New Anti-Glycative Lignans from the Defatted Seeds of Sesamum indicum

Seven known analogs, along with two previously undescribed lignan derivatives sesamlignans A (1) and B (2), were isolated from a water-soluble extract of the defatted sesame seeds (Sesamum indicum L.) by applying the chromatographic separation method. Structures of compounds 1 and 2 were elucidated based on extensive interpretation of 1D, 2D NMR, and HRFABMS spectroscopic data. The absolute configurations were established by analyzing the optical rotation and circular dichroism (CD) spectrum. Inhibitory effects against the formation of advanced glycation end products (AGEs) and peroxynitrite (ONOO−) scavenging assays were performed to evaluate the anti-glycation effects of all isolated compounds. Among the isolated compounds, (1) and (2) showed potent inhibition towards AGEs formation, with IC50 values of 7.5 ± 0.3 and 9.8 ± 0.5 μM, respectively. Furthermore, the new aryltetralin-type lignan 1 exhibited the most potent activity when tested in the in vitro ONOO− scavenging assay.


Introduction
Among naturally occurring bioactive polyphenolics, lignan is a class of diphenolic secondary metabolites that are distributed in the plant kingdom and biosynthesized by oxidative dimerization of two phenylpropanoid units [1]. This class of natural polyphenols possesses a broad range of structural diversity and biological potencies, including antitumor, antioxidant, anti-inflammatory, anti-neurodegenerative, and antiviral activities [2]. Recent interest in novel lignans originated from natural foodstuffs has continuing increase due to their biological benefits correlated with disease prevention and health promotion. Sesame (Sesamum indicum L.), one of the most important oilseed crops, is a rich source of lignans such as sesamol, sesamin, sesamolin, and sesaminol [3]. Sesame seeds produce a highly stable oil and provide several food nutritional benefits [4]. Studies have reported good antioxidant activities [5] as well as numerous valuable biological effects [6]. Sesame cake obtained from oil extraction is mainly used as a feed ingredient for domestic animals or is discarded. However, defatted sesame seeds are reported to exert various biological properties, including antioxidant [7], anti-diabetic [8,9], and inhibition of brain edema formation [10], and were found to be rich in lignan and polyphenolic constituents [11,12]. Especially, the major lignan glucoside (sesaminol triglucoside) isolated from defatted sesame cake exhibits potent biological activities such as radical scavenging and cytochrome P450 enzyme inhibition [13][14][15]. Current research interests in the biologically active lignans of S. indicum continue because of their inherent ability to prevent diseases and improve health [16,17].
Persistent hyperglycemia is a critical cause associated with the pathogenesis of diabetic complications [18], which can arise from protein kinase C (PKC) isoform activation, increased aldose reductase (AR)-related polyol pathway flux, increased hexosamine pathway

Inhibition of Formation of AGEs and ONOO − Scavenging Effects
All pure isolated compounds 1−9 were evaluated for their capacity to inhibit the formation of AGEs using aminoguanidine as the positive control (Table 2). Compared to the positive control aminoguanidine (IC 50 : 995.3 ± 3.6 µM), the most potent inhibitory effects against AGEs formation were exhibited by the new lignans sesamelignans A (1) and B (2) with IC 50 values of 7.5 ± 0.3 and 9.8 ± 0.5 µM, respectively. The IC 50 values of the furfuran-type lignans 3−7 were obtained in the range 17.8 to 65.8 µM for AGEs formation ability. The simple phenolic compounds 8 and 9 were considerably less effective compared to other lignan derivatives. In addition, we further evaluated the anti-oxidant effects of the isolated compounds using the previously reported ONOO − scavenging assay [40]. The novel aryltetralin lignan 1 showed maximum scavenging activity against the ONOO − scavenging assay (IC 50 : 8.1 ± 0.5 µM) compared to the positive control L -penicillamine (IC 50 : 15.0 ± 1.0 µM). Vanillic acid (9) has previously been described as a powerful ONOO − scavenging substance isolated from Panax ginseng, and our results are in agreement with those findings [41]. Although various lignan analogs from natural products have been reported as anti-glycation inhibitors [42], the current study is the first to validate the new aryltetralintype lignan 1 with potent inhibitory effects of AGEs formation and ONOO − scavenging activity. Taken together, our results indicate the potential to develop sesamelignan A (1) as a therapeutic for diabetic complications and related diseases.

Plant Material and Preparation
Sesame seeds (Sesamum indicum L.) were collected in June 2017 from Yecheon-gun, Republic of Korea, and identified by Prof. Tae Hoon Kim. A voucher specimen was deposited at the Natural Products Chemistry Laboratory of Daegu University. The dried sesame seeds (20 kg) were roasted in an electric frying pan (D-1692, Dongkwang oil machine Co., Seoul, Korea) at 300 • C for 12 min. Oil was extracted from the roasted sesame seeds using an electric oil squeezer (D-1880, Dongkwang oil machine Co., Seoul, Korea), and the remaining sesame byproducts were used in the experiment.

Evaluation of AGEs Formation Inhibitory Effects
Using a previously reported method [43] with minor modification, we evaluated the AGEs formation inhibitory potential of the isolated compounds. Briefly, the reaction mixture was prepared by adding 10 mg/mL BSA in 50 mM phosphate buffer (pH 7.4) containing 0.02% sodium azide to a sugar solution (200 mM D -fructose and 200 mM Dglucose). The reaction mixture (800 µL) was then combined with various concentrations of either the test compounds (200 µL) or the positive control (aminoguanidine) dissolved in 5% DMSO. After incubation at 37 • C for 7 days, the fluorescent reaction products were determined using an ELISA reader (Infinite F200; Tecan Austria GmBH, Grodig, Austria), with excitation and emission maxima at 350 and 450 nm, respectively. The concentration required for 50% inhibition (IC 50 value) of the fluorescence intensity was determined by linear regression analysis. All measurements were obtained in triplicate.

Evaluation of ONOO − Scavenging Activities
The ONOO − scavenging ability was evaluated by observing the extremely fluorescent dihydrorhodamine 123 (DHR 123) that is rapidly generated from non-fluorescent DHR 123 in the presence of ONOO − [40]. The dihydrorhodamine buffer (pH 7.4) comprises 50 mM sodium phosphate monobasic, 50 mM sodium phosphate dibasic, 90 mM sodium chloride, 5 mM potassium chloride, and 100 µM DTPA, and the final DHR 123 concentration used was 5.0 µM. The test sample was dissolved in 5% DMSO. The final fluorescent intensities of the treated samples were observed 5 min after treatment with and without the addition of authentic ONOO − (10 µM) dissolved in 0.3 N NaOH. The fluorescence intensity of the oxidized DHR 123 was estimated with a fluorescence ELISA reader at emission and excitation wavelengths of 530 and 480 nm, respectively. Results of the ONOO − scavenging effect were evaluated as the final fluorescence intensity minus the background fluorescence, determined by the detection of DHR 123 oxidation. The 50% inhibition (IC 50 value) was measured by linear regression analysis of the scavenging activity under the above assay conditions. L -Penicillamine was used as a positive control. All measurements were obtained in triplicate.

Statistical Analysis
Data for the in vitro analyses of AGEs formation and ONOO − scavenging activity were analyzed using the Proc GLM procedure of SAS software (version 9.3, SAS Institute Inc., Cary, NC, USA). The results are reported as the least square mean values and standard deviation. Statistical significance was considered at p < 0.05.

Conclusions
This paper reported two previously undescribed lignans (1 and 2) along with seven known compounds (3−9) isolated from the defatted sesame cake. The new chemical structures of 1 and 2 were characterized by detailed NMR, MS, and CD spectra data analysis. All compounds were evaluated for their inhibitory potential against AGEs formation and ONOO − scavenging properties. The unusual aryltetralin-type (1) and tertrahydrofurantype lignan (2) showed the most potent inhibitory effects of AGEs formation compared to the positive control. In addition, the newly discovered sesamlignan A (1) exhibited a maximum potency for ONOO − scavenging capacity. Thus, we propose that sesamlignans A and B have the potential to be developed as therapeutic agents for treating diabetic complications and related diseases.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/molecules28052255/s1, Figure S1: 1 H NMR spectrum of compound 1 in CD 3 OD. Figure S2: 13 C NMR spectrum of compound 1 in CD 3 OD: Figure S3. 1 H-1 H COSY spectrum of compound 1 in CD 3 OD. Figure S4: HSQC spectrum of compound 1 in CD 3 OD. Figure S5: HMBC spectrum of compound 1 in CD 3 OD. Figure S6: Expanded key HMBC correlation of compound 1 (H-7 to C-5). Figure S7: NOESY spectrum of compound 1 in CD 3 OD. Figure S8: HRFABMS spectrum of compound 1. Figure S9: CD spectrum of compound 1. Figure S10: 1 H NMR spectrum of compound 2 in CD 3 OD. Figure S11: 13 C NMR spectrum of compound 2 in CD 3 OD. Figure S12: 1 H-1 H COSY spectrum of compound 2 in CD 3 OD. Figure S13: HSQC spectrum of compound 2 in CD 3 OD. Figure S14: HMBC spectrum of compound 2 in CD 3 OD. Figure S15: NOESY spectrum of compound 2 in CD 3 OD. Figure S16: HRFABMS spectrum of compound 2. Figure S17: CD spectrum of compound 2. Figure S18: 1 H NMR spectrum of compound 3 in CD 3 OD. Figure S19: 13 C NMR spectrum of compound 3 in CD 3 OD. Figure S20: 1 H NMR spectrum of compound 4 in CD 3 OD. Figure S21: 13 C NMR spectrum of compound 4 in CD 3 OD. Figure S22: 1 H NMR spectrum of compound 5 in CD 3 OD. Figure S23: 1 H NMR spectrum of compound 6 in CD 3 OD: Figure S24: 13 C NMR spectrum of compound 6 in CD 3 OD. Figure S25: 1 H NMR spectrum of compound 7 in CD 3 OD. Figure S26: 13 C NMR spectrum of compound 7 in CD 3 OD. Figure S27: 1 H NMR spectrum of compound 8 in CD 3 OD. Figure S28: 13 C NMR spectrum of compound 8 in CD 3 OD. Figure S29: 1 H NMR spectrum of compound 9 in CD 3 OD. Figure S30: Chemical structures of the new compounds 1 and 2 isolated from defatted Sesame cake. Table S1: AGEs formation inhibitory effects on the fraction from defatted Sesame cake.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.