Capsinoid is biosynthesized from phenylalanine and valine in a non-pungent pepper, Capsicum annuum L. cv. CH-19 sweet

Article abstract of DOI:10.1271/bbb.50665

The biosynthetic pathway of capsinoid in 'CH-19 Sweet' was investigated. [³H]Valine and [¹⁴C]phenylalanine were injected into the fruits of the intact plant. Both of radioactivities were detected in capsinoid fractions. ¹⁴

Full text of DOI:10.1271/bbb.50665

Biosci. Biotechnol. Biochem., 70 (6), 1513–1516, 2006
Note
Capsinoid Is Biosynthesized from Phenylalanine and Valine
in a Non-Pungent Pepper, Capsicum annuum L. cv. CH-19 Sweet
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Kouzou SUTOH, Kenji KOBATA, Susumu YAZAWA, and Tatsuo WATANABE
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Graduate School of Nutritional and Environmental Sciences, University of Shizuoka,
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2-1 Yada, Shizuoka 422-8526, Japan
2
Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
Received December 12, 2005; Accepted February 2, 2006; Online Publication, June 23, 2006
[doi:10.1271/bbb.50665]
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The biosynthetic pathway of capsinoid in ‘CH-19
adenosylmethionine ([ C]-SAM) (sp. act., 52.7 mCi/
mmol) were purchased from Moravek Biochemicals
(Brea, CA, USA). Vanillin and dithiothreitol (DTT)
were purchased from Wako Pure Chemicals (Osaka,
Japan). Capsiate and dihydrocapsiate were enzymati-
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Sweet’ was investigated. [ H]Valine and [ C]phenyl-
alanine were injected into the fruits of the intact plant.
Both of radioactivities were detected in capsinoid
fractions. 14C radioactivity was observed in phenyl-
propanoid compounds, and in vanillin, vanillylamine,
vanillyl alcohol, and vanillic acid. We confirmed that
capsinoid is biosynthesized from phenylalanine and
valine.
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cally synthesized in our laboratory. The remaining
chemicals were obtained from Sigma-Aldrich Japan
(Tokyo, Japan).
For [ C]vanillin synthesis, [ C]-SAM (500 ml), 8 mM
,4-dihydroxybenzaldehyde (50 ml), 50 mM DTT (30 ml),
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Key words: Capsicum annuum; CH-19 Sweet; biosyn-
thetic pathway; tracer experiment; capsinoid
1.6 M MgCl (20 ml), catechol O-methyltransferase (0.3
mg, 300 U, Sigma), and 1 M phosphate buffer pH 9.7
2
(15 ml) were mixed into a 1.5 ml volume light-shielding
Recently, three homologs of nonpungent compounds,
capsiate, dihydrocapsiate, and nordihydrocapsiate, have
been isolated from the fruits of a nonpungent cultivar of
Treff microtube. The mix solution was incubated at
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37 C at 100 rpm for 48 h. [ C]Vanillin was purified by
high performance liquid chromatography (HPLC), as
described below, and gave 43% yield.
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Capsicum annuum cv. CH-19. They were fatty acid
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esters of vanillyl alcohol named capsinoid. We
conjectured that capsinoid and capsaicinoid have a
close relation in their biosynthetic pathway because
capsinoid has a remarkable structural resemblance to
capsaicinoid except for their center linkage: an amide
moiety in capsaicinoid and an ester moiety in capsinoid.
Moreover, ‘CH-19 Sweet’ plants have been selected and
fixed from a pungent cultivar of Capsicum annuum cv.
The experiment was carried out under 5,500 lux of
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fluorescent lamp illumination at 26 C. [ H]Valine and
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[ C]phenylalanine, each 2 mCi, or [ C]vanillin, 0.3
mCi, were injected directly into the loculus of fruits
using a 100 ml Hamilton microsyringe (#710). The fruits
were harvested at 1, 2, 3, 5, and 10 h after injection in
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the case of [ H]valine and [ C]phenylalanine, and 5 h
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after injection in the case of [ C]vanillin, and were
immediately freeze-dried.
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CH-19 which contains much capsaicinoid.
In the present study, an in vivo experiment was
performed by injection of radiolabelled valine and
phenylalanine into ‘CH-19 Sweet’ fruits of intact plants.
A further in vivo experiment administered radiolabelled
vanillin into ‘CH-19 Sweet’ and pungent cultivar of
Capsicum annuum cv. Takanotsume fruits.
The freeze-dried fruits were separated into placenta,
pericarp, and seeds, and each tissue was ground in a
mortar. The placenta was first extracted twice with 5 ml
of ethyl acetate each time for 1 h because capsinoid
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has high solubility and stability against ethyl acetate.
Subsequently, the residue was extracted twice with 5 ml
of 80% ethanol each time for 1 h to obtain phenolics.
The pericarp and seed were extracted twice with 5 ml of
80% ethanol each time to obtain phenolics. Each
extractant was evaporated to dryness, and placenta
extracts were dissolved in 300 ml of 80% ethanol for
HPLC. The pericarp and seeds extracts were dissolved
Fruits 20–30 d from anthesis (DFA) were used for
most experiments because the period of active accumu-
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lation of capsinoid is 15–30 DFA in summer.
All chemicals were of analytical reagent grade. L-
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[
alanine (sp. act., 396 mCi/mmol), and S-[methyl- C]-
3,4- H]Valine (sp. act., 30 Ci/mmol), L-[U- C]phenyl-
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To whom correspondence should be addressed. Tel: +81-54-264-5543; Fax: +81-54-264-5550; E-mail: watanbt@u-shizuoka-ken.ac.jp
Abbreviations: CAP, capsaicin; CST, capsiate; DC, dihydrocapsaicin; DCT, dihydrocapsiate; DTT, dithiothreitol; HPLC, high performance liquid
chromatography; SAM, S-adenosylmethionine; VOH, vanillyl alcohol

