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Natural products as sources of new antioxidants: Synthesis and antioxidant evaluation of Mannich bases of novel sesamol derivatives
Industrial Crops & Products 141 (2019) 111762
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Natural products as sources of new antioxidants: Synthesis and antioxidant evaluation of Mannich bases of novel sesamol derivatives
T
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Yong Guoa, Jiangping Fana, Lailiang Qua, Chongnan Baoa, Qian Zhanga, Hong Daib, , ⁎ Ruige Yanga, a Key Laboratory of Advanced Drug Preparation Technologies, Ministry of Education, School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, 450001, Henan Province, PR China b College of Chemistry and Chemical Engineering, Nantong University, Nantong, 226019, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Sesamol Mannich base Antioxidant activity Cytotoxicity Structure–activity relationship
Sesamol, a natural phenolic compound with good antioxidant activity, is a nonoil component of sesame seed oil. Two series of novel Mannich bases, 4-(aminoalkyl)-6-allyl-sesamols were synthesized from sesamol, substituted benzaldehydes and piperidine/morpholine via the one-pot reaction. An efficient way using toluene as solvent, catalyst-free and one-pot reaction for the synthesis of these sesamol derivatives was developed. The structures of all 4-(aminoalkyl)-6-allyl-sesamols were determined by spectral analysis. Compound 6f was unambiguously confirmed by X-ray diffraction further. The antioxidant assays have demonstrated that the sesamol derivative 7i displayed excellent antioxidant activity. The structure–activity relationships (SARs) of these sesamol derivatives were also observed. Further, the representative compounds 7a, 7g and 7i exhibited relative low toxicity to normal cells. Based on these characteristics, these sesamol derivatives will have great potential application in food and drugs.
1. Introduction Natural products have long been used for the benefit the humankind including use in foods, cosmetics, biological probes and medicines (Maestri et al., 2006). Natural antioxidants are generally classified as vitamins, phenolic compounds and flavonoids (Lee and Shibamoto, 2002). Sesamol (1, 1,3-benzodioxol-5-ol, Fig. 1), a natural compound and a derivative of phenol, is a major component of sesame seed oil (Aggarwal and Khurana, 2017). Compound 1 has been found to be a good antioxidant in lard and vegetable oils (Joshi et al., 2005). It possesses antioxidant activity that can scavenge intracellular reactive oxygen species. In addition to its antioxidant activities, compound 1 has been demonstrated to possess antimutagenic (Chen et al., 2015), antiinflammatory (Chavali et al., 2001), neuroprotective (Hou et al., 2006), antiplatelet and antiaging activities (Chang et al., 2010; Sharma and Kaur, 2006). Sesamol has a benzodioxole and a phenolic hydroxyl group in its structure. Although the benzodioxole and phenolic groups are responsible for the antioxidant activities of compound 1, the phenolic hydroxyl group and C-6 position are very easily oxidized and formed some oxidative dimers (I–IV) in nature, including in foods and related systems (Masuda et al., 2009). Masuda et al. (2010) further reported that these dimers had potent cytotoxic activity to rat ⁎
thymocytes. Hence, in order to maintain antioxidant properties of sesamol (the phenolic hydroxyl group must remain unchanged) and prevent C-6 position from oxidizing or polymerization, some reductive groups (such as double bond, triple bond and amino groups) could be introduced at C-4 or C-6 position of sesamol (Natella et al., 1999). In light of the above-mentioned results, and as far as we know, few research has focused on structural modifications of 1 for improvement its character of easy formation of dimers and for use as potential antioxidants. Herein, amino and allyl groups have been introduced at C-4 and C-6 positions of sesamol, and a series of novel 4-(aminoalkyl)-6allyl-sesamols were prepared via the one-pot reaction and evaluated for their preliminary antioxidant and cytotoxic activities. 2. Material and methods 2.1. Chemicals All reagents and solvents were of reagent grade or purified according to standard methods before use. Sesamol (C7H6O3, 98.0%), different substituted benzaldehydes (95.0%), morpholine (C4H9NO) and piperidine (C5H11N) were obtained from Macklin Biochemical Inc. (Shanghai, China). α-Tocopherol (VE), 2,2-diphenyl-1-picrylhydrazyl
Corresponding authors. E-mail addresses: [email protected] (H. Dai), [email protected] (R. Yang).
https://doi.org/10.1016/j.indcrop.2019.111762 Received 11 March 2019; Received in revised form 31 August 2019; Accepted 3 September 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.
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found 370.29. Data for 7a: yield: 61%, white solid; mp: 85–87 °C; IR cm-1: 3443, 2958, 2844, 1639, 1450, 1117, 1050; 1H NMR (400 MHz, CDCl3) δ: 11.37 (s, 1H, -OH), 7.46 (d, J = 6.4, 2H, -Ph), 7.27-7.32 (m, 3H, -Ph), 6.50 (s, 1H, H-7), 5.93-6.03 (m, 1H, H-2′), 5.76 (dd, J = 1.6, 18.4 Hz, 2H, -OCH2O-), 4.99-5.05 (m, 2H, H-1′), 4.57 (s, 1H, H-1′′), 3.75-3.79 (m, 4H, morpholine ring), 3.26-3.37 (m, 2H, H-3′), 2.46-2.62 (m, 4H, morpholine ring); MS (ESI) m/z calcd for C21H24NO4 ([M + H]+) 354.16, found 354.32. Data for 7b: yield: 52%, white solid; mp: 70–72 °C; IR cm-1: 3424, 3074, 2962, 2852, 1638, 1491, 1450, 1119, 1049; 1H NMR (400 MHz, CDCl3) δ: 11.43 (s, 1H, -OH), 7.55 (t, J =6.8 Hz, 1H, -Ph), 7.23-7.27 (m, 1H, -Ph), 7.03-7.11 (m, 2H, -Ph), 6.54 (s, 1H, H-7), 5.95-6.05 (m, 1H, H-2′), 5.76 (d, J =16.4 Hz, 2H, -OCH2O-), 5.02-5.12 (m, 3H, H-1′′ and H-1′), 3.69-3.75 (m, 4H, morpholine ring), 3.28-3.39 (m, 2H, H-3′), 2.46-2.69 (m, 4H, morpholine ring); MS (ESI) m/z calcd for C21H23FNO4 ([M + H]+) 372.15, found 372.27. Data for 7c: yield: 57%, yellow liquid; IR cm-1: 3431, 2964, 2894, 2852, 1650, 1510, 1449, 1119, 1049; 1H NMR (400 MHz, CDCl3) δ: 11.28 (s, 1H, -OH), 7.42-7.46 (m, 2H, -Ph), 6.97-7.01 (t, J =8.8 Hz, 2H, -Ph), 6.51 (s, 1H, H-7), 5.93-6.03 (m, 1H, H-2′), 5.76 (dd, J = 1.6, 18.4 Hz, 2H, -OCH2O-), 4.99-5.04 (m, 2H, H-1′), 4.55 (s, 1H, H-1′′), 3.75-3.79 (m, 4H, morpholine ring), 3.26-3.37 (m, 2H, H-3′), 2.42-2.62 (m, 4H, morpholine ring); MS (ESI) m/z calcd for C21H23FNO4 ([M + H]+) 372.15, found 372.24.
Fig. 1. Structures of sesamol (1) and its oxidative dimers (I–IV).
(DPPH), 2,6-di-tert-butyl-4-methylphenol (BHT) and 2,4,6-tripyridyl-striazine (TPTZ) were purchased from Aladdin Chemical Industrial, Inc. (Shanghai, China). 2.2. Instrument and Materials A melting-point (mp) instrument from Tech Instrument Co., Ltd. (Beijing, China) was used to measure the melting points (mp). Infrared spectra (IR) were recorded on a PE-1710 FT-IR spectrometer (PerkinElmer, Waltham, USA). 1H NMR spectra of all sesamol derivatives were determined by a Bruker Avance spectrometer (400 MHz). A SMART APEX II equipment (Bruker, Karlsruhe, Germany) was used to detect Xray crystallography. Electrospray ionization mass spectrometry (ESIMS) were measured on an Agilent 1100 LC/MSD SL instrument (Agilent, Palo Alto, USA). The Chinese Academy of Sciences Cell Bank (Shanghai, China) provided the normal rat kidney tubule epithelial (NRK-52E) and RAW264.7 cells.
