Flavonoids, a Potential New Insight of Leucaena leucocephala

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Agricultural and Environmental Chemistry

Flavonoids, a Potential New Insight of Leucaena leucocephala Foliage in Ruminant Health Ying-Chao Xu, Zhenru Tao, Yu Jin, Yunfei Yuan, Tina T.X. Dong, Karl W.K. Tsim, and Zhong-Yu Zhou J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02739 • Publication Date (Web): 28 Jun 2018 Downloaded from http://pubs.acs.org on June 29, 2018

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Journal of Agricultural and Food Chemistry

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Flavonoids, a Potential New Insight of Leucaena leucocephala Foliage in

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Ruminant Health

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Yingchao Xu†,§, Zhenru Tao†,§, Yu Jin†,§, Yunfei Yuan†, Tina T. X. Dong‡, Karl W. K. Tsim‡, and

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Zhongyu Zhou†,*

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†

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Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy

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of Sciences, Guangzhou, China

Key Laboratory of Plant Resources Conservation and Sustainable Utilization, Guangdong

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‡

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Science and Technology, Hong Kong, China

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§

Division of Life Science and Center for Chinese Medicine, The Hong Kong University of

University of Chinese Academy of Sciences, Beijing, China

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*Corresponding Author: Dr. Zhongyu Zhou, Key Laboratory of Plant Resources Conservation

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and Sustainable Utilization, Guangdong Provincial Key Laboratory of Applied Botany, South

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China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China; Phone: +86 20

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37086970; E-mail: [email protected].

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ACS Paragon Plus Environment


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ABSTRACT: We investigated the constituents of Leucaena leucocephala foliage collected from

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Guangdong province in China, and isolated seventeen diverse flavonoids (1-17), including

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flavones (5-9, 11, and 12), flavonols (1, 10 and 16), flavanone 4, flavanonol 15 and flavonol

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glycosides (2, 3, 13, 14 and 17). Flavonoids quercetin (1), quercetin-3-O-α-rhamnopyranoside

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(2), and myricetin-3-O-α-rhamnopyranoside (17) were the major flavonoids components in L.

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leucocephala leaves, at a total concentration of about 2.5% of dry matter. pHRE-Luc inductive

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activity to mimic the activation of erythropoietin (EPO) gene, anti-inflammatory, anti-diabetic,

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and antioxidant activities of isolated flavonoids (1-17) were evaluated. Flavonoids 7, 10 and 13

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could strongly induce the transcriptional activity of pHRE-Luc, which indicated their potential to

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induce the expression of EPO. Flavonoids 7, 10, 13, and 17 displayed strong anti-inflammatory

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activity, relatively equal to the positive control dexamethasone. Flavonoids 1, 2, 3, 11, 12, 16,

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and 17 showed stronger antioxidant activities of DPPH radical scavenging capacity than ascorbic

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acid. Flavonoids 1, 2 and 10 showed weak cellular antioxidant activities against tert-butyl

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hydroperoxide (tBHP) induced ROS formation. Flavonoid rhamnoside 2 and arabinoside 3

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undergone deglycosylation to the aglycone quercetin under anaerobic incubation with cattle

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rumen microorganisms. Furthermore, the potential health benefits for ruminant of flavonoids,

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which was rich in L. leucocephala foliage, was also discussed.

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Key Words: Leucaena leucocephala, forage, flavonoids, ruminant health, rumen fermentation

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INTRODUCTION

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Leucaena leucocephala belongs to the family Fabaceae, which is indigenous to Mexico, and

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now is widely distributed throughout the tropics and subtropics, including central America,

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Africa, Asia and northern Australia 1. L. leucocephala flowered at April to July and fruit ripened

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at August to October. It is a fast growing tropical legume and a high biomass yielding plant. L.

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leucocephala was acted as promising forage, because of researchers in Hawaii and tropical

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Australia have discovered that cattle feeding on L. leucocephala may appeared weight gains than

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those of cattle which feeding on the greatest pastures anywhere1. Further, leucaena leaf meal

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(LLM) were highly degradable in the rumen and LLM could be used to improve rumen ecology 2.

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L. leucocephala forage presented high level of crude protein (CP), high digestibility and

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voluntary intake of CP 3. The plant's drought-tolerance and hardiness made it a promising

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candidate of sustainable feed supplements for ruminants during both the dry and rainy seasons of

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the year 4. In the past decades, a great deal of work had been conducted on the poultry nutrition

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of L. leucocephala, due to its abundant of minerals, protein, and carotenes. L. leucocephala for

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nutritive value and forage productivity were extensively reviewed 3, 5.

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Previous chemical study on L. leucocephala seeds led to the isolation of gibberellins 6,

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5α,8α-epidioxy-(24ξ)-ergosta-6,22-dien-3β-ol 7, β-sitosterol 7,β-sitostenone 7, stigmastenone 7,

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lupeol 7, 3-dipalmitoyl-2-oleoylglycerol 7, linoleic acid 7, methylparaben 7, isovanillic acid 7,

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pheophytin-a 7, pheophorbide a methyl ester 7, methyl-132-hydroxy-(132-S)-pheophorbide-b 7, 32-

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hydroxy-(132-S)-pheophytin-a 7, and aristophyll-C 7. Gallocatechin with nitrification inhibitory

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activity, epigallocatechin, catechin, and epicatechin were isolated from the roots of L.

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leucocephala 8. Researchers have isolated polyphenolic compounds with antioxidant activity

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including flavonoids from the leaves of L. leucocephala

9-12

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. Mimosine has been detected in


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leaves, flowers, pods, seeds and roots, while asparagine was the most abundant amino acid in

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flowers, which was also detected in leaves, pods and seeds, but not roots 13.

