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ArticleCite This: ACS Omega 2018, 3, 17287−17294

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Accumulation and Subcellular Localization of Acteoside in Sesame
Plants (Sesamum indicum L.)
Yushiro Fuji, Takashi Ohtsuki, and Hiroshi Matsufuji*
Department of Food Bioscience and Biotechnology, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa,
Kanagawa 252-0880, Japan

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S Supporting Information*

ABSTRACT: The localization of acteoside and its precursors in
diﬀerent organs of sesame plant (Sesamum indicum L.), includingroots, stems (epidermis, cortex, and stele), leaves (blades andpetioles), ﬂower petals, and seeds (immature and mature) wasinvestigated. The highest acteoside content was found in leaf
blades (12.3% dry weight), followed by in petioles (3.1%) andpetals (2.7%) and at a trace levels or not detected in other organs.
In contrast, the known precursors of acteoside, cinnamate, pcoumarate, caﬀeate, tyramine, tyrosol, salidroside, L-3,4-dihydroxyphenylalanine, dopamine, and hydroxytyrosol were not detectedin any organs, even by multiple-reaction monitoring analysis byliquid chromatography−electrospray ionization tandem mass
spectrometry, despite acteoside being present at high levels in
the leaves. The leaves stained with diphenylborinic acid 2aminoethyl ester (DPBA), producing the acteoside−DPBA complex that was detected by ﬂuorescence at an excitation
wavelength of 365 nm, showed accumulation of acteoside at the veins and glandular trichomes. For the ﬁrst time, the leaf blade
organ was shown to contain a large amount of acteoside in the sesame plant, with acteoside synthesis in the leaf petioles and/or
blades and accumulation in the glandular trichomes with transport through the veins. However, tyrosine and phenylalanine
contents were very low with no downstream metabolites or known acteoside precursors detected in any organs.

■leaves, ﬂowers, and fruits,7 and it shows diﬀerent localization toother organs of diﬀerent plants. For example, in Echinacea
pallida, acteoside was shown to accumulate in ﬂowers morethan in leaves and roots.8 In Plantago majorL., the acteosidecontent was highest in ﬂower stalks (2.30 mg g−1 DW; 0.23%)
followed by seeds (2.28 mg g−1), young leaves (0.02 mg g−1),
and old leaves (0.006 mg g−1).9 Although there are nocomprehensive reports on the distribution of acteoside in S.
indicum L., Potterat et al.10 isolated acteoside from the root
bark of Sesamum angolense (Pedaliaceae family) andGrab̨ kowska et al.11 reported that the acteoside and isoacteoside contents were higher in leaves and stems than in roots in
Harpagophytum procumbens (Pedaliaceae family).
Recent pharmacological studies of acteoside have shown
extensive biological activities, such as antioxidant,12 antiinﬂammatory,13 and hepatoprotective activity,14 in addition toprotective properties against neurological diseases, such as
Alzheimer’s15,16 and Parkinson’s diseases.17 Therefore, acteoside is expected to be useful for pharmaceutical applications,but commercial-scale production remains unattainable becauseof the requirements for large-scale evidence-based human

INTRODUCTION
Sesame plants (Pedaliaceae family, Sesamum indicum L.) aregrown in the tropical or subtropical regions for their seeds,
which are one of the world’s most important oil crops. The
seeds have been well studied for their sesame-speciﬁc lignans,such as sesamin and sesamolin, and their chemical composition
and functional properties.1,2 In contrast, very little information
is available on the composition of the other organs of sesame
because cultivation has primarily been for seed production; theother parts, while large in quantity, are agricultural byproducts.3 Recently, dried powder of young sesame leaves
from plants grown to a height of 30−70 cm (about 40−60 days
after planting) was commercially produced as a health foodsupplement in Japan. In previous reports, we showed thatyoung sesame leaves contain three iridoids and eight
polyphenols, and among them, acteoside shows strong
antioxidant activity and accumulation in the leaves during
growth. The acteoside content reached a maximum of 12.9% of
dry weight content in leaves at the 4th stage (plant height, 74.5
± 9.7 cm), which is one of the highest levels reported in plants
in nature (Figure 1).4,5
Acteoside, also called verbascoside, is widely distributed in
dicotyledonous plants, which include over 150 plant species
belonging to 20 families and 77 genera.6 It is not speciﬁc to
any plant organ, being present in plant roots, bark, stems,© 2018 American Chemical Society

Received: October 14, 2018
Accepted: December 3, 2018
Published: December 13, 2018
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ArticleFigure 1. Chemical characterization of sesame leaves and changes in the major compound content at diﬀerent growth stages. (A) Representative
HPLC chromatogram of sesame leaves. (B) Chemical structures of polyphenols and iridoids in sesame leaves. (C) Changes in content at diﬀerent
growth stages.5

Figure 2. HPLC chromatograms of 60% MeOH extracts from diﬀerent organs of the sesame plant. I1, lamalbid; I2, sesamoside; I3, shanzhiside
methyl ester; P2, chlorogenic acid; Ped, pedaliin; Act, acteoside; and P6, isoacteoside.

corresponding genes remain to be identiﬁed and incorporated
into this system.
Sesame plants have enormous biosynthetic potential for
producing pharmaceutically valuable acteoside and may
therefore be an attractive bioproduction resource. In thisstudy, we clariﬁed the distribution of acteoside and its
precursors in diﬀerent organs of the sesame plant to gain
basic information on acteoside biosynthesis. Further, subcellular localization of acteoside by ﬂuorescence imaging with
diphenylborinic acid 2-aminoethyl ester (DPBA) was examined. Our ﬁndings may provide information that will lead tothe use of sesame leaves as natural sources of acteoside andmay be helpful for elucidating acteoside biosynthesis in sesame
plants.

studies. Extractions from plant sources have very low yield
because of the low content in plants (about 0.02−0.4% in mostplants, rarely ∼3.0%), variability in the plant growth
conditions, losses in the puriﬁcation steps to remove
contaminants, and so on. On the other hand, a complete
chemical synthesis of acteoside has been developed,18,19 but
the complex 15-step procedure required due to the speciﬁc
structure of acteoside also results in a low yield of 4.4−7.1%.
Recently, some biotechnological approaches using nongenetically modiﬁed plant cell cultures have been reported,20,21 but
eﬀective mass production and development of eﬃcient cells
have not yet been achieved because acteoside biosynthesis,
several downstream metabolites, key enzymes, and their
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ArticleTable 1. Contents (% DW) of Major Polyphenols and Iridoids in Diﬀerent Organs of the Sesame Planta
organ
leaf
compounds
Iridoidlamalbid
sesamosideshanzhiside methyl
ester
Polyphenol
chlorogenic acidpedaliin
acteosideisoacteoside

stem

root

ﬂower

seed

blade

petioleepidermis

cortex

stele

root

petal

immature

0.27 ± 0.20
1.37 ± 1.31
0.15 ± 0.08

1.29 ± 0.10
0.33 ± 0.28
0.86 ± 0.04

0.52 ± 0.01
0.80 ± 0.01
3.65 ± 0.01

0.34 ± 0.03
0.35 ± 0.01
0.51 ± 0.01

5.31 ± 0.02
2.63 ± 0.01
1.65 ± 0.03

0.06 ± 0.01
0.57 ± 0.01
0.19 ± 0.01

6.23 ± 0.20
3.40 ± 0.03
1.09 ± 0.20

7.04 ± 0.03
2.98 ± 0.02
0.62 ± 0.04

0.15 ± 0.05
1.37 ± 0.13
12.3 ± 0.97
0.82 ± 0.15

0.19 ± 0.01
1.34 ± 0.08
3.07 ± 0.48
0.38 ± 0.03

0.03 ± 0.01
0.91 ± 0.03
0.15 ± 0.03
0.28 ± 0.04

tr.
tr.
tr.
tr.

0.13 ± 0.05
tr.

0.35 ± 0.07
tr.
2.66 ± 1.70
0.59 ± 0.38

0.23 ± 0.01
tr.
0.84 ± 0.60
tr.

tr.

matureMean ± standard deviation (SD; n = 9); tr. <0.003%. Blanks are “not detected”. Cinnamate, p-coumarate, caﬀeate, tyrosol, salidroside, andhydroxytyrosol were not detected in all organs.

a

Table 2. Contents (μg g−1 DW) of Free Amino Acids and Amines in Diﬀerent Organs of the Sesame Planta
organ
root