2.4. Antioxidant activities of compounds 1, 6a–k and 7a–k 2.4.1. DPPH radical scavenging activity The DPPH radical scavenging activity of compounds 1, 6a–k and 7a–k was evaluated by a reported procedure (Wang et al., 2015) with little modifications, using the microplate reader. Briefly, the stock solutions were prepared by compounds 1, 6a–k, 7a–k, BHT and VE which were dissolved in MeOH. Then, the stock solutions were further diluted in MeOH to acquire required concentrations of 1.56-200 μg/mL in 96 well microplates. Diluted sample solution (100 μl) and the DPPH working solution (0.078 mg/mL, 100 μl) were put into each well of a 96-well microplate. After vortexing, the microplates were left in the dark for 30 min at 25 °C, and the absorbance (ABS) values of samples was recorded on a microplate reader at 517 nm. The positive controls were BHT and VE. The solvent MeOH was used as the blank control. Each experiment was repeated three times. The DPPH radical scavenging activity of compounds 1, 6a–k and 7a–k was evaluated as the following formula: DPPH radical scavenging activity (%) = (1 – B/A) × 100, where A represents the absorbance of the blank control, and B represents the absorbance of the tested groups. The radical scavenging capacity was assessed as the effective concentration (EC50) which defined as the concentration which resulted in 50% inhibition.
2.3. Synthesis of compounds 6a–k and 7a–k 5-Allyloxy-sesamol (2) and 6-allyl-sesamol (3) were synthesized according to previously reported procedure (Guo et al., 2016; Qu et al., 2017). To a solution of 6-allyl-sesamol (3, 0.5 mmol, 89.5 mg) in dry toluene under N2 was added morpholine/piperidine (0.55 mmol) and substituted benzaldehyde (0.55 mmol) in sequence. When the reaction was finished according to analytical thin-layer chromatography (TLC) analysis, solvent was concentrated in vacuo. We used preparative thinlayer chromatography (PTLC) to purify the mixture and obtained the title compounds 6a−k and 7a–k. Exemplary data of compounds 6a–c and 7a–c were listed as follows, other data of compounds 6d–k and 7d–k were listed in the Supplementary material. Data for 6a: yield: 62%, pale yellow solid; mp: 83–85 °C; IR cm-1: 3436, 2935, 2855, 1638, 1450, 1108, 1049; 1H NMR (400 MHz, CDCl3) δ: 12.09 (s, 1H, -OH), 7.46-7.53 (m, 2H, -Ph), 7.22-7.30 (m, 3H, -Ph), 6.47 (s, 1H, H-7), 5.95-6.05 (m, 1H, H-2′), 5.74 (dd, J = 1.2, 19.2 Hz, 2H, -OCH2O-), 5.00-5.04 (m, 2H, H-1′), 4.52 (s, 1H, H-1′′), 3.27-3.38 (m, 2H, H-3′), 2.29-2.38 (m, 3H, piperidine ring), 1.45-1.73 (m, 7H, piperidine ring); MS (ESI) m/z calcd for C22H26BrNO3 ([M + H]+) 352.18, found 352.25. Data for 6b: yield: 41%, white solid; mp: 90–91 °C; IR cm-1: 3434, 2962, 2944, 2881, 1639, 1491, 1456, 1099, 1047; 1H NMR (400 MHz, CDCl3) δ: 12.21 (s, 1H, -OH), 7.54 (s, 1H, -Ph), 7.20-7.25 (m, 1H, -Ph), 7.01-7.09 (m, 2H, -Ph), 6.51 (s, 1H, H-7), 5.97-6.07 (m, 1H, H-2′), 5.73 (d, J =17.6 Hz, 2H, -OCH2O-), 5.03-5.06 (m, 3H, H-1′ and H-1′′), 3.293.43 (m, 2H, H-3′), 2.07-2.55 (m, 3H, piperidine ring), 1.43-1.75 (m, 7H, piperidine ring); MS (ESI) m/z calcd for C22H25FNO3 ([M + H]+) 370.17, found 370.29. Data for 6c: yield: 42%, white solid; mp: 45–47 °C; IR cm-1: 3438, 2938, 2852, 1638, 1499, 1454, 1222, 1048; 1H NMR (400 MHz, CDCl3) δ: 12.00 (s, 1H, -OH), 7.42-7.52 (m, 2H, -Ph), 6.94-6.98 (m, 2H, -Ph), 6.48 (s, 1H, H-7), 5.94-6.04 (m, 1H, H-2′), 5.74 (d, J =17.6 Hz, 2H, -OCH2O-), 5.00-5.04 (m, 2H, H-1′), 4.51 (s, 1H, H-1′′), 3.27-3.37 (m, 2H, H-3′), 2.38-2.45 (m, 3H, piperidine ring), 1.25-1.63 (m, 7H, piperidine ring); MS (ESI) m/z calcd for C22H25FNO3 ([M + H]+) 370.17,
2.4.2. Ferric-reducing antioxidant power (FRAP) The FRAP assay was determined by the previous reports (Firuzi et al., 2005). The FRAP was based on the reducing capacity of tested compounds. The ferric ion (Fe3+) will be reduced by a potential antioxidant to the ferrous ion (Fe2+) and it can form a blue complex (Fe2+/ TPTZ). This complex has a great absorption at 593 nm. In short, the acetate buffer [NaOAc (3.1 g) and HOAc (20 mL) were dissolved in 1000 mL of water], FeCl3·H2O (20 mM) and a solution of 10 μM TPTZ in hydrochloric acid (40 μM) were mixed to prepared the FRAP reagent. This reagent was incubated at 37 °C for ten minutes. A 180 μl of FRAP reagent was added to 20 μl of each tested compounds 1, 6a–k, 7a–k, BHT, a-tocopherol solutions (200 μg/mL), then the mixture was incubated at 37 °C for four minutes. Absorbance values of the tested compounds were determined at 593 nm. MeOH (20 μl) was used as a blank. Fresh working solutions of FeSO4 (1.0 mmol/L) was used as the calibration. Values of FRAP are expressed as μmol Fe2+/mg tested 2
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pot reaction of compound 3, aromatic aldehydes and secondary amines without catalysts. Structures of compounds 6a–k and 7a–k were confirmed using spectral analysis of 1H NMR, IR, mp and ESI-MS. Moreover, three-dimensional steric structure of 6f was further determined by X-ray diffraction (Fig. 3). Publication no. CCDC (Cambridge Crystallographic Data Centre) of compound 6f was 1862039. This obviously proved that we have successfully prepared a series of 4-(aminoalkyl)-6allyl-sesamols 6a–k and 7a–k via the one-pot reaction. In addition, a probable reaction mechanism of this one-pot reaction is proposed. As shown in Fig. 4, different substituted benzaldehyde 4a–k reacted with secondary amines 5a/5a' to formed iminium ion 8, subsequent, the iminium carbon atom was attacked by the carbon atom of compound 3. Finally, a hydride shift leads to the formation of 4(aminoalkyl)-6-allyl-sesamols (6a–k and 7a–k).
compound (TC). All analyses were done in triplicate. 2.5. Cytotoxicity assay The cytotoxic activities of potent compounds 7a, 7g and 7i were screened in NRK-52E and RAW264.7 cells according to the Cell Counting Kit-8 (CCK-8) method (Xiong et al., 2016). In brief, 5 × 103 NRK-52E and RAW264.7 cells in 100 μL medium were seed to each of 96-well plates. Subsequently, these cells were incubation at 37 °C for 24 h, a 100 μL of fresh medium with the compounds 7a, 7g and 7i in different concentration was added to remove and replace the used culture medium. The negative control was only added fresh medium. After treatment for 24 h, the used culture medium was discarded, and then using the new culture medium to wash the used medium twice. At last, new medium containing 5% CCK-8 (100 μL) was added to each well. After cultured at 37 °C for a further 4 h, a Microplate Reader was used to record the ABS values of the tested compounds at 450 nm. Cell survival rate (activity %) was calculated as follows: Cell survival rate (%) = (ODexperiment – ODblank)/(ODnegative control – ODblank) × 100%, where OD is the abbreviation of optical density. Each compound was tested in triplicate. The results expressed as the inhibitory concentration (IC) of 50% values ± SD.
3.2. Antioxidant activities and structure–activity relationships (SARs) 3.2.1. DPPH radical scavenging activity The antioxidant activity of 1, 6a–k, 7a–k, commercial antioxidants BHT and VE was first assessed by their scavenging capacity against DPPH radical (Sammaiah et al., 2015). The amount of tested sesamol derivatives required to inhibit the radicals by 50% was calculated and expressed as their EC50 values. The antioxidant activity results of sesamol derivatives are shown in Fig. 5. The EC50 values of all 4-(aminoalkyl)-6-allyl-sesamols ranged from 7.81 to 24.31 μg/mL, which suggested that some of sesamol derivatives exhibited moderate to good antioxidant activity. The lead compound sesamol showed remarkable DPPH radical scavenging activity with EC50 value of 10.95 μg/mL, which was close to the commercial antioxidant VE. Some similar results were also confirmed by the previously reported (Erkan et al., 2008). Among all the sesamol derivatives, 7a, 7g and 7i displayed extraordinary DPPH radical scavenging activity with EC50 values of 9.25, 7.81 and 7.85 μg/mL respectively, which were higher than the standards BHT (EC50: 11.9 μg/mL) and VE (EC50: 10.76 μg/mL). In addition, compounds 7h and 7k showed comparable DPPH radical scavenging activity with EC50 values of 10.89 and 10.8 μg/mL respectively, in comparison with VE, but slightly higher than that of BHT. Based on the SARs of these prepared sesamol derivatives, in general, it was observed that 4-(aminoalkyl)-6-allyl-sesamols containing the morpholine were found to have more potent DPPH radical scavenging activity than that containing the piperidine. For example, the EC50 values of compounds 7a–k were all lower than that of 6a–k [e.g., 7a (EC50: 9.25 μg/mL) vs. 6a (EC50: 18.4 μg/mL); 7b (EC50: 12.33 μg/mL) vs. 6b (EC50: 24.31 μg/mL); 7c (EC50: 11.73 μg/mL) vs. 6c (EC50: 19.56 μg/mL)]. In addition, when R was -H or electron-donating group, the corresponding sesamol derivatives exhibited more potent DPPH radical scavenging activity than that R was electron-withdrawing
2.6. Statistical analysis All data are expressed as mean ± SD in triplicate analysis for all the experiments. All statistical analysis was processed by the SPSS 21.0 (SPSS Inc., Chicago, USA) software. 3. Results and discussion 3.1. Chemistry As shown in Fig. 2, firstly, compound 1 reacted with allylbromide using K2CO3 as base to easily prepare compound 2 in 93% yield. Then, Claisen rearrangement of compound 2 in N,N-dimethylaniline gave 6allyl-sesamol 3 in 90% yield. The detailed synthesis parameters of compounds 2 and 3 can be found in our previous studies (Guo et al., 2016; Qu et al., 2017). It is worth mentioning that stirring of 6-allylsesamol 3, piperidine and benzaldehyde in refluxing EtOH under a nitrogen atmosphere for 12 h afforded the target compound 6a in only 24.0% yield (Table 1, Entry 1). Inspired by this result, the reaction was optimized in terms of the different solvents. Solvents of EtOH, N,Ndimethylformamide (DMF), toluene, CH3CN and 1,4-dioxane were screened. The best yield of 62.0% of the title compound 6a was achieved in toluene. Finally, we used toluene as solvent for the synthesis of 4-(aminoalkyl)-6-allyl-sesamols (6a–k, 7a–k) through the one-
Fig. 2. Synthetic routes of Mannich bases, 4-(aminoalkyl)-6-allyl-sesamols (6a–k and 7a–k). 3
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Table 1 Screening of solvents for the synthesis of the 4-(aminoalkyl)-6-allyl-sesamols taking the model reaction of 6-allyl-sesamol, benzaldehyde and piperidinea.
b
Entry
Solvent
1 2 3 4 5
EtOH DMF Toluene CH3CN 1,4-Dioxane
a b c
(mL)
Time (h)
Isolated yield c(%)
12 12 12 12 12
24.0 31.4 62.0 40.2 33.5
Optimal reaction conditions: molar ratio of 3 (0.5 mmol)/4a (0.55 mmol)/5a (0.55 mmol) = 1 : 1.1 : 1.1, 80 °C. Ethanol (EtOH), N,N-dimethylformamide (DMF). Isolated yields after preparative thin-layer chromatography (PTLC).
Fig. 4. Possible mechanism for the preparation of Mannich bases, 4-(aminoalkyl)-6-allyl-sesamols (6a–k and 7a–k).
3.2.2. FRAP assay The ability of antioxidants to reduce Fe3+ to Fe2+ in the presence of TPTZ is the fundamental of the FRAP assay. The TPTZ–Fe2+ complex displays an intense blue colour which displays absorption at 593 nm (Benzie and Strain, 1996). The results of the reducing power of 1, 6a–k and 7a–k expressed as μmol Fe2+ /mg TC in comparison with VE and BHT are summarised in Fig. 6. On the basis of FRAP values, most of the title compounds except 7i had lower FRAP values than the sesamol, VE and BHT. The compound 7i had FRAP value of 9.6 μmol Fe2+ /mg TC, which was higher than the positive control VE and BHT. The FRAP values of other compounds ranged from 1.98 to 5.15 μmol Fe2+ /mg TC. These results may be bacause the mechanism of FRAP is different from that of DPPH radical scavenging activity (Bakar et al., 2009). Additionally, the precursor sesamol also showed good antioxidant activity with the FRAP value of 8.52 μmol Fe2+ /mg TC, which was in line with those reported by Joshi et al. (2005) who studied the effect of sesamol on Fe3+-induced lipid peroxidation. In general, in DPPH and FRAP assay, compound 7i showed excellent antioxidant activity than the precusor sesamol, the commercial antioxidants VE and BHT.
Fig. 3. X-ray crystal structure of compound 6f.
group. For instance, the EC50 values of compounds 6g (R = 4-CH3), 7g (R = 4-CH3), 6h (R = 4-OCH3) and 7h (R = 4-OCH3) were 12.49, 7.81, 15.11 and 10.89 μg/mL, respectively. Whereas, the EC50 values of compounds 6c (R = 4-F), 7c (R = 4-F), 6d (R = 4-Cl) and 7d (R = 4Cl) were 19.56, 11.73, 20.19 and 12.23 μg/mL, respectively. Moreover, the compounds 6i (R = 4-dimethylamino) and 7i (R = 4-dimethylamino) showed outstanding DPPH radical scavenging activity, which may be because the 4-dimethylamino was a strong electron-donating group in comparison of other groups (Coe et al., 2001). Literature reported that the increase in the electron-donating groups displayed an obvious increase in the antioxidant activity by further stabilizing the phenolic hydroxyl radical (Merkl et al., 2010). These results are somewhat in agreement with some previous reports of synthesized Mannich bases (Oloyede et al., 2014).
3.2.3. Cytotoxicity In consideration of the accompanied toxicity to normal mammalian cells is one of the major obstacles to the preparation of food additives from many promising antioxidant compounds, so the title compounds with potent antioxidant capacity were further evaluated for their cytotoxic activities against RAW264.7 and NRK-52E cells. As depicted in Fig. 7, compounds 7a, 7g and 7i showed relative low cytotoxicities to 4
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Fig. 5. DPPH radical scavenging activity of 4-(aminoalkyl)-6-allyl-sesamols (6a–k and 7a–k); Values represent means ± SD (DPPH: 2,2-diphenyl-1-picrylhydrazyl; BHT: EC50: effective concentration 50%; 2,6-di-tert-butyl-4-methylphenol).
and will have great potential application in food and drugs.
noncancerous RAW264.7 (Fig. 7a) and NRK-52E (Fig. 7b) cells, and the IC50 values were higher than 96.3 μg/mL. It suggested that these compounds could be new promising alternatives for antioxidants.
Acknowledgments 4. Conclusion The authors gratefully acknowledge the financial support by National Natural Science Foundation of China (21502176), Natural Science Foundation of Hennan Province, China (No. 182300410025), the China Postdoctoral Science Foundation (2019M652585), Key Science and Technology Program of Henan Province, China (192102110054) and Startup Research Fund of Zhengzhou University (145-32211180).
In general, two series of twenty-two novel Mannich bases, 4-(aminoalkyl)-6-allyl-sesamols 6a–k and 7a–k were prepared by the one-pot reaction and their structures were characterized by spectral analysis. In addition, a simple procedure for the synthesis of these 4-(aminoalkyl)6-allyl-sesamols by using toluene as solvent, catalyst-free and the onepot reaction methodology was developed. This method will be valuable for development of other Mannich bases. The structure of 6f was unambiguously confirmed by X-ray diffraction further. The antioxidant assays have demonstrated the sesamol derivative 7i displayed excellent antioxidant activity. Further, the representative compounds 7a, 7g and 7i exhibited relative low cytotoxicity against RAW264.7 and NRK-52E cells. The results suggest that these novel sesamol derivatives could offer an interesting solution for natural products-based antioxidants,
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2019.111762.
Fig. 6. Ferric-reducing antioxidant power (FRAP) of Mannich bases, 4-(aminoalkyl)-6-allyl-sesamols (6a–k and 7a–k); Values represent means ± SD (BHT: 2,6-ditert-butyl-4-methylphenol). 5
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Fig. 7. Relative cell viabilities of compounds 7a, 7g and 7i against RAW264.7 (a) and NRK-52E (b) cells; Values represent means ± SD.
References
isolated from various herbs and spices. J. Agri. Food Chem. 50, 4947–4952. https:// doi.org/10.1021/jf0255681. Joshi, R., Kumar, M.S., Satyamoorthy, K., Unnikrisnan, M.K., Mukherjee, T., 2005. Free radical reactions and antioxidant activities of sesamol: pulse radiolytic and biochemical studies. J. Agri. Food Chem. 53, 2696–2703. https://doi.org/10.1021/ jf0489769. Maestri, D.M., Nepote, V., Lamarque, A.L., Zygadlo, J.A., 2006. Natural products as antioxidants. Phytochemistry: Advances in research 37, 105–135. Masuda, T., Fujimoto, A., Oyama, Y., Maekawa, T., Sone, Y., 2009. Structure of cytotoxic products from Fe-catalyzed oxidation of sesamol in ethanol. Tetrahedron Lett. 50, 3905–3908. https://doi.org/10.1016/j.tetlet.2009.04.063. Masuda, T., Shingai, Y., Fujimoto, A., Unnikrisnan, M.K., Mukherjee, T., 2010. Identification of cytotoxic dimers in oxidation product from sesamol, a potent antioxidant of sesame oil. J. Agri. Food Chem. 58, 10880–10885. https://doi.org/10. 1021/jf103015j. Merkl, R., Hradkova, I., Filip, V., Drkal, J.S., 2010. Antimicrobial and antioxidant properties of phenolic acids alkyl esters. Czech J. Food Sci. 28, 275–279. Natella, F., Nardini, M., Felice, M.D., Scaccini, C., 1999. Benzoic and cinnamic acid derivatives as antioxidants: Structure-activity relation. J. Agri. Food Chem. 47, 1453–1459. https://doi.org/10.1021/jf980737w. Oloyede, G.K., Willie, I.E., Adeeko, O.O., 2014. Synthesis of Mannich bases: 2-(3Phenylaminopropionyloxy)-benzoic acid and 3-Phenylamino-1-(2, 4, 6-trimethoxyphenyl)-propan-1-one, their toxicity, ionization constant, antimicrobial and antioxidant activities. Food Chem. 165, 515–521. https://doi.org/10.1016/j.foodchem. 2014.05.119. Qu, L., Xu, H., Wang, X., Huang, M., Deng, L., Guo, Y., 2017. Application of Sustainable Natural Bioresources in Agriculture: Iodine-Mediated Oxidative Cyclization for Metal-Free One-Pot Synthesis of N-Phenylpyrazole Sarisan Analogues as Insecticidal Agents. ACS Omega. 2, 5974–5980. https://doi.org/10.1021/acsomega.7b01106. Wang, S., Wang, D., Liu, Z., 2015. Synergistic, additive and antagonistic effects of Potentilla fruticosa combined with EGb761 on antioxidant capacities and the possible mechanism. Ind. Crops Prod. 67, 227–238. https://doi.org/10.1016/j.indcrop.2015. 01.025. Sammaiah, A., Kaki, S.S., Manoj, G.N.V.T.S., Poornachandra, Y., Kumar, C.G., Prasad, R.B.N., 2015. Novel fatty acid esters of apocynin oxime exhibit antimicrobial and antioxidant activities. Eur. J. Lipid Sci. Tech. 117, 692–700. Sharma, S., Kaur, I.P., 2006. Development and evaluation of sesamol as an antiaging agent. Int. J. Dermatol. 45, 200–208. https://doi.org/10.1111/j.1365-4632.2004. 02537.x. Xiong, H., Zheng, H., Wang, W., Liang, J., Wen, W., Zhang, X., Wang, S., 2016. A convenient purification method for silver nanoclusters and its applications in fluorescent pH sensors for bacterial monitoring. Biosens. Bioelectron. 86, 164–168. https://doi. org/10.1016/j.bios.2016.06.038.
Aggarwal, K., Khurana, J.M., 2017. Synthesis of a novel 5a, 10a-dihydroxy-5aH-[1,3] dioxolo [4,5-f] indeno [1,2-b] benzofuran-10(10aH)-one their XRD, FTIR, NMR and DFT studies. J. Mol. Struct. 1130, 739–747. https://doi.org/10.1016/j.molstruc. 2016.11.008. Bakar, M.F.A., Mohamed, M., Rahmat, A., Fry, J., 2009. Phytochemicals and antioxidant activity of different parts of bambangan (Mangifera pajang) and tarap (Artocarpus odoratissimus). Food Chem. 113, 479–483. https://doi.org/10.1016/j.foodchem. 2008.07.081. Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: The FRAP assay. Anal. Biochem. 239, 70–76. https://doi.org/ 10.1006/abio.1996.0292. Chang, C.C., Lu, W.J., Chiang, C.W., Jayakumar, T., Ong, E.T., Hsiao, G., Fong, T.H., Chou, D.S., Sheu, J.R.J., 2010. Potent antiplatelet activity of sesamol in an in vitro and in vivo model: pivotal roles of cyclic AMP and p38 mitogen-activated protein kinase. J. Nutr. Biochem. 21, 1214–1221. https://doi.org/10.1016/j.jnutbio.2009.10.009. Chavali, S.R., Utsunomiya, T., Forse, R.A., 2001. Increased survival after cecal ligation and puncture in mice consuming diets enriched with sesame seed oil. Crit. Care Med. 29, 140–143. https://doi.org/10.1097/00003246-200101000-00028. Chen, W.Y., Chen, F.Y., Lee, A.S., Ting, K.H., Chang, C.M., Hsu, J.F., Lee, W.S., Sheu, J.R., Chen, C.H., Shen, M.Y., 2015. Sesamol reduces the atherogenicity of electronegative L5 LDL in vivo and in vitro. J. Nat. Prod. 78, 225–233. https://doi.org/10.1021/ np500700z. Coe, B.J., Harris, J.A., Clays, K., Persoons, A., Wostyn, K., Brunschwig, B.S., 2001. A comparison of the pentaammine (pyridyl) ruthenium (II) and 4-(dimethylamino) phenyl groups as electron donors for quadratic non-linear optics. Chem. Comm. 17, 1548–1549. https://doi.org/10.1039/B103543F. Erkan, N., Ayranci, G., Ayranci, E., 2008. Antioxidant activities of rosemary (Rosmarinus Officinalis L.) extract, blackseed (Nigella sativa L.) essential oil, carnosic acid. rosmarinic acid and sesamol. Food chem. 110, 76–82. https://doi.org/10.1016/j. foodchem.2008.01.058. Firuzi, O., Lacanna, A., Petrucci, R., Marrosu, G., Saso, L., 2005. Evaluation of the antioxidant activity of flavonoids by ferric reducing antioxidant power assay and cyclic voltammetry. BBA-Gen. Sub. 1721, 174–184. https://doi.org/10.1016/j.bbagen. 2004.11.001. Guo, Y., Qu, L., Wang, X., Huang, M., Jia, L., Zhang, Y., 2016. Iodine-catalyzed oxidative cyclisation for the synthesis of sarisan analogues containing 1,3,4-oxadiazole as insecticidal agents. RSC Adv. 6, 93505–93510. https://doi.org/10.1039/C6RA22343E. Hou, R.C., Chen, Y.S., Chen, C.H., Chen, Y.H., Jeng, K.C., 2006. Protective effect of 1,2,4benzenetriol on LPS-induced NO productionby BV2 microglial cells. J. Biomed. Sci. 13, 89–99. https://doi.org/10.1007/s11373-005-9039-5. Lee, K., Shibamoto, T., 2002. Determination of antioxidant potential of volatile extracts
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Natural products as sources of new antioxidants: Synthesis and antioxidant evaluation of Mannich bases of novel sesamol derivatives
T
⁎
Yong Guoa, Jiangping Fana, Lailiang Qua, Chongnan Baoa, Qian Zhanga, Hong Daib, , ⁎ Ruige Yanga, a Key Laboratory of Advanced Drug Preparation Technologies, Ministry of Education, School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou, 450001, Henan Province, PR China b College of Chemistry and Chemical Engineering, Nantong University, Nantong, 226019, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Sesamol Mannich base Antioxidant activity Cytotoxicity Structure–activity relationship
Sesamol, a natural phenolic compound with good antioxidant activity, is a nonoil component of sesame seed oil. Two series of novel Mannich bases, 4-(aminoalkyl)-6-allyl-sesamols were synthesized from sesamol, substituted benzaldehydes and piperidine/morpholine via the one-pot reaction. An efficient way using toluene as solvent, catalyst-free and one-pot reaction for the synthesis of these sesamol derivatives was developed. The structures of all 4-(aminoalkyl)-6-allyl-sesamols were determined by spectral analysis. Compound 6f was unambiguously confirmed by X-ray diffraction further. The antioxidant assays have demonstrated that the sesamol derivative 7i displayed excellent antioxidant activity. The structure–activity relationships (SARs) of these sesamol derivatives were also observed. Further, the representative compounds 7a, 7g and 7i exhibited relative low toxicity to normal cells. Based on these characteristics, these sesamol derivatives will have great potential application in food and drugs.
1. Introduction Natural products have long been used for the benefit the humankind including use in foods, cosmetics, biological probes and medicines (Maestri et al., 2006). Natural antioxidants are generally classified as vitamins, phenolic compounds and flavonoids (Lee and Shibamoto, 2002). Sesamol (1, 1,3-benzodioxol-5-ol, Fig. 1), a natural compound and a derivative of phenol, is a major component of sesame seed oil (Aggarwal and Khurana, 2017). Compound 1 has been found to be a good antioxidant in lard and vegetable oils (Joshi et al., 2005). It possesses antioxidant activity that can scavenge intracellular reactive oxygen species. In addition to its antioxidant activities, compound 1 has been demonstrated to possess antimutagenic (Chen et al., 2015), antiinflammatory (Chavali et al., 2001), neuroprotective (Hou et al., 2006), antiplatelet and antiaging activities (Chang et al., 2010; Sharma and Kaur, 2006). Sesamol has a benzodioxole and a phenolic hydroxyl group in its structure. Although the benzodioxole and phenolic groups are responsible for the antioxidant activities of compound 1, the phenolic hydroxyl group and C-6 position are very easily oxidized and formed some oxidative dimers (I–IV) in nature, including in foods and related systems (Masuda et al., 2009). Masuda et al. (2010) further reported that these dimers had potent cytotoxic activity to rat ⁎
thymocytes. Hence, in order to maintain antioxidant properties of sesamol (the phenolic hydroxyl group must remain unchanged) and prevent C-6 position from oxidizing or polymerization, some reductive groups (such as double bond, triple bond and amino groups) could be introduced at C-4 or C-6 position of sesamol (Natella et al., 1999). In light of the above-mentioned results, and as far as we know, few research has focused on structural modifications of 1 for improvement its character of easy formation of dimers and for use as potential antioxidants. Herein, amino and allyl groups have been introduced at C-4 and C-6 positions of sesamol, and a series of novel 4-(aminoalkyl)-6allyl-sesamols were prepared via the one-pot reaction and evaluated for their preliminary antioxidant and cytotoxic activities. 2. Material and methods 2.1. Chemicals All reagents and solvents were of reagent grade or purified according to standard methods before use. Sesamol (C7H6O3, 98.0%), different substituted benzaldehydes (95.0%), morpholine (C4H9NO) and piperidine (C5H11N) were obtained from Macklin Biochemical Inc. (Shanghai, China). α-Tocopherol (VE), 2,2-diphenyl-1-picrylhydrazyl
Corresponding authors. E-mail addresses: [email protected] (H. Dai), [email protected] (R. Yang).
https://doi.org/10.1016/j.indcrop.2019.111762 Received 11 March 2019; Received in revised form 31 August 2019; Accepted 3 September 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.
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found 370.29. Data for 7a: yield: 61%, white solid; mp: 85–87 °C; IR cm-1: 3443, 2958, 2844, 1639, 1450, 1117, 1050; 1H NMR (400 MHz, CDCl3) δ: 11.37 (s, 1H, -OH), 7.46 (d, J = 6.4, 2H, -Ph), 7.27-7.32 (m, 3H, -Ph), 6.50 (s, 1H, H-7), 5.93-6.03 (m, 1H, H-2′), 5.76 (dd, J = 1.6, 18.4 Hz, 2H, -OCH2O-), 4.99-5.05 (m, 2H, H-1′), 4.57 (s, 1H, H-1′′), 3.75-3.79 (m, 4H, morpholine ring), 3.26-3.37 (m, 2H, H-3′), 2.46-2.62 (m, 4H, morpholine ring); MS (ESI) m/z calcd for C21H24NO4 ([M + H]+) 354.16, found 354.32. Data for 7b: yield: 52%, white solid; mp: 70–72 °C; IR cm-1: 3424, 3074, 2962, 2852, 1638, 1491, 1450, 1119, 1049; 1H NMR (400 MHz, CDCl3) δ: 11.43 (s, 1H, -OH), 7.55 (t, J =6.8 Hz, 1H, -Ph), 7.23-7.27 (m, 1H, -Ph), 7.03-7.11 (m, 2H, -Ph), 6.54 (s, 1H, H-7), 5.95-6.05 (m, 1H, H-2′), 5.76 (d, J =16.4 Hz, 2H, -OCH2O-), 5.02-5.12 (m, 3H, H-1′′ and H-1′), 3.69-3.75 (m, 4H, morpholine ring), 3.28-3.39 (m, 2H, H-3′), 2.46-2.69 (m, 4H, morpholine ring); MS (ESI) m/z calcd for C21H23FNO4 ([M + H]+) 372.15, found 372.27. Data for 7c: yield: 57%, yellow liquid; IR cm-1: 3431, 2964, 2894, 2852, 1650, 1510, 1449, 1119, 1049; 1H NMR (400 MHz, CDCl3) δ: 11.28 (s, 1H, -OH), 7.42-7.46 (m, 2H, -Ph), 6.97-7.01 (t, J =8.8 Hz, 2H, -Ph), 6.51 (s, 1H, H-7), 5.93-6.03 (m, 1H, H-2′), 5.76 (dd, J = 1.6, 18.4 Hz, 2H, -OCH2O-), 4.99-5.04 (m, 2H, H-1′), 4.55 (s, 1H, H-1′′), 3.75-3.79 (m, 4H, morpholine ring), 3.26-3.37 (m, 2H, H-3′), 2.42-2.62 (m, 4H, morpholine ring); MS (ESI) m/z calcd for C21H23FNO4 ([M + H]+) 372.15, found 372.24.
Fig. 1. Structures of sesamol (1) and its oxidative dimers (I–IV).
(DPPH), 2,6-di-tert-butyl-4-methylphenol (BHT) and 2,4,6-tripyridyl-striazine (TPTZ) were purchased from Aladdin Chemical Industrial, Inc. (Shanghai, China). 2.2. Instrument and Materials A melting-point (mp) instrument from Tech Instrument Co., Ltd. (Beijing, China) was used to measure the melting points (mp). Infrared spectra (IR) were recorded on a PE-1710 FT-IR spectrometer (PerkinElmer, Waltham, USA). 1H NMR spectra of all sesamol derivatives were determined by a Bruker Avance spectrometer (400 MHz). A SMART APEX II equipment (Bruker, Karlsruhe, Germany) was used to detect Xray crystallography. Electrospray ionization mass spectrometry (ESIMS) were measured on an Agilent 1100 LC/MSD SL instrument (Agilent, Palo Alto, USA). The Chinese Academy of Sciences Cell Bank (Shanghai, China) provided the normal rat kidney tubule epithelial (NRK-52E) and RAW264.7 cells.
2.4. Antioxidant activities of compounds 1, 6a–k and 7a–k 2.4.1. DPPH radical scavenging activity The DPPH radical scavenging activity of compounds 1, 6a–k and 7a–k was evaluated by a reported procedure (Wang et al., 2015) with little modifications, using the microplate reader. Briefly, the stock solutions were prepared by compounds 1, 6a–k, 7a–k, BHT and VE which were dissolved in MeOH. Then, the stock solutions were further diluted in MeOH to acquire required concentrations of 1.56-200 μg/mL in 96 well microplates. Diluted sample solution (100 μl) and the DPPH working solution (0.078 mg/mL, 100 μl) were put into each well of a 96-well microplate. After vortexing, the microplates were left in the dark for 30 min at 25 °C, and the absorbance (ABS) values of samples was recorded on a microplate reader at 517 nm. The positive controls were BHT and VE. The solvent MeOH was used as the blank control. Each experiment was repeated three times. The DPPH radical scavenging activity of compounds 1, 6a–k and 7a–k was evaluated as the following formula: DPPH radical scavenging activity (%) = (1 – B/A) × 100, where A represents the absorbance of the blank control, and B represents the absorbance of the tested groups. The radical scavenging capacity was assessed as the effective concentration (EC50) which defined as the concentration which resulted in 50% inhibition.
2.3. Synthesis of compounds 6a–k and 7a–k 5-Allyloxy-sesamol (2) and 6-allyl-sesamol (3) were synthesized according to previously reported procedure (Guo et al., 2016; Qu et al., 2017). To a solution of 6-allyl-sesamol (3, 0.5 mmol, 89.5 mg) in dry toluene under N2 was added morpholine/piperidine (0.55 mmol) and substituted benzaldehyde (0.55 mmol) in sequence. When the reaction was finished according to analytical thin-layer chromatography (TLC) analysis, solvent was concentrated in vacuo. We used preparative thinlayer chromatography (PTLC) to purify the mixture and obtained the title compounds 6a−k and 7a–k. Exemplary data of compounds 6a–c and 7a–c were listed as follows, other data of compounds 6d–k and 7d–k were listed in the Supplementary material. Data for 6a: yield: 62%, pale yellow solid; mp: 83–85 °C; IR cm-1: 3436, 2935, 2855, 1638, 1450, 1108, 1049; 1H NMR (400 MHz, CDCl3) δ: 12.09 (s, 1H, -OH), 7.46-7.53 (m, 2H, -Ph), 7.22-7.30 (m, 3H, -Ph), 6.47 (s, 1H, H-7), 5.95-6.05 (m, 1H, H-2′), 5.74 (dd, J = 1.2, 19.2 Hz, 2H, -OCH2O-), 5.00-5.04 (m, 2H, H-1′), 4.52 (s, 1H, H-1′′), 3.27-3.38 (m, 2H, H-3′), 2.29-2.38 (m, 3H, piperidine ring), 1.45-1.73 (m, 7H, piperidine ring); MS (ESI) m/z calcd for C22H26BrNO3 ([M + H]+) 352.18, found 352.25. Data for 6b: yield: 41%, white solid; mp: 90–91 °C; IR cm-1: 3434, 2962, 2944, 2881, 1639, 1491, 1456, 1099, 1047; 1H NMR (400 MHz, CDCl3) δ: 12.21 (s, 1H, -OH), 7.54 (s, 1H, -Ph), 7.20-7.25 (m, 1H, -Ph), 7.01-7.09 (m, 2H, -Ph), 6.51 (s, 1H, H-7), 5.97-6.07 (m, 1H, H-2′), 5.73 (d, J =17.6 Hz, 2H, -OCH2O-), 5.03-5.06 (m, 3H, H-1′ and H-1′′), 3.293.43 (m, 2H, H-3′), 2.07-2.55 (m, 3H, piperidine ring), 1.43-1.75 (m, 7H, piperidine ring); MS (ESI) m/z calcd for C22H25FNO3 ([M + H]+) 370.17, found 370.29. Data for 6c: yield: 42%, white solid; mp: 45–47 °C; IR cm-1: 3438, 2938, 2852, 1638, 1499, 1454, 1222, 1048; 1H NMR (400 MHz, CDCl3) δ: 12.00 (s, 1H, -OH), 7.42-7.52 (m, 2H, -Ph), 6.94-6.98 (m, 2H, -Ph), 6.48 (s, 1H, H-7), 5.94-6.04 (m, 1H, H-2′), 5.74 (d, J =17.6 Hz, 2H, -OCH2O-), 5.00-5.04 (m, 2H, H-1′), 4.51 (s, 1H, H-1′′), 3.27-3.37 (m, 2H, H-3′), 2.38-2.45 (m, 3H, piperidine ring), 1.25-1.63 (m, 7H, piperidine ring); MS (ESI) m/z calcd for C22H25FNO3 ([M + H]+) 370.17,
2.4.2. Ferric-reducing antioxidant power (FRAP) The FRAP assay was determined by the previous reports (Firuzi et al., 2005). The FRAP was based on the reducing capacity of tested compounds. The ferric ion (Fe3+) will be reduced by a potential antioxidant to the ferrous ion (Fe2+) and it can form a blue complex (Fe2+/ TPTZ). This complex has a great absorption at 593 nm. In short, the acetate buffer [NaOAc (3.1 g) and HOAc (20 mL) were dissolved in 1000 mL of water], FeCl3·H2O (20 mM) and a solution of 10 μM TPTZ in hydrochloric acid (40 μM) were mixed to prepared the FRAP reagent. This reagent was incubated at 37 °C for ten minutes. A 180 μl of FRAP reagent was added to 20 μl of each tested compounds 1, 6a–k, 7a–k, BHT, a-tocopherol solutions (200 μg/mL), then the mixture was incubated at 37 °C for four minutes. Absorbance values of the tested compounds were determined at 593 nm. MeOH (20 μl) was used as a blank. Fresh working solutions of FeSO4 (1.0 mmol/L) was used as the calibration. Values of FRAP are expressed as μmol Fe2+/mg tested 2
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pot reaction of compound 3, aromatic aldehydes and secondary amines without catalysts. Structures of compounds 6a–k and 7a–k were confirmed using spectral analysis of 1H NMR, IR, mp and ESI-MS. Moreover, three-dimensional steric structure of 6f was further determined by X-ray diffraction (Fig. 3). Publication no. CCDC (Cambridge Crystallographic Data Centre) of compound 6f was 1862039. This obviously proved that we have successfully prepared a series of 4-(aminoalkyl)-6allyl-sesamols 6a–k and 7a–k via the one-pot reaction. In addition, a probable reaction mechanism of this one-pot reaction is proposed. As shown in Fig. 4, different substituted benzaldehyde 4a–k reacted with secondary amines 5a/5a' to formed iminium ion 8, subsequent, the iminium carbon atom was attacked by the carbon atom of compound 3. Finally, a hydride shift leads to the formation of 4(aminoalkyl)-6-allyl-sesamols (6a–k and 7a–k).
compound (TC). All analyses were done in triplicate. 2.5. Cytotoxicity assay The cytotoxic activities of potent compounds 7a, 7g and 7i were screened in NRK-52E and RAW264.7 cells according to the Cell Counting Kit-8 (CCK-8) method (Xiong et al., 2016). In brief, 5 × 103 NRK-52E and RAW264.7 cells in 100 μL medium were seed to each of 96-well plates. Subsequently, these cells were incubation at 37 °C for 24 h, a 100 μL of fresh medium with the compounds 7a, 7g and 7i in different concentration was added to remove and replace the used culture medium. The negative control was only added fresh medium. After treatment for 24 h, the used culture medium was discarded, and then using the new culture medium to wash the used medium twice. At last, new medium containing 5% CCK-8 (100 μL) was added to each well. After cultured at 37 °C for a further 4 h, a Microplate Reader was used to record the ABS values of the tested compounds at 450 nm. Cell survival rate (activity %) was calculated as follows: Cell survival rate (%) = (ODexperiment – ODblank)/(ODnegative control – ODblank) × 100%, where OD is the abbreviation of optical density. Each compound was tested in triplicate. The results expressed as the inhibitory concentration (IC) of 50% values ± SD.
3.2. Antioxidant activities and structure–activity relationships (SARs) 3.2.1. DPPH radical scavenging activity The antioxidant activity of 1, 6a–k, 7a–k, commercial antioxidants BHT and VE was first assessed by their scavenging capacity against DPPH radical (Sammaiah et al., 2015). The amount of tested sesamol derivatives required to inhibit the radicals by 50% was calculated and expressed as their EC50 values. The antioxidant activity results of sesamol derivatives are shown in Fig. 5. The EC50 values of all 4-(aminoalkyl)-6-allyl-sesamols ranged from 7.81 to 24.31 μg/mL, which suggested that some of sesamol derivatives exhibited moderate to good antioxidant activity. The lead compound sesamol showed remarkable DPPH radical scavenging activity with EC50 value of 10.95 μg/mL, which was close to the commercial antioxidant VE. Some similar results were also confirmed by the previously reported (Erkan et al., 2008). Among all the sesamol derivatives, 7a, 7g and 7i displayed extraordinary DPPH radical scavenging activity with EC50 values of 9.25, 7.81 and 7.85 μg/mL respectively, which were higher than the standards BHT (EC50: 11.9 μg/mL) and VE (EC50: 10.76 μg/mL). In addition, compounds 7h and 7k showed comparable DPPH radical scavenging activity with EC50 values of 10.89 and 10.8 μg/mL respectively, in comparison with VE, but slightly higher than that of BHT. Based on the SARs of these prepared sesamol derivatives, in general, it was observed that 4-(aminoalkyl)-6-allyl-sesamols containing the morpholine were found to have more potent DPPH radical scavenging activity than that containing the piperidine. For example, the EC50 values of compounds 7a–k were all lower than that of 6a–k [e.g., 7a (EC50: 9.25 μg/mL) vs. 6a (EC50: 18.4 μg/mL); 7b (EC50: 12.33 μg/mL) vs. 6b (EC50: 24.31 μg/mL); 7c (EC50: 11.73 μg/mL) vs. 6c (EC50: 19.56 μg/mL)]. In addition, when R was -H or electron-donating group, the corresponding sesamol derivatives exhibited more potent DPPH radical scavenging activity than that R was electron-withdrawing
2.6. Statistical analysis All data are expressed as mean ± SD in triplicate analysis for all the experiments. All statistical analysis was processed by the SPSS 21.0 (SPSS Inc., Chicago, USA) software. 3. Results and discussion 3.1. Chemistry As shown in Fig. 2, firstly, compound 1 reacted with allylbromide using K2CO3 as base to easily prepare compound 2 in 93% yield. Then, Claisen rearrangement of compound 2 in N,N-dimethylaniline gave 6allyl-sesamol 3 in 90% yield. The detailed synthesis parameters of compounds 2 and 3 can be found in our previous studies (Guo et al., 2016; Qu et al., 2017). It is worth mentioning that stirring of 6-allylsesamol 3, piperidine and benzaldehyde in refluxing EtOH under a nitrogen atmosphere for 12 h afforded the target compound 6a in only 24.0% yield (Table 1, Entry 1). Inspired by this result, the reaction was optimized in terms of the different solvents. Solvents of EtOH, N,Ndimethylformamide (DMF), toluene, CH3CN and 1,4-dioxane were screened. The best yield of 62.0% of the title compound 6a was achieved in toluene. Finally, we used toluene as solvent for the synthesis of 4-(aminoalkyl)-6-allyl-sesamols (6a–k, 7a–k) through the one-
Fig. 2. Synthetic routes of Mannich bases, 4-(aminoalkyl)-6-allyl-sesamols (6a–k and 7a–k). 3
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Table 1 Screening of solvents for the synthesis of the 4-(aminoalkyl)-6-allyl-sesamols taking the model reaction of 6-allyl-sesamol, benzaldehyde and piperidinea.
b
Entry
Solvent
1 2 3 4 5
EtOH DMF Toluene CH3CN 1,4-Dioxane
a b c
(mL)
Time (h)
Isolated yield c(%)
12 12 12 12 12
24.0 31.4 62.0 40.2 33.5
Optimal reaction conditions: molar ratio of 3 (0.5 mmol)/4a (0.55 mmol)/5a (0.55 mmol) = 1 : 1.1 : 1.1, 80 °C. Ethanol (EtOH), N,N-dimethylformamide (DMF). Isolated yields after preparative thin-layer chromatography (PTLC).
Fig. 4. Possible mechanism for the preparation of Mannich bases, 4-(aminoalkyl)-6-allyl-sesamols (6a–k and 7a–k).
3.2.2. FRAP assay The ability of antioxidants to reduce Fe3+ to Fe2+ in the presence of TPTZ is the fundamental of the FRAP assay. The TPTZ–Fe2+ complex displays an intense blue colour which displays absorption at 593 nm (Benzie and Strain, 1996). The results of the reducing power of 1, 6a–k and 7a–k expressed as μmol Fe2+ /mg TC in comparison with VE and BHT are summarised in Fig. 6. On the basis of FRAP values, most of the title compounds except 7i had lower FRAP values than the sesamol, VE and BHT. The compound 7i had FRAP value of 9.6 μmol Fe2+ /mg TC, which was higher than the positive control VE and BHT. The FRAP values of other compounds ranged from 1.98 to 5.15 μmol Fe2+ /mg TC. These results may be bacause the mechanism of FRAP is different from that of DPPH radical scavenging activity (Bakar et al., 2009). Additionally, the precursor sesamol also showed good antioxidant activity with the FRAP value of 8.52 μmol Fe2+ /mg TC, which was in line with those reported by Joshi et al. (2005) who studied the effect of sesamol on Fe3+-induced lipid peroxidation. In general, in DPPH and FRAP assay, compound 7i showed excellent antioxidant activity than the precusor sesamol, the commercial antioxidants VE and BHT.
Fig. 3. X-ray crystal structure of compound 6f.
group. For instance, the EC50 values of compounds 6g (R = 4-CH3), 7g (R = 4-CH3), 6h (R = 4-OCH3) and 7h (R = 4-OCH3) were 12.49, 7.81, 15.11 and 10.89 μg/mL, respectively. Whereas, the EC50 values of compounds 6c (R = 4-F), 7c (R = 4-F), 6d (R = 4-Cl) and 7d (R = 4Cl) were 19.56, 11.73, 20.19 and 12.23 μg/mL, respectively. Moreover, the compounds 6i (R = 4-dimethylamino) and 7i (R = 4-dimethylamino) showed outstanding DPPH radical scavenging activity, which may be because the 4-dimethylamino was a strong electron-donating group in comparison of other groups (Coe et al., 2001). Literature reported that the increase in the electron-donating groups displayed an obvious increase in the antioxidant activity by further stabilizing the phenolic hydroxyl radical (Merkl et al., 2010). These results are somewhat in agreement with some previous reports of synthesized Mannich bases (Oloyede et al., 2014).
3.2.3. Cytotoxicity In consideration of the accompanied toxicity to normal mammalian cells is one of the major obstacles to the preparation of food additives from many promising antioxidant compounds, so the title compounds with potent antioxidant capacity were further evaluated for their cytotoxic activities against RAW264.7 and NRK-52E cells. As depicted in Fig. 7, compounds 7a, 7g and 7i showed relative low cytotoxicities to 4
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Fig. 5. DPPH radical scavenging activity of 4-(aminoalkyl)-6-allyl-sesamols (6a–k and 7a–k); Values represent means ± SD (DPPH: 2,2-diphenyl-1-picrylhydrazyl; BHT: EC50: effective concentration 50%; 2,6-di-tert-butyl-4-methylphenol).
and will have great potential application in food and drugs.
noncancerous RAW264.7 (Fig. 7a) and NRK-52E (Fig. 7b) cells, and the IC50 values were higher than 96.3 μg/mL. It suggested that these compounds could be new promising alternatives for antioxidants.
Acknowledgments 4. Conclusion The authors gratefully acknowledge the financial support by National Natural Science Foundation of China (21502176), Natural Science Foundation of Hennan Province, China (No. 182300410025), the China Postdoctoral Science Foundation (2019M652585), Key Science and Technology Program of Henan Province, China (192102110054) and Startup Research Fund of Zhengzhou University (145-32211180).
In general, two series of twenty-two novel Mannich bases, 4-(aminoalkyl)-6-allyl-sesamols 6a–k and 7a–k were prepared by the one-pot reaction and their structures were characterized by spectral analysis. In addition, a simple procedure for the synthesis of these 4-(aminoalkyl)6-allyl-sesamols by using toluene as solvent, catalyst-free and the onepot reaction methodology was developed. This method will be valuable for development of other Mannich bases. The structure of 6f was unambiguously confirmed by X-ray diffraction further. The antioxidant assays have demonstrated the sesamol derivative 7i displayed excellent antioxidant activity. Further, the representative compounds 7a, 7g and 7i exhibited relative low cytotoxicity against RAW264.7 and NRK-52E cells. The results suggest that these novel sesamol derivatives could offer an interesting solution for natural products-based antioxidants,
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.indcrop.2019.111762.
