Functional Analyses on Antioxidant, Anti-inflammatory, and

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Functional analyses on antioxidant, anti-inflammatory and anti-proliferative effects of extracts and compounds from Ilex latifolia Thunb, a Chinese bitter tea Ting Hu, Xiao-Wei He, and Jian-Guo Jiang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf501670v • Publication Date (Web): 13 Aug 2014 Downloaded from http://pubs.acs.org on August 16, 2014

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Journal of Agricultural and Food Chemistry

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Functional analyses on antioxidant, anti-inflammatory and

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anti-proliferative effects of extracts and compounds from

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Ilex latifolia Thunb, a Chinese bitter tea

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Ting Hu, Xiao-Wei He, Jian-Guo Jiang*

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College of Food and Bioengineering, South China University of Technology, Guangzhou, 510640,

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China. *Author for correspondence (e-mail: [email protected]; phone +86-20-87113849; fax:

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+86-20-87113843)

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Abstract: Ilex latifolia Thunb (I. latifolia), widely distributed in China, has been used as

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functional food and drunk for a long time. This study was aimed to identify the bioactive

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constituents with antioxidant, antitumor and anti-inflammatory properties. I. latifolia was

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extracted with 95% ethanol, and then partitioned into four fractions, petroleum ether fraction,

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ethyl acetate fraction, n-butanol fraction, and water fraction, respectively. Results showed that the

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ethyl acetate fraction was found to have significant ferric reducing antioxidant power activity,

16

DPPH radical scavenging activity and oxygen radical absorbance capacity, cytotoxicity against

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human cervix carcinoma Hela cells and inhibitory effect on NO production in macrophage RAW

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264.7 cells. Five compounds were isolated from the ethyl acetate fraction and they were identified

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as ethyl caffeate (1), ursolic acid (2), chlorogenic acid (3), 3,4-di-O-caffeoylquinic acid methyl

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ester (4), 3,5-di-O-caffeoylquinic acid methyl ester (5), the last two of which were isolated for the

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first time from I. latifolia. Compounds 4 and 5 exhibited cytotoxicity actions against tumor cell

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line. Compound 3 showed the strongest anti-inflammatory activity than other compounds. The

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results obtained in this work might contribute to the understanding of biological activities of I.

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latifolia and further investigation on its potential application values for food and drug.

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Keywords: I. latifolia, antioxidant, antitumor, anti-inflammatory, ursolic acid, chlorogenic acid,

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3,4-di-O-caffeoylquinic acid methyl ester, 3,5-di-O-caffeoylquinic acid methyl ester

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ACS Paragon Plus Environment


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Abbreviations

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I. latifolia

Ilex latifolia Thunb

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DPPH

2, 2-Diphenyl-1-picrylhydrazyl

31

LPS

lipopolysaccharide

32

MTT

3-(4, 5-dimethylthiazol-z-yl)-2, 5-diphenyl tetrazolium bromide

33

GAE

gallic acid equivalent

34

TLC

thin-layer chromatography

35

HPLC

high performance liquid chromatography

36

DMSO

dimethyl sulfoxide

37

OD

optical density

38

4-CAME

3, 4-di-O-caffeoylquinic acid methyl ester

39

5-CAME

3, 5-di-O-caffeoylquinic acid methyl ester

40

95%EtOH

95% ethanol extract

41

PE

petroleum ether fraction

42

EA

ethyl acetate fraction

43

n-BuOH

n-butanol fraction

44

W

water fraction

45

5-FU

5-fluorouracil

46

TPC

total phenolic content

47

TE

Trolox equivalent

48

2


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Introduction

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Ilex latifolia Thunb (I. latifolia) is a bitter tea of Chinese origin. It has been consumed

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traditionally as a kind of tea in China and Southeastern Asia for a long history. The taste of I.

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latifolia is much bitter than that of tea made from the leaves of Camellia sinensis.1 The main

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species are I. latifolia, and I. cornuta, which belong to the same genus as mate (Ilex

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paraguariensis).2-4 I. latifolia has gained attention as a functional tea in the past decade because of

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its potential health and economic significance. Many beneficial functions of I. latifolia such as

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cardiovascular, antioxidant, antiobesity, antidiabetic, anti-inflammatory, hepatoprotective and

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neuroprotective effects have been reported.5, 6

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There is a growing interest in I. latifolia about its chemical composition and functions due to

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its applications in cancer chemotherapy and other various pharmacological effects, particularly

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antitumor and anti-inflammatory activities.7, 8 I. latifolia remains an underutilized natural resource.

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In order to develop its health-promoting compounds into new food ingredients or nutraceutical

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applications, more research is urgently needed for the isolation and identification of the

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compounds with efficient procedures from I. latifolia extracts.

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The main purpose of the present study was to isolate the bioactive constituents with

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antioxidant, antitumor and anti-inflammatory properties. The crude extract and four fractions

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(petroleum ether fraction, ethyl acetate fraction, n-butanol fraction, and water fraction), which

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partitioned from 95% ethanol extract of I. latifolia, were measured to compare their activity by the

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use of ferric reducing antioxidant power assay, DPPH radical scavenging assay, oxygen radical

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absorbance assay, cell proliferation assay against human cervix carcinoma Hela cell and inhibitory

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effect on NO production in LPS-induced RAW 264.7 macrophages. The activities of five

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compounds isolated from ethyl acetate fraction were also determined.

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Materials and methods

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Plant material. I. latifolia, derived from Wuzhishan city (Hainan province, China), was

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purchased from Forest Drugstore Chain Co., LTD of Guangzhou, China. Samples were air-dried

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under shade for one week and pulverized to powder. The dried materials were stored in a

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well-closed container for further use.

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Reagents. Methanol, HPLC grade water, Forlin-Ciocalteu phenol reagent, sodium 3



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carbonate were purchased from chemical reagent factory (Tianjing, China). DPPH (2,

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2-Diphenyl-1-picrylhydrazyl), LPS (lipopolysaccharide), MTT (3-(4, 5-dimethylthiazol-z-yl)-2,

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5-diphenyl tetrazolium bromide), N-1-naphtylethylenediamine dihydrochloride, sulfanilamide,

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5-fluorouracil, gallic acid and Trolox were purchased from Sigma Chemical Co. (USA). Human

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cervix carcinoma Hela cell line and mouse macrophage RAW 264.7 cell line were obtained from

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the cell bank of Chinese academy of sciences (CAS, Shanghai, China). Cell culture medium and

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all of the other materials required for cell culture were purchased from Life Technologies

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Corporation (United States). All the other chemicals were of analytical grade.

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Extraction, isolation and purification. The process of I. latifolia extraction and

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isolation is shown in Figure 1. The dried powder (3kg) of I. latifolia was extracted with 95%

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ethanol (3×10 L) for 3h at 80℃. The crude extract was evaporated to dryness under reduced

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pressure. The extract was suspended in water (1.5L) firstly, and then partitioned with petroleum

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ether, ethyl acetate, and n-butanol in sequence with the same volume (shake sharply to mixture,

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then stand for 4 hours at room temperature and atmosphere pressure every time), which yielded

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petroleum ether fraction (111.3 g), ethyl acetate fraction (456.2 g), n-butanol fraction (153.5 g),

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and water fraction (177.4 g), respectively (for extraction yields see Table 1). The ethyl acetate

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fraction was chosen for the further isolation for the sake of its better activity among the four

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fractions in the biological activity screening tests.

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It was dissolved in chloroform and applied to a silica gel (200-300 mesh) column (7.5 cm ×

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1.2 m) and eluted with a gradient of chloroform and methanol (100:0, 98:2, 95:5, 90:10, 80:20,

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50:50, 0:100, v/v, each 10 L). The eluates were pooled into 28 fractions (100:0-fractions 1-2,

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98:2-fractions

3-7,

95:5-fractions

8-12,

90:10-fractions

13-17,

80:20-fractions

18-22,

100

50:50-fractions 23-27, 0:100-fraction 28) based on thin-layer chromatography (TLC) and high

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performance liquid chromatography (HPLC). Fraction 6 (from 98:2 fraction) was loaded on a

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ODS column and eluted with a mixture of methanol and water (30%, 60%, 90%, 100%, v/v, each

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2.5 L) to obtain four fractions. The 30% methanol fraction was further purified by Sephadex

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LH-20 column chromatography and compound 1 was obtained (300 mg). The 100% fraction

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appears crystal, compound 2 was obtained (13 g). Fraction 17 was purified by Sephadex LH-20

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column chromatography eluted with methanol and compound 3 was obtained (5 g). Fraction 20

107

(from 90:10 fraction) was dissolved in chloroform and applied on a second silica gel column (5 4


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Journal of Agricultural and Food Chemistry 108

cm × 80 cm). The column was eluted with a stepwise mixture of chloroform and methanol (99:1,

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98:2, 95:5, 90:10, v/v, each 5 L), the 98:2 fraction continue to separate followed by

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semi-preparative HPLC using 48% methanol solution as the mobile phase, to yield compounds 4

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(1.2 g) and compound 5 (900 mg). The yields of extraction of five compounds (based on the initial

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3 kg of dried I. latifolia) are 0.01%, 0.43%, 0.17%, 0.04%, 0.03%, respectively.

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The isolated compounds and 95%EtOH extract were dissolved in methanol (approximately 2

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mg/mL) filtered through a 0.45µm micropore membrane before use and 20 µL were injected into

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the HPLC instrument for analysis. The relative contents in percentage of each compound in the

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initial extract were determined and calculated with area normalization method, which are 0.22%,

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2.09%, 1.07%, 0.35%, 0.28%, respectively. The purity of five compounds was detected by HPLC

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and all of the purity reaches above 95%.

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Determination of total phenolic content. A precisely weighed amount of samples

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was used for total phenolic content (TPC), which were dissolved in methanol solution. The TPC

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was determined by the Folin-Ciocalteu’s reagent method9. Briefly, the appropriate sample solution

122

was oxidized with the Folin-Ciocalteu reagent, and sodium carbonate was used to neutralize the

123

reaction. After a 30 min incubation period, absorbance was measured at 765 nm. The

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concentration was calculated using gallic acid as standard, and the results were expressed as

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milligrams gallic acid equivalents (GAE) per gram dry weight (DW), which were shown in Table

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1.

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Assays for ferric reducing antioxidant power (FRAP). Assays for ferric reducing

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antioxidant powder were measured by the method of Benzie and Strain,10 in which the absorbance

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at 593 nm was measured. Results were expressed as Trolox equivalents per gram of dry weight,

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TE/g DW.

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Assays for DPPH radical scavenging. Assays for DPPH radical scavenging were

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determined by the method of Villaño et al.11 with some modifications. Briefly, a total of 0.1 mL of

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sample in ethanol was added to 3.0 mL of DPPH ethanolic solution. Absorbance was measured at

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517 nm after 30 min at room temperature in the dark. All measurements were performed in

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triplicate. The percent DPPH scavenging ability was calculated as: DPPH scavenging ability =

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(Acontrol-Asample/Acontrol) × 100. IC50 values calculated denote the concentration of a sample required

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to decrease the absorbance at 517 nm by 50%. The lower the IC50 value the more powerful the 5



antioxidant capacity.

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Assays for oxygen radical absorbance capacity (ORAC). Assays for oxygen

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radical absorbance capacity were determined by the method of Dávalos et al.12 with some

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modifications. In brief, samples were diluted with NaH2PO4-Na2HPO4 solution (75 mM, pH 7.4),

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then placed on microplates along with fluorescein solution (39.9 µM) and AAPH solution (38.25

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mM). The plates were incubated in an automated microplate reader at 37 ℃ and read every min for

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70 min using a fluorescence detector with an excitation wavelength of 485 nm and emission

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wavelength at 520 nm. A calibration curve was prepared using Trolox and the results were

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expressed as Trolox equivalents per g of dry weight, TE/g DW.

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Cell culture. The cell lines used in this study were human cervix carcinoma Hela and mouse

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macrophage RAW 264.7. Cells were maintained and cultured in tissue culture flasks (25 cm2,

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Corning) containing DMEM (Gibco BRL) medium supplemented with 10% fetal bovine serum

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(FBS), 1% penicillin (100 IU/mL) and streptomycin (100 µg/mL) at 37°C in humidified incubator

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with 5% CO2. The medium was changed every 24-48 h.13

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Dried samples were firstly dissolved in DMSO, and then were diluted with the culture

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medium into different concentrations. The final concentration of DMSO in the culture medium

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was maintained at less than 0.5% (v/v) in order to avoid solvent toxicity.

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Antitumor activity. The effect of samples on the proliferation of Hela cell was tested by 3-(4,

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5-dimethylthiazol-z-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. MTT is captured by cells

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and reduced intracellularly in a mitochondrion-dependent reaction to yield formazan product. The

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ability of cells to reduce MTT provides an indication of their intactness and mitochondrial activity

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that serves as a measure of viability.14, 15 Exponential growth phase Hela cells were plated into

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96-well culture plates (5 × 104 cells/mL, 100µL/well) and incubated overnight. When the cells

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were attached to the plates, an equal volume of fresh media containing different concentrations of

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samples were added. After 24 h, the supernatant was discarded and cells were washed with PBS

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for two times. 200 µL of fresh media containing 20µL MTT (0.5 mg/mL) solution was added to

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each well followed by incubation for 4 h at 37 °C. After this period, the supernatant was removed

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and 150 µL of DMSO was added to dissolve formazan crystals. The absorbance was measured at

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490 nm after the plates were shaken for 8 min. 5-fluorouracil is a kind of natural antitumor drugs,

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and it shows strong effect in antitumor assays. It was very popular used as a positive control in 6


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antitumor activities. Therefore, 5-fluorouracil was used as a positive control in this work. The

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percentage of cell proliferation inhibition was calculated using the following formula 1:16

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Cell proliferation inhibition (%) = (1 −

ODsample ODcontrol

) × 100%

(1)

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Anti-inflammatory assays. The anti-inflammatory activities of total crude extraction

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fraction, ethyl acetate fraction, petroleum ether fraction, water fraction, n-butyl alcohol fraction,

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and isolated compounds were tested with RAW 264.7 macrophages in vitro. Firstly, cytotoxicity

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test should be performed by MTT assay in order to choose the samples for further investigation,

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which was no-toxic to normal cells. Whereafter, the samples with no toxicity could be done the

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anti-inflammatory activity below.

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Exponential growth phase RAW 264.7 cells were seeded into 96-well culture plates (1 × 105

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cells/mL, 100µL/well) and incubated overnight. When the cells were attached to the plates, an

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equal volume of fresh media containing different concentrations of samples were added. Then

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cells were stimulated with LPS (final concentration was 1 µg/mL). After 24 h, the supernatants

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were collected to measure NO content using the Griess reaction17 with minor modifications. In

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brief, 100 µL cell supernatants were added with 50 µL of 0.1% N-1-naphtylethylenediamine

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dihydrochloride in distilled water and 50 µL of 1% sulfanilamide in 5% H3PO4. The absorbance

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was measured at 540 nm after the plates were stand for 8 min at room temperature. Sodium nitrite

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standard curve was obtained and used to calculate the nitrite level in samples. NO inhibition rate

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was calculated using the following formula 2:18

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NO inhibition (%) =

ODLPS − ODLPS+ sample ODLPS − ODblank

× 100%

(2)

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Statistical Analysis. All the experiments were carried out 3 replications. The data were

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expressed as the mean ± standard deviation (SD). The SPSS statistical analysis software was used

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to perform analysis of variance (ANOVA). Each group treated was compared with the

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corresponding controls by means of Student’s F-test and correlation test. The significant difference

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was checked by Duncan’s multiple range tests and the level was set at p n-BuOH > 95% EtOH > W > PE,

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which was shown in Table 1. The TPC of ethyl acetate fraction was much higher than those of

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95% EtOH and the other fractions. It seems that ethyl acetate was the most suitable solvent to

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concentrate phenolic substance. Moreover, lower TPC was found in petroleum ether fraction.

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Dietary antioxidants have been shown to prevent the oxidation of biomolecules, which can be

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regarded as effective scavengers of detrimental free radicals.24, 25 Tea is a good source of dietary

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antioxidants, such as caffeoylquinic acids, tea polyphenols. The antioxidant properties of tea are

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primary owing to the phenolic content, and several epidemiological research have held the

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chemoprotective properties of polyphenols.26 In this work, the antioxidant activities of extract and

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fractions from I. latifolia have been evaluated in vitro. The crude extract (95% EtOH), as well as

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its four fractions of PE, EA, n-BuOH and W, were evaluated for antioxidant activities using the

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ferric reducing antioxidant power assay (FRAP), DPPH radical scavenging activity and oxygen

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radical absorbance capacity (ORAC) and the results were compared (Table 1). The activities

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decreased in the order of EA > n-BuOH >95% EtOH > W > PE, according to the FRAP and

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ORAC assays, with the exception of the rank order of n-BuOH and 95% EtOH in the DPPH assay.

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This trend was similar to that observed for the total polyphenol content. The ethyl acetate fraction

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of I. latifolia exhibited remarkable FRAP value (265.05 ± 1.5 µmol TE/g DW), free radical

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scavenging activity against DPPH (IC50=16.3 ± 3.3µg/ml) and ORAC value (2367 ± 88 µmol TE/g

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DW). By comparing 95% EtOH and its four fractions, the antioxidant power of 95% EtOH was

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less than those of EA and n-BuOH in the FRAP and ORAC assays, which may result from the

225

active components through condensation effects during the solvent-solvent partitioning progresses. 8


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The extract of I. latifolia was certified to contain a large number of caffeoylquinic acids, which

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contributed to the antioxidant activity.27 It is observed that I. latifolia is a beneficial herbal drink

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due to its antioxidant activity. Identification of isolated compounds. The structure identification of isolated compounds

229 230

was performed with MS, 1H-NMR and

13

231

reported spectral data, the chemical structures of five compounds were identified and shown in

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Figure 2. Spectroscopic data are described as below.

C-NMR analysis and comparison with the previously

233

Compound 1 was obtained as brown acicular crystal. The negative ESI-MS showed a

234

quasi-molecular ion at m/z 206.8 [M-H]-. 1H-NMR (CD3OD-d4, 400 MHz) δ ppm: 7.52 (1H, d,

235

J=15.9 Hz), 6.23 (1H, d, J=15.9Hz), 7.03 (1H, d, J=1.9 Hz), 6.77 (1H, d, J=8.4 Hz), 6.92 (1H, dd,

236

J=1.9, 8.4 Hz), 4.18 (2H, q, J=6.6 Hz), 1.30 (3H, t, J=6.6 Hz). 13C-NMR (CD3OD-d4, 101 MHz) δ

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ppm: 167.4 (C-1), 114.8 (C-2), 145.5 (C-3), 127.4 (C-4), 115.5 (C-5), 146.2 (C-6), 148.7 (C-7),

238

116.2 (C-8), 122.3 (C-9), 60.4 (C-1’), 14.5 (C-2’). Compared with the NMR and MS data given in

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reference,19 compoud 1 was identified as ethyl caffeate.

240

Compound 2 was obtained as white prismatic crystal. The negative ESI-MS showed a

241

quasi-molecular ion at m/z 455.2 [M-H]-. 1H-NMR (CD3Cl-d4, 400 MHz) δ ppm: 2.10 (1H, d,

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H-18), 3.13 (1H, t, H-3), 5.26 (1H, t, H-12), 0.80 (3H, d, J=7.6 Hz, H-29), 0.70 (3H, d, J=7.6 Hz,

243

H-30), 1.26, 1.10, 0.89, 0.86, 0.78 (5×3H, s). 13C-NMR (CD3Cl-d4, 101 MHz) δ ppm: 38.7 (C-1),

244

27.3 (C-2), 79.1 (C-3), 38.8 (C-4), 55.3 (C-5), 18.4 (C-6), 33.0 (C-7), 39.6 (C-8), 47.7 (C-9), 37.1

245

(C-10), 23.4 (C-11), 125.9 (C-12), 138.0 (C-13), 42.1 (C-14), 28.2 (C-15), 24.3 (C-16), 47.9

246

(C-17), 52.8 (C-18), 39.1 (C-19), 38.8 (C-20), 30.7 (C-21), 36.8 (C-22), 28.1 (C-23), 15.6 (C-24),

247

15.5 (C-25), 17.0 (C-26), 23.6 (C-27), 181.2 (C-28), 17.5 (C-29), 21.4 (C-30). These ESI-MS,

248

1

249

identified as ursolic acid. It was found to be the major constituent isolated from I. latifolia.

H-NMR and 13C-NMR data were similar to those in previous report,20 therefore, compoud 2 was

250

Compound 3 was obtained as white powder. The negative ESI-MS showed a

251

quasi-molecular ion at m/z 352.7 [M-H]-. 1H-NMR (CD3OD-d4, 400 MHz) δ ppm: 2.21 (dd,

252

J=13.6, 3.1 Hz), 2.07 (dd, J=13.6, 4.4 Hz), 4.20 (ddd, J=3.1, 4.4, 3.1 Hz), 3.76 (dd, J=8.5, 3.1 Hz),

253

5.37 (ddd, J=9.3, 4.8, 8.5 Hz), 2.11 (dd, J=13.6, 9.3 Hz), 2.26 (dd, J=13.6, 4.8 Hz), 7.08 (d, J=2.0

254

Hz), 6.81 (d, J=8.2 Hz), 6.98 (dd, J=8.2, 2.0 Hz), 7.59 (d, J=15.9 Hz), 6.29 (d, J=15.9 Hz).

255

13

C-NMR (CD3OD-d4, 101 MHz) δ ppm: 126.6 (C-1), 114.1 (C-2), 144.2 (C-3), 147.0 (C-4), 9



116.0 (C-5), 122.6 (C-6), 145.9 (C-7), 115.2 (C-8), 168.9 (C-9), 76.6 (C-10), 38.8 (C-11), 70.6

257

(C-12), 72.8 (C-13), 71.0 (C-14), 37.3 (C-15), 180.1 (C-16). These data were in good agreement

258

with the reported compound, chlorogenic acid.21

259

Compound 4 was obtained as yellow powder. The negative ESI-MS showed a

260

quasi-molecular ion at m/z 529 [M-H]-. 1H-NMR (DMSO-d6, 400 MHz) δ ppm: 6.82 (2H, brd,

261

J=8.1 Hz, H-5’, 5’’), 7.03 (2H, dd, J=8.1, 2.1 Hz, H-6’, 6’’), 7.07 (2H, d, J=2.1 Hz, H-2’, 2’’), 7.56

262

(1H, d, J=15.9 Hz, H-7’), 7.47 (1H, d, J=15.9 Hz, H-7’’), 6.32 (1H, d, J=15.9 Hz, H-8’), 6.19 (1H,

263

d, J=15.9 Hz, H-8’’), 5.32 (1H, m, H-3), 5.02 (1H, m, H-4), 4.20 (1H, m, H-5), 3.65 (3H, s, OCH3),

264

2.27 (2H, m, H-2, 6), 2.00 (2H, m, H-1, 7). 13C-NMR (DMSO-d6, 101 MHz) δ ppm: 73.2 (C-1),

265

37.8 (C-2), 67.9 (C-3), 72.4 (C-4), 65.5 (C-5), 36.8 (C-6), 173.5 (C-7), 125.6 (C-1’), 125.3 (C-1’’),

266

114.5 (C-2’, 2’’), 145.7 (C-3’), 145.5 (C-3’’), 148.7 (C-4’), 148.6 (C-4’’), 115.9 (C-5’), 114.9

267

(C-5’’), 121.5 (C-6’), 121.0 (C-6’’), 147.6 (C-7’), 147.1 (C-7’’), 113.9 (C-8’), 113.4 (C-8’’), 166.6

268

(C-9’), 165.3 (C-9’’), 52.1 (OCH3). Comparing the above data with the literature,22 compound 4

269

was identified as 3,4-di-O-caffeoylquinic acid methyl ester (4-CAME).

270

Compound 5 was obtained as yellow powder. The negative ESI-MS showed a

271

quasi-molecular ion at m/z 529 [M-H]-. 1H-NMR (DMSO-d6, 400 MHz) δ ppm: 2.09~2.33 (4H, m,

272

H-2, 6), 3.71 (3H, s, OCH3), 4.34 (1H, m, H-5), 5.10 (1H, dd, J=8.0, 3.2 Hz, H-4), 5.50 (1H, m,

273

H-3), 6.16 (1H, d, J=16.0 Hz, H-8’), 6.28 (1H, d, J=16.0 Hz, H-8’’), 6.74 (2H, d, J=8.0 Hz, H-5’,

274

5’’), 6.90 (1H, m, H-6’, 6’’), 7.00 (1H, d, J=2.0 Hz, H-2’), 7.02 (1H, d, J =2.0 Hz, H-2’’), 7.49 (1H,

275

d, J=16.0 Hz, H-7’), 7.59 (1H, d, J=16.0 Hz, H-7’’).

276

(C-7), 168.4 (C-9’’) , 167.9 (C-9’), 149.7 (C-4’, 4’’), 147.7 (C-7’’), 147.6 (C-7’), 146.7 (C-3’, 3’’),

277

127.7 (C-1’’), 127.5 (C-1’), 123.1 (C-6’, 6’’), 116.5 (C-5’’), 116.4 (C-5’), 115.1 (C-2’, 2’’), 114.7

278

(C-8’, 8’’), 75.8 (C-1), 74.9 (C-4), 69.0 (C-5), 68.6 (C-3), 53.0 (OCH3), 38.5 (C-2), 38.3 (C-6).

279

Compound 5 was identified as 3,5-di-O-caffeoylquinic acid methyl ester (5-CAME) according to

280

the literature.23

13

C-NMR (DMSO-d6, 101 MHz) δ: 177.5

281

Compounds 4 and 5 were isolated and identified from I. latifolia for the first time.

282

Evaluation of cytotoxicity against Hela cells. In this investigation, the 95% EtOH extract,

283

fractions and isolated compounds 1-5 from the I. latifolia were evaluated for their antiproliferative

284

activities against Hela human cervix carcinoma cell line using the MTT bioassay. Figure 3

285

indicated the morphological changes on the growth and survival of Hela cells as examined by 10


Page 10 of 23

Page 11 of 23


phase-contrast microscopy. The cell proliferation inhibition rates of samples are shown in figure 4.

287

Among the crude extract and four fractions, 95% EtOH and EA both exhibited the strongest

288

cytotoxicity (Fig.4A). Although the inhibition rate of extract and four fractions was lower than that

289

of 5-fluorouracil, the inhibition rate of 95% EtOH and EA fraction reached nearly 50% and 55% at

290

the concentration of 50 µg/mL. This result showed that the active ingredients on antitumor were

291

concentrated into EA fraction after the 95% EtOH was partitioned into four fractions.

292

The isolated compounds show significant cytotoxicity against Hela cell line except

293

chlorogenic acid (Fig.4B). When the concentration was below 4 µM, all compounds showed very

294

lower inhibition rate as compared to positive control. However, the inhibition rate of 4-CAME and

295

5-CAME increased significantly at the concentration of 6 to 10 µM. The inhibition rate of ursolic

296

acid rose with the increase of concentration, and reached about 28% at the max concentration.

297

While along with the further increase of concentration, the inhibition rate of chlorogenic acid was

298

changed a little. Ethyl caffeate also exhibited a poor effect at the concentration from 1 to 10 µM.

299

4-CAME and 5-CAME are derivatives of caffeoylquinic acid found in several plants. There

300

is little study of antitumor activity on these two compounds, but the physicochemical property of

301

caffeoylquinic acid was widely reported in the literature. Hu et al.28 reported that methyl

302

3,5-di-caffeoyl quinate inhibited proliferation in a dose-manner as detected by MTT, trypan blue

303

exclusion and flow cytometric assays using HT-29 cells. 5-Caffeoylquinic acid was used in

304

preparing anti-tumor medicine in form of tablet, capsule, slow-release tablet or used for inhibiting

305

growth and transfer of tumor. In our study, the two derivatives (4-CAME and 5-CAME) showed

306

significantly cytotoxicity against Hela cell line, which can be the potential antitumor drugs in

307

future.

308

Effect of the extract, fractions and compounds on NO production in RAW264.7

309

macrophages. MTT colorimetric assays were carried out to evaluate the toxic effect of

310

compounds on RAW 264.7 macrophage cell line, and the results were showed as relative cell

311

viability referred to control (equal to 100%). The cell viability all reached above 92% (Data not

312

shown). Generally speaking, it could be considered as non-toxicity that the survival rate was

313

higher than 90% when treated with samples.

314

Current research has demonstrated the participation of ROS in systems of inflammation.29 I.

315

latifolia was studied as potential inhibitors of NO production in inflammatory reactions. 11



Stimulation of LPS-induced RAW 264.7 cell leads to overproduction of NO and it could be

317

detected and quantified. Figure 5A displayed that crude extract and four fractions significantly

318

inhibited NO release and decreased in the order of EA (78.16%), 95% EtOH (62.78%), W

319

(56.63%), n-BuOH (35.11%) and PE (23.87%) at the concentration of 50 µg/mL. It was clear that

320

EA showed the most active anti-inflammatory effect among the crude extract and all fractions.

321

Many previous literatures showed that methanol/ethanol extracts or fractions had good

322

anti-inflammatory activity. Yang et al. 30 reported that ethyl acetate fraction of the seeds of Brucea

323

javanica showed significant decrease on NO production in LPS-induced RAW 264.7 macrophages.

324

In addition, Mariana et al. 31 reported that ethanol extract and hexane and ethyl acetate fractions

325

from Couroupita guianensis Aublet leaves were exhibited anti-inflammatory activity. In this study,

326

ethyl acetate fraction showed a better anti-inflammatory ability than that of 95% ethanol extract.

327

Inhibitory rates of compounds 1-5 on NO production decreased in a turn of chlorogenic acid

328

(EC50=4.15 µM), ursolic acid (EC50=6.49 µM), ethyl caffeate (EC50=7.72 µM), 4-CAME

329

(EC50=7.81 µM) and 5-CAME (EC50>10 µM). Among the five isolated compounds, ursolic acid

330

and chlorogenic acid showed strong anti-inflammatory ability on NO production in RAW 264.7

331

macrophages (Fig.5B). Chlorogenic acid was reported that it can efficiently inhibit LPS-induced

332

proinflammatory responses in hepatic stellate cells and the anti-inflammatory effect may be due to

333

the inhibition of LPS/ROS/NF-kB signaling pathway.32

334

According to the above results, ursolic acid showed the best radical quenching capacity and a

335

similar ferric reducing capacity than chlorogenic acid, whereas the latter showed the best

336

anti-inflammatory activity and the worst anti-proliferative effect. Thus it can be seen that different

337

compounds often has different biological activities. It may exert strong effect in a certain activity,

338

but very weak in others on the contrary. Chlorogenic acid showed a better antioxidant capacity and

339

anti-inflammatory activity, which may be related to its structure with a lot of hydroxyl groups.

340

However, the mechanism of antioxidant and anti-inflammatory activity in ursolic acid and

341

chlorogenic acid should be further investigated in the future study.

342

In conclusion, an efficient method for bioassay-guided preparative isolation was used for

343

identifying the antioxidant, antitumor and anti-inflammatory constituents in I. latifolia. Five

344

compounds were isolated and identified. Antioxidant, antitumor and anti-inflammatory effects of

345

extract, four fractions and the five isolated compounds were evaluated. The ethyl acetate fraction 12


Page 12 of 23

Page 13 of 23


was found to exhibit effectively antioxidant, antitumor and anti-inflammatory activities. In the

347

evaluation of cytotoxicity against tumor cell line, compounds 4-CAME and 5-CAME showed fine

348

activities at high concentrations in comparison to 5-fluorouracil. In addition, chlorogenic acid

349

showed stronger anti-inflammatory than others. These results indicated that after the fractional

350

extraction of 95% EtOH into four fractions, the active ingredients were concentrated into ethyl

351

acetate fraction. Compounds 4-CAME and 5-CAME are the bioactive chemical compositions

352

isolated from I. latifolia and they were isolated and identified from I. latifolia for the first time.

353

The results obtained in this work might contribute to understanding the biological activity and

354

further investigation of Ilex latifolia Thunb for food and drug application.

355

References

356

(1) Liu, L. X.; Sun, Y.; Laura, T.; Liang, X. F.; Ye, H.; Zeng, X. X. Determination of

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polyphenolic content and antioxidant activity of kudingcha made from Ilex kudingcha C.J.

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Tseng. Food Chem. 2009, 112, 35-41.

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(2) Filip, R.; Lopez, P.; Giberti, G.; Coussio, J.; Ferraro, G. Phenolic compounds in seven South American Ilex species. Fitoterapia 2001, 72, 774-778.

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(3) Bravo, L.; Goya, L.; Lecumberri, E. LC/MS characterization of phenolic constituents of mate

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(Ilex paraguariensis, St. Hil.) and its antioxidant activity compared to commonly consumed

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beverages. Food Res. Int. 2007, 40, 393-405.

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(4) Heck, C. I.; De Mejia, E. G. Yerba Mate tea (Ilex paraguariensis): A comprehensive review

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(5) Chen, