RESEARCH ARTICLE

Chemical characterization and biologicalactivity in young sesame leaves (Sesamum
indicum L.) and changes in iridoid andpolyphenol content at different growth stages
Yushiro Fuji1, Ayumi Uchida2, Katsunori Fukahori2, Makoto Chino1, Takashi Ohtsuki1,
Hiroshi Matsufuji1*a1111111111
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1 Department of Food Bioscience and Biotechnology, College of Bioresource Sciences, Nihon University,
Fujisawa, Kanagawa, Japan, 2 Wadaman Science Co., Ltd, Nijoden-cho, Nakagyo-ku, Kyoto, Japan* hmatsufu@brs.nihon-u.ac.jp

Abstract
OPEN ACCESS
Citation: Fuji Y, Uchida A, Fukahori K, Chino M,
Ohtsuki T, Matsufuji H (2018) Chemical
characterization and biological activity in young
sesame leaves (Sesamum indicum L.) andchanges in iridoid and polyphenol content at
different growth stages. PLoS ONE 13(3):e0194449. https://doi.org/10.1371/journal.
pone.0194449
Editor: Joseph J. Barchi, National Cancer Instituteat Frederick, UNITED STATES
Received: December 8, 2017
Accepted: March 2, 2018
Published: March 27, 2018
Copyright: © 2018 Fuji et al. This is an open accessarticle distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction inany medium, provided the original author andsource are credited.
Data Availability Statement: All relevant data arewithin the paper and its Supporting Information
files.
Funding: This study received financial support
from Wadaman Science Co., Ltd. The funderprovided support in the form of salaries for authors
AU and KF, but did not have any additional role inthe study design, data collection and analysis,
decision to publish, or preparation of theThree iridoids (lamalbid (I1), sesamoside (I2) and shanzhiside methyl ester (I3)) and sevenpolyphenols (cistanoside F (P1), chlorogenic acid (P2), pedalitin-6-O-laminaribioside (P3),pedaliin (P4), isoacteoside (P6), pedalitin (P7) and martynoside (P8)) were identified inyoung sesame leaves (Sesamum indicum L.) other than the acteoside (P5) reported previously. P3 was a new compound, and I1, I3, P2 and P8 were found in a species of Sesamum
for the first time. HPLC analyses revealed that the compounds I1 (0.29–1.75% of dry
leaves), I2 (0.38–0.87%), I3 (0.04–1.07%), P4 (0.01–2.05%) and P5 (0.13–4.86%) were
present primarily in young sesame leaves and were found in plants cultivated on different
farms (plant height, 30–70 cm). Of the identified compounds, P5 and P6 showed high 1,1diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, oxygen radical absorbance capacity(ORAC), and in vitro antiglycation activities. Given its content, P5 makes a major contribution to the biological activities of young sesame leaves. The compounds were examined at
six different growth stages of plants cultured in a greenhouse to determine the optimum harvest stage and for end-use assessment. P5 accumulated in the leaves during growth, andthe content reached a maximum of 12.9% of dry leaves in the 4th stage (plant height, 74.5
±9.7 cm), which is one of the highest percentages reported in plants from nature.

Introduction
Sesame (Pedaliaceae family, Sesamum indicum L.) is one of the most important oilseed crops
and is widely cultivated in tropical and subtropical areas such as Myanmar, India, China, andAfrica. In 2014, the production of sesame seeds was 6,235,530 t worldwide, with nearly 70% of
the world production being in Asia and about 25% in Africa [1]. Sesame is harvested between
90 and 150 days after planting. About 65% of the annual crop is processed into oil and 35% isused in food [2]. The seeds contain lignans such as sesamin and sesaminol and are highly valued as a traditional health and nutraceutical food. The seed oil is used for cooking.

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Chemical composition in sesame leaves

manuscript. Also, a part of this study was
financially supported by the Nihon University
President Grant Initiative. The funder provided
support in the form of salaries for authors MC, TO
and HM, but did not have any additional role in the
study design, data collection and analysis, decision
to publish, or preparation of the manuscript. The
specific roles of these authors are articulated in the‘author contributions’ section.
Competing interests: This study received financial
support from Wadaman Science Co., Ltd., theemployer of AU and KF. There are no patents,
products in development or marketed products to
declare. This does not alter our adherence to all the
PLOS ONE policies on sharing data and materials,
as detailed online in the guide for authors.