ﬂower

blade

petioleepidermis

cortex

stele

root

petal

immature

mature121.4 ± 2.4
40.6 ± 2.71167 ± 28.3

68.3 ± 1.5
32.4 ± 2.7
141.6 ± 1.6

47.2 ± 0.6
7.0 ± 0.1
78.6 ± 0.9

15.6 ± 0.5
4.0 ± 1.0
22.4 ± 1.2

46.8 ± 2.1
7.0 ± 0.1
93.4 ± 3.8

4.0 ± 0.9279 ± 7.9
tr.
tr.
11.1 ± 0.8
38.1 ± 0.5
37.2 ± 2.1
tr.
5.4 ± 1.0
18.8 ± 0.7
107.4 ± 1.6
31.9 ± 0.4
tr.
23.3 ± 1.7
234.9 ± 3.1

243.4 ± 8.1
tr.
2.1 ± 0.9

85.9 ± 1.8
tr.

249.6 ± 8.4
tr.
13.9 ± 1.5
18.8 ± 0.7
5.2 ± 0.1
18.0 ± 3.1
tr.
tr.
15.7 ± 0.6
32.2 ± 1.5
22.6 ± 1.2
tr.
17.5 ± 0.1

345.8 ± 4.5
315.4 ± 2.9
916.6 ± 15.6
19.4 ± 0.3
1934.1 ± 25.2
32.8 ± 0.6
64.7 ± 3.6
93.6 ± 0.8
230.6 ± 7.0
191.6 ± 3.5
43.8 ± 3.0
103.6 ± 3.1
168.2 ± 6.8
452.0 ± 12.4
168.8 ± 2.3
122.6 ± 5.8
169.2 ± 3.6
1387.3 ± 33.4

29.6 ± 1.1
62.0 ± 5.1
151.0 ± 2.0

1645.6 ± 41.3
tr.
3.1 ± 0.1
37.6 ± 1.4
62.0 ± 1.6
75.5 ± 3.5
tr.
25.9 ± 2.6
54.4 ± 1.4
200.2 ± 4.3
120.6 ± 4.4
10.2 ± 2.7
65.5 ± 0.4
357.5 ± 58.3

227.3 ± 10.4
103.6 ± 0.6
378.1 ± 9.9
19.3 ± 0.2
672.3 ± 30.4
22.7 ± 0.3
231.5 ± 8.3
103.7 ± 1.3
178.7 ± 4.9
122.4 ± 4.1
20.3 ± 1.0
214.5 ± 9.1
4025.0 ± 32.1
218.1 ± 49.4
126.7 ± 7.9
63.8 ± 2.1
209.4 ± 3.0
192.6 ± 8.4

leaf
compounds
Amino Acidalaninearginine
aspartic acidcysteineglutamic acidglycinehistidineisoleucine
leucinelysinemethionine
phenylalanine
prolineserinethreoninetyrosinevalineGABA
Amine
tyramine
L-DOPA
dopamine

stem

tr.
4.9 ± 0.8

16.1 ± 0.1
29.1 ± 1.4
12.7 ± 0.7
tr.

tr.
10.6 ± 0.7
2.8 ± 0.6
3.5 ± 0.1

45.7 ± 1.5

5.8 ± 0.1
tr.

9.9 ± 0.6
1.7 ± 1.2

seed

373.9 ± 5.1
tr.
18.6 ± 0.2
22.2 ± 0.2
24 ± 1.7
13.6 ± 0.8
tr.
37.4 ± 1.1
20.7 ± 0.2
15.4 ± 0.7
7.1 ± 0.1
14.5 ± 3.2
24.2 ± 0.5
tr.

Mean ± SD (n = 9); tr. <0.5 μg g−1. Blanks are “not detected”.

a■

RESULTS
Distribution of Acteoside and Related Compounds inDiﬀerent Organs. First, we examined the distribution of
acteoside and its related compounds in diﬀerent organs of thesesame plant. Figure 2 shows the representative highperformance liquid chromatography (HPLC) chromatogramsof 60% MeOH extracts from diﬀerent organs: leaves (blades
and petioles), stems (epidermis, cortex, and stele), ﬂower
petals, roots, and seeds (immature and mature). Major peaks
were detected and identiﬁed as lamalbid, sesamoside, shanzhiside methyl ester, chlorogenic acid, pedaliin, acteoside, andisoacteoside based on the comparison of retention times andUV and LC−ESI-MS spectra with authentic or isolated
compounds. These compounds were previously found insesame leaves (blades and petioles) and were present in