1514
K. SUTOH et al.
Fig. 1. The Conversion Rate (%) of [ H]Valine and [14C]Phenylalanine into Capsiate (CST) and Dihydrocapsiate (DCT) in Placenta Tissue in Vivo.
3
A, H CST; B, H DCT; C, 14C CST; D, 14C DCT. Lines show the average of data. Number of experiments: 1 h, 1; 2–10 h, 2–4. , CST;
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,
DCT.
in 500 ml of 80% ethanol for liquid scintillation count-
ing. To obtain the lignin-like substance fraction, the
residues of each tissue were extracted with a mixture of
widely among the various tissues of the fruits, but total
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recovery was in the same range of 45–55% in H and
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C. The total recovery in the fruits did not reach 100%.
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2
0 ml distilled water, 100 ml gracial acetic acid, and
50 mg NaClO2 at 70 C for 6 h.
HPLC was performed using a Shimadzu SCL-6B
It is thought that [ H]valine and [ C]phenylalanine
moved into other plant parts. But, some radioactivity of
H was detected in the lignin fraction of parts of leaves
and stems of ‘CH-19 Sweet’ plant (data not shown).
The conversion rate of [ H]valine and [ C]phenyl-
alanine into capsinoid, i.e., capsiate (CST) and dihy-
drocapsiate (DCT), was calculated by the radioactivity
of capsinoid fraction per total radioactivity detected in
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liquid chromatograph system with a Fluofix IEW 425
column (250 mm ꢁ 4:6 mm i.d., Wako Pure Chemicals)
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at 40 C. A cold standard sample was injected simulta-
neously with a hot sample to clarify the retention times
of compounds. Fractions of L-phenylalanine, cinnamic
acid, p-coumaric acid, ferulic acid, vanillin, capsiate,
and dihydrocapsiate were obtained under the following
conditions: eluent, acetonitrile and 0.1% aqueous tri-
fluoroacetic acid; gradient, 0–30 min, 7% acetonitrile,
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the placenta (Fig. 1). The conversion rate of H and
C
into CST and DCT increased up to 3 h after injection,
and showed the same values at 10 h. The higher
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conversion rate of C than of H at all time points
except for that into DCT at 10 h after injection suggests
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0–70 min, 7–100% acetonitrile linear gradient; flow
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rate 0–30 min, 0.5 ml/min, 30–70 min, 1 ml/min; detec-
tion, 0–50 min UV 236 nm, 50–70 min UV 280 nm.
Fractions of caffeic acid, vanillylamine, vanillyl alcohol,
and vanillic acid were obtained under the following
conditions: eluent, acetonitrile and 0.1% aqueous tri-
fluoroacetic acid; gradient 0–15 min, 0% acetonitrile,
15–30 min, 0–30% acetonitrile; flow rate, 1 ml/min;
detection, UV 236 nm.
The HPLC fractions, the pericarp and seed extracts,
and lignin-like substance extracts were dissolved in 5–
that [ C]phenylalanine is taken more easily in capsinoid
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fraction than [ H]valine. Although the natural ratio of
CST and DCT in the fruits of ‘CH-19 Sweet’ is about
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2:1, the incorporation ratio of [ H] and [ C] into CST
was lower than that of DCT in this in vivo experiment.
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Iwai et al. have reported that dihydrocapsaicin (DC)
content became higher than capsaicin (CAP) as an effect
of exogenously added vanillylamine and isocapric acid
in in vivo experiments, but that the natural abundance
ratios of CAP and DC were about 2:1. Therefore,
exogenously added precursors probably have some
effect on capsinoid biosynthesis.
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0 ml of Ultima Gold liquid scintillation counter cock-
tail (Perkin Elmer Japan, Yokohama, Japan) and their
radioactivities were measured by LSC-5100 (Aloka,
Tokyo, Japan).
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Figure 2 shows the conversion rate of [ C]phenyl-
alanine into phenylpropanoid compounds, vanillin,
vanillylamine, vanillyl alcohol, vanillic acid, CST,
and DCT in placenta tissue. The radioactivity of
Radioactivities in organic solvent-soluble and lignin-
like substance fractions of each tissue of ‘CH-19 Sweet’
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fruits after injection of [ H]valine and [ C]phenyl-
alanine were measured with a liquid scintillation
counter. Recovery of radioactivity at 1–10 h varied
[ C]phenylalanine decreased gradually after injection.
The conversion rate of all precursor candidates increased
up to 5 h after injection.

