Flavonoids, a Potential New Insight of Leucaena leucocephala

Jun 28, 2018 - Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese. Academy of Sciences, Guangzhou, China. ‡. Divisio...
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Article Cite This: J. Agric. Food Chem. 2018, 66, 7616−7626

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Flavonoids, a Potential New Insight of Leucaena leucocephala Foliage in Ruminant Health Yingchao Xu,†,§ Zhenru Tao,†,§ Yu Jin,†,§ Yunfei Yuan,† Tina T. X. Dong,‡ Karl W. K. Tsim,‡ and Zhongyu Zhou*,† †

J. Agric. Food Chem. 2018.66:7616-7626. Downloaded from pubs.acs.org by IOWA STATE UNIV on 01/28/19. For personal use only.

Key Laboratory of Plant Resources Conservation and Sustainable Utilization, Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China 510650 ‡ Division of Life Science and Center for Chinese Medicine, The Hong Kong University of Science and Technology, Hong Kong, China § University of Chinese Academy of Sciences, Beijing, China 100049 S Supporting Information *

ABSTRACT: We investigated the constituents of Leucaena leucocephala foliage collected from Guangdong province in China and isolated 17 diverse flavonoids (1−17), including flavones (5−9, 11, and 12), flavonols (1, 10, and 16), flavanone 4, flavanonol 15, and flavonol glycosides (2, 3, 13, 14, and 17). Flavonoids quercetin (1), quercetin-3-O-α-rhamnopyranoside (2), and myricetin-3-O-α-rhamnopyranoside (17) were the major flavonoids components in L. leucocephala leaves, at a total concentration of about 2.5% of dry matter. pHRE-Luc inductive activity to mimic the activation of erythropoietin (EPO) gene, anti-inflammatory, antidiabetic, and antioxidant activities of isolated flavonoids (1−17) were evaluated. Flavonoids 7, 10, and 13 could strongly induce the transcriptional activity of pHRE-Luc, which indicated their potential to induce the expression of EPO. Flavonoids 7, 10, 13, and 17 displayed strong anti-inflammatory activity, relatively equal to the positive control dexamethasone. Flavonoids 1, 2, 3, 11, 12, 16, and 17 showed stronger antioxidant activities of DPPH radical scavenging capacity than ascorbic acid. Flavonoids 1, 2, and 10 showed weak cellular antioxidant activities against tert-butyl hydroperoxide (tBHP) induced ROS formation. Flavonoid rhamnoside 2 and arabinoside 3 undergone deglycosylation to the aglycone quercetin under anaerobic incubation with cattle rumen microorganisms. Furthermore, the potential health benefits for ruminant of flavonoids, which was rich in L. leucocephala foliage, was also discussed. KEYWORDS: Leucaena leucocephala, forage, flavonoids, ruminant health, rumen fermentation

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dien-3β-ol,7 β-sitosterol,7 β-sitostenone,7 stigmastenone,7 lupeol,7 3-dipalmitoyl-2-oleoylglycerol,7 linoleic acid,7 methylparaben,7 isovanillic acid,7 pheophytin-a,7 pheophorbide a methyl ester,7 methyl-132-hydroxy-(132-S)-pheophorbide-b,7 32-hydroxy-(132-S)-pheophytin-a,7 and aristophyll-C.7 Gallocatechin with nitrification inhibitory activity, epigallocatechin, catechin, and epicatechin were isolated from the roots of L. leucocephala.8 Researchers have isolated polyphenolic compounds with antioxidant activity including flavonoids from the leaves of L. leucocephala.9−12 Mimosine has been detected in leaves, flowers, pods, seeds, and roots, while asparagine was the most abundant amino acid in flowers, which was also detected in leaves, pods, and seeds but not roots.13 About the medicinal properties, Li reported that the extract of L. leucocephala seeds and leaves had antidiabetic activities.14 The fraction of methanolic extract of L. leucocephala seeds showed inhibitory activities on α-glucosidase and aldose reductase.15 L. leucocephala was also widely cultivated in China and mainly distributed in the province of Guangdong, Guangxi, Fujian, Yunnan, and Hainan. However, only a few

INTRODUCTION Leucaena leucocephala belongs to the family Fabaceae, which is indigenous to Mexico, and now is widely distributed throughout the tropics and subtropics, including central America, Africa, Asia, and northern Australia.1 L. leucocephala flowered at April to July and fruit ripened at August to October. It is a fast growing tropical legume and a high biomass yielding plant. L. leucocephala was acted as promising forage, because of researchers in Hawaii and tropical Australia have discovered that cattle feeding on L. leucocephala have greater weight gains than those of cattle which feed on the greatest pastures anywhere.1 Further, leucaena leaf meal (LLM) were highly degradable in the rumen and LLM could be used to improve rumen ecology.2 L. leucocephala forage presented a high level of crude protein (CP), high digestibility, and voluntary intake of CP.3 The plant’s drought-tolerance and hardiness made it a promising candidate of sustainable feed supplements for ruminants during both the dry and rainy seasons of the year.4 In the past decades, a great deal of work had been conducted on the poultry nutrition of L. leucocephala, due to its abundant of minerals, protein, and carotenes. L. leucocephala for nutritive value and forage productivity were extensively reviewed.3,5 Previous chemical study on L. leucocephala seeds led to the isolation of gibberellins,6 5α,8α-epidioxy-(24ξ)-ergosta-6,22© 2018 American Chemical Society

Received: Revised: Accepted: Published: 7616

May 25, 2018 June 25, 2018 June 28, 2018 June 28, 2018 DOI: 10.1021/acs.jafc.8b02739 J. Agric. Food Chem. 2018, 66, 7616−7626

