Soymorphins, novel μ opioid peptides derived from soy β-conglycinin β-subunit, have anxiolytic activities

Article abstract of DOI:10.1271/bbb.70516

Based on the amino acid sequence YPFV found in the soy β-conglycinin β-subunit, which is common to an opioid peptide human β-casomorphin-4, peptides YPFVV, YPFVVN, and YPFVVNA were synthesized according to their primary structure. On guinea pig ileum (GPI

Full text of DOI:10.1271/bbb.70516

Biosci. Biotechnol. Biochem., 71 (10), 2618–2621, 2007
Communication
Soymorphins, Novel ꢀ Opioid Peptides Derived from Soy
ꢁ-Conglycinin ꢁ-Subunit, Have Anxiolytic Activities
y
Kousaku OHINATA, Shun AGUI, and Masaaki YOSHIKAWA
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University,
Gokasho Uji, Kyoto 611-0011, Japan
Received August 13, 2007; Accepted September 13, 2007; Online Publication, October 7, 2007
[doi:10.1271/bbb.70516]
Based on the amino acid sequence YPFV found in the
soy ꢁ-conglycinin ꢁ-subunit, which is common to an
1 µM
3 µM
10 µM
opioid peptide human ꢁ-casomorphin-4, peptides YPF-
VV, YPFVVN, and YPFVVNA were synthesized ac-
cording to their primary structure. On guinea pig ileum
(GPI) assay, they showed opioid activity (IC50 = 6.0, 9.2
and 13 ꢀM respectively) more potent than human ꢁ-
casomorphins, and were named soymorphins-5, -6, and
-7, respectively. Their opioid activities on mouse vas
deferens (MVD) assay were less potent than on GPI
assay, suggesting that they are selective for the ꢀ opioid
receptor. Human ꢁ-casomorphin-4 and soymorphin-5
were released from the soy 7S fraction (ꢁ-conglycinin)
by the action of gastrointestinal proteases. Soymor-
phins-5, -6, and -7 had anxiolytic activities after oral
administration at doses of 10–30 mg/kg in the elevated
plus-maze test in mice.
1 min
Fig. 1. Typical Opioid Activity of Soymorphin-5 (YPFVV) on
Guinea Pig Ileum (GPI) Assay.
Ileum-contracting activity by electrical stimulation was dose-
dependently inhibited by soymorphin-5 at doses of 1–10 mM.
Key words: opioid peptide; ꢀ opioid receptor; anxiolytic
effect; soy protein; ꢁ-conglycinin ꢁ-subunit
GPI assay. Figure 1 indicates the typical suppressive
effect of YPFVV on electrically stimulated contractions
on GPI assay. YPFVVN and YPFVVNA also dose-
dependently inhibited ileum contractions (data not
shown). These results suggest that YPFVV, YPFVVN,
and YPFVVNA have opioid activities; they were named
soymorphins-5, -6, and -7 respectively. The opioid
activities of synthetic peptides derived from the soy ꢁ-
conglycinin ꢁ-subunit are summarized in Table 1. The
IC50 values of soymorphins-5, -6, and -7 on GPI assay
were 6.0, 9.2, and 13 mM respectively, and their opioid
activities were more potent than those of human ꢁ-
casomorphin-4, -5, and -6 (IC50 = 20, 14, and 25 mM,
respectively). There are three types of opioid receptors:
ꢀ, ꢃ, and ꢄ.5,6) The opioid activities of soymorphins were
larger on GPI assay than those on MVD assay,
suggesting that these peptides are selective for the ꢀ-
opioid receptor. The affinities for the ꢀ-opioid receptor
were 17, 39, and 47 mM respectively, and the rank order
of their affinities for the ꢀ receptor was consistent with
that of the opioid activities on GPI assay. Taking all this
Opioid is a chemical substance that has morphine-like
action. An opioid peptide, bovine ꢁ-casomorphin-7
(YPFPGPI), isolated from casein peptone, was the first
example of bioactive peptides released from food
proteins.1) We have reported that human ꢁ-casomor-
phin-4 (YPFV) and related peptides derived from human
ꢁ-casein also have opioid activities, though they are less
potent than their bovine counterparts.2) Of three soy ꢁ-
conglycinin subunits (ꢂ, ꢂ0, and ꢁ),3) the ꢁ-subunit has
the human ꢁ-casomorphin-4 sequence (YPFV). Longer
peptides, YPFVV, YPFVVN, and YPFVVNA, were
synthesized according to the primary structure of the ꢁ-
conglycinin ꢁ-subunit by the Fmoc strategy, and their
opioid activities were tested by guinea pig ileum (GPI)
and mouse vas deferens (MVD) assay, as previously
described.4) All experiments were approved by the
Kyoto University Ethics Committee for Animal Re-
search Use.
