2024 Volume 72 Issue 12 Pages 1105-1109
Sesame (Sesamum indicum L.) is an important oilseed crop, and its seeds are a source of edible oil and widely used as a nutritious food that is beneficial to health in oriental countries. Phytochemical and biological investigations of the seeds have been well reported; however, those of the leaves have been limited. To explore the potential value of sesame leaves, we focused on their antihypertensive potency. Oral administration of sesame leaf extract significantly reduced blood pressure in spontaneously hypertensive rats. Next, we examined the angiotensin I-converting enzyme (ACE)-inhibitory activity of sesame leaves, stems, and seeds and observed that the inhibitory potencies of leaves and seeds were stronger than those of stems. Acteoside and pedaliin, the major compounds in the leaves, as well as exhibited ACE inhibitory activity. Furthermore, we determined the content of these compounds in the leaves, stems, and seeds using LC/MS. The contents of both compounds in the leaves were higher than those in the stems and seeds. These results suggest that sesame leaf extract can mitigate hypertension, at least in part, via the inhibition of ACE activity by acteoside and pedaliin, suggesting that sesame leaves may have the potential to be used for treating hypertension.
Hypertension is a chronic health problem affecting approximately one billion people worldwide.1) Hypertension prevalence remains high in Japan; over 60% of men aged ≥50 years and over 58% of women aged ≥60 years had hypertension in 2016.2) Problems associated with hypertension include heart disease and several other health-related diseases. Uncontrolled hypertension can lead to or progress to other serious complications and is a risk factor for cardiac arrhythmias, cerebral stroke, and renal failure.3) Angiotensin I-converting enzyme (ACE) is a key regulator of the renin-angiotensin-aldosterone system.4) ACE catalyzes the conversion of inactive angiotensin I into the potent vasoconstrictor angiotensin II, which increases blood pressure by increasing peripheral resistance, heart rate, and cardiac output. Thus, ACE inhibitors, such as captopril, enalapril, and lisinopril, are widely used in the treatment of hypertension. Therefore, ACE inhibitors are commonly used as medical drugs to treat hypertension.
Sesame (Sesamum indicum L.), of the Pedaliaceae family, is an annual plant known as sesame and is one of the most important oilseed crops grown in several countries, including Africa, China, and South America.5) Total sesame seed production has exceeded 6.0 million tons in recent years.6) Sesame seeds are used in food, feed, medicines, and industrial applications.7) The seeds are highly nutritious (oil 40–60%, protein 15–25%), and the bioactivities of natural antioxidants in the seeds, such as sesamin and sesaminol, have been extensively studied.8–10) Sesame leaves are a byproduct of sesame oil production and are consumed in cold dishes, pancakes, or noodles in China.11) Traditionally, sesame leaves, known as “青蘘” in traditional Chinese medicine, have been recognized as a vegetable specialty in a few areas of China.12) Phytochemical and biological investigations of leaves are limited compared with those of seeds.13–17) Leaf extracts can effectively inhibit high-fat diet-induced obesity by activating adenosine monophosphate-activated protein kinase (AMPK) in visceral adipose tissue.18) Ethanolic sesame leaf extract has been shown to relieve ulcer injuries and considerably decrease ulcer scores in a dose-dependent manner.19) The leaf extract inhibited the growth of human colon cancer cells by inducing apoptosis and G2/M cell cycle arres.20) However, no studies have investigated the antihypertensive and ACE-inhibitory activities of sesame leaves.
Acteoside is a major phenylethanol glycoside found in young sesame leaves.15) Acteoside, which was first isolated from Verbascum sinuatum L. in 1950, has several pharmacological activities, including anticancer,21) hepatoprotective,22) antimicrobial,23) and antidiabetic activities.24) Pedaliin is a major flavonoid glycoside found in sesame leaves.15) Pedaliin exhibits radical-scavenging and myeloperoxidase-inhibitory activities.17,25) Pedaliin also exhibits growth-inhibitory, anti-invasive, and anti-migratory effects in human colon cancer cells.26) However, few studies have investigated the biological functions of pedaliin.
We previously reported that the leaf extract of Rehmannia glutinosa Liboschitz forma hueichingensis inhibited ACE activity and had greater inhibitory potency than the root extract.27) Bioassay-guided fractionation of the active fraction revealed acteoside as the major active compound that inhibited ACE. Therefore, in this study, we aimed to evaluate the antihypertensive and ACE-inhibitory activities of sesame leaf extracts. Here, we examined whether the oral administration of sesame leaves regulates blood pressure in spontaneously hypertensive rats (SHR). Next, the ACE-inhibitory activities of the leaves, stems, and seeds were examined. Finally, the ACE-inhibitory activities of acteoside and pedaliin were evaluated, and the contents of these compounds in each part were determined using LC/MS to determine their efficacy. To the best of our knowledge, this is the first study to describe the antihypertensive activity of sesame leaf extracts.
SHR, whose systemic blood pressure increases with age, are frequently used animal models for investigating essential hypertension in humans.28) Myanmar is the world’s second largest producer of sesame followed by Sudan. Therefore, we first used sesame leaves cultivated in Myanmar to examine the antihypertensive activity using SHR. Normal feed or sample feed that included the extract of sesame leaves at 0.8% was administrated for four weeks, and the blood pressure of the rats was measured at 0, 7, 14, 21, and 28 d. Captopril (50 mg/kg body weight) was used as the positive control. As shown in Fig. 1, sesame leaf extract administration significantly inhibited the increase of blood pressure on days 14, 21, and 28. At the 28 d, the changes of the blood pressure from each initial value on SHRs were as follows: +32.7 ± 7.2 (control group), +9.7 ± 6.2 (sesame leaves group), and −4.2 ± 2.8 (captopril group). Body weight and food intake were not significantly different between groups (data not shown). These data suggest that sesame leaves reduce blood pressure in SHRs.
Each sample was mixed with food and free-fed to 9-week-old male SHR. Blood pressure was measured once a week and is shown as the change in the initial blood values. The data are expressed as mean ± S.E.M. (n = 6) (* p < 0.05, ** p < 0.01, compared with the blood pressure of the control group on each day). BP: blood pressure.
Next, the ACE-inhibitory activities were examined. In order to assess differences between cultivation areas, we used sesame cultivated in Vietnam in addition to Myanmar. The samples were sesame leaves and stems cultivated in Vietnam and Myanmar, and unroasted and roasted sesame seeds cultivated in Myanmar. The ACE-inhibitory activities of each sample were measured at two concentrations (0.625 and 1.25 mg/mL). As shown in Fig. 2, the leaf extract inhibited ACE more strongly than the stem extracts from Vietnam and Myanmar. The ACE-inhibitory effect of 1.25 mg/mL of stems and leaves was recorded as follows: 63.4 ± 11.0 and 95.3 ± 0.8% (stems and leaves cultivated in Vietnam, respectively), 37.3 ± 4.1 and 70.3 ± 1.8% (stems and leaves cultivated in Myanmar, respectively). The inhibitory effects of the seeds unroasted and roasted at 1.25 mg/mL were 67.9 ± 1.9 and 79.2 ± 5.9%, respectively, suggesting the roasting process slightly increased the ACE inhibition. The inhibitory effect of Myanmar leaves was almost equivalent to that of the seeds. In this study, we could not obtain the seeds cultivated in Vietnam, but the ACE-inhibitory activity of seeds of Vietnam should be tested in the future.
The percentage of ACE inhibition tested for each extract at 0.625 and 1.25 mg/mL. Captopril (Cap) was used as a positive control (12.5 ng/mL). The data are expressed as mean ± S.E.M. of three independent experiments (n = 6).
Previous phytochemical investigations demonstrated that acteoside and pedaliin are a major phenylethanol glycoside and a flavonoid glycoside, respectively, in sesame leaves13–15) (Fig. 3A). Furthermore, our previous study reported that leaf extract of R. glutinosa f. hueichingensis has ACE-inhibitory activity, and bioassay-guided fractionation of the active fraction revealed acteoside was potent inhibitor of ACE.27) Therefore, in this study, we focused on acteoside and pedaliin to elucidate the active compounds that inhibit ACE activity in sesame leaves. As shown in Fig. 3B, both acteoside and pedaliin at 100 and 200 µM showed ACE-inhibitory activities, and the inhibitory effect of pedaliin (59.1 ± 3.6% at 100 µM, and 78.9 ± 2.2% at 200 µM) was slightly stronger than those of acteoside (52.8 ± 2.5% at 100 µM, and 65.0 ± 2.6% at 200 µM).
Percentage of ACE inhibition tested with acteoside or pedaliin at 100 and 200 µM. The data are expressed as mean ± S.E.M. of three independent experiments (n = 6).
The acteoside and pedaliin contents of each extract were quantified using LC/MS. Five micromolar concentrations of each sample (1–30 µM) were injected into the LC/MS system, and the amounts of acteoside and pedaliin were calculated based on peak areas and calibration curves. Typical SIM chromatograms (Positive-Ion ESI-MS) of acteoside and pedaliin on each part of S. indicum were shown in Supplementary Fig. S1. The linear regression equations for each calibration curve were y = 1125.2x (acteoside) and y = 46335x (pedaliin), where x and y represent the analyte concentration and peak area, respectively.