Article

Journal of Agricultural and Food Chemistry

E3-1−E3-15. Fraction E3-11, from the elution with MeOH/H2O (60:40), was separated on Sephadex LH-20 column chromatography with the elution of CHCl3/MeOH (1:4, v/v), to yield fractions E3-111−E3-11-12, and pure compound 4 (9.6 mg) was obtained from E311-9. E3-11-4 was subjected to Sephadex LH-20 column chromatography eluted with MeOH to obtain E3-11-4-2, followed by preparative HPLC with a Shim-pack PRC-ODS C-18 column (5 μm, 20 mm × 250 mm) using 60% methanol in water (v/v) as a mobile phase at the flow rate of 6 mL/min to obtain a mixture of 8 and 9 (28.3 mg, tR = 78 min). E3-11-5 was further purified by preparative HPLC using 50% methanol in water (v/v) as a mobile phase at the flow rate of 6 mL/min to obtain 5 (7.8 mg, tR = 78 min) and 6 (9.4 mg, tR = 110 min). Fraction E3-12, from the elution with MeOH/H2O (70:30), was separated with Sephadex LH-20 column chromatography with the elution of CHCl3/MeOH (1:4, v/v) to obtain pure 7 (553 mg) and 12 (8.3 mg). Fraction E4 (30 g) eluted with CHCl3/MeOH (90:10) and was separated on MPLC using MeOH/H2O (10:100−100:0, v/v) eluant to give fractions E4-1−E4-13. Fraction E4-6 was applied on Sephadex LH-20 column chromatography with the elution of CHCl3/MeOH (1:4, v/v) to obtain 11 (5 mg). Fraction E4-7 was applied on Sephadex LH-20 column chromatography eluted with MeOH to provide 10 (830 mg). Fraction E7 (25 g), eluted with CHCl3/MeOH (80:20), was subjected to MPLC using a MeOH/H2O (10:100−100:0, v/v) eluant to give fractions E7-1−E7-19. Fraction E7-8 was divided into seven fractions by Sephadex LH-20 column chromatography eluted with CHCl3/MeOH (1:4, v/v), and one fraction E7-18-6 of seven was further purified by Sephadex LH-20 column chromatography eluted with MeOH and preparative HPLC using 50% methanol in water (v/ v) at the flow rate of 6 mL/min to provide 13 (860 mg, tR = 18.6 min) and 14 (410 mg, tR = 20 min). Fraction E7-10 was separated on Sephadex LH-20 column chromatography eluted with MeOH to obtain 16 (7 mg). Fraction E7-13 was separated on Sephadex LH-20 (CHCl3/MeOH, 1:1, v/v) and silica gel (CHCl3/MeOH, 40:1, v/v) column chromatography to provide 15 (16.6 mg). Fraction E10 (50 g) eluted with CHCl3/MeOH (70:30) was separated on MPLC using MeOH/H2O (10:100−100:0, v/v) eluant to give fractions E10-1−E10-12. Fraction E10-5 was purified by Sephadex LH-20 column chromatography with the elution of CHCl3/ MeOH (1:4, v/v) to obtain 3 (23 mg). Fraction E10-7 was separated on Sephadex LH-20 column chromatography eluted with CHCl3/ MeOH (1:1, v/v) to obtain 2 (13 g). Fraction E10-8 was subjected to Sephadex LH-20 column chromatography eluted with CHCl3/MeOH (1:4, v/v) to obtain 1 (22 g). Fraction E16 (16 g) eluted with CHCl3/MeOH (60:40) and MPLC was applied using MeOH/H2O (10:100−100:0, v/v) eluant to give fractions E16-1−E16-12. Fraction E16-9 was further applied on Sephadex LH-20 (CHCl3/MeOH, 1:4, v/v) and silica gel (CHCl3/ MeOH, 7:1, v/v) column chromatography to obtain 17 (9 g). Content of Flavonoids Determination by UPLC−QQQ-MRM MS/MS. The UPLC−QQQ tandem mass spectrometry experiments was performed with an Agilent RRLC 1200 series system (Waldron, Germany) and Agilent QQQ-MS/MS system equipped with an ESI ion source. The liquid chromatography was carried out on an Agilent Zorbax Eclipse Plus C18 (RRHD, 50 × 2.1 mm, 1.8 μm) column at 25 °C. Analysis was completed with a gradient elution of 0.1% formic acid and 3% acetonitrile in water (A)−0.1% formic acid in acetonitrile (B) within 10 min. The gradient program was 2% B → 4% B at 0−2 min; 4% B → 90% B at 2−8 min; 90% B → 90% B at 8−10 min at a flow rate of 0.3 mL min−1 with a sample injection volume of 2.0 μL. The MS/MS was selected in positive mode based on the Optimizer program, which is an automated method development tool to generate and optimize MRM transitions in Agilent Mass Hunter Workstation. Other parameters were set as following: the temperature was 325 °C; the drying gas at the flow rate was 10 L/min; capillary voltage at 4 000 V; nebulizer pressure at 35 psig; delta electromultiplier voltage at 400 V. The collision energy values and fragmentor voltage were adjusted to obtain the highest abundance.

reports studied the chemical composition of L. leucocephala cultivated in China.16,17 We investigated the constituents of L. leucocephala foliage collected from Guangdong province in China and isolated 17 diverse flavonoids (1−17). Flavonoids displayed a wide range of biological activities. It was reported that flavonoids from Radix astragali induced the expression of erythropoietin in cultured human embryonic kidney 293 T (HEK293T) fibroblast cells.18,19 The anti-inflammatory properties of flavonoids have been well studied.20 The extract of L. leucocephala seeds and leaves was reported to have antidiabetic activities.14,15 Flavonoids were well-known for their antioxidant capacity. Therefore, pHRE-Luc inductive activity to mimic the activation of EPO gene, antiinflammatory, antidiabetic, and antioxidant activities of EtOH extracts and EtOAc fraction as well as isolated flavonoids (1− 17) were evaluated. The bioavailability of flavonoid glycosides was studied through the metabolism of flavonoids 2 and 3 under anaerobic incubation with rumen microorganisms. Furthermore, the potential health benefits for ruminant of flavonoids, which was rich in L. leucocephala foliage, was also discussed.

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MATERIALS AND METHODS

General Experimental Procedures. 1H and 13C NMR spectra were recorded in CD3OD or DMSO-d6 on a Bruker DRX-500 NMR (Bruker Biospin Gmbh, Rheistetten, Germany) instrument using the residual solvent peak as reference, spectrometers operating at 500 MHz for 1H and 125 MHz for 13C, respectively. ESIMS and ESIMS/ MS were collected on an MDS SCIEX API 2000 LC/MS/MS instrument. Medium pressure liquid chromatography (MPLC) was carried out on a CXTH P3000 instrument (Beijing Chuang Xin Tong Heng Science and Technology Co., Ltd., Beijing, China) equipped with a UV 3000 UV−vis detector and a C-18 column (50 μm, 50 m × 500 mm). HPLC analysis was conducted with two Shimadzu LC20AT pumps, a Shimadzu SPD-M20A diode array detector, and a Shimadzu SIL-20A auto sampler using an Agilent Zorbax SB-Aq column (5 μm, 4.6 mm × 250 mm). For column chromatography, silica gel (80−100 mesh and 200−300 mesh Qingdao Haiyang Chemical Co., Qingdao, China), Sephadex LH-20 (Pharmacia Fine Chemical Co., Ltd., Oppsala, Sweden) was performed. Thin-layer chromatography (TLC) was conducted on precoated silica gel plates (HSGF254, Yantai Jiang you Silica Gel Development Co., Ltd., Yantai, China) and spot detection was performed by spraying 10% H2SO4 in ethanol, followed by heating. Analytical grade ethyl acetate, chloroform, methanol, petroleum ether (bp 60−90 °C), n-butanol were purchased from Tianjin Fuyu Fine Chemical Industry Co. (Tianjin, China). 1,1-diphenyl-2-picryhydrazyl (DPPH), cobalt chloride (CoCl2), and dipeptidyl-peptidase 4 (DPP4) inhibitor screening kit were purchased from Sigma-Aldrich. Reagents for cell cultures were obtained from Invitrogen Technologies (Carlsbad, CA). Plant Material. The L. leucocephala leaves were collected from Guangzhou, China, in July 2016 and identified by Dr. Zhongyu Zhou. The voucher specimen (No. ZZY20160702) was deposited at the Laboratory of Phytochemistry at the South China Botanical Garden, Chinese Academy of Sciences. Extraction and Isolation. The L. leucocephala foliage were collected and dried with the exposure under the sun. The dried foliage (23 kg) were powdered and extracted three times with 95% EtOH (50 L) at room temperature for 3 days each time. The EtOH extracts was concentrated in vacuo using rotary evaporators and suspended in H2O and then sequentially extracted with petroleum ether, EtOAc, and n-butanol. The EtOAc fraction (700 g) was subjected to silica gel column chromatography, eluted with CHCl3/MeOH (from 100:0 to 0:100, v/ v) to give fractions E1−E17. Fraction E3 (14 g) eluted with CHCl3/ MeOH (95:5) and was further applied on MPLC using a decreasing polarity of MeOH/H2O (10:100−100:0, v/v) eluant to give fractions 7617

DOI: 10.1021/acs.jafc.8b02739 J. Agric. Food Chem. 2018, 66, 7616−7626

Article

Journal of Agricultural and Food Chemistry

Figure 1. Chemical structures of 17 isolated flavonoids from L. leucocephala foliage. penicillin, and 100 μg/mL streptomycin in a 37 °C, 5% CO2, and water saturated incubator. Since all compounds had a safe concentration at 50 μg/mL or 10 μM on HEK293T cells, a same concentration of 50 μg/mL of EtOH extracts and EtOAc fraction and 10 μM of compounds 1, 2, 7, 10, 13, 14, and 17 were chosen for MTT assay on RAW 264.7 macrophage. For anti-inflammatory activity assay, RAW 264.7 macrophage (3 × 104 cells/ml) were pretreated with different extracts or flavonoids for 3 h, followed by stimulation with LPS (0.1 μg/mL) for an additional 24 h. The supernatants of cells were analyzed for the levels of TNF-α and IL-6 by enzyme linked immunosorbent assay (ELISA) using commercial TNF-α and IL-6 detecting kits (R&D Systems, Inc., Minneapolis, USA). DMSO (0.1%) and dexamethasone (10 μM) were used as vehicle and positive controls, respectively. All values were given as mean ± SEM (n = 3). Data analysis involved Student’s t test. Dipeptidyl-peptidase 4 (DPP4) Inhibitor Screening Assay. Before DPP4 inhibitor screening assay, since the test compounds were dissolved in DMSO, DMSO was assessed for an uninhibited concentration on DPP4 enzyme. DPP4 was found to be free at a concentration of 0.2% DMSO. DPP4 inhibitor screening assay was performed using a commercial kit (Sigma-Aldrich, St. Louis, MO) according to the kit introduction. In brief, 12.5 μL of different compounds or extracts and 25 μL of DPP4 enzyme were added into each well of the 96 well plate, which was incubated for 10 min at 37 °C in the dark. After incubation, 12.5 μL of DPP4 substrate was added into each reaction well. Immediately, the fluorescence (FLU, λex = 360/λem = 460 nm) was measured on a microplate reader in

We used the software of Agilent Mass Hunter Workstation for data acquisition, processing, and analysis. pHRE-Luc Activity Assay. For cell cultures, human embryonic kidney (HEK) 293T fibroblast cell from American Type Culture Collection (ATCC) were maintained in dulbecco’s modified eagle medium (DMEM) added with 10% fetal bovine serum (FBS), 100 IU/mL penicillin, and 100 μg/mL streptomycin. Cultures were placed in a water saturated 5% CO2 incubator at 37 °C. Safe concentrations of EtOH extracts, EtOAc fraction, and selected compounds were identified by methyl thiazolyl tetrazolium (MTT) assay.21 HEK293T fibroblast cell is an excellent in vitro model in studying the physiological regulation of EPO expression, which is sensitive to hypoxia stress. The DNA construct of luciferase reporter (pHRE-Luc) and vector were generated as described previously.18 Briefly, cultured HEK293T cells (3 × 104 cells/mL) were seeded into 12-well plates and transfected with pHRE-Luc by the calcium phosphate precipitation method. L. leucocephala leave extracts were applied onto transfected HEK293T cells. After 1 day, the cell lysates were collected for luciferase assay. The luciferase activity was evaluated in Tropix TR717TM Microplate Luminometer (Bedford, MA), and the activity was expressed as absorbance (up to 560 nm) per mg of protein. The authentication of pHRE-Luc was confirmed by its activation in exposure to application of CoCl2 at 100 μM, which was frequently used to mimic the effect of hypoxia. Anti-Inflammatory Activity Assay. For cell culture, the murine RAW 264.7 macrophage from ATCC was cultured in DMEM medium supplemented with 10% heated-inactivated FBS, 100 IU/mL 7618

DOI: 10.1021/acs.jafc.8b02739 J. Agric. Food Chem. 2018, 66, 7616−7626

Journal of Agricultural and Food Chemistry

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kinetic mode for 20 min at 37 °C. The enzyme control contained 0.2% DMSO instead of the compound solution. For inhibitory rates calculation, the fluorescence for each well versus time was plotted. Two time points (T1 and T2) in the linear range of the plot were chosen and the slope for each well between T1 and T2 were obtained.

Article

RESULTS

Seventeen flavonoids were isolated and identified, including quercetin (1), quercetin-3-O-α-rhamnopyranoside (2), quercetin-3-O-α-arabinofuranose (3), naringenin (4), geraldone (5), 7,3′-dihydroxy-4′-methoxyflavone (6), apigenin (7), chrysoeriol (8), diosmetin (9), kaempferol (10), luteolin (11), 3′,4′,7-trihydroxyflavone (12), juglanin (13), kaempferol-3-O-α-rhamnopyranoside (14), (+) taxifolin (15), myricetin (16), and myricetin-3-O-α-rhamnopyranoside (17). To the best of our knowledge, flavonoids 1, 4, 5, 6, 9, 10, and 12−15 were isolated from L. leucocephala for the first time, and 4 and 15 were the first record of 2,3-dihydroflavones in this plant. The structure of 17 flavonoids (1−17) were shown in Figure 1. Spectroscopic Data of Seventeen Flavonoids. Quercetin (1). Yellow amorphous powder. 1H NMR (500 MHz, CD3OD): δ ppm 6.18 (1H, d, J = 1.6 Hz, H-6), 6.39 (1H, d, J = 1.9 Hz, H-8), 6.89 (1H, d, J = 8.5 Hz, H-2′), 7.73 (1H, d, J = 2.1 Hz, H-5′), 7.63 (1H, dd, J = 8.5, 2.1 Hz, H-6′). 13C NMR (125 MHz, CD3OD): δ ppm 148.03 (C-2), 137.20 (C-3), 177.33 (C-4), 162.47 (C-5), 99.28 (C-6), 165.63 (C-7), 94.45 (C-8), 158.23 (C-9), 104.50 (C-10), 124.44 (C-1′), 116.01 (C-2′), 146.21 (C-3′), 148.76 (C-4′), 116.24 (C-5′), 121.69 (C-6′). ESI-MS m/z 303 [M + H]+, 341 [M + K]+. The structure was also confirmed with comparison with published data.25 Quercetin-3-O-α-rhamnopyranoside (2). Yellow amorphous powder. 1H NMR (500 MHz, CD3OD): δ ppm 6.20 (1H, d, J = 2.1 Hz, H-6), 6.37 (1H, d, J = 2.1 Hz, H-8), 7.34 (1H, d, J = 2.1 Hz, H-2′), 6.91 (1H, d, J = 8.3 Hz, H-5′), 7.31 (1H, dd, J = 8.3, 2.1 Hz, H-6′), 5.35 (1H, d, J = 1.4 Hz, H-1″), 4.22 (1H, dd, J = 3.3, 1.4 Hz, H-2″), 3.75 (1H, dd, J = 9.5, 3.3 Hz, H-3″), 3.41 (1H, m, H-4′′), 3.15 (1H, m, H-5′′), 0.94 (3H, d, J = 6.2 Hz, H-6″). 13C NMR (125 MHz, CD3OD): δ ppm 159.31 (C-2), 136.23 (C-3), 179.65 (C-4), 163.21 (C-5), 99.82 (C-6), 165.89 (C-7), 94.71 (C-8), 158.53 (C-9), 105.90 (C-10), 122.85 (C-1′), 116.93 (C-2′), 146.41 (C-3′), 149.80 (C-4′), 116.36 (C-5′), 122.96 (C-6′), 103.54 (C-1″), 71.89 (C-2′′), 72.11 (C-3′′), 72.02 (C-4′′), 73.25 (C-5′′), 17.65 (C6′′). ESI-MS positive m/z 359 [M + H]+, 381 [M + Na]+. The structure was also confirmed with comparison with published data.26 Quercetin-3-O-α-arabinofuranose (3). Yellow amorphous powder. 1H NMR (500 MHz, CD3OD): δ ppm 6.21 (1H, d, J = 2.1 Hz, H-6), 6.40 (1H, d, J = 2.1 Hz, H-8), 7.53 (1H, d, J = 2.1 Hz, H-2′), 6.91 (1H, d, J = 8.4 Hz, H-5′), 7.50 (1H, dd, J = 8.4, 2.1 Hz, H-6′), 5.47 (1H, s, H-1″), 4.33 (1H, d, J = 3.0 Hz, H-2″), 3.91 (1H, m, H-3′′), 3.87 (1H, m, H-4′′), 3.50 (2H, m, H-5′′). 13C NMR (125 MHz, CD3OD): δ ppm 158.60 (C-2), 134.92 (C-3), 179.88 (C-4), 163.08 (C-5), 99.95 (C-6), 166.25 (C-7), 94.82 (C-8), 159.34 (C-9), 105.58 (C-10), 122.95 (C-1′), 116.84 (C-2′), 146.37 (C-3′), 149.67 (C-4′), 116.45 (C-5′), 123.11 (C-6′), 109.54 (C-1″), 83.32 (C-2′′), 78.72 (C-3′′), 88.04 (C-4′′), 62.56 (C-5′′). ESI-MS negative m/z 433 [M − H]−. The structure was also confirmed with comparison with published data.12 Naringenin (4). White needle crystal. 1H NMR(500 MHz, CD3OD): δ ppm 5.33 (1H, dd, J = 12.9, 3.0 Hz, H-2), 3.10 (1H, dd, J = 17.1, 12.9 Hz, H-3α), 2.69 (1H, dd, J = 17.1, 3.0 Hz, H-3β), 5.88 (1H, d, J = 2.1 Hz, H-6), 5.89 (1H, d, J = 2.0 Hz, H-8), 7.31 (2H, d, J = 8.5 Hz, H-2′, 6′), 6.82 (2H, d, J = 8.5 Hz, H-3′, 5′). 13C NMR (125 MHz, CD3OD): δ ppm 80.47 (C-2), 44.03 (C-3), 197.75 (C-4), 165.49 (C-5), 97.06

Slope = (FLU2 − FLU1)/(T2 − T1) = ΔFLU/minute Relative Inhibition (%) = (SlopeEC − SlopeSM)100/SlopeEC where SlopeSM = the slope of the sample inhibitor SlopeEC = the slope of the enzyme control DPPH Radical Scavenging Assay. The DPPH radical scavenging activity was carried out according to the procedures as previously described.22 DPPH was freshly prepared in methanol at a concentration of 0.1 mM. Test compounds were preliminary screened at 50 μM, and those which had more than 50% DPPH radical scavenging activity were further experimented for SC50 (the concentration of sample required to scavenge 50% of DPPH radicals) determination. For further screening, test compounds were dissolved in methanol and diluted 2-fold to six concentrations (from 1.5615 to 50 μM). A volume of 20 μL of the compound solution and 180 μL of the 0.1 mM DPPH solution were mixed in 96-well plates. Ascorbic acid was dissolved in methanol and used as a positive control. The control contained methanol instead of the compound solution, and the blank contained methanol in place of the DPPH solution. Each reaction was repeated in triplicate. The plates were incubated at 37 °C for 30 min in the dark. The absorbance (OD) reading in each well was taken at 517 nm on a microplate reader. The inhibitory rates of DPPH radicals were calculated according to the formula inhibition (%) = [1 − (OD treated − OD blank)/OD control] × 100. The SC50value was obtained through the software of SPSS 16.0. Finally, the data presented are means ± SD of three determinations. Cellular Reactive Oxygen Species (ROS) Formation Level. ROS formation level was measured according to literature procedures.23 In detail, RAW 264.7 cells were grown in 96-well plates (3 × 104 cells/mL) for 24 h incubation; the cells were then preincubated with 50 μg/mL of EtOH extracts and EtOAc fraction, and 10 μM flavonoids 1, 2, 7, 10, 13, 14, and 17 for 24 h. The cells were stained with 50 μM of dichlorodi-hydrofluorescein diacetate (DCFH-DA) for 1 h and subsequently incubated with tBHP (100 μM) for 30 min to induce the ROS formation. DCF fluorescence intensities were measured in an Envision 2104 Multilabel Reader (PerkinElmer Inc.) at an excitation and emission wavelength of 485 and 535 nm, respectively. Metabolism of Flavonoid Glycosides by Cattle Rumen Microorganisms in Vitro. Fresh cattle rumen liquid was obtained from healthy cattle which had not taken antibiotics for at least 3 months prior to the study and had no history of gastrointestinal disorders. The method of coincubation of flavonoid glycosides with rumen liquid was according to a reference with a minor adjustment,24 which was to mimic rumen fermentation. In detail, 2 mg flavonoid glycoside (2 or 3) was dissolved with 10 μL of DMSO and added into 5 mL cattle rumen liquid. A volume of 10 μL of DMSO and 5 mL cattle rumen liquid was set as a blank control. The mixture was incubated at 37 °C in an anaerobic condition for 24 h. The cultured mixture was extracted with water saturated n-butanol three times. The extracts was evaporated, and the residue was dissolved in methanol (1 mL) and filtered through a 0.45 μm membrane filter for HPLC analysis. Analysis was completed with a gradient elution of water (A)−methanol (B) within 40 min. The gradient program was 10% B at 0−5 min; 10% B → 100% B at 5−35 min; 100% B at 35−40 min at a flow rate of 1 mL min−1. UV absorption was monitored at 254 nm. For comparison, flavonoid glycosides 2 and 3, and their aglycone 1 were also analyzed by HPLC in the same batch. 7619

DOI: 10.1021/acs.jafc.8b02739 J. Agric. Food Chem. 2018, 66, 7616−7626

Article

Journal of Agricultural and Food Chemistry

118.14 (C-6′), 55.76 (−OCH3). ESI-MS positive m/z 323 [M + Na]+, 339 [M + K]+, 301 [M + H]+. ESI-MS negative m/z 299 [M − H]−, 599 [2M − H]−. The structure was also confirmed with comparison with published data.31 Kaempferol (10). Yellow amorphous powder. 1H NMR (500 MHz, CD3OD): δ ppm 8.09 (2H, d, J = 8.6 Hz, H-2′, 6′), 6.92 (2H, d, J = 8.6 Hz, H-3′, 5′), 6.20 (1H, d, J = 1.9 Hz, H6), 6.40 (1H, d, J = 1.9 Hz, H-8). 13C NMR (125 MHz, CD3OD): δ ppm 148.01 (C-2), 137.12 (C-3), 177.34 (C-4), 158.24 (C-5), 99.26 (C-6), 165.57 (C-7), 94.46 (C-8), 123.73 (C-1′), 130.67 (C-2′, 6′), 116.29 (C-3′, 5′), 160.54 (C-4′). The structure was also confirmed with comparison with published data.32 Luteolin (11). Yellow amorphous powder. 1H NMR (500 MHz, DMSO-d6): δ ppm 6.66 (1H, s, H-3), 6.19 (1H, d, J = 2.1 Hz, H-6), 6.44 (1H, d, J = 2.1 Hz, H-8), 7.41 (1H, dd, J = 8.2, 2.3 Hz, H-2′), 6.89 (1H, d, J = 8.2 Hz, H-3′), 7.39 (1H, d, J = 2.3 Hz, H-6′). 13C NMR (125 MHz, DMSO-d6): δ ppm 163.87 (C-2), 103.69 (C-3), 181.63 (C-4), 157.26 (C-5), 98.80 (C-6), 164.09 (C-7), 93.81 (C-8), 161.46 (C-9), 103.69 (C-10), 118.97 (C-1′), 113.36 (C-2′), 145.71 (C-3′), 149.67 (C-4′), 115.99 (C-5′), 121.49 (C-6′). The structure was also confirmed with comparison with published data.33 3′,4′,7-Trihydroxyflavone (12). Yellow amorphous powder. 1 H NMR (500 MHz, CD3OD): δ ppm 6.63 (1H, s, H-3), 7.97 (1H, d, J = 8.8 Hz, H-5), 6.90−6.96 (3H, m, H-6, 8, 5′), 7.40 (2H, m, H-2′, 6′). 13C NMR (125 MHz, CD3OD): δ ppm 166.06 (C-2), 105.20 (C-3), 180.27 (C-4), 127.74 (C-5), 116.23 (C-6), 164.81 (C-7), 103.46 (C-8), 159.65 (C-9), 117.25 (C-10), 124.03 (C-1′), 114.16 (C-2′), 147.02 (C-3′), 150.77 (C-4′), 116.79 (C-5′), 120.20 (C-6′). The structure was also confirmed with comparison with published data.34 Juglanin (13). Yellow amorphous powder. 1H NMR (500 MHz, CD3OD): δ ppm 6.20 (1H, d, J = 2.1 Hz, H-6), 6.39 (1H, d, J = 2.1 Hz, H-8), 7.95 (2H, d, J = 8.5 Hz, H-2′, H-6′), 6.92 (2H, d, J = 8.5 Hz, H-3′, H-5′), 5.48 (1H, s, H-1″), 4.32 (1H, d, J = 2.9 Hz, H-2″), 3.91 (1H, m, H-3′′), 3.81 (1H, m, H-4′′), 3.48 (2H, m, H-5′′). 13C NMR (125 MHz, CD3OD): δ ppm 158.55 (C-2), 134.94 (C-3), 179.90 (C-4), 163.06 (C5), 99.89 (C-6), 165.99 (C-7), 94.80 (C-8), 159.34 (C-9), 105.67 (C-10), 122.79 (C-1′), 131.96 (C-2′, 6′), 116.50 (C-3′, 5′), 161.53 (C-4′), 109.65 (C-1″), 83.34 (C-2′′), 78.65 (C3′′), 88.03 (C-4′′), 62.55 (C-5′′). The structure was also confirmed with comparison with published data.32 Kaempferol-3-O-α-rhamnopyranoside (14). Yellow amorphous powder. 1H NMR (500 MHz, CD3OD): δ ppm 7.75 (2H,d, J = 8.4 Hz, H-2′, 6′), 6.93 (2H, d, J = 8.4 Hz, H-3', 5'), 6.19 (1H, s, H-6), 6.36 (1H, s, H-8), 5.38 (1H, d, J = 1.5 Hz, H-1″), 4.23 (1H, dd, J = 3.3, 1.7 Hz, H-2″), 3.72 (1H, m, H3′′), 3.34 (2H, m, H-4′′, 5′′), 0.93 (3H, d, J = 5.4 Hz, H-6″). 13 C NMR (125 MHz, CD3OD): δ ppm 159.22 (C-2), 136.19 (C-3), 179.56 (C-4), 163.15 (C-5), 99.81 (C-6), 165.79 (C-7), 94.75 (C-8), 158.48 (C-9), 105.91 (C-10), 122.62 (C-1′), 131.88 (C-2′, 6′), 116.49 (C-3′, 5′), 161.51 (C-4′), 103.47 (C1″), 73.19 (C-2′′), 72.11 (C-3′′), 72.00 (C-4′′), 71.90 (C-5′′), 17.64 (C-6′′). The structure was also confirmed with comparison with published data.35 (+) Taxifolin (15). White needles. 1H NMR (500 MHz, CD3OD): δ ppm 4.91 (1H, d, J = 11.5 Hz, H-2), 4.50 (1H, d, J = 11.5 Hz, H-3), 5.88 (1H, d, J = 2.1 Hz, H-6), 5.92 (1H, d, J = 2.1 Hz, H-8), 6.97 (1H, d, J = 2.1 Hz, H- 2′), 6.80 (1H, d, J = 8.2 Hz, H-5′), 6.85 (1H, dd, J = 2.1 Hz, 8.2 Hz, H-6′. 13C NMR (125 MHz, CD3OD): δ ppm 85.09 (C-2), 73.66 (C-3),

(C-6), 166.41 (C-7), 96.18 (C-8), 164.87 (C-9), 103.34 (C10), 131.09 (C-1′), 129.01 (C-2′, 6′), 116.32 (C-3′, 5′), 159.01 (C-4′). The structure was also confirmed with comparison with published data.27 Geraldone (5). White amorphous powder. 1H NMR (500 MHz, DMSO-d6): δ ppm 6.83 (1H, s, H-3), 7.86 (1H, d, J = 8.7 Hz, H-5), 6.90 (1H, dd, J = 8.7, 2.2 Hz, H-6), 6.99 (1H, d, J = 2.2 Hz, H-8), 7.54 (1H, d, J = 2.2 Hz, H-2′), 6.93 (1H, d, J = 8.9 Hz, H-5′), 7.54 (1H, dd, J = 8.9, 2.2 Hz, H-6′), 3.89 (3H, s, -OCH3). 13C NMR (125 MHz, DMSO-d6): δ ppm 162.36 (C-2), 104.83 (C-3), 176.33 (C-4), 126.40 (C-5), 114.78 (C6), 162.59 (C-7), 102.54 (C-8), 157.39 (C-9), 116.08 (C-10), 122.16 (C-1′), 110.02 (C-2′), 147.99 (C-3′), 150.20 (C-4′), 115.74 (C-5′), 119.92 (C-6′), 55.94 (−OCH3). ESI-MS positive m/z 307 [M + Na]+, 323 [M + K]+, 285 [M + H]+. ESI-MS negative m/z 283.1 [M − H]−, 567 [2M − H]−. The structure was also confirmed with comparison with published data.28 7,3′-Dihydroxy-4′-methoxyflavone (6). Yellow amorphous powder. 1H NMR (500 MHz, DMSO-d6): δ ppm 6.67 (1H, s, H-3), 7.86 (1H, d, J = 8.7 Hz, H-5), 6.91 (1H, dd, J = 8.7, 2.2 Hz, H-6), 6.95 (1H, d, J = 2.2 Hz, H-8), 7.42 (1H, d, J = 2.3 Hz, H-2′), 7.08 (1H, d, J = 8.6 Hz, H-5′), 7.51 (1H, dd, J = 8.5, 2.3 Hz, H-6′), 3.86 (3H, s,-OCH3). 13C NMR (125 MHz, DMSO-d6): δ ppm 162.61 (C-2), 105.10 (C-3), 176.18 (C-4), 126.45 (C-5), 114.83 (C-6), 157.35 (C-7), 102.36 (C-8), 162.81 (C-9), 116.07 (C-10), 123.65 (C-1′), 112.78 (C-2′), 146.73 (C-3′), 150.68 (C-4′), 112.17 (C-5′), 118.24 (C-6′), 55.72(−OCH3). ESI-MS positive m/z 307 [M + Na]+, 323 [M + K]+, 285 [M + H]+. ESI-MS negative m/z 283 [M−H]−, 567 [2M−H]−. The structure was also confirmed with comparison with published data.29 Apigenin (7). Yellow amorphous powder. 1H NMR (500 MHz, CD3OD): δ ppm 6.60 (1H, s, H-3),6.21 (1H, d, J = 2.1 Hz, H-6), 6.46 (1H, d, J=2.1H, H-8), 7.86 (2H, d, J = 8.8 Hz, H-2′, 6′), 6.93 (2H, d, J = 8.8 Hz, H-3′, 5′). 13C NMR (125 MHz, CD3OD): δ ppm 166.30 (C-2), 103.85 (C-3), 183.91 (C-4), 159.45 (C-5), 100.16 (C-6), 166.13 (C-7), 95.07 (C-8), 163.23 (C-9), 105.30 (C-10), 123.29 (C-1′), 129.45 (C-2′, 6′), 117.03 (C-3′, 5′), 162.76 (C-4′). The structure was also confirmed with comparison with published data.12 Chrysoeriol (8). Yellow amorphous powder. 1H NMR (500 MHz, DMSO-d6): δ ppm 12.97 (1H, s, 5-OH), 6.89 (1H, s, H3), 6.20 (1H, d, J = 2.1 Hz, H-6), 6.51 (1H, d, J = 2.1 Hz, H8), 7.56 (1H, overlapped, H-2′, 6′), 6.94 (1H, d, J = 8.9 Hz, H5′), 3.90 (3H, s, -OCH3). 13C NMR (125 MHz, DMSO-d6): δ ppm 163.65 (C-2), 103.20 (C-3), 181.77 (C-4), 161.42 (C-5), 98.81 (C-6), 164.14 (C-7), 94.03 (C-8), 157.31 (C-9), 103.73 (C-10), 121.52 (C-1′), 110.21 (C-2′), 148.01 (C-3′), 150.71 (C-4′), 115.75 (C-5′), 120.34 (C-6′), 55.96 (−OCH3). ESIMS positive m/z 323 [M + Na]+, 339 [M + K]+, 301 [M + H]+. ESI-MS negative m/z 299 [M − H]−, 599 [2M − H]−. The structure was also confirmed with comparison with published data.30 Diosmetin (9). Yellow amorphous powder. 1H NMR (500 MHz, DMSO-d6): δ ppm 12.93 (1H, s, 5-OH), 6.74 (1H, s, H3), 6.20 (1H, d, J = 2.0 Hz, H-6), 6.47 (1H, d, J = 2.0 Hz, H8), 7.43 (1H, d, J = 2.3 Hz, H-2′), 7.09 (1H, d, J = 8.5 Hz, H5′), 7.56 (1H, overlapped, H-6′), 3.87 (3H, s, -OCH3). 13C NMR (125 MHz, DMSO-d6): δ ppm 163.50 (C-2), 103.51 (C-3), 181.66 (C-4), 161.45 (C-5), 98.61 (C-6), 164.14 (C-7), 93.90 (C-8), 157.31 (C-9), 103.69 (C-10), 123.01 (C-1′), 112.94 (C-2′), 146.01 (C-3′), 151.12 (C-4′), 112.18 (C-5′), 7620

DOI: 10.1021/acs.jafc.8b02739 J. Agric. Food Chem. 2018, 66, 7616−7626

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Figure 2. Isolated flavonoids from L. leucocephala foliage induces pHRE-Luc in transfected HEK293T cells. (A) The cell viability of different concentration of flavonoids 1, 2, 7, 10, 13, 14, and 17 on HEK293T cells. Values are expressed as the percentage of vehicle control, and they are in mean ± SD, where n = 3, each with triplicate samples. *p < 0.05, **p < 0.01, ***p < 0.001, as compared to the vehicle control. (B) A luciferasereporter containing six HREs and a downstream luciferase-reporter gene, namely as pHRE-Luc, was used as a study tool (upper panel). Cultured HEK293T cells, transfected with pHRE-Luc, were treated with CoCl2 (100 μM, positive control) and 10 μM flavonoids for 24 h. The cell lysates were subjected to luciferase assay. Values are expressed as the percentage of increase to basal reading (untreated culture), and they are in mean ± SEM, where n = 3, each with triplicate samples. *p < 0.05, **p < 0.01, ***p < 0.001, as compared to the vehicle control.

flavonoids were determined by UPLC-QQQ MS/MS by comparing with isolated standards using multiple reactions monitoring (MRM) mode, with a retention time at 5.299, 5.303, 6.549, 5.519, 5.503, 5.509, and 5.055 min, respectively (Figure S1). Two suitable transition pairs were chosen for acquisition in MRM mode for compounds 1, 2, 7, 10, 13, 14, 17, and internal standard vulpinic acid, as listed in Table S1. The contents of these compounds in L. leucocephala leaves extract were determined by an established UPLC-MS method, according to the established calibration curves (Tables S2 and S3). Their quantitative content of flavonoids 1, 2, 7, 10, 13, 14, and 17 were 11.2, 7.4, 0.2, 0.6, 0.9, 0.2, and 5.8 g/kg dry matter weight, respectively. Flavonoids 1, 2, and 17 were the major flavonoids components in L. leucocephala leaves, at a total concentration of about 2.5% of dry matter. pHRE-Luc Transcriptional Inductive Activity. It was reported that flavonoids from Radix astragali induced the expression of erythropoietin in cultured HEK293T fibroblast cells.18,19 EtOH extracts, EtOAc fraction and flavonoids 1, 2, 7, 10, 13, 14, and 17 were tested for inductive luciferase activity of HRE. The concentration of 10 μM was selected as all tested compounds showed larger than 90% cell viability at 10 μM (Figure 2A), and 50 μg/mL concentration was determined for EtOH extracts and EtOAc fraction in the same way by MTT assay (data not shown). CoCl2, served as a positive control, induced the luciferase activity with 71% increase at 100 μM (Figure 2B). EtOH extracts and EtOAc fraction showed no

198.38 (C-4), 165.28 (C-5), 97.30 (C-6), 168.8 (C-7), 96.27 (C-8), 164.48 (C-9), 101.83 (C-10), 129.85 (C-1′), 115.88 (C-2′), 147.11 (C-3′), 146.29 (C-4′), 116. 08 (C-5′), 120. 89 (C-6′). The structure was also confirmed with comparison with published data.36 Myricetin (16). White amorphous powder. 1H NMR (500 MHz, CD3OD): δ ppm 6.18 (1H, d, J = 2.1 Hz, H-6), 6.38 (1H, d, J = 2.1 Hz, H-8), 7.34 (2H, s, H-2′, 6′). 13C NMR (125 MHz, CD3OD): δ ppm 148.00 (C-2), 137.34 (C-3), 177.26 (C-4), 162.47 (C-5), 99.21 (C-6), 165.56 (C-7), 94.37 (C-8), 158.19 (C-9), 104.48 (C-10), 123.09 (C-1′), 108.52 (C-2′, 6′), 146.70 (C-3′, 5′), 136.92 (C-4′). The structure was also confirmed with comparison with published data.25 Myricetin-3-O-α-rhamnopyranoside (17). Yellow amorphous powder. 1H NMR (500 MHz, DMSO-d6): δ ppm 12.69 (1H, s, 5-OH), 6.20 (d, J = 2.1 Hz, H-6), 6.39 (d, J = 2.1 Hz, H-8), 6.89 (2H, s, H-2′, 6′), 5.20 (d, J = 1.5 Hz, H-1″), 0.84 (3H, J = 6.2 Hz, H-6″). 13C NMR (125 MHz, DMSOd6): δ ppm 157.45 (C-2), 134.29 (C-3), 177.77 (C-4), 161.28 (C-5), 98.66 (C-6), 164.21 (C-7), 93.52 (C-8), 156.39 (C-9), 104.01 (C-10), 119.61 (C-1′), 107.92 (C-2′, 6′), 145.77 (C-3′, 5′), 136.46 (C-4′), 101.95 (C-1″), 70.29 (C-2″), 70.54 (C-3″), 71.29 (C-4″), 70.02 (C-5″), 17.5 (C-6″). The structure was also confirmed with comparison with published data.25 Flavonoids Content Determination by UPLC-MS. Since the isolated amount of 1, 2, 7, 10, 13, 14, and 17 were rather higher than other flavonoids, the contents of these 7621

DOI: 10.1021/acs.jafc.8b02739 J. Agric. Food Chem. 2018, 66, 7616−7626

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Dexamethasone (10 μM), used as positive control, decrease the secretion of IL-6 and TNF-α by 53% and 15%, respectively (Figure 3B). EtOAc fraction was found to reduce secretion of IL-6 by 52% at 50 μg/mL, while EtOH extracts displayed no decreasing activity on IL-6 secretion at 50 μg/mL. Flavonoids 7, 10, 13, and 17 were found to strongly reduce secretion of IL-6 by 54%, 46%, 52%, and 54% relative to vehicle at the concentration of 10 μM, respectively, which were relatively equal to the positive control dexamethasone (10 μM). Flavonoids 1 could weakly decrease the secretion of IL-6 by 25%. However, EtOH extracts, EtOAc fraction, and all tested flavonoids displayed no inhibited activity against TNF-α secretion. DPP4 Inhibitory Activity. It was reported that the extract of L. leucocephala seeds and leaves had antidiabetic activities.14 The fraction of methanolic extract of L. leucocephala seeds was found to show inhibitory activities on α-glucosidase and aldose reductase.15 Since the predominant component of seeds was mimosine, this compound was tested the effects on streptozotocin-induced diabetic mice and it was not the active compounds responsible for antidiabetic activities of seeds.37 The major components of leaves were flavonoids, which indicated that flavonoids could be active compounds responsible for antidiabetic activities of L. leucocephala leaves extracts. Inhibitors of DPP4 inhibit the degradation of glucosedependent insulinotropic polypeptide and glucagon-like peptide-1 and have emerged as oral antidiabetic agents.38 EtOH extracts and EtOAc fraction, as well as all isolated flavonoids (1−17) were therefore evaluated the antidiabetic via dipeptidyl-peptidase 4 (DPP4) assay. Unfortunately, EtOH extracts (100 μg/mL), EtOAc fraction (100 μg/mL), and all tested flavonoids (1−17) at 20 μM appeared no significantly DPP4 inhibitory activity compared with vehicle. The antidiabetic active component of L. leucocephala seeds and leaves still stayed unknown. Antioxidant Activities of DPPH Radical Scavenging Capacity. Flavonoids were well-known for their antioxidant capacity. Antioxidant activities of EtOH extracts, EtOAc fraction, and all isolated flavonoids (1−17) were tested on DPPH radical scavenging capacity. EtOH extracts and EtOAc fraction displayed 36.6 ± 1.0% and 88.6 ± 1.4% DPPH radical scavenging activity at 12.5 μg/mL, respectively (Table 1). The DPPH radical scavenging activity of SC50 of 1, 2, 3, 11, 12, 16, 17 and VC were shown in Table 1. Flavonoids 1, 2, 3, 11, 12, 16, and 17 showed stronger DPPH radical scavenging activity than positive control of ascorbic acid. The results indicated potent antioxidant activity of flavonoids 1, 2, 3, 11, 12, 16, and 17. In general, antioxidant activity of flavonoids relies on their structures and substitution pattern of hydroxyl groups. We may conclude from our results that the critical requirement for effective radical scavenging is adjacent 3′,4′-dihydroxy group in ring-B of flavones (11 and 12) and flavonols (1, 2, 3, 16, and 17). Our result is consistent with the previous report, that C2− C3 double bond and adjacent to the C4-oxo function group were significant for antioxidant activity of flavonoids.39 This explained that flavanonol 15 with adjacent 3′,4′-dihydroxy in ring B displayed no DPPH scavenging activity. Cellular Antioxidant Activities Against tBHP Induced ROS Formation. Beside the chemical antioxidant activity of DPPH radical scavenging capacity, we also tested cellular antioxidant activity against tBHP induced ROS formation on RAW 264.7 macrophage. EtOH extracts (50 μg/mL) and

pHRE-Luc inductive activity at 50 μg/mL. Flavonoids 7, 10, and 13 could strongly induce the transcriptional activity of pHRE-Luc with 102%, 127%, and 50% increase at 10 μM, respectively, while 14 and 17 showed transcriptional inductive activity of pHRE-Luc at 10 μM without statistical significance (Figure 2B). Flavonoids 1 and 2 showed no increase of luciferase activity at 10 μM (Figure 2B). Anti-Inflammatory Activity. The anti-inflammatory properties of flavonoids have been well studied.20 Extracts and flavonoids were treated on RAW 264.7 macrophage and cytokines levels of IL-6 and TNF-α were calculated using ELISA kits to evaluate the anti-inflammatory activity. Since all compounds selected for pHRE-Luc activity assay had a safe concentration at 50 μg/mL or 10 μM on HEK293T cells, a same concentration of 50 μg/mL of EtOH extracts and EtOAc fraction, and 10 μM of compounds 1, 2, 7, 10, 13, 14, and 17 was chosen for MTT assay on RAW 264.7 macrophage. The MTT results showed that RAW 264.7 macrophage had more than 85% cell viability at 50 μg/mL or 10 μM (Figure 3A).

Figure 3. Flavonoids isolated from L. leucocephala foliage suppresses the LPS-induced pro-inflammatory cytokines secretion in cultured RAW 264.7 macrophage. (A) The cell viability of EtOH extracts (50 μg/mL), EtOAc fraction (50 μg/mL), and flavonoids 1, 2, 7, 10, 13, 14, and 17 at 10 μM concentration on RAW 264.7 macrophage. The MTT results showed that RAW 264.7 macrophage had more than 85% cell viability at 50 μg/mL or 10 μM. (B) Cultured RAW 264.7 macrophages were pretreated with EtOH extracts (50 μg/mL), EtOAc fraction (50 μg/mL), and 10 μM concentration of different flavonoids for 3 h. Then, LPS (0.1 μg/mL) was applied onto the cultures for 24 h to mimic the chronic inflammation. The protein expressions of pro-inflammatory cytokines were revealed by ELISA method. Dexamethasone (Dex, 10 μM) served as positive control of suppressor. Data are expressed mean ± SEM, where n = 4, each with triplicate samples. Statistical comparison was made with the vehicle control; **p < 0.01; ***p < 0.001. 7622

DOI: 10.1021/acs.jafc.8b02739 J. Agric. Food Chem. 2018, 66, 7616−7626

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flavonoids 1−3 showed peaks at retention time of 28.372, 26.415, and 26.257 min, respectively. Both metabolites of flavonoids 2 and 3 after anaerobic incubation with cattle rumen microorganisms showed peaks at 28.424 min, which was not observed in the blank control. The UV absorption profiles of degradation metabolites of flavonoids 2 and 3 at 28.424 min were the same with flavonoid 1 at 28.372 min (Figure S2). Therefore, the glycosidic bonds in flavonoids 2 and 3 were degraded under the anaerobic condition with cattle rumen liquid. Rumen liquid contained variety of microorganisms, which might catalyze the deglycosylation reaction.

Table 1. DPPH Radical Scavenging Activity of EtOH Extracts, EtOAc Fraction and Flavonoids 1, 2, 3, 11, 12, 16, 17, and VC flavonoids

SC50 (μM)a

% of scavenging activityb

EtOH extracts EtOAc fraction 1 2 3 11 12 16 17 VC (positive control)

n.a. n.a. 7.8 ± 0.41 9.788 ± 0.82 6.3 ± 0.04 5.7 ± 0.49 12.88 ± 0.33 7.03 ± 0.03 12.03 ± 0.24 20.32 ± 0.34

36.6 ± 1.0 88.6 ± 1.4 n.a. n.a. n.a. n.a. n.a. n.a. n.a. n.a.