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About the medicinal properties, Li reported that the extract of L. leucocephala seeds and

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leaves had anti-diabetic activities 14. The fraction of methanolic extract of L. leucocephala seeds

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showed inhibitory activities on α-glucosidase and aldose reductase 15. L. leucocephala was also

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widely cultivated in China and mainly distributed in the province of Guangdong, Guangxi,

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Fujian, Yunnan, and Hainan. However, only a few reports studied on the chemical composition

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of L. leucocephala cultivated in China 16, 17. We investigated the constituents of L. leucocephala

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foliage collected from Guangdong province in China, and isolated seventeen diverse flavonoids

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(1-17). Flavonoids displayed a wide range of biological activities. It was reported that flavonoids

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from Radix astragali induced the expression of erythropoietin in cultured human embryonic

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kidney 293 T (HEK293T) fibroblast cells

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have been well studied 20. The extract of L. leucocephala seeds and leaves was reported to have

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anti-diabetic activities

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Therefore, pHRE-Luc inductive activity to mimic the activation of EPO gene, anti-inflammatory,

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anti-diabetic, and antioxidant activities of EtOH extracts and EtOAc fraction, as well as isolated

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flavonoids (1-17) were evaluated. The bioavailability of flavonoid glycosides was studied

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through the metabolism of flavonoids 2 and 3 under anaerobic incubation with rumen

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microorganisms. Furthermore, the potential health benefits for ruminant of flavonoids, which

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was rich in L. leucocephala foliage, was also discussed.

14, 15

18, 19

. The anti-inflammatory properties of flavonoids

. Flavonoids were well known for their antioxidant capacity.

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MATERIALS AND METHODS General Experimental Procedures. 1H, and 13C NMR spectra were recorded in CD3OD or

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DMSO-d6 on a Bruker DRX-500 NMR (Bruker Biospin Gmbh, Rheistetten, Germany)

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instrument using the residual solvent peak as reference, spectrometers operating at 500 MHz for

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1

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SCIEX API 2000 LC/MS/MS instrument. Medium pressure liquid chromatography (MPLC) was

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carried out on a CXTH P3000 instrument (Beijing Chuang Xin Tong Heng Science and

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Technology Co., Ltd, Beijing, China) equipped with a UV 3000 UV–vis Detector and a C-18

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column (50 µm, 50 × 500 mm). HPLC analysis was conducted with two Shimadzu LC-20AT

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pumps, a Shimadzu SPD-M20A diode array detector and a Shimadzu SIL-20A auto sampler

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using an Agilent Zorbax SB-Aq column (5 µm, 4.6 mm × 250 mm). For column

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chromatography, silica gel (80–100 mesh and 200-300 mesh Qingdao Haiyang Chemical Co.,

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Qingdao, China), Sephadex LH-20 (Pharmacia Fine Chemical Co., Ltd., Oppsala, Sweden) was

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performed. Thin-layer chromatography (TLC) was conducted on precoated silica gel plates

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(HSGF254, Yantai Jiang you Silica Gel Development Co., Ltd., Yantai, China) and spot detection

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was performed by spraying 10% H2SO4 in ethanol, followed by heating. Analytical grade ethyl

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acetate, chloroform, methanol, petroleum ether (b.p. 60–90° C), n-butanol were purchased from

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Tianjin Fuyu Fine Chemical Industry Co. (Tianjin, China). DPPH, cobalt chloride (CoCl2), and

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DPP4 (dipeptidyl-peptidase 4) inhibitor screening kit were purchased from Sigma-Aldrich.

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Reagents for cell cultures were obtained from Invitrogen Technologies (Carlsbad, CA).

H, and 125 MHz for

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C, respectively. ESIMS and ESIMS/MS were collected on an MDS

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Plant Material. The L. leucocephala leaves were collected from Guangzhou, China, in July

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2016, and identified by Dr. Zhongyu Zhou. The voucher specimen (No. ZZY20160702) was

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deposited at the Laboratory of Phytochemistry at the South China Botanical Garden, Chinese

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Academy of Sciences.

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Extraction and Isolation. The L. leucocephala foliage were collected and dried with the

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exposure under the sun. The dried foliage (23 kg) were powdered, and extracted three times with

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95% EtOH (50 L) at room temperature for three days each time. The EtOH extracts was

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concentrated in vacuo using rotary evaporators and suspended in H2O and then sequentially

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extracted with petroleum ether, EtOAc and n-butanol.

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The EtOAc fraction (700 g) was subjected to silica gel column chromatography, eluted with

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CHCl3/MeOH (from 100:0 to 0:100, v/v) to give fractions E1–E17. Fraction E3 (14 g) eluted

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with CHCl3/MeOH (95:5), was further applied on MPLC using a decreasing polarity of

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MeOH/H2O (10:100–100:0, v/v) eluant to give fractions E3-1–E3-15. Fraction E3-11, from the

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elution with MeOH/H2O (60:40), was separated on Sephadex LH-20 column chromatography

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with the elution of CHCl3/MeOH (1:4, v/v), to yield fractions E3-11-1–E3-11-12, and pure

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compound 4 (9.6 mg) was obtained from E3-11-9. E3-11-4 was subjected to Sephadex LH-20

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column chromatography eluted with MeOH to obtain E3-11-4-2, followed by preparative HPLC

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with a Shim-pack PRC-ODS C-18 column (5 µm, 20 mm × 250 mm) using 60% methanol in

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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

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mg, tR = 78 min). E3-11-5 was further purified by preparative HPLC using 50% methanol in

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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

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6 (9.4 mg, tR = 110 min). Fraction E3-12, from the elution with MeOH/H2O (70:30), was

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separated on Sephadex LH-20 column chromatography with the elution of CHCl3/MeOH (1:4,

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v/v), to obtain pure 7 (553 mg), and 12 (8.3 mg).

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Fraction E4 (30 g) eluted with CHCl3/MeOH (90:10), was separated on MPLC using

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MeOH/H2O (10:100–100:0, v/v) eluant to give fractions E4-1–E4-13. Fraction E4-6 was applied

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on Sephadex LH-20 column chromatography with the elution of CHCl3 /MeOH (1:4, v/v) to

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obtain 11 (5 mg). Fraction E4-7 was applied on Sephadex LH-20 column chromatography eluted

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with MeOH to provide 10 (830 mg).

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Fraction E7 (25 g), eluted with CHCl3/MeOH (80:20), was subjected to MPLC using a

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MeOH/H2O (10:100–100:0, v/v) eluant to give fractions E7-1–E7-19. Fraction E7-8 was divided

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into seven fractions by Sephadex LH-20 column chromatography eluted with CHCl3/MeOH (1:4,

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v/v), and one fraction E7-18-6 of seven ones was further purified by Sephadex LH-20 column

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chromatography eluted with the MeOH and preparative HPLC using 50% methanol in water

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(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

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min). Fraction E7-10 was separated on Sephadex LH-20 column chromatography eluted with

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MeOH to obtain 16 (7 mg). Fraction E7-13 was separated on Sephadex LH-20 (CHCl3/MeOH,

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1:1, v/v) and silica gel (CHCl3/MeOH, 40:1, v/v) column chromatography to provide 15 (16.6

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mg).