Biosynthetic Pathway of Capsinoid in ‘CH-19 Sweet’
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Fig. 2. The Conversion Rate (%) of [14C]Phenylalanine into Phenylpropanoid (A) and Vanillyl Compounds (B) in Placenta Tissue in Vivo.
Number of experiments: 1 h, 1; 2–10 h, 2–4. Phe, phenylalanine; Cin, cinnamic acid; Cou, p-coumaric acid; Caf, caffeic acid; Fer, ferulic acid;
V, vanillin; VA, vanillylamine; VOH, vanillyl alcohol; Vacid, vanillic acid.
Table 1. The Conversion Rate (%) of [14C]Vanillin into Vanillyl-
amine (VA), Vanillyl Alcohol (VOH), Vanillic Acid (Vacid), Capsiate
whereas vanillyl alcohol was produced in both pungent
and sweet pepper fruits. This result suggests that
(CST), Dihydrocapsiate (DCT), Capsaicin (CAP) and Dihydrocapsai-
cin (DC) at 5 h in Placenta Tissue of ‘CH-19 Sweet’ or ‘Takanotsume’
Fruits in Vivo
capsinoid biosynthesis in sweet fruit was caused by a
defect in vanillylamine synthesis from vanillin. Since
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the radioactivity of C was detected in the vanillyl-
CH-19 Sweet
Takanotsume
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amine fraction in the [ C]phenylalanine injection
experiment, however, we are now trying to identify
genes encoding capsinoid or capsaicinoid synthetase
to clarify what gene mutation accounts for capsinoid
production in ‘CH-19 Sweet’ pepper.
Compound
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2
V
VA
0.73
nd
2.02
nd
1.04
3.38
15.27
2.66
nd
2.15
7.25
8.70
5.64
nd
VOH
Vacid
CST
DCT
CAP
DC
10.03
4.73
3.77
3.35
nd
0.58
2.33
12.03
5.14
nd
In conclusion, the early part of the biosynthetic
pathway of capsinoid is the same as that of capsaicinoid.
nd
nd
0.13
0.08
8.97
4.69
nd
nd
Acknowledgments
Duplicate data are shown as Nos. 1 and 2, separately.
nd, not detected.
We thank Dr. Yuhji Yamamoto, Ms. Kumiko Arai,
and Dr. Tadahiro Tadokoro of Tokyo University of
Agriculture for technical advise on HPLC measurement
of SAM and for providing us with an authentic SAM.
As Fig. 1 and 2 show, capsinoid is synthesized from
valine and phenylalanine as starting precursor of the
biosynthetic pathway in a manner similar to capsaici-
noid, and phenylpropanoid pathway compounds, viz.,
cinnamic acid, p-coumaric acid, caffeic acid, and ferulic
acid, participate in capsinoid synthesis. Furthermore,
vanillin appears to be a key precursor as a branch point
in the biosynthesis pathway between capsinoid and
capsaicinoid.
References
1
)
Kobata, K., Todo, T., Yazawa, S., Iwai, K., and
Watanabe, T., Novel capsaicinoid-like substances, cap-
siate and dihydrocapsiate, from the fruits of a nonpungent
cultivar, CH-19 Sweet, of pepper (Capsicum annuum L.).
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2
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Kobata, K., Sutoh, K., Todo, T., Yazawa, S., Iwai, K., and
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Nat. Prod., 62, 335–336 (1999).
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the correlation between capsinoid and capsaicinoid in
biosynthesis, an additional experiment was performed
14
by injection of [ C]vanillin into ‘CH-19 Sweet’ or
pungent cultivar of Capsicum annuum ‘Takanotsume’
3) Yazawa, S., Suetome, N., Okamoto, K., and Namiki, T.,
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(in Japanese), 58, 601–607 (1989).
14
fruits of intact plants. [ C]Vanillin was not incorporated
into phenylpropanoid compounds at all in either ‘CH-19
14
Sweet’ or ‘Takanotsume.’ In ‘CH-19 Sweet,’ [ C]va-
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14
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14
capsinoid. It is interesting that [ C]vanillin was con-
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K. SUTOH et al.
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