diﬀerent organs, although additional unknown peaks weredetected in ﬂower petals (Unk-F1 and Unk-F2), roots (UnkR1 and Unk-R2), and immature seeds (Unk-IM1 and UnkIM2) but were not identiﬁed in this study. Sesamin andsesamolin, but not cinnamate, p-coumarate, caﬀeate, tyrosol,
salidroside, or hydroxytyrosol, which are downstream metabolites derived from tyrosine and phenylalanine by theshikimate pathway, were detected in all organs and even in
analysis of multiple-reaction monitoring (MRM) analysis withliquid chromatography−electrospray ionization tandem mass
spectrometry (LC−ESI-MS/MS).
The breakdown of the major compounds in diﬀerent organs
is given in Table 1. The leaf blade had the most acteoside (12.3± 1.0%), followed by petioles (3.1 ± 0.5%). This composition
breakdown was almost in agreement with that of the sesame
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Articleleaves grown in a greenhouse at the College of Bioresource
Sciences, Nihon University in 2016.5 Acteoside was present inlow levels in the epidermis of the stem and roots (0.15 ± 0.03
and 0.13 ± 0.05%, respectively) but not in the stele. It was alsopresent in the ﬂower petals (2.7 ± 1.7%) and immature seeds(0.84 ± 0.60%) but not in the mature seeds. Isoacteoside, anisomer of acteoside, showed almost the same distribution as
acteoside, although at lower levels than acteoside. Thus, large
amounts of acteoside in the leaves are assumed to besynthesized in the leaf petiole and/or blades but not in thestem and roots. Additionally, the breakdown in the ﬂower
petals suggested that the acteoside-biosynthesized system otherthan in the leaves would also exist in the ﬂower petals.
Other iridoids, lamalbid, sesamoside, and shanzhiside methyl
ester, were present mainly in stems, ﬂower petals, andimmature seeds but with diﬀerent distribution proﬁles ineach organ: lamalbid and sesamoside were present mainly inimmature seeds (7.04 ± 0.03 and 2.98 ± 0.02%, respectively),
ﬂower petals (6.23 ± 0.20 and 3.40 ± 0.03%, respectively), andstele (5.31 ± 0.02 and 2.63 ± 0.01%, respectively), whileshanzhiside methyl ester was found in the epidermis of thestem (3.65 ± 0.01%). In addition, sesamoside was present inlarger amounts in leaf blades than in petioles, but lamalbid andshanzhiside methyl ester were found more so in petioles thanin leaf blades. Pedaliin, a ﬂavonoid glycoside, was present inleaf blades (1.37 ± 0.13%), petioles (1.34 ± 0.08%), andepidermis (0.91 ± 0.03%) but was not detectable in otherorgans.
The free amino acid content as well as the amine precursorsof acteoside [tyramine, L-3,4-dihydroxyphenylalanine (LDOPA), and dopamine] in diﬀerent organs is summarized inTable 2. The units are expressed as μg g−1 (1 μg g−1 =0.0001%). For amino acids, glutamic acid was the major aminoacid in various organs (85.9−1934.1 μg g−1), except for rootand petals, and proline was dominant in roots (5.8 ± 0.1 μgg−1) and petals (4025.0 ± 32.1 μg g−1). γ-Aminobutyric acid
(GABA) was found to be present in higher amounts inimmature seeds (1387.3 ± 33.4 μg g−1), leaf blades (357.5 ±58.3 μg g−1), and petioles (234.9 ± 3.1 μg g−1) than in otherorgans. However, tyrosine and phenylalanine contents as wellas acteoside precursors derived from the shikimate pathway
were very low (tr. −122.6 μg g−1 and tr. −214.5 μg g−1,
respectively) as expected, and no tyramine, L-DOPA, or
dopamine, which are downstream metabolites of tyrosine andphenylalanine, were detected in any organs.
Localization of Acteoside in Leaf Blades. To investigate
the localization of acteoside in leaf blades, we ﬁrst subdivided
leaf blades horizontally or vertically and assayed the acteosidecontent. As a result, no diﬀerences were found among
subdivided leaf blades (data not shown), suggesting that
acteoside is distributed uniformly in whole leaves.
Next, we attempted to determine subcellular localization
using a DPBA ﬂuorescence stain method. DPBA (also called
Naturstoﬀ reagent or Neu’s reagent) reacts with compounds
such as ﬂavonoids containing o-diphenolic and 5-hydroxy-4keto groups.22 Before staining leaf blades with DPBA, theﬂuorescence responses of the major chemical compounds insesame leaves were tested against DPBA and compared withthose of authentic standards and isolated compounds. As
shown in Figure 3, controls, quercetin−DPBA and rutin−
DPBA complexes, showed yellow ﬂuorescence in accordancewith previous reports.22−24 Acteoside−DPBA complexes
showed sky blue ﬂuorescence upon UV irradiation at 365