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Fraction E10 (50 g) eluted with CHCl3/MeOH (70:30), was separated on MPLC using

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MeOH/H2O (10:100–100:0, v/v) eluant to give fractions E10-1–E10-12. Fraction E10-5 was

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purified by Sephadex LH-20 column chromatography with the elution of CHCl3/MeOH (1:4,

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v/v) to obtain 3 (23 mg). Fraction E10-7 was separated on Sephadex LH-20 column

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chromatography eluted with CHCl3/MeOH (1:1, v/v) to obtain 2 (13 g). Fraction E10-8 was

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subjected to Sephadex LH-20 column chromatography eluted with CHCl3/MeOH (1:4, v/v) to

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obtain 1 (22 g).

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Fraction E16 (16 g) eluted with CHCl3/MeOH (60:40), was applied MPLC using

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MeOH/H2O (10:100–100:0, v/v) eluant to give fractions E16-1–E16-12. Fraction E16-9 was

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further applied on Sephadex LH-20 (CHCl3/MeOH, 1:4, v/v) and silica gel (CHCl3/MeOH, 7:1,

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v/v) column chromatography to obtain 17 (9 g).

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Content of Flavonoids Determination by UPLC-QQQ-MRM MS/MS. The UPLC-QQQ

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tandem mass spectrometry system was performed with an Agilent RRLC 1200 series system

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(Waldron, Germany) and Agilent QQQ-MS/MS system equipped with an ESI ion source. The

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liquid chromatography was carried out on an Agilent Zorbax Eclipse Plus C18 (RRHD, 50 ×

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2.1mm, 1.8 µm) column at 25 ˚C. Analysis was completed with a gradient elution of 0.1% formic

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acid and 3% acetonitrile in water (A) - 0.1% formic acid in acetonitrile (B) within 10 min. The

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gradient program was 2% B→4% B at 0 – 2 min; 4% B→90% B at 2–8 min; 90% B→90% B at

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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

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was selected in positive mode based on the Optimizer program, which is an automated method

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development tool to generate and optimize MRM transitions in Agilent Mass Hunter Workstation.

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Other parameters were set as following: the temperature was 325 ˚C; the drying gas at the flow

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rate was 10 L/min; capillary voltage at 4,000 V; nebulizer pressure at 35 psig; delta electro multi-

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plier voltage at 400 V. The collision energy values and fragmentor voltage were adjusted to

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obtain the highest abundance. We used the software of Agilent Mass Hunter Workstation for data

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acquisition, processing and analysis.

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pHRE-Luc Activity Assay. For cell cultures, human embryonic kidney (HEK) 293T

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fibroblast cell from American Type Culture Collection (ATCC) were maintained in dulbecco's

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modified eagle medium (DMEM) added with 10% fetal bovine serum (FBS), 100 IU/mL

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penicillin, and 100 µg/mL streptomycin. Cultures were placed in a water saturated 5% CO2

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incubator at 37 oC. Safe concentration of EtOH extracts, EtOAc fraction, and selected

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compounds, were identified by methyl thiazolyl tetrazolium (MTT) assay

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fibroblast cell is an excellent in vitro model in studying the physiological regulation of EPO

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expression, which is sensitive to hypoxia stress. The DNA construct of luciferase reporter

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(pHRE-Luc) and vector were generated as described previously

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. HEK293T

. Briefly, cultured HEK293T

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cells (3 x 104 cells/mL) were seeded into 12-well plates and transfected with pHRE-Luc by

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calcium phosphate precipitation method. L. leucocephala leaves extracts was applied onto

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transfected HEK293T cells. After 1 day, the cell lysates were collected for luciferase assay. The

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luciferase activity was evaluated in Tropix TR717TM Microplate Luminometer (Bedford, MA),

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and the activity was expressed as absorbance (up to 560 nm) per mg of protein. The

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authentication of pHRE-Luc was confirmed by its activation in exposing to application of CoCl2

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at 100 µM, which was frequently used to mimic the effect of hypoxia.

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Anti-inflammatory Activity Assay. For cell culture, the murine RAW 264.7 macrophage

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from ATCC was cultured in DMEM medium supplemented with 10% heated-inactivated FBS,

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100 IU/mL penicillin, and 100 µg/mL streptomycin in a 37 oC, 5% CO2 and water saturated

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incubator. Since all compounds had a safe concentration at 50 µg/mL or 10 µM on HEK293T

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cells, a same concentration of 50 µg/mL of EtOH extracts and EtOAc fraction, and 10 µM of

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compounds 1, 2, 7, 10, 13, 14, and 17 were chosen for MTT assay on RAW 264.7 macrophage.

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For anti-inflammatory activity assay, RAW 264.7 macrophage (3×104 cells/ml) were pretreated

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with different extracts or flavonoids for 3h, followed by stimulated with LPS (0.1 µg/ml) for an

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additional 24 h. The supernatants of cells were analysed for the levels of TNF-α and IL-6 by

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enzyme linked immunosorbent assay (ELISA) using commercial TNF-α and IL-6 detecting kits

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(R&D Systems, Inc., Minneapolis, USA). DMSO (0.1%) and dexamethasone (10 µM) were used

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as vehicle and positive controls, respectively. All values were given as mean ± SEM (n = 3).

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Data analysis involved Student’s t-test.

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Dipeptidyl-peptidase 4 (DPP4) Inhibitor Screening Assay. Before DPP4 inhibitor

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screening assay, since the test compounds were dissolved in DMSO, DMSO was assessed for an

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uninhibited concentration on DPP4 enzyme. DPP4 was found to be free at a concentration of

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0.2% DMSO. DPP4 inhibitor screening assay was performed using a commercial kit (Sigma-

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Aldrich, St. Louis, MO) according to the kit introduction. In brief, 12.5 µL of different

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compounds or extracts and 25 µL DPP4 enzyme were added into each well 96 wells plate, which

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was incubated for 10 minutes at 37 oC in the dark. After incubation, 12.5 µL DPP4 substrate was

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added into each reaction well. Immediately, the fluorescence (FLU, λex = 360/λem = 460 nm)

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was measured on a microplate reader in kinetic mode for 20 minutes at 37 oC. The enzyme

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control contained 0.2% DMSO instead of the compound solution. For inhibitory rates calculation,

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the fluorescence for each well versus time was plotted. Two time points (T1 and T2) in the linear

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range of the plot were chosen and the slope for each well between T1 and T2 were obtained.

