on Proteinase Activities.
_________________________________________________________________________
Enzyme*1
Substrate*2
Reaction
Inhibition (%)*3
bufferKA
PDA_________________________________________________________________________
Aspartic protease
Human renin(0.5 µg/ml)

Porcine renin(0.5 µg/ml)

Porcine pepsin(0.5 µg/ml)

Nma-Ile-His-Pro-PheHis-Leu-Val-Ile-HisThr-Lys(Dnp)-r-r-NH2(20 µM)
Nma-His-Pro-Phe-HisLeu-Leu-Val-TyrLys(Dnp)-r-r-NH2(20 µM)
MOCAc-Gly-Lys-ProIle-Leu-Phe-Phe-ArgLeu-Lys(Dnp)-r-NH2(20 µM)

Buffer A*4

79.0

90.8

Buffer A*4

74.4

88.1

Buffer B*5

N. I.

N. I.

Serine protease
Bovine trypsin
Pro-Phe-Arg-MCA
Buffer C*6
N. I.
N. I.
(1.0 µg/ml)(0.2 mM)
Subtilisin
Suc-Ala-Ala-Ala-pNA
Buffer D*7
N. I.
N. I
(10 ng/ml)(0.2 mM)
Cysteine protease
PapainPro-Phe-Arg-MCA
Buffer E*8
N. I.
N. I.
(10 ng/ml)(0.2 mM)
Bromeline
Pro-Phe-Arg-MCA
Buffer E*8
18.4
9.9(2.0 µg/ml)(0.2 mM)
Metallo proteaseRabbit ACE
Hip-His-Leu
Buffer F*9
N. I.
N. I.
(1 mU/ml)(2.0 mM)
Aminopeptidase M
Leu-MCA
Buffer G*10
N. I.
N. I
(0.2 µg/ml)(0.2 mM)___________________________________________________________________________*1
Values in parentheses indicate final enzyme concentrations; *2 Values in parentheses indicate
final substrate concentrations; *3 The ratios of enzyme inhibition in the presence of 0.15 mM
of KA or PDA are indicated; *4 50 mM Na-phospahte, pH 6.5, 0.1 M NaCl, and 0.02% Tween
20; *5 0.1 M Na-citrate, pH 3.0, 0.1 M NaCl, and 0.02% Tween 20; *6 50 mM Tris-HCl, pH 7.5,
and 0.02% Tween 20; *7 50 mM Tris-HCl, pH 8.0, and 0.02% Tween 20; *8 20 mM Na-phosphate,
pH 6.0, and 0.02% Tween 20; *9 0.1 M HEPES, pH7.5, 0.3 M NaCl, and 0.01% Triton X-100;*1050 mM Tris-HCl, pH 7.5, and 0.02% Tween 20.
N.I., no inhibition.

5

Effects of KA and PDA on renin and otherproteinases
The crude methanol extract of A. cordata
inhibited rh-renin activity in a dose-dependent
manner with IC50 value of 130 µg/ml. The isolated
KA and PDA inhibited rh-renin activity strongly
with IC50 values of 14.0 and 15.6 µg/ml (46.3 and51.2 µM), respectively (Fig. 3).
Table 1 shows the inhibitory spectra of KA
and PDA. KA and PDA strongly inhibited rh-renin
and porcine renin activities. KA and PDA also
weakly inhibited cysteine proteinase bromeline
but they had no effects on pepsin, bovine trypsin,
subtilisin, papain, rabbit ACE, or aminopeptidase
M. These results indicate that KA and PDA are
renin-specific inhibitors from A. cordata.
Some physiological functions of KA andPDA have been reported. These compounds
exhibit pharmacological activities such as
analgesic, sedative, and anti-inflammatory effects[15, 17]. Moreover, the effects of PDA and its
related compounds on large-conductance K+
channels were examined using human embryonickidney 293 cells. The measurement of BKαβ1
opening under whole-cell voltage clamp showedthat these compounds activated BKαβ1 in asimilar manner [18]. On the other hand, effects ofthese compounds on proteinases have not beenreported.
In conclusion, this is the first demonstrationof renin inhibitory activities of KA and PDA. To
understand the anti-hypertensive effects of KA and
PDA, animal experiments using spontaneously
hypertensive rats or Dahl salt-sensitive rats arenecessary.

References[1] Persson, P. B. (2003) J. Physiol., 552, 667-671.
[2] Studdy, P. R., Lapworth, R., and Bird, R.
(1983) J. Clin. Pathol., 36, 938-947.
[3] Sleight, P. (2002) Eur. Heart J. (Suppl. A), 4,
A53-A57.
[4] Vercruysse, L., Camp, J. V., and Smagghe , G.
(2005) J. Agric. Food Chem., 53, 8106-8115.
[5] Wu, J., Aluko, R. E., and Nakai, S. (2006) J.
Agric. Food Chem., 54, 732-738.
[6] Guang, C., and Phillips, R. D. (2009) J. Agric.
Food Chem., 57, 5113-5120.
[7] Takahashi, S., Ogasawara, H., Watanabe, T.,
Kumagai, M., Inoue, H., and Hori, K. (2006)
Biosci. Biotechnol. Biochem., 70, 2913-2918 .
[8] Takahashi, S, Hori, K, Kumagai, M, andWakabayashi S. (2007) J. Biol. Macromol., 7,49-54.
[9] Takahashi, S, Hori, K, Shinbo, M, Hiwatashi,
K, Gotoh, T, and Yamada, S. (2008) Biosci.
Biotechnol. Biochem., 72, 3232-3236.
[10] Takahashi, S., Hori, K., Hokari, M., Gotoh, T.,
and Sugiyama, T. (2010) Biomed. Res., 31,
155-159.
[11] Takahashi, S., Tokiwano, T., Hata, K.,
Kodama, I., Hokari, M., Suzuki, N.,
Yoshizawa, Y., and Gotoh, T. (2010) Biosci.
Biotechnol. Biochem., 74, 1713-1715.
[12] Takahashi, S., Tokiwano, T., Suzuki, N.,
Kodama, I., Yoshizawa, Y., and Gotoh, T.
(2010) J. Biol. Macromol., 10, 83-91.
[13] Takahashi, S., Ohsawa, T., Miura, R., andMiyake, Y. (1983) J. Biochem., 93, 265-274.
[14] Takahashi, S., Hata, K., Kikuchi, K-I., andGotoh, T. (2007) Biosci. Biotechnol.
Biochem., 71, 2610-2613.
[15] Okuyama, E., Nishimura, S., and Yamazaki,
M. (1991) Chem. Pharm. Bull., 39, 405-407.
[16] Kim, Y. H., Chung, B.S., and Sankawa, U.
(1988) J. Nat. Prod. 51, 1080-1083.
[17] Lim, H., Jung, H. A., Choi, J. S., Kim, Y. S.,
Kang, S. S., and Kim, H. P (2009) Arch.

Acknowledgments
This study was supported in part by aGrant-in-Aid for Scientific Research (No.
20380081) from the Japan Society for the
Promotion of Science.

6836-846.

Pharm. Res., 32, 1237-1243.
[18] Imaizumi, Y., Sakamoto, K., Yamada, A.,
Hotta, A., Ohya, S., Muraki, K., Uchiyama,M., and Ohwada, T. (2002) Mol. Pharm., 62,

Communicated by Ohnishi Masatake7