Anti-inflammatory and antioxidant jasmonates and flavonoids from lychee seeds

Journal of Functional Foods 54 (2019) 74–80

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Anti-inflammatory and antioxidant jasmonates and flavonoids from lychee seeds ⁎

Xuzhe Donga, Yuying Huanga, Yihai Wanga,b, , Xiangjiu Hea,b, a b

T

⁎

School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China Guangdong Engineering Research Center for Lead Compounds & Drug Discovery, Guangzhou 510006, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Lychee seeds Methyl jasmonate Flavonoids Anti-inflammatory Antioxidant

Lychee (Litchi chinensis Sonn.) is a famous tropical fruit. While enjoying its delicacy, it also produces a lot of lychee seeds as waste. In this study, bioactive phytochemical study of the seeds of lychee led to the isolation and identification of four methyl jasmonate analogs (1–4), one lignanoside (5), together with fifteen flavonoids (6–20). Their chemical structures were established on the basis of spectroscopic analysis including HR-ESI-MS, 1D and 2D NMR spectra. The anti-inflammatory and antioxidant activities were evaluated for the isolated compounds. Compounds 2 and 3 exhibited notable inhibition on NO production induced by lipopolysaccharides (LPS) in macrophages cell line RAW 264.7. Compound 5 and some flavonoids revealed potent antioxidant capacities. The results disclosed that the methyl jasmonates in lychee seeds may be partially responsible for antiinflammatory activity of lychee and could be served as anti-inflammatory agent in functional food.

1. Introduction Lychee (Litchi chinensis Sonn.) is a tropical and subtropical evergreen plant which belongs to the only species of the genus Litchi in Sapindaceae family. It is prevalent in global market as an important fruit attributing to its delicious arils and abundant nutritional value. Lychee can be consumed as fresh and deep-processed products, whereas its seeds are mainly discarded as waste except a trifling amount is applied as traditional Chinese medicine to treat epigastric pain, testicular swelling and pain (Lin et al., 2015; Xu, Xie, Hao, Jiang, & Wei, 2011). Previous pharmacological studies revealed that lychee seeds possessed anti-hyperglycemic, anti-hyperlipidemic, anti-platelet aggregation, antitumor, antiviral, and antioxidant effects (Chen, Wu, Gu, & Chen, 2007), although it has been reported to contain plant toxin a-(Methylene cyclopropyl) glycine can lead to hypoglycemia and death (Gray & Fowden, 1962; Islam et al., 2017; Qiu et al., 2018). Additionally, recent research provided that lychee seeds also exhibited anti-influenza virus and reducing visceral obesity properties (Gangehei et al., 2010; Jin et al., 2009), significantly ameliorated the learning and memory effects in the model mice (Choi et al., 2014; Ye et al., 2013). In recent years,

the chemical studies have suggested that lychee seeds contained large quantities of bioactive phytochemicals, such as oligosaccharides, phenolics, flavonoids (Ding, Wang, Zhao, & Du, 2006; Xu et al., 2011), which could be utilized as a natural antioxidant agent for health care (Prasad et al., 2009). In the continuing programs to seek antitumor and anti-inflammatory agents from medicinal plants, fruits and vegetables (Cheng, Yi, Wang, Huang, & He, 2017; Feng, Wang, Yi, Yang, & He, 2016; Qiao, Wang, Xiang, Wang, & He, 2015), phytochemical study on the seeds of lychee has been carried out. Herein, we reported the isolation, purification, structural elucidation and bioactive evaluation of a lignanoside, four methyl jasmonates, as well as fifteen flavonoids from lychee seeds. 2. Materials and methods 2.1. General experimental procedures Optical rotations were measured on a Rudolph II digital polarimeter (Rudolph Inc., Hackettstown, NJ, USA). IR spectra (4000–450 cm−1)

Abbreviations: COSY, correlated spectroscopy; DPPH, 1,1-diphenyl-2-picrylhydrazyl; DMSO, dimethyl sulfoxide; ESI-MS, electrospray ionization mass spectrometry; FRAP, ferric ion reducing antioxidant power; HMBC, 1H-detected heteronuclear multiple-bond correlation; HSQC, heteronuclear single-quantum coherence; HPLC, high performance liquid chromatography; HR-ESI-Q-TOF-MS, high-resolution electrospray ionization quadrupole time-of-flight mass spectrometry; MPLC, medium pressure liquid chromatography; LPS, lipopolysaccharides; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NMR, nuclear magnetic resonance; NO, nitric oxide; ODS, octadecylsilane; RAW 264.7, murine macrophage ⁎ Corresponding authors at: School of Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China. E-mail addresses: [email protected] (Y. Wang), [email protected] (X. He). https://doi.org/10.1016/j.jff.2018.12.040 Received 29 August 2018; Received in revised form 25 November 2018; Accepted 29 December 2018 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.

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the preparative HPLC (22% MeOH/H2O) to yield compounds 6 (tR = 15 min, 46.0 mg) and 7 (tR = 18 min, 9.7 mg). Fraction E12-7 (547.9 mg) was purified with the preparative HPLC (36% MeOH/H2O) to yield compounds 5 (tR = 21 min, 12.8 mg), 8 (tR = 18 min, 3.4 mg) and 11 (tR = 26 min, 6.8 mg). Fraction E12-8 (251.1 mg) was purified with the preparative HPLC (47% MeOH/H2O) to get compounds 9 (tR = 19 min, 24.4 mg) and 16 (tR = 14 min, 3.0 mg). Fraction E14 (15.96 g) was subjected to an ODS MPLC eluted with MeOH/H2O to obtain 10 subfractions, of which E14-2 (3.02 g) was separated via a silica gel C.C. with CHCl3-MeOH (100:0 to 1:1, v/v) to afford 7 subfractions, of which E14-2-3 (1.42 g) was purified with the preparative HPLC (2.3 mL/min, 31% MeOH/H2O) to afford compounds 18 (tR = 21 min, 134.0 mg) and 19 (tR = 33 min, 84.0 mg). Compounds 17 (tR = 44 min, 7.4 mg) and 20 (tR = 18 min, 17.8 mg) were obtained by the preparative HPLC (2.8 mL/min, 35% MeOH/H2O) from fraction E14-2-5 (101.5 mg). Fraction E15 (7.5 g) was subjected to an ODS MPLC eluted with MeOH/H2O (10%-70%, v/v), followed by a Sephadex LH-20 column eluted with MeOH/H2O (50%-90%, v/v) and then purified with the preparative HPLC (2.8 mL/min, 18% MeOH/H2O) to afford compounds 12 (tR = 23 min, 10.5 mg), 13 (tR = 21 min, 9.8 mg), 14 (tR = 47 min, 12.1 mg) and 15 (tR = 32 min, 5.7 mg). (1R,2R,2′Z)-2-[5′-(Acetyloxy)-2-penten-1-yl]-3-oxo-cyclopentaneacetic acid methyl ester (1), yellow oil. [α ]26 D −62.0 (c = 0.35, MeOH). IR (KBr): 2956, 1739, 1640, 1438, 1372, 1240, 1171 cm−1. 1H NMR (400 MHz, DMSO-d6) and 13C NMR (100 MHz, DMSO-d6) spectroscopic data were listed in Table 1. HR-ESI-MS [M + Na]+ m/z 305.1360 (calcd for C15H22O5Na, 305.1359). (1R,2R,2′Z)-2-(5-Methoxy-5-oxo-2-penten-1-yl)-3-oxo-cyclopentaneacetic acid methyl (2), yellow oil. [α ]26 D −29.4 (c = 0.32, MeOH). IR (KBr): 2954, 1737, 1437, 1374, 1263, 1168 cm−1. 1H NMR (400 MHz, DMSOd6) and 13C NMR (100 MHz, DMSO-d6) spectroscopic data were indicated in Table 1. HR-ESI-MS [M + Na]+ m/z 291.1204 (calcd for C14H20O5Na, 291.1203). (1R,2R,2′E)-3-Hydroxy-2-(5-methoxy-5-oxo-2-penten-1-yl)-cyclopentaneacetic acid methyl ester (3), colorless oil. [α ]26 D +36.3 (c = 0.32, MeOH). IR (KBr): 3678, 2948, 1736, 1635, 1438, 1372, 1268, 1172 cm−1. 1H NMR (400 MHz, DMSO-d6) and 13C NMR (100 MHz, DMSO-d6) spectroscopic data were listed in Table 1. HR-ESI-MS [M + Na]+ m/z 293.1368 (calcd for C14H22O5Na, 293.1359). (−)−(8S,7′S, 8′S)-Burselignan-9′-O-α-L-arabinoside (5), white powder [α ]26 D +35.1 (c = 0.22, MeOH). IR (KBr): 3678, 3059, 2924, 1738, 1605, 1513, 1438, 1276 cm−1. The UV spectrum showed absorptions at 204 and 284 nm. 1H NMR (400 MHz, DMSO-d6) and 13C NMR (100 MHz, DMSO-d6) spectroscopic data were provided in Table 2. HR-ESI-MS [M + Na]+ m/z 515.1887 (calcd for C25H32O10Na, 515.1888).

were recorded on a Perkin-Elmer Spectrum 100 FT-IR spectrometer (Perkin-Elmer Inc., Waltham, MA, USA) with KBr pellets. UV spectra were acquired with a PharmaSpec UV-1700 spectrophotometer (Shimadzu, Tokyo, Japan). NMR spectra were obtained on a Bruker Avance III-400 MHz NMR spectrometer (Bruker Inc., Falanden, Switzerland) in DMSO-d6 with residual peaks at δH 2.50 and δC 39.5 as chemical shift references. High-resolution electrospray ionization mass spectrometry (HR-ESI-MS) data were conducted par LC-MS on an Acquity UPLC-Q-TOF Micro-mass spectrometer (Waters Corp., Milford, MA, USA). Preparative HPLC were performed on a HPLC system composed of a Dynamax model HPXL solvent delivery system (Rainin Instrument Co. Inc., Woburn, MA, USA) equipped with a Shodex RI201H refractive index detector (Tokyo, Japan). A C18 column (Cosmosil 5C18-AR-II, 5 μm, 10 ID × 250 mm, Nacalai Tesque, Kyoto, Japan) was applied for preparative purposes. Gas chromatography for sugar analysis was carried out on a Thermo Trace-1300 GC (Thermo Fisher Scientific, Waltham, MA, USA). Fluorescence plate reader (SpectraMax M-2) was from Molecular Devices (Sunnyvale, CA, USA). 2.2. Chemicals and reagents Sephadex LH-20 (Amersham Biosciences, Uppsala, Sweden), silica gel (Anhui Liangchen Silicon Material Co. Ltd., Lu′an, China) and ODS (40–60 μm, Merck KGaA, Darmstadt, Germany) were applied for column chromatography (C.C.). Methanol for HPLC was purchased from Oceanpak Alexative Chemical Co., Ltd. (Gothenburg, Sweden). Deuterated solvents for NMR experiments, sugar reagents for GC–MS analysis, LPS and MTT were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All other analytical chemicals and reagents were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). 2.3. Plant material Lychee seeds (20 kg) were purchased from Qingping herbal market, Guangzhou, on Aug. 2015 and were identified as Litchi chinensis Sonn. by Prof. X. J. He of Guangdong Pharmaceutical University. A voucher specimen (GDPU-NPR-2015010) was deposited at the Lead Compounds Laboratory, Guangdong Pharmaceutical University. 2.4. Extraction and isolation The dried lychee seeds (20 kg) were crushed and refluxed with 70% ethanol (65 L × 3) and 4 h for each extraction. The ethanol extract was concentrated under vacuum to afford 12 L ethanol-free extracting solution, and then sequentially partitioned with cyclohexane, chloroform, ethyl acetate and n-butanol, respectively. The EtOAc-soluble fraction (120.11 g) was subjected to a silica gel C.C. eluted gradiently with CHCl3-MeOH (100:0 to 2:1, v/v) to get fraction E1-E17. Fraction E4 (2.1 g) was separated by a silica gel C.C. using cyclohexane-ethyl acetate (100:0 to 1:1, v/v) as eluent to obtain 13 subfractions, of which E4-10 (198 mg) was fractionated by a Sephadex LH20 column (CHCl3/MeOH 2:1) and then purified with the preparative HPLC (45% MeOH/H2O) to get compounds 1 (tR = 25 min, 20.0 mg) and 2 (tR = 15 min, 40.0 mg). Subfraction E4-12 (198 mg) was separated by a silica gel C.C. using cyclohexane-ethyl acetate (100:0 to 2:1, v/v) and then purified by the preparative HPLC (25% MeOH/H2O) to yield compound 3 (tR = 15 min, 7.0 mg). Fraction E8 (3.92 g) was separated with a silica gel C.C. eluted with chloroform-ethyl acetate (100:1 to 2:1, v/v), through a Sephadex LH-20 column (CHCl3/MeOH 2:1) and then purified with the preparative HPLC (35% MeOH/H2O) to afford compound 4 (tR = 25 min, 57.0 mg). Fraction E12 (5.81 g) was separated over a silica gel C.C. eluted with CHCl3-MeOH (100:1 to 1:1, v/v) to get 10 subfractions, of which E12-6 (1.17 g) was subjected to an ODS MPLC eluted with MeOH/H2O (10%−50%, v/v), passed through a Sephadex LH-20 column (MeOH/H2O 4:1, v/v) and then purified with

2.5. Acid hydrolysis and GC–MS analysis Compound 5 (1.1 mg) was dissolved in 2 M HCl (5 mL) and heated at 90 °C for 2 h. The sugar obtained from the hydrolysates was turned to aldononitrile peracetates and analyzed by GC–MS as previously described protocol (Feng et al., 2016). 2.6. NO inhibitory assay Anti-inflammatory effects of the crude extracts and isolated compounds from lychee seeds were evaluated via measuring the accumulation of NO in the culture supernatant induced by lipopolysaccharide (LPS) in macrophages cell line RAW 264.7 utilizing Griess reaction described in the literature (Wang, Xiang, Yi, & He, 2017). 2.7. Antioxidant assay DPPH radical scavenging activity was evaluated by measuring the DPPH radical scavenging utilizing the DPPH assay as previously 75

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Table 1 1 H and 13C NMR data of compounds 1–3 measured in DMSO-d6. 1

2 δH (J in Hz)

δH (J in Hz)

2.17, dd (10.1, 5.5) 1.98, dt (10.8, 5.4)

δC

1 2 3 4

37.3 d 52.9 d 218.4 s 37.2 t

2.06, m 1.97, ov – 2.21, m

37.3 d 52.9 d 218.3 s 37.1 t

5

26.4 t

2.31, d (5.6)

26.6 t

1′ 2′ 3′ 4′ 5′ 6′ 7′ 1′′

24.9 t 128.8 d 126.6 d 26.6 t 63.2 t 170.3 s 20.6 q 38.0 t

24.9 t 129.5 d 123.0 d 32.1 t 171.5 s 51.2 q

2′′ 3′′

172.3 s 51.3 q

2.25, t (5.1) 5.38 (10.9) 5.40 (10.9) 2.09, d (8.7); 1.45, m 3.98, t (6.8) – 1.98, ov 2.66, dd (15.5,4.5); 2.37, m – 3.60, s

a b c

, mult.

b,c

3

δCa, mult.

No.

a

b,c

2.21–2.24, m; 2.08–2.13, m 1.38–1.50, m; 2.02–2.08, m 2.26, dd (8.3, 4.9) 5.47, dd (12.3, 5.3) 5.53, dd (12.3, 5.3) 3.13, d (6.9) – 3.59, s

37.9 t

2.34, dd, (15.6,9.0); 2.68, dd (15.6, 4.4) – 3.61, s

172.3 s 51.5 q

δCa,

mult.

δH (J in Hz)b,c

38.2 d 50.3 d 71.8 d 33.2 t

2.00, 1.26, 3.93, 1.71,

m dd (9.6, 4.8) s m; 1.48,m

28.8 t 25.3 t 132.1 d 121.3 d 32.1 t 171.8 s 51.1 q

1.10–1.18, m; 1.85–1.95, m 2.04, d (7.3); 2.09, t (3.8) 5.60, dd (15.7, 7.6) 5.46, dd, (15.7, 7.1) 3.13, d (6.9) – 3.59, s

38.6 t

2.13, m; 2.47, ov

173.1 s 51.5 q

– 3.57, s

Measured at 100 MHz. Measured at 400 MHz. Assignments were reconfirmed by HSQC, HMBC and 1H–1H COSY experiments.

to the modified procedure (Xiang, Wang, Yi, & He, 2016). Briefly, the FRAP reagent was prepared by mixing 100 mL of sodium acetic buffer (0.3 M, pH 3.6), 10 mL of TPTZ solution (10 mM TPTZ in 40 mM HCl) and 10 mL of FeCl3 (20 mM). Then, 40 μL of the test sample was added to 200 μL of FRAP reagent in the well of the microplate, and incubated at 37 °C for 10 min. The absorbance of the mixture was measured at 600 nm. FeSO4 solutions ranging from 50 μM to 300 μM were used to perform the calibration curve. The antioxidant efficiency of the test sample was calculated with reference to the standard curve.

Table 2 1 H and 13C NMR data of compound 5 measured in DMSO-d6. 5 No.

δCa,

1 2 3 4 5 6 7 8

127.1 s 132.7 s 116.3 d 144.1 s 145.5 s 111.8 d 32.6 t 37.7 d

9

62.7 t

1′ 2′ 3′ 4′

136.9 s 113.8 d 147.2 s 144.5 s

mult.

δH (J in Hz)b,c

No.

δCa,

– – 6.08, – – 6.60, 2.71, 1.89, 1.6) 3.58, m – 6.78, – –

s d (7.3) dd (9.4,

5′ 6′ 7′ 8′ 9′ 3′-OCH3 5-OCH3 9′-O-Ara

115.4 d 121.1 d 45.7 d 44.1 d 67.1 t 55.5 q 55.6 q

6.68, 6.48, 4.02, 1.71, 3.84, 3.70, 3.71,

m; 3.49,

1

104.2 d

3.90, d (6.5)

2 3 4 5

70.7 d 72.6 d 67.7 d 65.4 t

3.35, m 3.32, m 3.59–3.61, m 3.32, m; 3.64, dd (12.1, 2.7)

s

mult.

δH (J in Hz)b,c d (8.1) dd (1.6, 8.1) d, (10.7) t (10.5) dd (7.7); 2.96, m s s

3. Results and discussion 3.1. Structure elucidation of the isolated compounds

d (1.6)

Lychee seeds were extracted with 70% ethanol, fractionated with different solvents, and then successively chromatographed on silica gel, ODS, Sephadex LH-20 and HPLC to obtain four new compounds (1–3, 5), as well as sixteen known compounds (Fig. 1). Compound 1 was obtained as yellow oil, [α ]26 D −62.0 (c = 0.35 in MeOH). Its HR-ESI-MS at m/z 305.1360 [M + Na]+ (calcd for C15H22O5Na 305.1359) was corresponding to the molecular formula of C15H22O5. The IR spectrum gave the characteristic absorptions at 2956 (CeH), 1739 (C]O) and 1640 (C]C) cm−1. In the 13C NMR, there were 15 carbons corresponding to one ketone (δC 218.4), one carboxyl (δC 172.3), one acetyl group (δC 170.3, 20.6), two olefinic (δC 128.8, 126.6), one oxymethylene (δC 63.2), five methylenes (δC 38.0, 37.2, 26.6, 26.4 and 24.9), two methines (δC 52.9 and 37.3), and one methoxyl (δC 51.3). In the 1H NMR spectrum, two olefinic proton signals at δH 5.38 (1H, d, J = 10.9 Hz) and 5.40 (1H, d, J = 10.9 Hz) indicated the presence of a cis carbon-carbon double bond. Its 1H–1H COSY spectrum demonstrated the presence of a proton sequence as shown in Fig. 2. In the HMBC spectrum, the correlations of δH 2.06 (H1)/1.97 (H-2) with δC 218.4 (C-3), δH 2.21 (H-4) with δC 37.3 (C-1), δH 2.31 (H-5) with δC 52.9 (C-2), indicated the presence of a cyclopentanone unit. These NMR data of 1 were considerably similarly to those of compound 4 ((1R,2R,2′Z)-methyl 12-hydroxyjasmonate) (Nakamura, Miyatake, Inomata, & Ueda, 2008) except for the acetyl group (δC 170.3, 20.6). The acetylation position was deduced to be on C-5′ based on the downfield shift of C-5′ (+0.9 ppm) and upfield shift of C-4′ (−5.2 ppm) compared to those of compound 4. This was further supported by the long range correlations of δH 1.98 (H-7′) with δC 170.3 (C-

a

Measured at 100 MHz. Measured at 400 MHz. c Assignments were reconfirmed by HSQC, HMBC and 1H–1H COSY experiments. b

described (Pan, Yi, Wang, Chen, & He, 2016). Briefly, DPPH was prepared in methanol at the concentration of 0.2 mM, and 100 μL of this solution was mixed with 100 μL of the test sample (dissolved in methanol) at the concentration of 2.5–400 μM. These solutions were mixed and incubated at 25 °C in the dark for 0.5 h. Finally, the OD value was determined at 510 nm using a plate reader. The superoxide anion radical scavenging activity was measured to estimate the antioxidant capacity of isolated compounds according to the reported protocol (Feng et al., 2016). The 1000 μL reaction mixture contained 445 μL of Tris-HCl (pH 8.1, 50 mM), 250 μL of NADH (0.15 mM), 50 μL of PMS (0.03 mM), 250 μL of NBT (0.10 mM), and 5 μL of the test sample dissolved in Tris-HCl (50 mM, pH 8.1). The reaction was conducted for 5 min at 37 °C and initiated by the addition of PMS. Ascorbic acid was used as positive control. The ferric reducing antioxidant capacity (FRAP) was performed to elucidate the total antioxidant activity of isolated compounds according 76

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Fig. 1. Chemical structures of the compounds isolated from lychee seeds.

6′)/63.2 (C-5′), and δH 3.98 (H-5′) with δC 170.3 (C-6′) in the HMBC spectrum. As previously report (Holbrook & Moloney, 1997), methyl jasmonate analogs existed four possible stereoisomeric forms: (+)-(1R, 2S)isomer and (−)-(1S, 2R) -isomer were a pair of optical isomers with the two side chains in the cis arrangement relative to the plane of the cyclopentane. Another pair of optical isomers were (−)-(1R, 2R) and (+)-(1S, 2S)-isomer, with the two side chains in the trans arrangement. In the 1H NMR spectrum of 1, H-1 and H-2 resonated at δH 2.06 and 1.97, respectively. Resonances for H-1 and H-2 of the 1,2-cis isomer were published at δH 2.80 and 2.35–2.45, whereas the 1,2-trans isomer exhibited H-1 at δH 2.24 and H-2 at δH 1.91 (Chi et al., 2012; Kikuzaki, Miyajima, & Nakatani, 2008). Thus, compound 1 was proposed to be 1,2-trans configuration. In the trans-configuration, the (1S, 2S) stereoisomeric was confirmed by the right-handed (+) specific rotation, and

(1R, 2R) stereoisomeric displayed left-handed (−) specific rotation (Fig. 3). Supported by the above evidence and the specific rotation ([α ]26 D −62.0, c = 0.35 MeOH), the structure of compound 1 was eventually elucidated to be (1R,2R,2′Z)-2-[5′-(acetyloxy)-2- penten-1yl]-3-oxo-cyclopentaneacetic acid methyl ester. Herein, it was the first time to determine the stereochemistry at C-1 and C-2 of 1 as (1R,2R)isomer although the planer structure were previously published (Jimenezaleman, Machado, Baldwin, & Boland, 2017). Compound 2, [α ]26 D −29.4 (c = 0.32, MeOH), was obtained as yellow oil. The molecular formula was determined to be C14H20O5 based on the HR-ESI-MS at m/z 291.1204 [M + Na]+ (calcd for C14H20O5Na 291.1203). The 1H and 13C NMR spectra of 2 (Table 1) were similar to those of 1. In the HMBC, the correlations of δH 3.59 (H6′)/3.13 (H-4′) with δC 171.5 (C-5′), δH 3.13 (H-4′) with δC 129.5 (C2′)/123.0 (C-3′) suggested that compound 2 had a eCOOCH3 moiety 77

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Fig. 2. Selected 1H–1H COSY and HMBC and correlations of compounds 1–3, 5.

unit and a methyl 2-trans-pentenoate moiety at C-1 and C-2, respectively. The carbon skeleton of 3 was similar to cucurbic acid. For cucurbic acid, the resonances for H-1 and H-2 of the 1,2-cis isomer were at δH 2.60–2.06 and δH 2.15 (Holbrook & Moloney, 1997; Nakamura et al., 2008), whereas the 1,2-trans isomer showed H-1 signal at δH 1.84–2.15 and H-2 at δH 1.42–1.52, respectively (Carvajal et al., 2011; Dathe et al., 1991; Fujita, Terato, & Nakayama, 1996). Meanwhile, the cis isomer arrangement at C-2 and C-3 was an unfavorable configuration and never appeared as a major natural component. On the contrary, 2,3-trans isomer was a preferential configuration and the major natural component. In the 1H NMR spectrum of 3, H-1, H-2 and H-3 were resonated at δH 1.98, 1.20–1.30 and 3.94, respectively, which were consistent with those of methyl 3-epi-2-isocucurbate in the literature (Dathe et al., 1991; Fujita et al., 1996). Therefore, the structure of 3 was determined to be (1R,2R,2′E)-3-hydroxy-2-(5-methoxy-5-oxo-2-penten1-yl)- cyclopentaneacetic acid methyl ester. Compound 5 was purified as white powder, [α ]26 D +35.1 (c = 0.22, MeOH). The molecular formula was determined to be C25H32O10 on the basis of the ion at m/z 515.1887 [M + Na]+ (calcd for C25H32O10Na 515.1888) in the HR-ESI-MS. The IR spectrum revealed the presence of hydroxyl (3678 cm−1) and benzene (1605, 1513 and 1438 cm−1). In the 1H NMR, signals at δH 6.78 (1H, d, J = 1.6 Hz, H-2′), 6.68 (1H, d, J = 8.1 Hz, H-5′) and 6.48 (1H, d, J = 8.1, 1.6 Hz, H-6′) revealed the presence of 1,3,4-trisubstituted benzene group. Meanwhile, there were two methoxyls at δH 3.70 (3H, s) and 3.71 (3H, s). The 13C NMR spectrum showed 25 carbons (Table 2), which corresponded to two benzene groups (δC 147.2, 145.5, 144.5, 144.1, 136.9, 132.7, 127.1, 121.1, 116.3, 115.4, 113.8 and 111.8), five saccharide moiety carbons (δC 104.2, 72.6, 70.7, 67.7 and 65.4), two methoxyls (δC 55.6 and 55. 5), three methines (δC 45.7, 44.1 and 37.7), one methylene (δC 32.6) and two oxygenated methylenes (δC 67.1 and 62.7). Those NMR spectroscopic data indicated compound 5 as a 2,7′-cyclolign- 9,9′-diol system lignan (Zhang et al., 2016). The anomeric proton signals at δH 3.90 (1H, d, J = 6.5 Hz) was corresponding to an anomeric carbon at δC 104.2 in HSQC spectrum and the acid hydrolysis and GC analysis suggested that the sugar was L-arabinose. The HMBC correlation of δH 2.96 (H-9′) with δC 104.2 (CAra-1) confirmed that the L-arabinose was

Fig. 3. Four stereoisomers of methyl jasmonate analogs.

linked to C-4′, while 1 had a eCH2OCOCH3 unit located at C-4′. In the 1 H NMR spectrum of 2, H-1 and H-2 resonated at δH 2.17 and 1.98, which disclosed the planar structure of compound 2 to be trans isomer. The specific rotation at [α ]26 D −29.4 (c = 0.32, MeOH) indicated the (1R,2R) absolute configuration for 2. Consequently, the structure of 2 was determined as (1R,2R,2′Z)-2-(5-methoxy-5-oxo-2-penten-1-yl)-3oxo-cyclopentaneacetic acid methyl ester. Compound 3, [α ]26 D +36.3 (c = 0.32, MeOH), was obtained as colorless oil. The molecular formula was drawn to be C14H22O5 on the basis of its HR-ESI-MS at m/z 293.1368 [M + Na]+ (calcd for C14H22O5Na 293.1359). The IR spectrum revealed the presence of hydroxyl (3600–3000 cm−1), carboxylic carbonyl (1736 cm−1), and an olefinic (1635 cm−1) function groups. The NMR spectra of 3 were similar to those of 2, and the main differences were listed in Table 1. In the 1H NMR spectrum, two olefinic proton signals at δH 5.60 (1H, dd, J = 15.7, 7.6 Hz) and 5.46 (1H, dd, J = 15.7, 7.1 Hz) indicated the presence of a trans carbon-carbon double bond. The HMBC correlations of δH 2.04 (H-1′) with δC 50.3 (C-2)/71.8 (C-3), δH 2.13 (H-1′′) with δC 38.2 (C-1)/50.3 (C-2) disclosed that compound 3 was comprised of a disubstituted cyclopentanol skeleton correlating with a eCH2COOCH3 78

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located at C-9′. In the NOESY spectrum, an enhancement of the proton signal of H-2′ on irradiation of H-8′ and H-7′, the enhancement of H-6′ on irradiation of H-8′ and H-7′, which demonstrated that H-7′ and H-8′ were on the same side while H-7′/H-8 were on the different side. Furthermore, the S-configuration of C-7′ was confirmed by the CD spectrum which exhibited negative Cotton effect at 292 nm and positive Cotton effect at 273 nm. Ultimately, the structure of compound 5 was elucidated to be (+)-(8S,7′S,8′S)- burselignan-9′-O-α-L-arabinoside. Herein, the stereochemistry at H-8, H-7′ and H-8′ of 5 were elucidated to be (8S,7′S,8′S)-isomer for the first time although the planar structure was previously published (Medvedeva, Ivanova, Volkova, & Medvedev, 1987). Sixteen known compounds were identified by comparison of the NMR data with reported data as (1R,2R,2′Z)-methyl 12-hydroxyjasmonate (4) (Nakamura et al., 2008), (−)-catechin (6), (−)-epicatechin (7) (Nechepurenko et al., 2008), naringenin-7-O-β-D-glucopyranoside (8) (Liu & Wu, 2009), pinocembrin 7-O-rutinoside (9) (Xu et al., 2011), naringenin 7-O-β-D- rutinoside (10) (Shimoda et al., 2010), quercetin (11) (Cheng, Zhou, & Tan, 2001), quercetin-3-O-β-Dglucopyranoside (12) (Jin et al., 2009), quercetin-3-O-xyloside (13) (Zhu et al., 2013), quercetin-3-O-β-D-galactoside (14), isovitexin (15) (Jayasinghe, Balasooriya, Bandara, & Fujimoto, 2004), phlorizin (16) (Zhang et al., 2012), phloretin rutinoside (17) (Qin, Xing, Zhou, & Yao, 2015), 2α,3α-epoxy-5,7,3′,4′- tetrahydroxyflavan-(4β-8-epicatechin) (18), 2α,3α-epoxy-5,7,3′,4′-tetrahydroxyflavan (4β-8-catechin) (19), (4α-8-epicatechin) (20) 2β,3β-epoxy-5,7,3′,4′-tetrahydroxyflavan(Wang & Lou, 2011), respectively.

Table 4 Antioxidant activities of compounds 1–20.a,b

3.2. Anti-inflammatory activities

11–13 and 15 also exhibited stronger antioxidant capacities compared to ascorbic acid (29.97 ± 1.12 μM). Among the new compounds, only compound 5 showed antioxidant activity with the IC50 value of 34.98 ± 0.46 μM, while 1–3 exhibited the IC50 values larger than 300 μM and were considered as inactive. In the PMS/NADH-NBT assay, compounds 11, 12 and 18 exhibited obvious superoxide radical scavenging activity with the IC50 values under 100 μM. Compounds 6, 7, 13, 17, 19 and 20 also performed moderate antioxidant capacities compared to ascorbic acid (222.10 ± 5.60 μM). Among the new compounds, only 5 showed the IC50 value of 272.34 ± 6.52 μM and confirmed as weak superoxide radical scavenging ability. In accordance with DPPH values and PMS/NADH-NBT values, compounds 11 and 18–20 disclosed prominent antioxidant activity with FRAP values above 2.5 mM FeSO4/g. Compound 5, by contrasted with ascorbic acid (1.05 ± 0.05 mM FeSO4/g), revealed preferable total antioxidant activity with FRAP values of 3.07 ± 0.23 mM FeSO4/g, as well as compounds 6, 7, 12, 13 and 15 showed moderate antioxidant capacity in the FRAP assay. These results evaluated with three different antioxidant methods may be elucidate by the diverse mechanisms of dissimilar assays. It revealed that combined various methods for screening bioactivity is required for a comprehensive assessment.

Compd.

DPPH (IC50, μM)

PMS/NADH-NBT (IC50, μM)

FRAP (mmol/g)d

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Ascorbic acidc

> 300 > 300 > 300 > 300 34.98 ± 0.46 32.20 ± 0.28 34.12 ± 8.23 > 300 > 300 0.39 ± 0.13 11.46 ± 2.34 19.20 ± 3.71 25.17 ± 0.55 143.32 ± 1.77 24.66 ± 1.20 > 300 232.86 ± 2.52 5.24 ± 1.16 8.53 ± 0.36 7.06 ± 0.40 29.97 ± 1.12

> 300 > 300 > 300 > 300 272.34 ± 6.52 163.97 ± 4.23 195.83 ± 11.98 > 300 > 300 288.49 ± 7.62 92.11 ± 3.33 92.34 ± 5.99 118.34 ± 4.15 > 300 272.35 ± 8.27 > 300 > 300 72.38 ± 6.97 118.53 ± 5.72 115.07 ± 3.57 222.10 ± 5.60

< 0.5 < 0.5 < 0.5 < 0.5 3.07 ± 1.87 ± 1.32 ± < 0.5 < 0.5 < 0.5 3.55 ± 1.34 ± 1.56 ± < 0.5 1.14 ± < 0.5 0.50 ± 3.52 ± 2.71 ± 2.61 ± 1.05 ±

a b c d

Compounds 1–5 and four extracts were measured for the anti-inflammatory effects against NO production induced by LPS in RAW264.7 cells. The MTT assay was utilized to estimate the cytotoxicity of compounds to RAW 264.7 macrophage cells. All the tested samples showed no cytotoxicity against RAW 264.7 macrophage cells at their effective concentration. The results were summarized in Table 3. Compounds 2 and 3 exhibited pronounced inhibitions on NO production with the IC50 values of 61.25 ± 3.25 μM and 43.56 ± 2.17 μM. Other compounds exhibited weak inhibitory effect with IC50 values larger than 100 μM. As previously described (Carvajal et al., 2011), the antitumor effects of jasmonates can be explained by the effect of the stereo configuration and the optimal lipophilicity and the number of hydroxyl group. Following the same line of comparison between the capacities resulted from the jasmonate and derivative in tumor cell, we would be assumed that the hydroxyl groups present in jasmonates are also an significant factor in anti-inflammatory effects against NO production induced by LPS in RAW 264.7 cells; the structural difference could be making the difference in potential of its activity between jasmonates. This may explain why the anti-inflammatory capacity of compound 3 was stronger than compounds 1, 2 and 4.

0.23 0.17 0.10

3.08 0.14 0.12 0.20 0.04 0.23 0.10 0.15 0.05

Values are the mean ± SD; n = 3. Test concentrations ranged from 6.25 to 100 μM. Ascorbic acid was utilized as positive control. The results of the FRAP assay were calculated as mM FeSO4/(g of powder).

4. Conclusion 3.3. Antioxidant activity In conclusion, four methyl jasmonate analogs (1–4), one lignanoside (5), together with fifteen flavonoids (6–20) were isolated and identified from lychee seeds. Compounds 2 and 3 exhibited notable inhibition on NO production induced by lipopolysaccharides (LPS) in macrophages cell line RAW 264.7. Compound 5 and some flavonoids revealed potent

The DPPH, PMS/NADH-NBT and FRAP assays were applied to evaluate the antioxidant capacities of compounds 1–20 (Table 4). In the DPPH assay, compounds 10, 18–20 exhibited noteworthy DPPH radical scavenging activity with the IC50 values under 10 μM. Compounds

Table 3 Anti-inflammatory activities of compounds 1–5 on LPS-induced NO production in RAW264.7.a Compd.

1

2

3

4

5

Indomethacinb

IC50 (μM)

> 100

61.25 ± 3.25

43.56 ± 2.17

> 100

> 100

34.47 ± 2.86

a b

Values are the mean ± SD; n = 3. Indomethacin was used as positive control. 79

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antioxidant capacities. The results disclosed that the methyl jasmonates in lychee seeds may be partially responsible for anti-inflammatory activity of lychee and could be served as anti-inflammatory agent in functional food. The mechanism of action of these compounds needs further study, and the toxic components of lychee seeds also should be given sufficient attention.

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Acknowledgements This study was supported by Guangdong Province Universities and Colleges Pearl River Scholar Funded Scheme (2017), and the project (2015A020211027) of Guangdong Provincial of Science and Technology Department. Conflict of interest All authors read and approved the final manuscript. The authors declare that there are no conflicts of interest. Ethics statement I declare that our research did not include any human subjects and animal experiments. Appendix A. Supplementary material The original spectra of new compounds 1–3 and 5, including UV, IR, H, 13C NMR, 2D-NMR, HR-ESI-MS and CD were provided as supplementary data. Supplementary data to this article can be found online at https://doi.org/10.1016/j.jff.2018.12.040. 1

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