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Slope = (FLU2–FLU1)/(T2–T1) = ∆FLU/minute

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Relative Inhibition (%) = (SlopeEC–SlopeSM)100/SlopeEC

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where:

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SlopeSM = the slope of the sample inhibitor

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SlopeEC = the slope of the enzyme control

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DPPH Radical Scavenging Assay. The DPPH radical scavenging activity was carried out

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according to the procedures as previously described 22. DPPH was freshly prepared in methanol

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at a concentration of 0.1 mM. Test compounds were preliminary screened at 50 µM, and those

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which had more than 50% DPPH radical scavenging activity were further experimented for SC50

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(the concentration of sample required to scavenge 50% of DPPH radicals) determination. For

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further screening, test compounds were dissolved in methanol and diluted 2-fold to six

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concentrations (from 1.5615 to 50 µM). 20 µL of the compound solution and 180 µL of the 0.1

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mM DPPH solution were mixed in 96-well plates. Ascorbic acid was dissolved in methanol and

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used as a positive control. The control contained methanol instead of the compound solution, and

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the blank contained methanol in place of the DPPH solution. Each reaction was repeated in

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triplicate. The plates were incubated at 37 °C for 30 min in the dark. The absorbance (OD)

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reading in each well was taken at 517 nm on a microplate reader. The inhibitory rates of DPPH

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radicals were calculated according to the formula inhibition (%) = [1 − (OD treated – OD

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blank)/OD control] × 100. The SC50value was obtained through the software of SPSS 16.0.

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Finally, the data presented are means ± SD of three determinations.

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Cellular Reactive Oxygen Species (ROS) Formation Level. ROS formation level was

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measured according to literature procedures 23. In detail, RAW 264.7 cells were grown in 96-well

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plates (3×104 cells/mL) for 24 h incubation; the cells were then pre-incubated with 50 µg/mL of

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EtOH extracts and EtOAc fraction, and 10 µM flavonoids 1, 2, 7, 10, 13, 14 and 17 for 24 h. The

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cells were stained with 50 M of dichlorodi-hydrofluorescein diacetate (DCFH-DA) for 1 h and

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subsequently incubated with tBHP (100 µM) for 30 min to induce the ROS formation. DCF

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fluorescence intensities were measured in an Envision 2104 Multilabel Reader (PerkinElmer

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Inc.) at an excitation and emission wavelength of 485 nm and 535 nm, respectively.

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Metabolism of Flavonoid Glycosides by Cattle Rumen Microorganisms in vitro. Fresh

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cattle rumen liquid was obtained from healthy cattle which had not taken antibiotics for at least

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three months prior to the study and had no history of gastrointestinal disorders. The method of

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co-incubation of flavonoid glycosides with rumen liquid was according to a reference with a

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minor adjustment24, which was to mimic rumen fermentation. In detail, 2 mg flavonoid glycoside

242

(2 or 3) was dissolved with 10 µL DMSO, and added into 5 mL cattle rumen liquid. 10 µL

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DMSO and 5 mL cattle rumen liquid was set as a blank control. The mixture was incubated at

244

37 °C in an anaerobic condition for 24 h. The cultured mixture was extracted with water

245

saturated n-butanol three times. The extracts was evaporated, and the residue was dissolved in

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methanol (1 mL) and filtered through a 0.45 µm membrane filter for HPLC analysis. Analysis

247

was completed with a gradient elution of water (A) - methanol (B) within 40 min. The gradient

248

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

249

rate of 1 mL min-1. UV absorption was monitored at 254 nm. For comparison, flavonoid

250

glycosides 2 and 3, and their aglycone 1 were also HPLC analyzed in the same batch.

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RESULTS

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Seventeen flavonoids were isolated and identified, including quercetin (1), quercetin-3-O-α-

253

rhamnopyranoside (2), quercetin-3-O-α-arabinofuranose (3), naringenin (4), geraldone (5), 7,3′-

254

dihydroxy-4′-methoxyflavone (6), apigenin (7), chrysoeriol (8), diosmetin (9), kaempferol (10),

255

luteolin (11), 3′,4′,7-trihydroxyflavone (12), juglanin (13), kaempferol-3-O-α-rhamnopyranoside

256

(14), (+) taxifolin (15), myricetin (16), and myricetin-3-O-α-rhamnopyranoside (17). To the best

257

of our knowledge, flavonoids 1, 4, 5, 6, 9, 10, and 12-15 were isolated from L. leucocephala for

258

the first time, and 4 and 15 were the first record of 2,3-dihydroflavones in this plant. The

259

structure of seventeen flavonoids (1-17) were shown in Figure 1. Spectroscopic Data of Seventeen Flavonoids. Quercetin (1): Yellow amorphous powder.

260 261

1

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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,

263

H-6′).

264

(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′),

265

116.01 (C-2′), 146.21 (C-3′), 148.76 (C-4′), 116.24 (C-5′), 121.69 (C-6′). ESI-MS positive m/z

266

301 [M+H]+, 341 [M+K]+. The structure was also confirmed with comparison with published

267

data 25.

268

H-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-

13

C-NMR (125 MHz, CD3OD): δ ppm 148.03 (C-2), 137.20 (C-3), 177.33 (C-4), 162.47

Quercetin-3-O-α-rhamnopyranoside (2): Yellow amorphous powder. 1H-NMR (500 MHz,

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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

270

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 =

271

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

272

(1H, m, H-4′′), 3.15 (1H, m, H-5′′), 0.94 (3H, d, J = 6.2 Hz, H-6′′).

273

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-

274

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

275

(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′′),

276

73.25 (C-5′′), 17.65 (C-6′′). ESI-MS positive m/z 359 [M+H]+, 381 [M+Na]+. The structure was

277

also confirmed with comparison with published data 26.

13

C-NMR (125 MHz,

278

Quercetin-3-O-α-arabinofuranose (3): Yellow amorphous powder. 1H-NMR (500 MHz,

279

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

280

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′′),

281

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-

282

NMR (125 MHz, CD3OD): δ ppm 158.60 (C-2), 134.92 (C-3), 179.88 (C-4), 163.08 (C-5), 99.95

283

(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′),

284

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

285

(C-3′′), 88.04 (C-4′′), 62.56 (C-5′′). ESI-MS negative m/z 433 [M–H]–. The structure was also

286

confirmed with comparison with published data 12.

287

Naringenin (4): White needle crystal. 1H-NMR(500 MHz, CD3OD): δ ppm 5.33 (1H, dd, J =

288

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),

289

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′),

290

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-

291

3), 197.75 (C-4), 165.49 (C-5), 97.06 (C-6), 166.41 (C-7), 96.18 (C-8), 164.87 (C-9), 103.34 (C-

13



292

10), 131.09 (C-1′), 129.01 (C-2′, 6′), 116.32 (C-3′, 5′), 159.01 (C-4′). The structure was also

293

confirmed with comparison with published data 27.

294

Geraldone (5): White amorphous powder. 1H-NMR (500 MHz, DMSO-d6): δ ppm 6.83 (1H,

295

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,

296

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,

297

H-6′), 3.89 (3H, s, -OCH3). 13C-NMR (125 MHz, DMSO-d6): δ ppm 162.36 (C-2), 104.83 (C-3),

298

176.33 (C-4), 126.40 (C-5), 114.78 (C-6), 162.59 (C-7), 102.54 (C-8), 157.39 (C-9), 116.08 (C-

299

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′),

300

55.94 (-OCH3). ESI-MS positive m/z 307 [M+Na]+, 323 [M+K]+, 285 [M+H]+. ESI-MS negative

301

m/z 283.1 [M–H]–, 567 [2M–H]–. The structure was also confirmed with comparison with

302

published data 28.

303

7,3′-Dihydroxy-4′-methoxyflavone (6): Yellow amorphous powder. 1H-NMR (500 MHz,

304

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,

305

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′),

306

7.51 (1H, dd, J = 8.5, 2.3 Hz, H-6′), 3.86 (3H, s,-OCH3).

307

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

308

(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′),

309

112.17 (C-5′), 118.24 (C-6′), 55.72(-OCH3). ESI-MS positive m/z 307 [M+Na]+, 323 [M+K]+,

310

285 [M+H]+. ESI-MS negative m/z 283 [M–H]–, 567 [2M–H]–. The structure was also confirmed

311

with comparison with published data 29.

13

C-NMR (125 MHz, DMSO-d6): δ

312

Apigenin (7): Yellow amorphous powder. 1H-NMR (500 MHz, CD3OD): δ ppm 6.60 (1H, s,

313

H-3), 6.46 (1H, d, J = 2.1 Hz, H-8), 6.21 (1H, d, J = 2.1 Hz, H-6), 7.86 (2H, d, J = 8.8 Hz, H-2′,

314

6′), 6.93 (2H, d, J = 8.8 Hz, H-3′, 5′).

13

C-NMR (125 MHz, CD3OD): δ ppm 166.30 (C-2),

14


Page 14 of 45

Page 15 of 45


315

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),

316

105.30 (C-10), 123.29 (C-1′), 129.45 (C-2′, 6′), 117.03 (C-3′, 5′), 162.76 (C-4′). The structure

317

was also confirmed with comparison with published data 12.

318

Chrysoeriol (8): Yellow amorphous powder. 1H-NMR (500 MHz, DMSO-d6): δ ppm 12.97

319

(1H, s, 5-OH), 6.89 (1H, s, H-3), 6.20 (1H, d, J = 2.1 Hz, H-6), 6.51 (1H, d, J = 2.1 Hz, H-8),

320

7.56 (1H, overlapped, H-2′, 6′), 6.94 (1H, d, J = 8.9 Hz, H-5′), 3.90 (3H, s, -OCH3).

321

(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-

322

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

323

(C-3′), 150.71 (C-4′), 115.75 (C-5′), 120.34 (C-6′), 55.96 (-OCH3). ESI-MS positive m/z 323

324

[M+Na]+, 339 [M+K]+, 301 [M+H]+. ESI-MS negative m/z 299 [M–H]–, 599 [2M–H]–. The

325

structure was also confirmed with comparison with published data 30.

13

C-NMR

326

Diosmetin (9): Yellow amorphous powder. 1H-NMR (500 MHz, DMSO-d6): δ ppm 12.93

327

(1H, s, 5-OH), 6.74 (1H, s, H-3), 6.20 (1H, d, J = 2.0 Hz, H-6), 6.47 (1H, d, J = 2.0 Hz, H-8),

328

7.43 (1H, d, J = 2.3 Hz, H-2′), 7.09 (1H, d, J = 8.5 Hz, H-5′), 7.56 (1H, overlapped, H-6′), 3.87

329

(3H, s, -OCH3). 13C-NMR (125 MHz, DMSO-d6): δ ppm 163.50 (C-2), 103.51 (C-3), 181.66 (C-

330

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

331

(C-1′), 112.94 (C-2′), 146.01 (C-3′), 151.12 (C-4′), 112.18 (C-5′), 118.14 (C-6′), 55.76 (-OCH3).

332

ESI-MS positive m/z 323 [M+Na]+, 339 [M+K]+, 301 [M+H]+. ESI-MS negative m/z 299 [M–

333

H]–, 599 [2M–H]–. The structure was also confirmed with comparison with published data 31.

334

Kaempferol (10): Yellow amorphous powder. 1H-NMR (500 MHz, CD3OD): δ ppm 8.09

335

(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, H-6), 6.40

336

(1H, d, J = 1.9 Hz, H-8).

337

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′,

13

C-NMR (125 MHz, CD3OD): δ ppm 148.01 (C-2), 137.12 (C-3),

15



Page 16 of 45

338

6′), 116.29 (C-3′, 5′), 160.54 (C-4′). The structure was also confirmed with comparison with

339

published data 32.

340

Luteolin (11): Yellow amorphous powder. 1H-NMR (500 MHz, DMSO-d6): δ ppm 6.66 (1H,

341

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

342

Hz, H-2′), 6.89 (1H, d, J = 8.2 Hz, H-3′), 7.39 (1H, d, J = 2.3 Hz, H-6′).

343

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

344

(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′),

345

149.67 (C-4′), 115.99 (C-5′), 121.49 (C-6′). The structure was also confirmed with comparison

346

with published data 33.

13

C-NMR (125 MHz,

347

3′,4′,7-Trihydroxyflavone (12): Yellow amorphous powder. 1H-NMR (500 MHz, CD3OD): δ

348

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,

349

H-2′, 6′).