Fig. 6. Ferric-reducing antioxidant power (FRAP) of Mannich bases, 4-(aminoalkyl)-6-allyl-sesamols (6a–k and 7a–k); Values represent means ± SD (BHT: 2,6-ditert-butyl-4-methylphenol). 5
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Fig. 7. Relative cell viabilities of compounds 7a, 7g and 7i against RAW264.7 (a) and NRK-52E (b) cells; Values represent means ± SD.
References
isolated from various herbs and spices. J. Agri. Food Chem. 50, 4947–4952. https:// doi.org/10.1021/jf0255681. Joshi, R., Kumar, M.S., Satyamoorthy, K., Unnikrisnan, M.K., Mukherjee, T., 2005. Free radical reactions and antioxidant activities of sesamol: pulse radiolytic and biochemical studies. J. Agri. Food Chem. 53, 2696–2703. https://doi.org/10.1021/ jf0489769. Maestri, D.M., Nepote, V., Lamarque, A.L., Zygadlo, J.A., 2006. Natural products as antioxidants. Phytochemistry: Advances in research 37, 105–135. Masuda, T., Fujimoto, A., Oyama, Y., Maekawa, T., Sone, Y., 2009. Structure of cytotoxic products from Fe-catalyzed oxidation of sesamol in ethanol. Tetrahedron Lett. 50, 3905–3908. https://doi.org/10.1016/j.tetlet.2009.04.063. Masuda, T., Shingai, Y., Fujimoto, A., Unnikrisnan, M.K., Mukherjee, T., 2010. Identification of cytotoxic dimers in oxidation product from sesamol, a potent antioxidant of sesame oil. J. Agri. Food Chem. 58, 10880–10885. https://doi.org/10. 1021/jf103015j. Merkl, R., Hradkova, I., Filip, V., Drkal, J.S., 2010. Antimicrobial and antioxidant properties of phenolic acids alkyl esters. Czech J. Food Sci. 28, 275–279. Natella, F., Nardini, M., Felice, M.D., Scaccini, C., 1999. Benzoic and cinnamic acid derivatives as antioxidants: Structure-activity relation. J. Agri. Food Chem. 47, 1453–1459. https://doi.org/10.1021/jf980737w. Oloyede, G.K., Willie, I.E., Adeeko, O.O., 2014. Synthesis of Mannich bases: 2-(3Phenylaminopropionyloxy)-benzoic acid and 3-Phenylamino-1-(2, 4, 6-trimethoxyphenyl)-propan-1-one, their toxicity, ionization constant, antimicrobial and antioxidant activities. Food Chem. 165, 515–521. https://doi.org/10.1016/j.foodchem. 2014.05.119. Qu, L., Xu, H., Wang, X., Huang, M., Deng, L., Guo, Y., 2017. Application of Sustainable Natural Bioresources in Agriculture: Iodine-Mediated Oxidative Cyclization for Metal-Free One-Pot Synthesis of N-Phenylpyrazole Sarisan Analogues as Insecticidal Agents. ACS Omega. 2, 5974–5980. https://doi.org/10.1021/acsomega.7b01106. Wang, S., Wang, D., Liu, Z., 2015. Synergistic, additive and antagonistic effects of Potentilla fruticosa combined with EGb761 on antioxidant capacities and the possible mechanism. Ind. Crops Prod. 67, 227–238. https://doi.org/10.1016/j.indcrop.2015. 01.025. Sammaiah, A., Kaki, S.S., Manoj, G.N.V.T.S., Poornachandra, Y., Kumar, C.G., Prasad, R.B.N., 2015. Novel fatty acid esters of apocynin oxime exhibit antimicrobial and antioxidant activities. Eur. J. Lipid Sci. Tech. 117, 692–700. Sharma, S., Kaur, I.P., 2006. Development and evaluation of sesamol as an antiaging agent. Int. J. Dermatol. 45, 200–208. https://doi.org/10.1111/j.1365-4632.2004. 02537.x. Xiong, H., Zheng, H., Wang, W., Liang, J., Wen, W., Zhang, X., Wang, S., 2016. A convenient purification method for silver nanoclusters and its applications in fluorescent pH sensors for bacterial monitoring. Biosens. Bioelectron. 86, 164–168. https://doi. org/10.1016/j.bios.2016.06.038.
Aggarwal, K., Khurana, J.M., 2017. Synthesis of a novel 5a, 10a-dihydroxy-5aH-[1,3] dioxolo [4,5-f] indeno [1,2-b] benzofuran-10(10aH)-one their XRD, FTIR, NMR and DFT studies. J. Mol. Struct. 1130, 739–747. https://doi.org/10.1016/j.molstruc. 2016.11.008. Bakar, M.F.A., Mohamed, M., Rahmat, A., Fry, J., 2009. Phytochemicals and antioxidant activity of different parts of bambangan (Mangifera pajang) and tarap (Artocarpus odoratissimus). Food Chem. 113, 479–483. https://doi.org/10.1016/j.foodchem. 2008.07.081. Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: The FRAP assay. Anal. Biochem. 239, 70–76. https://doi.org/ 10.1006/abio.1996.0292. Chang, C.C., Lu, W.J., Chiang, C.W., Jayakumar, T., Ong, E.T., Hsiao, G., Fong, T.H., Chou, D.S., Sheu, J.R.J., 2010. Potent antiplatelet activity of sesamol in an in vitro and in vivo model: pivotal roles of cyclic AMP and p38 mitogen-activated protein kinase. J. Nutr. Biochem. 21, 1214–1221. https://doi.org/10.1016/j.jnutbio.2009.10.009. Chavali, S.R., Utsunomiya, T., Forse, R.A., 2001. Increased survival after cecal ligation and puncture in mice consuming diets enriched with sesame seed oil. Crit. Care Med. 29, 140–143. https://doi.org/10.1097/00003246-200101000-00028. Chen, W.Y., Chen, F.Y., Lee, A.S., Ting, K.H., Chang, C.M., Hsu, J.F., Lee, W.S., Sheu, J.R., Chen, C.H., Shen, M.Y., 2015. Sesamol reduces the atherogenicity of electronegative L5 LDL in vivo and in vitro. J. Nat. Prod. 78, 225–233. https://doi.org/10.1021/ np500700z. Coe, B.J., Harris, J.A., Clays, K., Persoons, A., Wostyn, K., Brunschwig, B.S., 2001. A comparison of the pentaammine (pyridyl) ruthenium (II) and 4-(dimethylamino) phenyl groups as electron donors for quadratic non-linear optics. Chem. Comm. 17, 1548–1549. https://doi.org/10.1039/B103543F. Erkan, N., Ayranci, G., Ayranci, E., 2008. Antioxidant activities of rosemary (Rosmarinus Officinalis L.) extract, blackseed (Nigella sativa L.) essential oil, carnosic acid. rosmarinic acid and sesamol. Food chem. 110, 76–82. https://doi.org/10.1016/j. foodchem.2008.01.058. Firuzi, O., Lacanna, A., Petrucci, R., Marrosu, G., Saso, L., 2005. Evaluation of the antioxidant activity of flavonoids by ferric reducing antioxidant power assay and cyclic voltammetry. BBA-Gen. Sub. 1721, 174–184. https://doi.org/10.1016/j.bbagen. 2004.11.001. Guo, Y., Qu, L., Wang, X., Huang, M., Jia, L., Zhang, Y., 2016. Iodine-catalyzed oxidative cyclisation for the synthesis of sarisan analogues containing 1,3,4-oxadiazole as insecticidal agents. RSC Adv. 6, 93505–93510. https://doi.org/10.1039/C6RA22343E. Hou, R.C., Chen, Y.S., Chen, C.H., Chen, Y.H., Jeng, K.C., 2006. Protective effect of 1,2,4benzenetriol on LPS-induced NO productionby BV2 microglial cells. J. Biomed. Sci. 13, 89–99. https://doi.org/10.1007/s11373-005-9039-5. Lee, K., Shibamoto, T., 2002. Determination of antioxidant potential of volatile extracts
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