The peptides were found to have opioid activities on
y
To whom correspondence should be addressed. Tel: +81-774-38-3725; Fax: +81-774-38-3774; E-mail: yosikawa@kais.kyoto-u.ac.jp
Abbreviations: i.p., intraperitoneal; p.o., per os; GPI, guinea pig ileum; MVD, mouse vas deferens; LAP, leucine aminopeptidase

Soymorphins Have Anxiolytic Activity
2619
Table 1. Opioid Activities of Soymorphin-5, -6, and -7 Derived from the Soy ꢁ-Conglycinin ꢁ-Subunit by Guinea Pig Ileum (GPI) and Mouse Vas
Deferens (MVD) Assays, and Their Affinities for the ꢀ-Opioid Receptor
Opioid activity (IC50, mM)
Affinity (IC50, mM)
ꢀ receptor
Peptide
Sequence
GPI (ꢀ)
MVD (ꢃ)
Soymorphin-5
Soymorphin-6
Soymorphin-7
YPFVV
YPFVVN
YPFVVNA
6.0
9.2
50
32
50
17
39
47
13
ꢀ Receptor binding assay was performed essentially as described previously.4,9) The membrane from CHO-K1 cells expressed with human ꢀ opioid receptor
(PerkinElmer Life Sciences, Boston, MA) was used with 1 nM of [3H]-DAMGO (American Radiolabeled Chemicals, St. Louis, MO) as a radioligand. Non-specific
binding was determined in the presence of 10 mM DAMGO. The IC50 value is the concentration that inhibited the binding of radioligands by 50%.
Table 2. Enzymatic Release of Soymorphins from the 7S Fraction (ꢁ-conglycinin) of Soy Protein and the Peptide Fragment Corresponding to the ꢁ-
Conglycinin ꢁ-Subunit (318–333) by Pancreatic Elastase and Leucine Aminopeptidase (LAP)
Peptide yield (mol %)
Substrate
YPFV
YPFVV
YPFVVN
YPFVVNA
VIPAAYPFVVNATSNL
Soy ꢁ-conglycinin
ND
4.8
36.5
9.1
ND
ND
ND
ND
ND, Not detected. A synthetic peptide fragment (0.1 mg/ml) corresponding to the soy ꢁ-conglycinin ꢁ-subunit (318–333) or 10 mg/ml of the 7S fraction (ꢁ-
conglycinin) was digested with elastase (E/S ¼ 1=20, 4 h) and LAP (E/S ¼ 1=7, 2 h) at 25 ꢀC at pH 7.5. The digest of the fragment peptide was analyzed using an
HPLC system and 492 protein sequencer (Applied Biosystems), as previously described.9) For the 7S fraction digest, the LC/MS system (Mariner, Applied
Biosystems) was used. Separation was performed with a linear gradient of acetonitrile in water containing 0.1% formic acid and 0.01% trifluoroacetic acid (TFA) from
5 to 95% (v/v) for 8 min at a flow rate of 0.2 ml/min using an ODS column, YMC Pack ProC18 (50 mm ꢁ 2:0 mm, YMC., Kyoto, Japan). Peptide fragments were
detected by UV absorption spectrometer and electrospray MS in positive ion mode. Soymorphin concentrations in the digest were quantified based on the peak heights
of synthetic soymorphins.
together, it was found that soymorphins derived from the
soy ꢁ-conglycinin ꢁ-subunit are more potent ꢀ-opioid
peptides than are human ꢁ-casomorphins.
these conditions. We also digested the soy 7S fraction
(ꢁ-conglycinin), which was kindly provided by Fuji Oil
Co., Ltd. (Osaka Japan), with pancreatic elastase
followed by LAP (Table 2). Human ꢁ-casomorphin-4
and soymorphin-5 were detected at 4.8 and 9.1%. They
might have been produced in the gastrointestinal tract by
digestive enzymes.
It is known that opioids are associated with emotional
behavior.11) Hence we tested whether soymorphins have
anxiolytic activities using the elevated plus-maze test
in mice, as previously described.11,12) Intraperitoneally
(i.p.) administered soymorphins-5, -6, and -7 at doses of
3–10 mg/kg increased the percentage (%) of time spent
in the open arms (Fig. 2a–c), indicating that these
peptides have anxiolytic activities. The dose-response
curves for soymorphin-5 and -6 were bell-shaped. On
the other hand, YPFV (human ꢁ-casomorphin-4 or
soymorphin-4) and YPFVE (human ꢁ-casomorphin-5)
did not show an anxiolytic effect at doses of 10–100
mg/kg after i.p. administration (data not shown). For
anxiolytic activity, Val5 in soymorphin-5 might be better
than Glu5 in human ꢁ-casomorphin-5.
Orally administered soymorphins-5, -6, and -7 also
increased the percentage of time spent in open arms
(Fig. 2d–f), suggesting that they are orally active
peptides having anxiolytic activities. This suggests that
at least a part of soymorphin might be absorbed intact.
The minimum effective doses for anxiolytic activities of
soymorphins-5 and 6 after oral administration (10 mg/
kg) were 3-fold higher than those after i.p. injection
(3 mg/kg). In contrast, the minimum effective dose of
soymorphin-7 (30 mg/kg) after oral administration was
Opioid peptides having a Tyr-Pro-aromatic amino
acid sequence are selective for the ꢀ receptor. This is
consistent with the fact that soymorphins containing
the YPF sequence are ꢀ-selective. Casomorphins (e.g.,
human ꢁ-casomorphin-7, YPFVEPI),2) hemorphin
(YPWT) derived from hemoglobin,7) and endomor-
phin-1 and 2 (YPWF-NH2 and YPFF-NH2 respectively)5)
containing the Tyr-Pro-aromatic amino acid sequence in
these molecules, are also ꢀ-selective opioid peptides, of
animal origin. In contrast, opioid peptides of plant
origin, such as gluten exorphin4,8) and rubiscolin,9,10)
with the Tyr-Pro-non-aromatic amino acid sequence, are
selective for the ꢃ receptor. Soymorphins were the first
ꢀ-selective opioid peptides of plant origin to be
identified.
To determine whether soymorphins are released from
the soy ꢁ-conglycinin ꢁ-subunit containing the amino
acid sequence of YPFV (human ꢁ-casomorphin-4 or
soymorphin-4), a model peptide corresponding to the ꢁ-
conglycinin ꢁ-subunit (318–333) was synthesized and
digested with gastrointestinal proteases. Ala0-soymor-
phin-5 (AYPFVV), which is released from the model
peptide after digestion of pancreatic elastase (Elastin
Products, Owensville, MO) has less potent opioid
activity than soymorphin-5. Ala0-soymorphin-5 was
quantitatively converted to soymorphin-5 by digestion
of leucineamino peptidase (LAP, Sigma-Aldrich, St.
Louis, MO) with an overall yield of 36.5% (Table 2),
but, soymorphins-4, -6, and -7 were not released under

2620
K. OHINATA et al.
a. Soymorphin-5 (i.p.)
b. Soymorphin-6 (i.p.)
***
c. Soymorphin-7 (i.p.)
***
**
30
*
*
*
30
*
20
10
0
20
10
0
20
10
0
cont
3
10 30
cont
3
10
30
cont
3
10 30
SM-5 (mg/kg)
SM-6 (mg/kg)
SM-7 (mg/kg)
d. Soymorphin-5 (p.o.)
e. Soymorphin-6 (p.o.)
f. Soymorphin-7 (p.o.)
*
***
**
30
20
10
0
**
30
20
10
0
20
10
0
cont 10 30 100
SM-5 (mg/kg)
cont
3
10 30
cont
10
30
SM-6 (mg/kg)
SM-7 (mg/kg)
Fig. 2. Anxiolytic Activities of Soymorphins (SM)-5, -6, and -7 after i.p. and Oral Administration at Doses of 3–100 mg/kg in the Elevated Plus-
Maze Test in Male ddY Mice.
Soymorphin-5 (a,d), -6 (b,e), or -7 (c,f) was intraperitoneally (i.p., a–c) or orally (p.o., d–f) administered 30 min before the tests. Values are
presented as means ꢂ SEM (a–c: n ¼ 11{21, d–f: n ¼ 7{18). Analysis of variance (ANOVA) followed by Fisher’s tests were used to assess
differences among groups. ꢃ p < 0:05, ꢃꢃ p < 0:01, ꢃꢃꢃ p < 0:001 as compared with the saline control group.
10-fold higher than that after i.p. injection (3 mg/kg).
This might be explained by the larger molecular size of
soymorphin-7, possibly decreasing the ability to pene-
trate the gut-blood barrier. In addition, soymorphins-5,
-6, and -7 did not change total entry in the elevated plus-
maze test (data not shown), suggesting that soymorphins
did not change locomotor activity under our experimen-
tal conditions. Soymorphins-5, -6, and -7 did not show
any anti-nociceptive effect under these conditions (data
not shown). Therefore, the dose requirement for anx-
iolytic activity is different from analgesia.
We have reported that rubiscolin-6, a ꢃ-opioid peptide
derived from a large subunit of spinach D-ribulose-1,5-
bisphosphate carboxylase/oxygenase (Rubisco), has
anxiolytic activity at a dose of 100 mg/kg after oral
administration, and that its anxiolytic activity was
mediated through ꢅ1 and dopamine D1 receptors.12)
Soymorphin-5-induced anxiolytic activity was not in-
hibited by a D1 receptor antagonist SCH23390 (data not
shown), suggesting that the mechanism of anxiolytic
activity of ꢀ opioid soymorphin-5 might be different
from that of ꢃ opioid rubiscolin-6. It has been reported
that the stress-induced increase in norepinephrine
release in the CNS was attenuated by ꢀ agonists such
as morphine and ꢁ-endorphin.13) Further investigation
should reveal the mechanism of anxiolytic activity of ꢀ-
opioid soymorphins.
(e.g., cholesterol- and triglyceride-lowering effects),14,15)
some of which have been attributed to soy proteins and
peptides.14) Recently, it was reported that the intake of
soy protein and its digestion suppress stress levels in
humans.16) The anxiolytic effects of soymorphins de-
rived from soy protein might contribute to the stress-
lowering effects of soy. To the best of our knowledge,
anxiolytic peptides, ‘‘soymorphins,’’ are the first exam-
ple of ꢀ-opioid peptides derived from soy protein.
In conclusion, soymorphins-5, -6, and -7, correspond-
ing to the amino acid sequence in the ꢁ-conglycinin ꢁ-
subunit of soy protein, have opioid activities and are
selective for the ꢀ-opioid receptor in GPI and MVD
assays. We also identified enzymatic condition for
releasing soymorphins from soy protein by proteases
present in the gastrointestinal tract. Soymorphins
showed anxiolytic-like activities in the elevated plus-
maze test in mice, which were present when they were
administered orally (10–30 mg/kg) or i.p. (3 mg/kg).
Acknowledgments
This work was supported in part by Grants-in-Aid for
Scientific Research from the Japan Society for the
Promotion of Science, to MY and KO, and by a
PROBRAIN grant from the Bio-Oriented Technology
Research Advancement Institution, to MY.
Many bioactive peptides derived from soy protein
have been identified, and we have also reported an
immunostimulating peptide soymetide derived from soy
ꢁ-conglycinin.14) It is well-known that soy has many
beneficial effects in preventing lifestyle-related diseases
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