Figure 3. Fluorescence response of DPBA complexes under UV
irradiation at 365 nm (A) or visible light (B). (a) Acteoside−DPBA
complex. (b) Caﬀeate−DPBA complex. (c) Hydroxytyrosol−DPBA
complex. (d) Pedalitin−DPBA complex. (e) Pedaliin−DPBA
complex. (f) Quercetin−DPBA complex. (g) Rutin−DPBA complex.

nm (maximum excitation (Ex) and emission (Em) wavelengthsof 400 and 475 nm), similar to that of the caﬀeate−DPBA
complex (Ex/Em = 390/470 nm) but diﬀerent from theultramarine color of the hydroxytyrosol−DPBA complex (Ex/Em = 335/385 nm), suggesting that the caﬀeoyl unit ofacteoside predominantly reacts with DPBA to produce a sky
blue ﬂuorescence complex. On the other hand, the pedalitin−
DPBA complex shows orange ﬂuorescence upon UV
irradiation at 365 nm (Ex/Em = 455/570 nm), whilepedaliin−DPBA shows a dark color despite the maximum
excitation and emission wavelengths being almost the same,
suggesting that the ﬂuorescence intensity of the ﬂavonoid
glycoside is weakened compared to that of the aglycone−
DPBA complex. No ﬂuorescence was observed in the mixturesof iridoids and DPBA (data not shown). Further, when the
mixture of acteoside and pedaliin in the concentration ratio(12:1 w/w) in the leaf blade was reacted with DPBA, theDPBA complex showed sky blue ﬂuorescence (Figure S1). On
the basis of these results, the DPBA method could be utilized
to examine subcellular localization of acteoside in sesame
leaves without the interference of other compounds (iridoids
and polyphenols) being in them.
The sesame leaf blade stained with DPBA after eliminating
chlorophyll showed sky blue ﬂuorescence in the whole leaf
upon UV radiation at 365 nm, while the leaf blade without
DPBA did not show ﬂuorescence (Figure 4A,B). Moreover,ﬂuorescence microscopy observations revealed that sky blue
ﬂuorescence occurred in veins and glandular trichomes (Figure
4C,D). Thus, for the ﬁrst time, acteoside was demonstrated to