In contrast to sesame seeds, very little information is available on sesame leaves, probably
because sesame is primarily cultivated for its seeds and the leaves are considered agriculturalsolid waste [3]. However, in Africa the leaves and shoots are used as vegetables, and the leaves
contain useful amount of nutrients such as amino acids [4]. In China, decoctions of sesame
leaves are used for various traditional and medicinal purposes, such as pain relief [5]. In Nigeria and many other tropical areas, the leaves of Sesamum radiatum are used for the treatmentof catarrh, eye pain, bruises, and erupted skin lesions [6]. Given the potential physiological
function of sesame leaves, the dried powder of young sesame leaves from plants 30–70 cm tall(about 40–60 days after planting) is commercially sold as a health food supplement in Japan.
Commercial products containing powdered young sesame leaves claim that the powder
provides much more folic acid, potassium, riboflavin, and polyphenols than young barley
leaves or kale. The Dictionary of Chinese Materia Medica, Japanese Edition [5] claims that thedried leaves of S. indicum contain 0.3% of the polyphenol pedaliin (6-hydroxy-luteolin
7-methyl ether 6-glucoside; pedalitin-6-O-glucoside), although no source is cited. Morita [7]reported the presence of pedaliin in sesame leaves based on its recrystallization from methanol
extract, and Jain [8] reported 0.5% pedaliin in cultured sesame tissue. However, there are few
reports regarding other polyphenol components in sesame leaves. Konan et al. [9] reported
that the leaves of S. radiatum are rich in phenolic compounds, phenols, lignans, and flavonoids, and Kwak et al. [10] reported that the leaves of S. indicum show a higher total polyphenol content than do perilla leaves, although the composition and amount were not reported.
Hata et al. [11] concluded that trace levels of lignans are present in S. indicum leaves (0.5–2.6 μg/g dry weight; 0.00005–0.00026%). We found that young sesame leaves contain morethan eight polyphenols, with acteoside (P5 in this study), a phenylethanoid, being one of the
major components [12].
In this study, we identified three iridoids and seven polyphenols other than acteoside, andevaluated in vitro antioxidant (DPPH and ORAC) and antiglycation activity of the identified
compounds. In addition, we investigated the content of major components in young sesame
leaves from plants cultivated on different farms in Japan, and the change in composition of theleaves at six different growth stages to determine the optimum harvest stage and for end-use
assessment. Finally, we identified and quantified the compounds in the leaves.

Materials and methods
Chemicals
Caffeic acid, hydroxytyrosol, chlorogenic acid (3-caffeoylquinic acid, P2), acteoside (verbascoside, P5), and isoacteoside (isoverbascoside, P6) were obtained from Sigma-Aldrich Co.
(St. Louis, MO, USA), BP Biochemicals Inc. (San Diego, CA, USA) or ChromaDex Inc. (Irvine,
CA, USA). DPPH (1,1-diphenyl-2-picrylhydrazyl) and aminoguanidine hydrochloride werefrom Wako Chemical Industries (Osaka, Japan), and fluorescein sodium salt, AAPH (2,20 -azobis(2-amidinopropane) dihydrochloride) and bovine serum albumin (BSA) were from SigmaAldrich Co. All reagents were of HPLC or special grade, purchased from Wako Chemical
Industries, and were used without further purification. MeOH-d4 and DMSO-d6 wereobtained from Isotech Inc. (Hatfield, PA, USA).

Sesame leaf samples
The sesame variety Myanmar Black Sesame (Lignan-Rich Black Goma) was cultivated in private open-field farms in different regions (Kagoshima, Shimane, Kumamoto, and Miyazaki) in
2013–2016 in Japan, and young leaves were harvested at about 40–60 days after planting (plantheight, about 30–70 cm) with the permission of the owners. This research field did not involve

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Chemical composition in sesame leaves

endangered or protected species. The leaves were washed with water, blanched for 60 s at 90 to
95 ˚C, dried for about 14 h at 40 to 60 ˚C, ground into a powder and passed through a 1.0 mm
sieve, then dried with superheated steam to inactivate microorganisms. Dried powder ofyoung sesame leaves was obtained from Wadaman Co. Ltd. (Kyoto, Japan).
Sesame plants were also grown in June 2015 (N.U. 2015) or from May to August 2016 (N.
U. 2016) in a greenhouse at the College of Bioresource Sciences, Nihon University. For N.U.
2015, the leaves were collected from eight plants about 40 days after planting (plant height,
about 30 cm). In N.U. 2016, the seeds were sown on May 9, 2016 in seed trays, and young seedlings were transplanted when plants reached the stage of 2–3 true leaves on May 22, 2016
(plant height, about 5 cm) to 3 L pots filled with red clay (1.5 L) and Metro-Mix-360 (1.5 L,
Sun Gro Horticulture Canada, Vancouver, Canada) containing fertilizer (6 g/pot; N:P2O5:
K2O:MgO = 6:40:6:15). The pots were put in an unheated greenhouse (variation in topsoil
temperature throughout the experiment is presented in supporting information S1 Fig). Leaves
were collected from 8–9 plants at six different growth stages (1st stage, June 7, 7.2±1.6 cm(n = 9); 2nd, June 30, 30.84.3 cm (n = 9); 3rd, July 14, 57.5±4.3 cm (n = 9); 4th, July 14, 74.5
±9.7 cm (n = 8); 5th, July 21, 81.9±11.9 cm (n = 8); 6th, August 4, 125.9±4.3 cm (n = 8). Freshand dry weight and number of leaves are summarized in supporting information S1 Table,
and the studied growth stages are illustrated in S2 Fig). On July 14, the leaves were collectedfrom two groups with or without buds and flowers. Any buds, flowers, capsules, or stem apices
were removed to evaluate the chemical composition of leaves at various growth stages. The
leaves were lyophilized and ground using a mortar and pestle. All samples of powder were
kept at –20 ˚C until used.