350

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-

351

1′), 114.16 (C-2′), 147.02 (C-3′), 150.77 (C-4′), 116.79 (C-5′), 120.20 (C-6′). The structure was

352


13

C-NMR (125 MHz, CD3OD): δ ppm 166.06 (C-2), 105.20 (C-3), 180.27 (C-4),

353

Juglanin (13): Yellow amorphous powder. 1H-NMR (500 MHz, CD3OD): δ ppm 6.20 (1H, d,

354

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,

355

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′′),

356

3.81 (1H, m, H-4′′), 3.48 (2H, m, H-5′′).

357

134.94 (C-3), 179.90 (C-4), 163.06 (C-5), 99.89 (C-6), 165.99 (C-7), 94.80 (C-8), 159.34 (C-9),

358

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′′),

359

83.34 (C-2′′), 78.65 (C-3′′), 88.03 (C-4′′), 62.55 (C-5′′). The structure was also confirmed with

360

comparison with published data 32.

13

C-NMR (125 MHz, CD3OD): δ ppm 158.55 (C-2),

16


Page 17 of 45


361

Kaempferol-3-O-α-rhamnopyranoside (14): Yellow amorphous powder. 1H-NMR (500 MHz,

362

CD3OD): δ ppm 6.93 (2H, d, J = 8.4 Hz, H-3′, 5′), 7.75 (2H,d, J = 8.4 Hz, H-2′, 6′), 6.36 (1H, s,

363

H-8), 6.19 (1H, s, H-6), 5.38 (1H, d, J = 1.5 Hz, H-1′′), 4.23 (1H, dd, J = 3.3, 1.7 Hz, H-2′′), 3.72

364

(1H, m, H-3′′), 3.34 (2H, m, H-4′′, 5′′), 0.93 (3H, d, J = 5.4 Hz, H-6′′).

365

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-

366

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′),

367

161.51 (C-4′), 103.47 (C-1′′), 73.19 (C-2′′), 72.11 (C-3′′), 72.00 (C-4′′), 71.90 (C-5′′), 17.64 (C-

368

6′′). The structure was also confirmed with comparison with published data 35.

13

C-NMR (125 MHz,

369

(+) Taxifolin (15): White needles. 1H-NMR (500 MHz, CD3OD): δ ppm 4.91 (1H, d, J =

370

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

371

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,

372

8.2 Hz, H-6′.

373

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′),

374

115.88 (C-2′), 147.11 (C-3′), 146. 29 (C-4′), 116. 08 (C-5′), 120. 89 (C-6′). The structure was

375


376

13

C-NMR (125 MHz, CD3OD): δ ppm 85.09 (C-2), 73.66 (C-3), 198.38 (C-4),

Myricetin (16): White amorphous powder. 1H-NMR (500 MHz, CD3OD): δ ppm 6.18 (1H, 13

377

d, J = 2.1 Hz, H-6), 6.38 (1H, d, J = 2.1 Hz, H-8), 7.34 (2H, s, H-2′, 6′).

C-NMR (125 MHz,

378

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-

379

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′),

380

136.92 (C-4′). The structure was also confirmed with comparison with published data 25.

381

Myricetin-3-O-α-rhamnopyranoside (17): Yellow amorphous powder. 1H-NMR (500 MHz,

382

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

383

(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,

17



Page 18 of 45

384

DMSO-d6): δ ppm 157.45 (C-2), 134.29 (C-3), 177.77 (C-4), 161.28 (C-5), 98.66 (C-6), 164.21

385

(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′,

386

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

387

(C-6″). The structure was also confirmed with comparison with published data 25.

388

Flavonoids Content Determination by UPLC-MS. Since the isolated amount of 1, 2, 7,

389

10, 13, 14, and 17 were rather higher than other flavonoids, the contents of these flavonoids were

390

determined by UPLC-QQQ MS/MS by comparing with isolated standards using multiple

391

reactions monitoring (MRM) mode, with a retention time at 5.299 min, 5.303 min, 6.549 min,

392

5.519 min, 5.503 min, 5.509 min, and 5.055 min, respectively (Figure S1). Two suitable

393

transition pairs were chosen for acquisition in MRM mode for compounds 1, 2, 7, 10, 13, 14, 17

394

and internal standard vulpinic acid, as listed in Table S1. The contents of these compounds in L.

395

leucocephala leaves extract were determined by an established UPLC-MS method, according to

396

the established calibration curves (Tables S2 and S3). Their quantitative content of flavonoids 1,

397

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,

398

respectively. Flavonoids 1, 2 and 17 were the major flavonoids components in L. leucocephala

399

leaves, at a total concentration of about 2.5% of dry matter.

400

pHRE-Luc Transcriptional Inductive Activity. It was reported that flavonoids from Radix 18, 19

401

astragali induced the expression of erythropoietin in cultured HEK293T fibroblast cells

402

EtOH extracts, EtOAc fraction and flavonoids 1, 2, 7, 10, 13, 14, and 17 were tested for

403

inductive luciferase activity of HRE. The concentration of 10 µM was selected as all tested

404

compounds showed larger than 90% cell viability at 10 µM (Figure 2A), and 50 µg/mL

405

concentration was determined for EtOH extracts and EtOAc fraction in the same way by MTT

406

assay (data not shown). CoCl2, served as a positive control, induced the luciferase activity with

18


.

Page 19 of 45


407

71% increase at 100 µM (Figure 2B). EtOH extracts and EtOAc fraction showed no pHRE-Luc

408

inductive activity at 50 µg/mL. Flavonoids 7, 10 and 13 could strongly induce the transcriptional

409

activity of pHRE-Luc with 102%, 127%, and 50% increase at 10 µM, respectively, while 14 and

410

17 showed transcriptional inductive activity of pHRE-Luc at 10 µM without statistical

411

significance (Figure 2B). Flavonoids 1 and 2 showed no increase of luciferase activity at 10 µM

412

(Figure 2B).

413

Anti-inflammatory Activity. The anti-inflammatory properties of flavonoids have been 20

414

well studied

415

levels of IL-6 and TNF-α were calculated using ELISA kits to evaluate the anti-inflammatory

416

activity. Since all compounds selected for pHRE-Luc activity assay had a safe concentration at

417

50 µg/mL or 10 µM on HEK293T cells, a same concentration of 50 µg/mL of EtOH extracts and

418

EtOAc fraction, and 10 µM of compounds 1, 2, 7, 10, 13, 14, and 17 was chosen for MTT assay

419

on RAW 264.7 macrophage. The MTT results showed that RAW 264.7 macrophage had more

420

than 85% cell viability at 50 µg/mL or 10 µM (Figure 3A). 10 µM Dexamethasone, used as

421

positive control, decrease the secretion of IL-6 and TNF-α by 53% and 15%, respectively

422

(Figure 3B). EtOAc fraction was found to reduce secretion of IL-6 by 52% at 50 µg/mL, while

423

EtOH extracts displayed no decreasing activity on IL-6 secretion at 50 µg/mL. Flavonoids 7, 10,

424

13, and 17 were found to strongly reduce secretion of IL-6 by 54%, 46%, 52%, and 54% relative

425

to vehicle at the concentration of 10 µM, respectively, which were relatively equal to the positive

426

control dexamethasone (10 µM). Flavonoids 1 could weakly decrease the secretion of IL-6 by

427

25%. However, EtOH extracts, EtOAc fraction and all tested flavonoids displayed no inhibited

428

activity against TNF-α secretion.

429

. Extracts and flavonoids were treated on RAW 264.7 macrophage and cytokines

DPP4 Inhibitory Activity. It was reported that the extract of L. leucocephala seeds and

19



Page 20 of 45

430

leaves had anti-diabetic activities14. The fraction of methanolic extract of L. leucocephala seeds

431

was found to show inhibitory activities on α-glucosidase and aldose reductase

432

predominant component of seeds was mimosine, this compound was tested the effects on

433

streptozotocin-induced diabetic mice and it was not the active compounds responsible for anti-

434

diabetic activities of seeds 37. The major components of leaves were flavonoids, which indicated

435

that flavonoids could be active compounds responsible for anti-diabetic activities of L.

436

leucocephala leaves extracts. Inhibitors of DPP4 inhibit the degradation of glucose-dependent

437

insulinotropic polypeptide and glucagon-like peptide-1 and have emerged as oral anti-diabetic

438

agents

439

therefore evaluated the anti-diabetic via dipeptidyl-peptidase 4 (DPP4) assay. Unfortunately,

440

EtOH extracts (100 µg/mL), EtOAc fraction (100 µg/mL), and all tested flavonoids (1-17) at 20

441

µM appeared no significantly DPP4 inhibitory activity compared with vehicle. The anti-diabetic

442

active component of L. leucocephala seeds and leaves still stayed unknown.

38

15

. Since the

. EtOH extracts and EtOAc fraction, as well as all isolated flavonoids (1-17) were

443

Antioxidant Activities of DPPH Radical Scavenging Capacity. Flavonoids were well

444

known for their antioxidant capacity. Antioxidant activities of EtOH extracts, EtOAc fraction,

445

and all isolated flavonoids (1-17) were tested on DPPH radical scavenging capacity. EtOH

446

extracts and EtOAc fraction displayed 36.6±1.0 % and 88.6±1.4 % DPPH radical scavenging

447

activity at 12.5 µg/mL, respectively (Table 1). The DPPH radical scavenging activity of SC50 of

448

1, 2, 3, 11, 12, 16, 17 and VC were shown in Table 1. Flavonoids 1, 2, 3, 11, 12, 16, and 17

449

showed stronger DPPH radical scavenging activity than positive control of ascorbic acid. The

450

results indicated potent antioxidant activity of flavonoids 1, 2, 3, 11, 12, 16, and 17.

451

In general, antioxidant activity of flavonoids relies on their structures and substitution

452

pattern of hydroxyl groups. We may conclude from our results that the critical requirement for

20


Page 21 of 45


453

effective radical scavenging is adjacent 3′,4′-dihydroxy group in ring-B of flavones (11 and 12)

454

and flavonols (1, 2, 3, 16 and 17). Our result is consistent with previous report, that C2–C3

455

double bond and adjacent to the C4–oxo function group were significant for antioxidant activity

456

of flavonoids

457

displayed no DPPH scavenging activity.

39

. This explained that flavanonol 15 with adjacent 3′,4′-dihydroxy in ring B

458

Cellular Antioxidant Activities Against tBHP Induced ROS Formation. Beside the

459

chemical antioxidant activity of DPPH radical scavenging capacity, we also tested cellular

460

antioxidant activity against tBHP induced ROS formation on RAW 264.7 macrophage. EtOH

461

extracts (50 µg/mL) and EtOAc fraction (50 µg/mL), as well as flavonoids 1, 2 and 10 all at 10

462

µM has been found to inhibit the ROS formation by 36%, 56%, 14%, 20% and 23%,

463

respectively, while flavonoids 7, 13, 14 and 17 showed no antioxidant activities against ROS

464

formation (Figure 4).

465

Metabolism of Flavonoid Glycosides 2 and 3 by Cattle Rumen Microorganisms in vitro.

466

Among these active flavonoids in our study, compounds 2, 13 and 17 are flavonoid rhamnosides

467

or arabinoside. They cannot undergo a digestion by lactase phloridzin hydrolase or β-glucosidase

468

42

469

animal. As shown in Figure 5, flavonoids 1-3 showed peaks at retention time of 28.372 min,

470

26.415 min and 26.257 min, respectively. Both metabolites of flavonoids 2 and 3 after anaerobic

471

incubation with cattle rumen microorganisms showed peaks at 28.424 min, which was not

472

observed in the blank control. The UV absorption profiles of degradation metabolites of

473

flavonoids 2 and 3 at 28.424 min were the same with flavonoid 1 at 28.372 min (Figure S2).

474

Therefore, the glycosidic bonds in flavonoids 2 and 3 were degraded under the anaerobic

475

condition with cattle rumen liquid. Rumen liquid contained variety of microorganisms, which

, which affect the bioavailability and absorption of these active flavonoids in the body of

21



476

might catalyze the deglycosylation reaction.

477

DISCUSSION

478

We isolated five C3-glycosides of flavonoids (2, 3, 13, 14, and 17), of which sugar parts

479

included arabinose and rhamnose without exception. However, previous literatures reported

480

glycosides of flavonoids contained glucuronides9, 11, galactose 10, and glucose 10, and glycosides

481

occurred at C-7

482

may be related to regional area of L. leucocephala cultivated.