Figure 4. Fluorescence imaging of sesame leaves stained with DPBA.
(A) Sesame leaves stained with (left) or without (right) DPBA undervisible light. (B) Sesame leaves stained with (left) or without (right)
DPBA upon UV irradiation at 365 nm. (C) Microscopic photographof veins (left) and trichomes (right) under visible light. (D)Fluorescence microscopy photograph of veins (left) and trichomes(right) under UV irradiation at 365 nm.
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Articleprecursors such as hydroxytyrosol, tyrosol, p-coumarate,caﬀeate, or hydroxytyrosol glycoside were found in olive
fruits26−28 and lilac ﬂowers,29 which contain a large amount ofacteoside (up to 9.0% in Coratina and Rocciola olive; less than
3.0% in other olive cultivars; 2.48% in lilac). Despite sesame
leaves having accumulated larger amounts of acteoside (about12%) than olive fruits and lilac ﬂowers, we could not identify
acteoside precursors in any organs of sesame plants. Thus,
there are two possible explanations for acteoside biosynthesis
in sesame plants: one is that synthesis occurs by a new,currently unknown pathway and the other is that key enzymes
are activated in the biosynthesis pathway. Further investigationis needed to clarify these possible explanations. So far, Liu etal.30 reported that production of acteoside increased 2- to 3fold when a cell suspension culture of Cistanche salsa was fed a
combination of phenylalanine and tyrosine. To clarify the
related genome, several transcriptional studies on acteosidebiosynthesis have been conducted on the cultured cells of Olea
europaea,31 Cistanche deserticola,32 and Rehmannia glutinosa.33
We plan to examine the production of acteoside byincorporating isotope-labeled amino acids and the major
genomic determinants of acteoside biosynthesis in sesame
plants using RNA-seq technology in vitro and in vivo.
This and our previous studies show that no acteoside is
detected in stele of the stem and provide some support for the
hypothesis that the acteoside biosynthesis system in sesame
plants is localized in leaf petioles and ﬂower petals. Althoughacteoside is present in many dicotyledonous plants, the organof accumulation diﬀers by plant species and the role ofacteoside is not fully elucidated. Glandular trichomes
accumulate a variety of secondary metabolites, such as
terpenes, fatty acid derivatives, alkaloids, and ﬂavonoids,
most of which protect the plant from abiotic and biotic
stresses.34,35 Therefore, acteoside also may protect the sesame
plant owing to its strong antioxidant activity. However, theextraordinarily high amounts that were found to accumulate in
the leaves (12.3% in this study; 12.9% in a previous study) areamong the highest levels reported in plants in nature. From theﬁndings that the major polyphenols in sesame leaves werelimited to acteoside and pedalitin (pedalitin-6-O-glucoside), a
ﬂavonoid biosynthesis-related gene may be present at a lower
copy number than in other plants, disrupted, or missing,resulting in the accumulation of a large amount of acteoside.
Considering another possibility, iridoids were found to be
major constituents in leaves in a previous report,5 and they
were present at higher levels in stems, suggesting that they aretransported to leaves through the steles of stems. Although thebiosynthesis pathways of lamalbid, sesamoside, and shanzhiside
methyl ester have been reported,36 there is no information
about the site of iridoid biosynthesis in S. indicum L. Further
investigation is required to unravel the complexity of thebiochemical pathways, such as transcriptome analysis to obtainrobust genomic data and identiﬁcation of the key enzyme.
In summary, we conclude that sesame plants have excellent
biosynthetic potential for acteoside. To use this bioproduction
resource of pharmaceutically valuable acteoside and sesame
plants, further investigation to elucidate the acteoside biosynthesis pathway is needed, such as experiments utilizing isotope
incorporation and in vitro and in vivo molecular biologicalapproaches.

pass thorough the veins and accumulate in the glandular
trichomes of sesame leaves.

■

DISCUSSION
To clarify the distribution of acteoside and its related
compounds in the sesame plant, exhaustive HPLC or LC−
ESI-MS/MS analyses for polyphenols, iridoids, amino acids,
and acteoside precursors were performed on all organs of thesesame plant, including leaves (leaf blades and petioles), stems(epidermis, cortex and stele), roots, ﬂower petals, and seeds(immature and mature). Acteoside was present in leaf blades at
the highest levels (12.3%), followed by in the petioles (3.1%).
Also, ﬂuorescence imaging observation of leaf blades stainedwith DPBA revealed that acteoside was present in the veinsand in the glandular trichomes of leaves. To the best of ourknowledge, there is no data for the subcellular localization of
acteoside in natural plants and we here demonstrate for theﬁrst time that acteoside accumulates in the glandular trichomesof sesame leaves. Thus, one can assume that acteoside insesame leaves is synthesized in the petioles and/or leaf blades
and accumulates in the glandular trichomes following passage
through the veins. However, our ﬁnding that known acteosideprecursors were not found or were detected at low levels in allorgans of sesame plants raises the question of how and fromwhich compounds acteoside is biosynthesized.
The three chemical components of acteoside are caﬀeate,saccharide (glucose and rhamnose), and hydroxytyrosol
aglycon. Although the downstream pathway of acteosidebiosynthesis remains to be fully elucidated, the components
of caﬀeate and hydroxytyrosol (or salidroside) are known to bebiosynthesized from phenylalanine and tyrosine, respectively,
which are derived from the shikimate pathway.25 The caﬀeoyl
moiety of acteoside is biosynthesized from phenylalanine via a
cinnamate pathway, whereas the hydroxytyrosol moiety isbiosynthesized from tyrosine with tyramine and/or dopamineintermediates (Figure 5). In survey studies, some acteoside