Extraction and isolation
Dried powder of young sesame leaves (0.5 kg) was defatted with n-hexane (1.0 L × 2), thenextracted with 80% EtOH (1.0 L × 2) for 1 h at room temperature. This procedure was conducted three times to obtain 6.0 L of EtOH extracts from 1.5 kg of powder. The extracts wereconcentrated to 450 mL and the solution (150 mL × 3) was loaded onto an HP-20 column(5.5 × 53 cm) and eluted with MeOH (1.5 L × 3) to remove most of the chlorophyll. The
MeOH effluent was evaporated in vacuo, and the residual aqueous solution was successively
partitioned with EtOAc and n-BuOH. The n-BuOH phase was allowed to stand overnight at –20 ˚C, then the supernatant (Fr. BS, 55 g) and precipitate (Fr. BP, 5 g) were separated using filter paper. Half of Fr. BS was redissolved in 80% EtOH and heated at 80 ˚C for 24 h under mild
alkaline conditions (pH 8.0 with 0.01 mol/L ammonium acetate) to hydrolyze most of theacteoside (P5) and obtain a mixture of I1, I2 and I3. The other half of Fr. BS was loaded onto a
Chemco LC-SORB ODS glass column (2.4 × 36 cm, Chemco Plus Scientific Co. Ltd., Osaka,
Japan) and eluted with 70% MeOH containing 0.1% HCOOH to afford a brown fraction (Fr.
BS-B). Isolation of each compound was performed using a preparative HPLC or TLC as follows, and the purity (%) of isolated compound was estimated by HPLC analysis.
The iridoids lamalbid (I1, 11 mg, 99%), sesamoside (I2, 8 mg, 98%) and shanzhiside methyl
ester (I3, 11 mg, 99%) were isolated from the alkaline hydrolysate of Fr. BS using recycling preparative HPLC (Senshu Scientific SSI1300 instrument, Waters XBridge C18 column, 10 × 250
mm, 10–30% MeOH containing 0.1% HCOOH). The polyphenols cistanoside F (P1, 2.6 mg,98%), pedalitin-6-O-laminaribioside (P3, 4.7 mg, 99%) and pedalitin (P7, 11 mg, 97%) wereisolated from Fr. BS-B using recycling preparative HPLC (GL Science Inertsil PREP-ODS column, 20 × 250 mm or Waters XBridge C18 column, 10 × 250 mm, 30–40% MeOH containing
0.1% HCOOH). Pedaliin (pedalitin-6-O-glucoside, P4, 32 mg, 99%) was isolated from Fr.
BP using recycling preparative HPLC (XBridge C18 column, 40% MeOH containing 0.1%

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Chemical composition in sesame leaves

HCOOH). Martynoside (P8, 7.6 mg, 99%) was isolated from Fr. BS using preparative TLC(Merck silica gel 60, CHCl3/MeOH/H2O (70:30:5 (by vol)), RF: 0.90) and recycling preparative
HPLC (XBridge C18 column, 40% MeOH containing 0.1% HCOOH).

HPLC or LC-ESI-MS/MS analysis
HPLC was conducted using an Agilent HP-1100 series instrument, a UV-Vis detector (Tokyo,
Japan), and a Waters XBridge C18 column (4.6 × 150 mm, 5 μm) eluted with a linear MeCN
gradient in 0.1% HCOOH (5 to 35% MeCN in 15 min, 35 to 100% MeCN in 25 min, and 100
to 5% MeCN in 1 min) at a flow rate of 0.8 mL/min at 40 ˚C. The sample injection volume was
10 μL and the eluent was monitored at 254 and 340 nm. For LC-ESI-MS/MS, a Waters Quattro
Premier XL mass spectrometer coupled to an ACQUITY UPLC system with a Waters
AQUITY photodiode array detector was used (Tokyo, Japan). Compounds were separated ona UPLC system with a Waters BEH C18 column (2.1 × 50 mm, 1.7 μm) and a linear MeCN
gradient in 0.1% HCOOH (5 to 35% MeCN in 3 min, 35 to 100% MeCN in 5 min, and 100 to
5% MeCN in 1 min) at a flow rate of 0.3 mL/min at 40 ˚C. The sample injection volume was
1 μL. A triple quadrupole mass spectrometer with an electrospray ionization source was used
for mass determination. The ion source conditions were optimized as follows: source temperature, 120 ˚C; capillary voltage, 3.5 kV; desolvation temperature, 400 ˚C; flow rate of desolvation
gas, 850 L/h; flow rate of cone gas, 50 L/h. The mass spectrometer was operated in both thepositive and negative ion modes, with a scan range from m/z 200 to 1200. For MS/MS experiments, different collision energies were used in the negative ion mode depending on the compounds studied.

Identification of isolated compounds
High-resolution mass spectra were measured by direct infusion in electrospray negative ion
mode using a Thermo Q-Exactive Orbitrap mass spectrometer (Waltham, MA, USA). 1H- and
13
C-NMR spectra were measured using a JEOL ECA-500 spectrometer (Tokyo, Japan) at 500
MHz and 150 MHz, respectively in methanol-d4 or DMSO-d6 with tetramethylsilane as aninternal standard. The signals in the 1H- and 13C-NMR spectra of the isolated compounds
were assigned on the basis of chemical shifts and the results of 2D NMR (DQF-COSY,NOESY, HSQC-TOCSY, and HMBC) studies. Optical rotation was measured with a JASCO
P-1020 polarimeter (Tokyo, Japan).
26:0
I1, lamalbid. White amorphous powder; UV-λmax: 237 nm; ½aD –72.0˚ (I1.0, H2O); positive ESI-MS: m/z 867 [2M+Na]+, 445 [M+Na]+, 423 [M+H]+; negative ESI-MS: m/z 421[M-H]-, 259 [aglycone]-; 1H- and 13C-NMR data, see S2 Table.
26:1
I2, sesamoside. White amorphous powder; UV- λmax: 232 nm; ½aD –72.3˚ (c 0.58,++
MeOH); positive ESI-MS: m/z 863 [2M+Na] , 443 [M+Na] , 421 [M+H]+; negative ESI-MS:
m/z 419 [M-H]-, 257 [aglycone]-; 1H- and 13C-NMR data, see S2 Table.
25:3
I3, shanzhiside methyl ester. White amorphous powder; UV-λmax: 237 nm; ½aD –142.4˚+++(c 0.38, MeOH); positive ESI-MS: m/z 835 [2M+Na] , 430 [M+Na] , 407 [M+H] ; negative
ESI-MS: m/z 405 [M-H]-, 243 [aglycone]-; 1H- and 13C-NMR data, see S2 Table.
25:3
P1, cistanoside F. Yellowish amorphous powder; UV-λmax: 298, 331 nm; ½aD –29.5˚(I1.0, MeOH); positive ESI-MS: m/z 511 [M+Na]+, 489 [M+H]+, 471, 325, 163; negative
ESI-MS: m/z 487 [M-H]-, 179 [M-Glc-Rhm-H]-; 1H- and 13C-NMR data, see S3 Table.
P2, chlorogenic acid (3-caffeoylquinic acid) (commercial authentic sample). UV-λmax:
297, 326 nm; positive ESI-MS: m/z 355 [M+H]+, 163; negative ESI-MS: m/z 353 [M-H]-, 191[M-Caf]-.

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Chemical composition in sesame leaves

P4, pedalitin-6-O-glucoside (pedaliin). Yellowish amorphous powder; UV-λmax: 271, 344 nm;
26:6½aD –4.3˚ (c 0.27, dioxane/EtOH = 1/9); positive ESI-MS: m/z 479 [M+H]+, 317 [M-Glc+H]+;
negative ESI-MS: m/z 477 [M-H]-, 315 [M-Glc-H]-; 1H- and 13C-NMR data, see Table 1.
25:9
P5, acteoside (commercial authentic sample). UV-λmax: 293, 335 nm; ½aD –85.2˚ (I1.0,
MeOH); positive ESI-MS: m/z 647 [M+Na]+, 625 [M+H]+, 471, 163; negative ESI-MS: m/z 623[M-H]-.
26:7
P6, isoacteoside (commercial authentic sample). UV-λmax: 292, 327 nm; ½aD –40.5˚ (c0.45, MeOH); positive ESI-MS: m/z 647 [M+H]+, 625 [M+H]+, 325, 163; negative ESI-MS: m/z
623 [M-H]-, 461 [M-Caf]-, 161.
P7, pedalitin. Yellowish amorphous powder; UV-λmax: 282, 344 nm; positive ESI-MS: m/z
317 [M+H]+, 302; negative ESI-MS: m/z 315 [M-H]-, 300; 1H- and 13C-NMR data, see Table 1.
26:0
P8, martynoside. Yellowish amorphous powder; UV-λmax: 288, 330 nm; ½aD –45.3˚ (I1.0,
MeOH); positive ESI-MS: m/z 653 [M+H]+, 485, 339; negative ESI-MS: m/z 651 [M-H]-; 475,
193, 175; 1H- and 13C-NMR data, see S3 Table.
Table 1. NMR spectroscopic data for compounds P3, P4 and P7.
pos. P3 (DMSO-d6)
δC

P4 (DMSO-d6)
δ H (mult, J in Hz)

δC

P7 (DMSO-d6)
δ H (mult, J in Hz)

δCδ H (mult, J in Hz)

2

164.0

164.0

163.8

3

102.6 6.75 (s)102.6 6.74 (s)102.3 6.70 (s)

4

182.0

182.0

181.9

4a

104.7

104.7

104.8

5

151.5

151.5

146.0

6

127.8

127.9

129.7

7

158.2

158.3

154.1

8

91.4 6.89 (s)91.4 6.88 (s)90.9 6.87 (s)

8a

152.4

152.4

149.4

10

121.3

121.3

121.5

20

113.4 7.45 (s)113.4 7.45 (s)113.3 7.45 (s)

30

145.6

145.6

145.6

40

149.6

149.6

149.5

50

115.8 6.90 (d, 9.0)

115.8 6.90 (d, 8.0)

115.8 6.90 (d, 8.0)06118.9 7.46 (d, 9.0)

118.9 7.46 (d, 8.0)

118.7 7.44 (d, 8.0)

7-OCH356.4 3.92 (s)56.4 3.91 (s)56.1 3.92 (s)5-OH

13.1 (s)13.1 (s)12.7 (s)GlcA-100

101.0 5.16 (d, 7.0)

101.8 5.05 (d, 7.0)

200

72.8 3.47 (m)74.0 3.22 (m)300

87.6 3.45 (m)76.4 3.22 (m)400

68.0 3.30 (m)69.8 3.12 (m)500

76.7 3.20 (m)77.1 3.06 (m)00

60.5 3.60 (brd, 10.5)

60.7 3.60 (dd, 3.0, 11.0)

6

3.45 (m)GlcB-1‴

3.41 (m)103.9 4.35 (d, 8.0)

2‴

73.7 3.08 (m)3‴75.9 3.21 (m)4‴

70.0 3.05 (m)5‴76.8 3.18 (m)6‴61.0 3.70 (brd, 11.0)
3.37 (m)https://doi.org/10.1371/journal.pone.0194449.t001

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Chemical composition in sesame leaves

Content of iridoids and polyphenols
The concentration of each compound in sesame leaves was measured by HPLC. Dry sesameleaf powder was extracted twice with 1 mL (for ~25 mg samples) or 50 mL 60% MeOH (for~100 mg samples) using a sonicating bath (15 min). After passing the extracts through a0.45 μm PVDF membrane filter, the filtrate was subjected to HPLC. Calibration curves wereconstructed using authentic or isolated compounds (0.005–0.5 mg/mL). The amount of eachanalyzed compound in the leaves was expressed as percentage of dry leaf weight.