9, 10

, which were not isolated in our study. The different chemical composition

483

L. leucocephala leaves provided an excellent source of high protein and mineral elements as

484

livestock and poultry fodder for the tropics 3, 5. The chemical composition of leucaena forage and

485

leucaena leaf meal were summarized 3. The leaf meal has an average CP value of 29.2% and the

486

forage [leaf (petiole and blade) and stem] 22.0% of dry matter 3. Proteins were previously

487

considered as the major nutritive value of leucaena forage. However, in this study, we throw the

488

sight on the link of productive flavonoids in leucaena leaves and health benefits for ruminant.

489

Diverse flavonoids (1-17) were isolated from L. leucocephala foliage. Quercetin (1), quercetin-3-

490

O-α-rhamnopyranoside (2), and myricetin-3-O-α-rhamnopyranoside (17) were the major

491

flavonoids components in L. leucocephala leaves. The contents of 1, 2, and 17 were 11.2, 7.4,

492

and 5.8 g/kg dried matter weight, respectively, and a total concentration of 1, 2, and 17 was about

493

2.5% of dry matter. Bioactivities of isolated flavonoids (1-17) were evaluated. Flavonoids 7, 10

494

and 13 could strongly induce the transcriptional activity of HRE, which indicated their potential

495

to induce the expression of erythropoietin. Flavonoids 7, 10, 13, and 17 displayed strong anti-

496

inflammatory activity, relatively equal to the positive control dexamethasone. Flavonoids 1, 2, 3,

497

11, 12, 16 and 17 showed stronger antioxidant activities of DPPH radical scavenging capacity

498

than ascorbic acid. Flavonoids 1, 2 and 10 showed weak cellular antioxidant activities against

22


Page 22 of 45

Page 23 of 45


499

tBHP induced ROS formation. EtOAc fraction showed stronger antioxidant activities than EtOH

500

extracts both on DPPH radical scavenging capacity and inhibitory effect against tBHP induced

501

ROS formation, which might be related to high content of flavonoids in EtOAc fraction.

502

It was well known that flavonoids displayed broad spectrum of bioactivities

40

, including

503

estrogenic activity, anti-inflammatory activity, antifungal activity, and anti-HIV-1 activity. The

504

potential nutritional effects and health benefits of quercetin on poultry as a replacer for

505

traditional immune boosters and growth promoters was reviewed

506

flavonoids in our study, compounds 2, 13 and 17 are flavonoid rhamnosides or arabinoside. They

507

cannot undergo a digestion by lactase phloridzin hydrolase or β-glucosidase 42, which affect the

508

bioavailability and absorption of these active flavonoids in the body of animal. However our

509

experiment has found that flavonoid glycosides 2 and 3 undergone deglycosylation to the

510

aglycone quercetin under anaerobic incubation with cattle rumen microorganisms, which was to

511

mimic rumen fermentation. It was suggested that the first ring-fission product of quercetin under

512

gut microbiota is 3,4-dihydroxyphenylacetic acid, which is subsequently subjected to

513

dehydroxylation to form 3-hydroxyphenylacetic acid, followed by further catabolism into

514

hippuric or benzoic acids, all of which can be absorbed by enterocytes

515

desaminotyrosine (DAT), a degradation product of flavonoids, which could be produced by

516

human enteric bacteria, was reported to protect from influenza through type I interferon

517

Similar beneficial function may also take place at gastrointestinal system in the body of ruminant

518

when feeding forage are rich in flavonoids. In this point of view, beside abundant crude protein

519

as animal nutrition, productive flavonoids in L. leucocephala leaves are merit for ruminant

520

health. The health benefits of plant flavonoids for animal is promising for further study.

521

Especially, degradation product of flavonoids after rumen fermentation may play an important

23


41

. Among these active

43

. Recently,

44

.


522

role in health promotion of ruminants.

523 524

ABBREVIATION

525

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

526

pressure liquid chromatography; DPP4, dipeptidyl-peptidase 4; HRE, hypoxia responsive

527

element; DPPH, 1,1-diphenyl-2-picrylhydrazyl; ROS, reactive oxygen species; FBS, fetal bovine

528

serum; EPO, erythropoietin; tBHP, tert-butyl hydroperoxide; LLM, leucaena leaf meal; CP,

529

crude protein; NMR, nuclear magnetic resonance; MTT, methyl thiazolyl tetrazolium; HEK,

530

human embryonic kidney; ATCC, American Type Culture Collection; DMEM, dulbecco's

531

modified eagle medium; ELISA, enzyme linked immunosorbent assay; DCFH-DA, 2,7-

532

dichlorodi-hydrofluorescein diacetate.

533 534

SUPPORTING INFORMATION

535

Representative LC-QQQ-MRM MS/MS chromatograms of L. leucocephala leaves extract

536

(Figure S1). The UV absorption profiles of flavonoid 1, and degradation metabolites of

537

flavonoids 2 and 3 after incubation with cattle rumen liquid (Figure S2). Mass spectra properties

538

of flavonoids in L. leucocephala leaves extract and IS (Table S1). Calibration curves, LOD and

539

LOQ of flavonoids quantitated using UPLC-MS/MS (Table S2). Recovery rate of 7 flavonoids

540

quantitated using UPLC-MS/MS (Table S3). These materials are available free of charge via the

541

Internet at http://pubs.acs.org.

542 543

FUNDING

544

This research was supported by National Natural Science Foundation of China (31470424),

24


Page 24 of 45

Page 25 of 45


545

Youth Innovation Promotion Association CAS to Zhongyu Zhou (2016310), Guangdong Special

546

Support Program (2015TQ01R054), and Hong Kong Scholar Program to Zhongyu Zhou

547

(XJ2015044).

548 549

ACKNOWLEDGMENTS

550

We thank Kun Jia (College of Veterinary Medicine, South China Agricultural University) for his

551

help to obtain cattle rumen liquid.

552 553

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672 673 674

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675

Figure legend

676

Figure 1. The chemical structures of seventeen isolated flavonoids from L. leucocephala

677

foliage.

678 679

Figure 2. Isolated flavonoids from L. leucocephala foliage induces pHRE-Luc in transfected

680

HEK293T cells.

681

(A): The cell viability of different concentration of flavonoids 1, 2, 7, 10, 13, 14, and 17 on

682

HEK293T cells. Values are expressed as the percentage of vehicle control, and they are in Mean

683

± SD, where n = 3, each with triplicate samples. *p < 0.05, **p < 0.01, ***p

Flavonoids, a Potential New Insight of Leucaena leucocephala

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