Figure 5. Proposed biosynthesis pathway of acteoside described inprevious reports.25
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ArticleEXPERIMENTAL SECTION
Chemical Reagents. Acteoside was obtained from BP
Biochemicals Inc. (San Diego, CA, USA). Pedalitin, pedaliin,
lamalbid, sesamoside, and shanzhiside methyl ester wereisolated previously.5 Hydroxytyrosol, DPBA, chloral hydrate,
glycerol, and amino acid mix standard (consisting of 17 aminoacids) were obtained from Sigma-Aldrich Co. (St. Louis, MO,
USA). Caﬀeate, quercetin, rutin, and GABA were from Wako
Chemical Industries (Osaka, Japan). AccQ•Tag Ultra
precolumn derivatization kit including borate buﬀer and
AQC derivatizing reagent were from Waters Co. (Milford,
MA, USA). All regents were used without further puriﬁcation.
Plant Materials. The sesame plants (S. indicum L. variety
Myanmar Black Sesame) were cultivated in a greenhouse at theCollege of Bioresource Sciences, Nihon University in June2017 as described in our previous report.5 Fresh leaves, stems,roots, and petals were collected at about 80 days after planting(plant height, about 70 cm; n = 9). The leaf blades and petioles
from leaves and the epidermis, cortex, and stele from stems
were separated manually and collected. Immature (white) andmature (black) seeds were collected at about 80−90 days
(plant height, 70−90 cm; n = 9) and about 90−110 days
(plant height, 120−150 cm; n = 9) after planting, respectively.
The plant organs are illustrated in Figure S2. After collection,organ samples were immediately frozen in liquid nitrogen,lyophilized, and ground using a mortar and pestle and thenkept at −20 °C until analysis. On the other hand, fresh leaf
samples (n = 3) for ﬂuorescence observation were collectedfrom plants with a height of about 70 cm and immediately
stained with DPBA as described below.
Polyphenol, Iridoid, and Acteoside Precursor Analysis. Polyphenol and iridoid analyses were performed inaccordance with methods described in our previous study.5 In
this study, the available aromatic precursors of acteoside(cinnamate, p-coumarate, caﬀeate, tyrosol, salidroside, andhydroxytyrosol), sesamin, and sesamolin were analyzed under
the same conditions. In brief, each dried organ (about 25 mg)
was extracted twice with 1 mL of 60% MeOH in a sonicating
bath (15 min, room temperature). The extracts were ﬁlteredthrough a 0.22 or 0.45 μm PVDF membrane ﬁlter andanalyzed with HPLC and/or LC−ESI-MS/MS. HPLC wasconducted using a Shimadzu Prominence system (Kyoto,
Japan) with a photodiode array detector and a Waters XBridge
C18 column (4.6 × 150 mm, 5 μm) and eluted with a linear
MeCN gradient in 0.1% HCOOH (5−35% MeCN in 15 min,35−100% MeCN in 25 min, and 100−5% MeCN in 1 min) ata ﬂow rate of 0.8 mL min−1 at 40 °C. The sample injection
volume was 10 μL, and the eluent was monitored at 234 and
340 nm. For LC−ESI-MS/MS, a Waters Quattro Premier XL
mass spectrometer was coupled to an ACQUITY UPLC
system. The compounds were separated on an ultraperformance liquid chromatography system with a Waters HSS T3
column (2.1 × 50 mm, 1.7 μm) and a linear MeCN gradient in
0.1% HCOOH (5−35% MeCN in 3 min, 35−100% MeCN in
5 min, and 100−5% MeCN in 1 min) at a ﬂow rate of 0.3 mL
min−1 at 40 °C. The ion source conditions were optimized asfollows: negative electrospray ionization; source temperature,
150 °C; capillary voltage, 3.5 kV; desolvation temperature, 500°C; ﬂow rate of desolvation gas, 1000 L h−1; and ﬂow rate ofcone gas, 50 L h−1. For MRM experiments to detect aromatic
precursors of acteoside, speciﬁc transitions of parent andproduct ions were monitored as shown in Figure S3.

Calibration curves were constructed using authentic or isolated
compounds (0.5−250 nmol mL−1). The amount of eachanalyzed compound in the organs was expressed as percentage
of dry organ weight.
Amino Acid and Amine Analysis. Seventeen aminoacids, GABA, and available amine precursors of acteoside(tyramine, L-DOPA, and dopamine) in diﬀerent organ samples
were analyzed by LC−ESI-MS/MS after derivatization with 6aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC)
using a Waters AccQ•Fluor reagent kit according to the
manufacturer’s instructions. In brief, each dried organ (about
25 mg) was extracted twice with 1 mL of ultrapure water in asonicating bath (15 min). The water extracts were ﬁlteredthrough a 0.22 μm PVDF membrane ﬁlter and directly used forderivatization. Aliquots (10 μL) of both extracts or the
standard solution were mixed with 70 μL of AccQ•Tag borate
buﬀer and 20 μL of AQC reagent. After heating at 55 °C for 10
min to ﬁnalize the derivatization reaction, 1 μL of the sample
was injected into the LC−ESI-MS/MS system with a Waters
AccQ•Tag Ultra column (2.1 × 100 mm, 1.7 μm). The
applied solvent system consisted of eluent (A), which is a
speciﬁed dilution of Waters AccQ•Tag Ultra Eluent A, andeluent (B), which is Waters AccQ•Tag Ultra Eluent B. The
column temperature was set to 55 °C, and gradient elution wasapplied with a ﬂow rate of 0.7 mL min−1 as follows: 0−0.54
min, 0.1% B; 0.54−5.74 min, 0.1−9.1% B; 5.74−7.74 min,9.1−21.2% B; 7.74−8.04 min, 21.2−59.6% B; 8.04−8.05 min,59.6−90% B; 8.05−8.64 min, 90−90% B; 8.64−8.73 min, 90−
0.1% B; and 8.73−9.50 min, 0.1−0.1% B. For MRM
experiments, monitoring speciﬁc transitions of parent andproduct ions are summarized in Table S1. The amount of eachanalyzed compound in the organs was expressed as μg of g-dry
organ weight.
Fluorescence Spectra of Selected Compounds with
DPBA. The ﬂuorescence derivatization method was performed
according to a previous report22 with some modiﬁcations. The
mixture (1.5 mL) in 50% dimethyl sulfoxide (DMSO) solution
containing isolated compounds (10 μM in the ﬁnal oflamalbid, sesamoside, shanzhiside methyl ester, acteoside,pedaliin, or pedalitin) or positive control (10 μM of quercetinor rutin) and DPBA (100 μM) were incubated at room
temperature for 10 min under dark conditions. Primary
ﬂuorescence of the mixture was observed with the naked eye
under UV irradiation (365 nm) using a transilluminator, andexcitation and emission spectra of the mixture were obtainedon a JASCO FP-6500 spectroﬂuorometer.
Fluorescence Microscopy of Sesame Leaf Tissue
Stained with DPBA. Leaf organs were ﬁxed according toprevious reports37,38 with some modiﬁcations. Fresh sesame
leaves were immersed in ethanol containing 10% acetic acid for
24 h at room temperature, followed by sequential 20 min soaks
in 90, 50, and 30% ethanol. After rinsing with water, the leaves
were exposed to a clearing reagent (8 g chloral hydrate, 1 mL
glycerol, and 2 mL water) for 1 h to remove chlorophyll. Afterrinsing with water again, the leaves were stained in 1 mM
DPBA-DMSO solution for 10 min. The stained leaf tissue wasobserved under a ﬂuorescence microscope with a U-MNU(365 nm) exciter ﬁlter (Olympus BX51, Olympus, Tokyo,
Japan).
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ASSOCIATED CONTENT

S Supporting Information*

The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsomega.8b02798.

■

Fluorescence response of the DPBA complex when the
mixture of acteoside and pedaliin in the concentration
ratio (12:1 w/w) in the leaf blade was reacted with
DPBA; illustration of diﬀerent organs in the sesame
plant used in this study; MRM chromatograms ofacteoside and its related compounds; and MRM
conditions of free amino acids and amine precursors ofacteoside (tyramine, L-DOPA, and dopamine) (PDF).

AUTHOR INFORMATION

Corresponding Author*E-mail: hmatsufu@brs.nihon-u.ac.jp.
ORCID

Hiroshi Matsufuji: 0000-0001-9190-1434
Notes

The authors declare no competing ﬁnancial interest.

■

ACKNOWLEDGMENTS
We are grateful to Prof. Satoshi Kubota of the Department ofAgricultural Bioscience, College of Bioresource Sciences,
Nihon University for valuable discussions and technicalsupport in the cultivation of S. indicum L. We are also thankful
to the General Research Institute of Nihon University for LC−
ESI-MS/MS measurements. This study was ﬁnancially
supported by the Nihon University President Grant Initiativeand JSPS KAKENHI Grant Number JP15K12247.

■

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