As shown in Fig. 4A, the acteoside content in leaves cultivated in Vietnam (25.5 mg/g) and Myanmar (21.0 mg/g) was significantly higher than that in stems (Vietnam: 17.0 mg/g, Myanmar: 10.6 mg/g). The acteoside content of the seeds (unroasted: 0.4 mg/g, roasted: 0.6 mg/g) was lower than that of the leaves and stems. Similarly, pedaliin was found in the leaves (Vietnam; 23.3 mg/g, Myanmar; 14.0 mg/g) more than in the stems (Vietnam; 5.6 mg/g, Myanmar; 5.7 mg/g), and little in the seeds (unroasted: 0.1 mg/g, roasted: 0.1 mg/g).
Each sample was extracted with 70% EtOH. Each extract was injected into the LC/MS system, and the acteoside and pedalilin contents were calculated based on the peak area and calibration curves. Data are expressed as mean ± S.E.M. (n = 3). Differences between the content of leaves and stems or seeds were determined using the Tukey–Kramer test, and ** p < 0.01 was considered statistically significant.
The sesame leaves cultivated in Vietnam, which showed the strongest ACE-inhibitory activity, contain acteoside and pedaliin at the content of 25.5 and 23.3 mg/g, respectively (Fig. 4A). The ACE-inhibitory activities of 1.25 mg/mL of leaves cultivated in Vietnam was 95.3 ± 0.8% (Fig. 2). In addition, the ACE-inhibitory activities of acteoside and pedaliin were as follows: acteoside: 52.8 ± 2.5 and 65.0 ± 2.6% at 100 and 200 µM, respectively, pedaliin: 59.1 ± 3.6 and 78.9 ± 2.2% at 100 and 200 µM, respectively (Fig. 3B). From these results, in case of the leaves cultivated in Vietnam, 1.25 mg/mL of leaf extract contains 0.051 and 0.061 µmol of acteoside and pedaliin, respectively, suggesting that the degrees of contribution on the the ACE-inhibition by acteoside and pedaliin were 22.8 and 44.1%, respectively. Thus, these two compounds seem to be major active compounds in the leaves exerting ACE-inhibitory activity. However, the possibility remains that other compounds also contribute on the activity; therefore, further study is needed.
Although acteoside and pedaliin are active constituents specific to leaves and stems, small amounts of acteoside and pedaliin contributed little to ACE-inhibitory activity in sesame seeds (Figs. 2, 4). In sesame seeds, sesamin, a major agricon lignan, has been reported to exhibit antihypertensive effects.29–31) In addition, the sesamin content in the leaves was 1/5000 or less than that in the seeds.32) Therefore, sesamin may be an active compound in the seeds with ACE-inhibitory activity. A previous study reported that the acteoside and pedaliin contents in sesame leaves depend on the growth stage.13) Therefore, further studies on the growth stage based on the amounts of these two constituents may lead to leaves with stronger ACE-inhibitory activity.
This report demonstrates the new functional properties of sesame leaves. The leaf extract showed an antihypertensive effect on SHR. The leaf extract inhibited ACE activity and had greater inhibitory potency than the stem and seed extracts. In addition, acteoside and pedaliin showed ACE-inhibitory activities, and their concentrations in the leaves were higher than those in the stems and seeds. These results suggest that acteoside and pedaliin are responsible for the inhibitory and antihypertensive effects of sesame leaves. Based on the above reports, sesame leaves represent a valuable material with ACE inhibitory activity and have the potential for use as a commercial food material. This study contributes to the development of evidence for the effective use of sesame leaves.
Dried leaves, stems, and seeds of S. indicum harvested in Vietnam (March, 2023) and Myanmar (January, 2023) were obtained from the Institute of Pharma Food Co., Ltd. (Kyoto, Kapan). Authentication was done by Dr. Johji Yamahara. A voucher specimen was deposited in the Department of Pharmacognosy, Faculty of Pharmaceutical Sciences, Nagasaki International University.
Reagents and ChemicalsMeOH and MeCN were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). ACE-Kit WST was purchased from Dojindo Laboratories (Kumamoto, Japan). Pedaliin (purity 99.96%) and acteoside (purity >97.0%) were obtained from TargetMol (Boston, MA, U.S.A.) and Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan), respectively. All other chemicals were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan).
ExtractionDried leaves, stems, unroasted seeds, and roasted seeds of S. indicum were extracted with 70% EtOH overnight at room temperature three times. The solvents were evaporated in vacuo, and the residue was used as the extraction sample. The yield of each extract was 22.7% from the leaves (Vietnam), 19.3% from the leaves (Myanmar), 20.7% from the stems (Vietnam), 20.2% from the stems (Myanmar), 5.4% from the unroasted seeds, and 3.9% from the roasted seeds.
Antihypertensive AssayAntihypertensive effects of the samples were evaluated in SHR (Japan SLC Inc., Hamamatsu, Japan). SHR were randomly divided into three groups with six rats in each group: control, sesame leaf, and captopril groups. In the sesame leaf group, the pulverized sesame leaf powder was mixed with food (8 mg/g) and fee-fed to a 9-week-old male SHR throughout the experiment. Captopril (50 mg/kg body weight) was used as the positive control.3) Blood pressure was measured once a week using a Softron BP-98AL noninvasive blood pressure instrument (Softron, Tokyo, Japan). The animal experiments in this study were carried out by the Institute of Pharma Food Co., Ltd. as contract research, which was commissioned by the authors, and these experiments were conducted in accordance with the regulations of the Institute of Pharma Food.
ACE Inhibition AssayACE inhibition assays were performed as described previously.27) Briefly, 50 µL of samples were mixed with 100 µL of ACE solution (10 mU/mL) in 96-well black microtiter plates (Nunc, Roskilde, Denmark). After 10 min of incubation at 37 °C, 25 µL of 25 mM hippuryl-histidyl-leucine was added and further incubated at 37 °C for 40 min. Then, 50 µL of 1 M NaOH was added, followed by 10 µL of 0.2% O-phthalaldehyde. After 15 min of incubation at room temperature, 15 µL of 3.6 M phosphate was added, and the fluorescence intensity (excitation at 365 nm and emission at 465 nm) was measured using a Beckman DTX 880 plate reader (Fullerton, CA, U.S.A.).
LC/MS AnalysisEach extract was dissolved in dimethyl sulfoxide (DMSO) and centrifuged at 12000 rpm for 10 min. The supernatant was diluted with MeOH to a final concentration of 1 mg/mL. They were filtered (Millex-LG, 0.20 µm, Merck KGaA, Darmstadt, Germany) and used as analytical samples. The standard solutions were prepared as follows: acteoside and pedaliin were dissolved in DMSO and diluted with MeOH to reach final concentrations of 30, 20, 10, 5, and 1 µM. Calibration curves were prepared based on the peak area when injecting a 5.0 µL aliquot of each standard solution into the LC/MS system.
LC/MS Instruments and ConditionsElectrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) analysis was performed using a Xevo G2-XS Q-TOF-MS with an H-Class UPLC system (Waters Corp., Milford, MA, U.S.A.). LC/MS analyses were conducted using Waters ACQUITY UPLC BEH C18 column (1.7 µm, 100 × 2.1 mm i.d.). The mobile phase of a linear MeCN gradient (10 to 100% MeCN in 13 min, 100% MeCN for 2 min, and 10% MeCN for 5 min) at a flow rate of 0.3 mL/min and 40 °C was used. Injection volume was 5.0 µL, and exact mass analysis was performed in positive electrospray in sensitivity analysis mode with SIM. The operating parameters were set as follows: scan range 50–1500 Da, source temperature 120 °C, desolvation temperature 600 °C, cone gas flow 50 L/h, desolvation gas flow 1000 L/h, capillary voltage 3.0 kV, cone voltage 40.0 V, and source offset voltage 80 V. Data acquisition was achieved using MS. Leucine enkephalin was used as the lock mass that generated a reference ion in the positive mode at m/z 556.2771 and introduced by a lock spray at 10 µL/min for accurate mass acquisition. Under the SIM mode, peaks corresponding to molecular ions for each compound were observed at the following retention times: acteoside, m/z 647 [M + Na]+, tR 3.9 min; and pedaliin, m/z 479 [M + H]+, tR 3.7 min. The standard curves of acteoside and pedaliin were constructed, and their linearity range and correlation (R2) were calculated with five standard solutions (1–30 µM). The standard curves were constructed by plotting concentration (µM) on the horizontal axis and peak area (µV × s) on the vertical axis.
Statistical AnalysisThe results are expressed as the means ± standard error of the mean (S.E.M.) for each condition. Statistical analyses were performed using a one-way ANOVA, followed by Dunnett’s test or the Tukey–Kramer test using GraphPad Prism 6 software. Differences were considered statistically significant at p < 0.05.
We are grateful to the Institute of Pharma Food Co., Ltd. for their help with the collection of the leaves, stems, and seeds of S. indicum. This research was supported by JSPS KAKENHI Grant Numbers: 24K23284 and 23K06206.
The authors declare no conflict of interest.
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