***c ***c ***d ***d ***d ***d ***d ***d ***d ***,e *f

■

DISCUSSION We isolated five C3-glycosides of flavonoids (2, 3, 13, 14, and 17), of which sugar parts included arabinose and rhamnose without exception. However, previous literature reported glycosides of flavonoids contained glucuronides,9,11 galactose,10 and glucose,10 and glycosides occurred at C-7,9,10 which were not isolated in our study. The different chemical composition may be related to regional area of L. leucocephala cultivated. L. leucocephala leaves provided an excellent source of high protein and mineral elements as livestock and poultry fodder for the tropics.3,5 The chemical composition of leucaena forage and leucaena leaf meal were summarized.3 The leaf meal has an average CP value of 29.2% and the forage [leaf (petiole and blade) and stem] 22.0% of dry matter.3 Proteins were previously considered as the major nutritive value of leucaena forage. However, in this study, we throw the sight on the link of productive flavonoids in leucaena leaves and potential health benefits for ruminant. Diverse flavonoids (1−17) were isolated from L. leucocephala foliage. Quercetin (1), quercetin-3-O-αrhamnopyranoside (2), and myricetin-3-O-α-rhamnopyranoside (17) were the major flavonoids components in L. leucocephala leaves. The contents of 1, 2, and 17 were 11.2, 7.4, and 5.8 g/kg dried matter weight, respectively, and a total concentration of 1, 2, and 17 was about 2.5% of dry matter. Bioactivities of isolated flavonoids (1−17) were evaluated. Flavonoids 7, 10, and 13 could strongly induce the transcriptional activity of HRE, which indicated their potential to induce the expression of erythropoietin. Flavonoids 7, 10, 13, and 17 displayed strong anti-inflammatory activity, relatively equal to the positive control dexamethasone. Flavonoids 1, 2, 3, 11, 12, 16, and 17 showed stronger antioxidant activities of DPPH radical scavenging capacity than ascorbic acid. Flavonoids 1, 2, and 10 showed weak cellular antioxidant activities against tBHP induced ROS formation. EtOAc fraction showed stronger antioxidant activities than EtOH extracts both on DPPH radical scavenging capacity and inhibitory effect against tBHP induced ROS formation, which might be related to high content of flavonoids in EtOAc fraction. It was well-known that flavonoids displayed broad spectrum of bioactivities,40 including estrogenic activity, anti-inflammatory activity, antifungal activity, and anti-HIV-1 activity. The potential nutritional effects and health benefits of quercetin on poultry as a replacer for traditional immune boosters and growth promoters was reviewed.41 Among these active flavonoids in our study, compounds 2, 13, and 17 are flavonoid rhamnosides or arabinoside. They cannot undergo a digestion by lactase phloridzin hydrolase or β-glucosidase,42 which affect the bioavailability and absorption of these active flavonoids in the body of animal. However, our experiment has

a

The concentration of sample required to scavenge 50% of DPPH radicals. Data are expressed as mean ± SD, where n = 3. bThe percentage of DPPH radical scavenging activity of extracts at 12.5 μg/ mL. cIndicates statistically difference (p < 0.001) compared to control at 12.5 μg/mL. dIndicates statistically difference (p < 0.001) compared to control at all six tested concentration (50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.725 μM, and 1.5615 μM). eIndicates statistically difference (p < 0.001) compared to control at four higher tested concentration (50 μM, 25 μM, 12.5 μM, and 6.25 μM). fIndicates statistically difference (p < 0.05) compared to control at tested concentration of 3.725 μM. n.a. not applicable.

EtOAc fraction (50 μg/mL) as well as flavonoids 1, 2, and 10 all at 10 μM has been found to inhibit the ROS formation by 36%, 56%, 14%, 20%, and 23%, respectively, while flavonoids 7, 13, 14, and 17 showed no antioxidant activities against ROS formation (Figure 4).

Figure 4. Flavonoids isolated from L. leucocephala foliage inhibits the tBHP Induced ROS Formation in cultured RAW 264.7 macrophage. Cultured RAW 264.7 macrophages were pretreated with EtOH extracts (50 μg/mL), EtOAc fraction (50 μg/mL), and 10 μM of different flavonoids for 24 h. The results were in % of inhibitory effect against ROS formation relative to the control (with tBHP alone). Data are expressed as mean ± SEM, where n = 4, each with five repeated samples. Statistical comparison was made with the vehicle control; **p < 0.01; ***p < 0.001.

Metabolism of Flavonoid Glycosides 2 and 3 by Cattle Rumen Microorganisms in Vitro. Among these active flavonoids in our study, compounds 2, 13, and 17 are flavonoid rhamnosides or arabinoside. They cannot undergo a digestion by lactase phloridzin hydrolase or β-glucosidase,42 which affect the bioavailability and absorption of these active flavonoids in the body of animal. As shown in Figure 5, 7623

DOI: 10.1021/acs.jafc.8b02739 J. Agric. Food Chem. 2018, 66, 7616−7626

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Journal of Agricultural and Food Chemistry

Figure 5. HPLC profile of flavonoids 1−3 and degradation metabolites of 2 and 3 after anaerobic incubation with cattle rumen liquid, and the cattle rumen liquid as blank control. (A) Flavonoid 1 showed a peak at 28.372 min. (B) Flavonoid 2 showed a peak at 26.415 min. (C) Flavonoid 3 showed a peak at 26.257 min. (D) Flavonoid 2 after anaerobic incubation with cattle rumen liquid showed a peak at 28.424 min. (E) Flavonoid 3 after anaerobic incubation with cattle rumen liquid showed a peak at 28.424 min. (F) The cattle rumen liquid as blank control showed no peak at about 28.4 min.

found that flavonoid glycosides 2 and 3 undergo deglycosylation to the aglycone quercetin under anaerobic incubation with cattle rumen microorganisms, which was to mimic rumen fermentation. It was suggested that the first ring-fission product of quercetin under gut microbiota is 3,4-dihydroxyphenylacetic acid, which is subsequently subjected to dehydroxylation to form 3-hydroxyphenylacetic acid, followed by further catabolism into hippuric or benzoic acids, all of which can be absorbed by enterocytes.43 Recently, desaminotyrosine (DAT), a degradation product of flavonoids, which could be produced by human enteric bacteria, was reported to protect from influenza through type I interferon.44 Similar beneficial function may also take place at gastrointestinal system in the body of ruminant when feeding forage are rich in flavonoids. In this point of view, beside abundant crude protein as animal nutrition, productive flavonoids in L. leucocephala leaves are merit for ruminant health. The health benefits of plant flavonoids for animal is promising for further study. Especially, the degradation product of flavonoids after rumen fermentation may play an important role in health promotion of ruminants.

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(Table S1); calibration curves, LOD, and LOQ of flavonoids quantitated using UPLC−MS/MS (Table S2); and recovery rate of seven flavonoids quantitated using UPLC−MS/MS (Table S3) (PDF)

AUTHOR INFORMATION

Corresponding Author

*Phone: +86 20 37086970. E-mail: [email protected]. ORCID

Karl W. K. Tsim: 0000-0003-4808-1674 Zhongyu Zhou: 0000-0001-9221-6455 Funding

This research was supported by National Natural Science Foundation of China (Grant 31470424), Youth Innovation Promotion Association CAS to Zhongyu Zhou (Grant 2016310), Guangdong Special Support Program (Grant 2015TQ01R054), Hong Kong Scholar Program to Zhongyu Zhou (Grant XJ2015044), and the Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, CAS (AB2018003). Notes

ASSOCIATED CONTENT

The authors declare no competing financial interest.

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.8b02739. Representative LC−QQQ-MRM MS/MS chromatograms of L. leucocephala leaves extract (Figure S1); UV absorption profiles of flavonoid 1 and degradation metabolites of flavonoids 2 and 3 after incubation with cattle rumen liquid (Figure S2); mass spectra properties of flavonoids in L. leucocephala leaves extract and IS

ACKNOWLEDGMENTS We thank Kun Jia (College of Veterinary Medicine, South China Agricultural University) for his help to obtain cattle rumen liquid.

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ABBREVIATIONS USED TLC, thin-layer chromatography; MPLC, medium pressure liquid chromatography;; HPLC, high-pressure liquid chroma7624

DOI: 10.1021/acs.jafc.8b02739 J. Agric. Food Chem. 2018, 66, 7616−7626

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tography; DPP4, dipeptidyl-peptidase 4; HRE, hypoxia responsive element; DPPH, 1,1-diphenyl-2-picrylhydrazyl; ROS, reactive oxygen species; FBS, fetal bovine serum; EPO, erythropoietin; tBHP, tert-butyl hydroperoxide; LLM, leucaena leaf meal; CP, crude protein; NMR, nuclear magnetic resonance; MTT, methyl thiazolyl tetrazolium; HEK, human embryonic kidney; ATCC, American Type Culture Collection; DMEM, dulbecco’s modified eagle medium; ELISA, enzyme linked immunosorbent assay; DCFH-DA, 2,7-dichlorodihydrofluorescein diacetate.

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DOI: 10.1021/acs.jafc.8b02739 J. Agric. Food Chem. 2018, 66, 7616−7626