Antioxidant activity
DPPH assays were performed according to our previous report [12] with modifications toallow the use of 96-well plates. Authentic and isolated compounds were dissolved in 60%
MeOH (except P4, which was dissolved in DMSO) and diluted with 60% MeOH as necessary.
Standard (Trolox, 20 μL) or sample (20 μL) was mixed with 80 μL 100 mmol/L Tris-HCl buffer(pH 7.4) and 100 μL 0.20 mmol/L DPPH in EtOH solution in a 96-well plate. The decrease inabsorbance was monitored at 520 nm for 30 min using a Tecan microplate spectrophotometer(Kanagawa, Japan). Results are expressed as μmol Trolox equivalent (TE)/g or mol TE/mol.
Oxygen radical absorbance capacity (ORAC) assays were performed according to themethod of Watanabe et al. [13] with some modifications. Authentic and isolated compounds
were dissolved in acetone/water/acetic acid (70:29.5:0.5 (by vol); AWA) and diluted with assay
buffer (75 mmol/L KH2PO4/K2HPO4, pH 7.4) as necessary. Standard (Trolox, 20 μL) or sample (20 μL) was mixed with 200 μL 94.4 nmol/L sodium fluorescein solution, preincubated at
37 ˚C for 5 min in a 96-well plate, then incubated with 31.7 μmol/L AAPH solution (75 μL) at
37 ˚C. The fluorescence (excitation, 485 nm; emission, 528 nm) was monitored every 2 minfor 90 min using a Bio-Tek Synergy 2 multi-mode microplate reader (Winooski, VT, USA).
The net area under the curve (AUC) was calculated by subtracting the AUC for the blank fromthat of each sample. The ORAC value for each sample was calculated using a standard curve
for Trolox (6.25, 12.5, 25, and 50 μmol/L). Results are expressed as μmol TE/g or mol TE/mol.

Antiglycation activity
Inhibition of BSA glycation in vitro was assayed according to the method of Tsuji-Naito et al.
[14] with some modifications. BSA (10 mg/mL) was mixed with 50 mmol/L glucose and fructose in 50 mmol/L phosphate buffer solution (pH 7.4) under sterile conditions. The solution(1.5 mL) was incubated in the presence or absence (as a control) of isolated compounds inwater or DMSO (0.001 to 0.015 mg/mL in the final) for 6 days at 37 ˚C in the dark. For theblank, phosphate buffer was used instead of sugar solution. The degree of BSA glycation wasmeasured using a JASCO FP-6500 spectrofluorometer (Tokyo, Japan) at an excitation wavelength of 370 nm and an emission wavelength of 440 nm. The inhibitory activity (%) was calculated as follows:
Inhibitory activity ð%Þ ¼ ½ðFLcontrol

FLblank ÞðFLsample

FLblank Þ=ðFLcontrol

FLblank Þ  100

The concentration (μmol/L) of samples required to inhibit 50% of BSA glycation is defined as
the IC50 value.

Results and discussion
Identification of iridoids and polyphenols in young sesame leaves
Typical HPLC chromatograms and photodiode array spectra of young sesame leaves areshown in Fig 1. Similar spectra were obtained for compounds I1, I2, and I3, for P1, P2, P5,

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Chemical composition in sesame leaves

Fig 1. UPLC-PDA chromatograms (A) and UV spectral data (B) of young sesame leaves.
https://doi.org/10.1371/journal.pone.0194449.g001

P6, and P8, and for P3, P4, and P7, suggesting that the compounds in each of these groups
have similar chromophores. Based on the chemical structure of acteoside (P5), we firstattempted to identify the unknown compounds using available authentic caffeoyl derivatives
and hydroxytyrosol. The structures of P2 and P6 were identified as chlorogenic acid (3-caffeoylquinic acid) and isoacteoside, respectively, based on comparison of retention times, UV
and LC-ESI-MS spectra with authentic compounds. On the other hand, no caffeic acid orhydroxytyrosol was detected in young sesame leaves, even in analysis by multiple reaction
monitoring methods with LC-ESI-MS/MS.
For the compounds isolated in this study, the LC-ESI-MS and the 1H- and 13C-NMR dataof I1, I2, I3, P1, P4, P7 and P8 were shown to be identical to those of lamalbid, sesamoside,shanzhiside methyl ester [15–17], cistanoside F [18], pedaliin (pedalitin-6-O-glucoside), pedalitin [7,19] and martynoside [20,21], respectively (Fig 2).
Compound P3 was isolated as a yellowish amorphous powder; UV-λmax: 271, 344 nm;
26:6½aD –19.6˚ (I1.0, dioxane/EtOH = 1/9). The LC-ESI-MS data obtained in positive and negative modes for compound P3 showed molecular-related ion peaks at m/z 641 [M+H]+ and 639

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Chemical composition in sesame leaves

Fig 2. Iridoids and polyphenols found in young sesame leaves.
https://doi.org/10.1371/journal.pone.0194449.g002[M-H]-, respectively. Also, high-resolution-ESI-MS (negative) ([M-H]-) data of m/z 639.1583(calcd for I28H31O17; 639.1560) indicated that P3 has a molecular formula of I28H32O17. Compared to pedalitin (P7) and pedaliin (P4), the molecular weight of P3 was 324 and 162 mu
larger, respectively. In addition, a peak corresponding to P7 appeared when isolated P4 or a
mixture containing P3 was hydrolyzed under acidic conditions. These findings suggested that
P3 is the diglycoside of pedalitin.
The 1H- and 13C-NMR data of P3 suggested the presence of pedalitin and two sugar units(Table 1; NMR spectral data are shown in supporting information S3 Fig). The typical coupling constants (7.0 and 8.0 Hz) of the anomeric proton signals at δ 5.16 and δ 4.35, respectively, and the results of 2D-NMR (HSQC-TOCSY and NOESY) experiments, suggested thatthe two sugar units were β-form of glucopyranosides. Also, glycosidation-induced 13C NMR
shifts in the A ring of the aglycone when compared with P7 were observed, and similar chemical shifts were also observed for P4. Furthermore, correlations between H-1’’ of glucose A (thePLOS ONE | https://doi.org/10.1371/journal.pone.0194449 March 27, 20188 / 15

Chemical composition in sesame leaves

anomeric proton signal at δ 5.16) and C-6 of the pedalitin unit (δ 127.8) were observed in the
HMBC spectrum. In addition, correlation between H-1‴ of glucose B (δ 4.35) and I3’’ of glucose A (δ 87.6) was observed, suggesting that laminaribiose unit would be glycosylated to C-6of the pedalitin unit. There are several reports of flavonoid glycosides containing a laminaribiose moiety, and the NMR data for the laminaribiose moiety of P3 were almost the same as
the data in these previous reports [22,23]. We therefore concluded that P3 is pedalitin-6-Olaminaribioside (Fig 2). To the best of our knowledge, pedalitin-6-O-laminaribioside is a newcompound.
Potterat et al. [16] reported the isolation of acteoside (P5), sesamoside (I2) and other iridoids (phlomiol, pulchelloside-I, and 6β-hydroxyipolamiide) from the root bark of Sesamum
species (S. angolense W.), and Takase et al. [24] identified an iridoid, sesinoside, in illuminatedseedlings of S. indicum L. Suzuki et al. [25] reported obtaining cistanoside F (P1), acteoside(P5), isoacteoside (P6), decaffeoyl acteoside, and campneoside I and II from a water extract of
S. indicum whole plants, although they did not mention whether the harvested plants contained leaves, stems, roots, flowers, or seeds, nor the age at harvest. The various iridoids andpolyphenols are presumably produced in different parts of sesame plants at different growthstages. Of the compounds identified in this study, I1, I3, P2 and P8 were found in a species of
Sesamum for the first time.

Iridoid and polyphenol contents of young sesame leaves cultivated on
several farms
In general, it is well-known that the content of secondary compounds produced in plants varies with the development stage of the plant and between leaves, stems and roots, and also
depends on environmental factors such as time of day, weather, and soil nutrient conditions.
Thus, chemical components were examined in young leaves cultivated on different farms andin a greenhouse. Typical HPLC chromatograms obtained are shown in supporting information S4 Fig. No other large peaks corresponding to unidentified compounds were observed inany chromatograms. The composition of the leaves is summarized in Table 2. Lamalbid (I1),
sesamoside (I2), shanzhiside methyl ester (I3), pedaliin (P4), and acteoside (P5) were major
compounds in all young sesame leaves used in this study, although the content differed depending on the region and harvest year: I1, 0.29–1.75%; I2, 0.38–0.87%; I3, 0.04–1.07%; P4,
0.01–2.05%; P5, 0.13–4.86%. Olivier et al. [26] reported that climate and geology did not play amajor role in the production of acteoside or isoacteoside in Lippia javanica from different
localities. The factors determining production of secondary compounds in sesame leaves andthe losses in sterilization processes are still being evaluated, and further investigations areneeded before harvesting young sesame leaves for use as a health food supplement.

Antioxidant and antiglycation activities of iridoids and polyphenols inyoung sesame leaves
Table 3 summarizes the antioxidant and antiglycation activities of each of the iridoids andpolyphenols isolated from young sesame leaves. Of the compounds, acteoside (P5) and isoacteoside (P6) had high DPPH radical scavenging activity, ORAC activity, and inhibitory activityof BSA glycation, while iridoids (I1, I2 and I3) had no antioxidant and antiglycation activities.
There are plenty of reports on the antioxidant and antiglycation activities of the compounds
identified in this study, particularly acteoside and isoacteoside [27–32], and the activities weobserved were almost similar to previous reports. Given the amount present of the compounds
and their activities, acteoside (P5) makes a major contribution to the antioxidant and antiglycation activities of young sesame leaves. In addition, several researchers have reported that

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Chemical composition in sesame leaves

Table 2. Content (%) of compounds in young sesame leaves cultivated in different regions in harvest years 2013 through 2016.
2013
Kagoshima

Kumamoto

Shimane

2014

2015

Kagoshima

Kagoshima

I1

0.35 ± 0.009

0.89 ± 0.199

0.69 ± 0.006

0.62 ± 0.199

0.45 ± 0.199

I2

0.57 ± 0.009

0.74 ± 0.115

0.85 ± 0.017

0.96 ± 0.115

0.38 ± 0.115

I3

0.17 ± 0.005

0.12 ± 0.028

0.69 ± 0.012

0.62 ± 0.028

0.23 ± 0.028

P1

0.03 ± 0.002

0.02 ± 0.006

0.04 ± 0.001

0.01 ± 0.006

0.01 ± 0.006

P2

0.06 ± 0.001

0.12 ± 0.010

0.03 ± 0.001

0.01 ± 0.010

0.00 ± 0.010

P3

0.16 ± 0.004

0.10 ± 0.009

0.05 ± 0.003

0.03 ± 0.009

0.03 ± 0.009

P4

1.66 ± 0.038

1.00 ± 0.062

0.41 ± 0.011

0.16 ± 0.062

0.33 ± 0.062

P5

1.20 ± 0.039

2.47 ± 0.133

1.16 ± 0.027

0.54 ± 0.017

0.13 ± 0.017

P6

0.27 ± 0.012

0.22 ± 0.017

0.09 ± 0.002

0.03 ± 0.001

0.04 ± 0.001

P7

n.d.c

0.01 ± 0.001

0.01 ± 0.001

n.d.

0.02 ± 0.002

P8

n.d.

0.09 ± 0.013

0.04 ± 0.001

0.01 ± 0.000

0.01 ± 0.001

ShimaneMiyazaki (1)b

Miyazaki (2)b

2015
N.U.a

2016
Kagoshima

I1

0.15 ± 0.019

0.55 ± 0.014

0.29 ± 0.002

1.37 ± 0.002

1.75 ± 0.009

I2

0.58 ± 0.046

0.51 ± 0.073

0.58 ± 0.004

0.50 ± 0.004

0.87 ± 0.001

I3

0.54 ± 0.008

0.11 ± 0.015

0.04 ± 0.001

0.67 ± 0.001

1.07 ± 0.004

P1

0.02 ± 0.000

0.03 ± 0.001

0.07 ± 0.001

0.02 ± 0.001

0.03 ± 0.000

P2

0.26 ± 0.001

0.03 ± 0.000

0.02 ± 0.000

n.d.

0.00 ± 0.000

P3

0.08 ± 0.004

0.07 ± 0.000

0.13 ± 0.001

0.04 ± 0.001

0.05 ± 0.000

P42.05 ± 0.094

0.01 ± 0.001

0.70 ± 0.002

0.37 ± 0.002

0.60 ± 0.002

P5

4.86 ± 0.047

1.32 ± 0.005

0.46 ± 0.004

0.13 ± 0.004

0.19 ± 0.002

P6

0.31 ± 0.030

0.02 ± 0.003

0.05 ± 0.002

0.01 ± 0.002

0.03 ± 0.001

P7

n.d.

0.32 ± 0.002

0.02 ± 0.001

n.d.

n.d.

P8

n.d.

0.02 ± 0.001

0.04 ± 0.001

n.d.

n.d.

a

N.U. was cultured in a greenhouse at Nihon University, Kanagawa and others were done on open-field farms in several regions.

bMiyazaki (1) and (2) were cultured at different area in Miyazaki prefecture.
n.d., not detected.

chttps://doi.org/10.1371/journal.pone.0194449.t002

acteoside inhibit the formation of advanced glycation end products (AGEs) in vivo [31, 33].
Thus, young sesame leaves with a high content of acteoside likely inhibit the formation ofAGEs, although further investigation is needed to verify the antiglycation effect in vivo.

Changes in chemical composition at different growth stages
The contents were examined at six different growth stages of the plants to determine optimum
harvest stage of sesame leaves. Fig 3 shows changes in the content of major extracted components in the leaves during growth in a greenhouse. Interestingly, the acteoside (P5) content
in the leaves increased remarkably during growing, and reached a maximum of 12.9% at the
4th stage (plant height, 74.5±9.7 cm). On the other hand, the iridoid (I1–I3) and pedaliin(P4) content in the leaves increased slightly up to the 2nd stage, and afterward decreased
moderately.
Acteoside is one of the phenylethanoid glycosides, which are widely distributed in the plant
kingdom; most have been isolated from medicinal plants. Studies have shown that acteosidehas various biological activities, such as antioxidative, anti-inflammatory [34], hepatoprotective [35] and neuroprotective [36] activities. Recent studies have also shown that acteoside

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Chemical composition in sesame leaves

Table 3. Antioxidant and antiglycation activities of the isolated compounds from sesame leaves.
DPPH

ORAC

Inhibition of BSA glycation

TEAC (μmol TE/g)

TEAC (μmol TE/g)

IC50 value (μM)

I1

n.a.a

n.a.

n.a.

I2

n.a.

n.a.

n.a.

I3

n.a.

n.a.

n.a.

P1

4100 ± 310 [2.85]b11360 ± 220 [5.55]

345.1 ± 42.2

P23780 ± 290 [1.34]

22600 ± 2280 [8.01]

28.1 ± 0.06

P32020 ± 77 [1.29]

8210 ± 1200 [5.26]

132.7 ± 15.0

P42010 ± 130 [0.96]

24410 ± 1760 [9.29]

41.8 ± 0.57

P55470 ± 120 [3.41]

23600 ± 3480 [14.73]

10.0 ± 1.2

P65700 ± 590 [3.56]

18520 ± 2190 [11.57]

11.1 ± 0.14P72030 ± 57 [0.64]

12380 ± 310 [3.92]

159.7 ± 2.4P8910 ± 59 [0.60]8240 ± 260 [4.73]

n.a.

CAc

16230 ± 620 [2.93]

36560 ± 1910 [6.59]

117.6 ± 9.3

HTc12180 ± 300 [2.20]

45310 ± 1480 [8.10]

44.2 ± 4.0

AGc

–

–

549.7 ± 74.8

The assays were carried out in at least triplicate and results are presented as means ± SD (n = 3 or 6).
abcn.a., no activity detected.
The value in box brackets shows TEAC (mol TE/mol).
CA, caffeic acid; HT, hydroxytyrosol; AG, aminoguanidine.

https://doi.org/10.1371/journal.pone.0194449.t003

would be a potential therapeutic compound for Alzheimer’s [37,38] and Parkinson’s [39] diseases. On the other hand, it has been pointed out that many issues remain unresolved withrespect to effective clinical applications of acteoside; large scale evidence-based human study
and mass production for pharmaceutical applications [40].
He et al. [41] reported that more than 150 plant species belonging to 20 families and 77 genera contain acteoside. The acteoside content varies within plant species of the same family or
genus, and high content has been reported in the leaves of Verbascum nigrum (3.03%) and Verbascum xanthophoenicem (1.58%) [42], the stems of Cistanche tubulosa (0.86–2.54%) and Cistanche desericola (0.48–2.11%) [43,44], and the aerial parts of Plantago lanceolata (2.17%) [45].
To the best of our knowledge, the accumulation of up to 12.9% acteoside in the leaves of S.
indicum is one of the highest reported amounts in plants. It is interesting that the content in
the leaves reached a maximum when the flowers began to open (4th stage), and then decreased
to 3.45% at the 6th stage. This raises the question of why sesame synthesizes so much acteosideand what the accumulated acteoside is used for. The biosynthetic pathway of acteoside has still
not been fully elucidated; in particular, several key enzymes and the genes encoding them arestill undiscovered [40]. On the other hand, the early steps are known; acteoside is biosynthesized from tyrosine and phenylalanine through caffeic acid and hydroxytyrosol [46,47]. In thisstudy, no caffeic acid or hydroxytyrosol was detected in any of the harvested leaves. Further
qualitative and quantitative analyses of different parts of whole plants of S. indicum are therefore in progress to learn more about the biosynthesis of acteoside.

Conclusions
Young sesame leaves contain three iridoids (I1–I3) and eight polyphenols (P1–P8). P3 was a
new compound and I1, I3, P2 and P8 were found in a Sesamum species for the first time.
Acteoside (P5) makes a major contribution to the biological activity of young sesame leaves

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Chemical composition in sesame leaves

Fig 3. Changes in contents of major components in sesame leaves at different growth stages. Plots of the figure presents lamalbid (I1, open triangle), sesamoside (I2,
open circle), shanzhiside methyl ester (I3, open square), pedaliin (P4, solid triangle) and acteoside (P5, solid circle).
https://doi.org/10.1371/journal.pone.0194449.g003

based on its amount and the activity of individual identified compounds. When major components were examined at six growth stages, the acteoside content in the leaves increased remarkably during growth and reached a maximum of 12.9% at the 4th stage. This is one of thehighest levels reported in plants in nature. Our results indicate that the amount of compounds
in the leaves of S. indicum L. depends on the growth stage, and the quantification of acteosideand the stage of the leaves at harvest should be considered when young sesame leaves aredesired with high biological activity as a health food supplement, or for other end-use applications of adult or old sesame leaves as a natural source of acteoside.

Supporting information
S1 Fig. Variation in topsoil temperature throughout the experiment. The thick line indicates daily maximum temperature and thin line indicates daily minimum temperature.
(TIF)
S2 Fig. Sesame plants at the studied growth stages.
(TIF)
S3 Fig. NMR spectra of pedalitin-6-O-laminaribioside (P3).
(TIF)
S4 Fig. Typical HPLC patterns of young sesame leaves cultivated in different regions.
(TIF)

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Chemical composition in sesame leaves

S1 Table. Fresh and dry weight and number of leaves at different growth stages.
(DOCX)S2 Table. NMR spectroscopic data for compounds I1, I2 and I3.
(DOCX)S3 Table. NMR spectroscopic data for compounds P1 and P8. a Our previous report [12]. b
The value in box brackets shows signal data in α-forms of P1-gluose moiety.
(DOCX)

Acknowledgments
We are grateful to Prof. Satoshi Kubota, Department of Agricultural Bioscience, College of
Bioresource Sciences, Nihon University, for valuable discussions and technical support in thecultivation of S. indicum. We are also thankful to Tomoyo Kiuchi, General Research Instituteof Nihon University, for high-resolution MS measurements.

Author Contributions
Conceptualization: Ayumi Uchida, Hiroshi Matsufuji.
Data curation: Yushiro Fuji, Makoto Chino, Takashi Ohtsuki.
Funding acquisition: Hiroshi Matsufuji.
Investigation: Yushiro Fuji, Makoto Chino.
Methodology: Yushiro Fuji, Takashi Ohtsuki.
Project administration: Hiroshi Matsufuji.
Resources: Ayumi Uchida, Katsunori Fukahori.
Supervision: Katsunori Fukahori, Hiroshi Matsufuji.
Visualization: Yushiro Fuji.
Writing – original draft: Yushiro Fuji.
Writing – review & editing: Takashi Ohtsuki, Hiroshi Matsufuji.

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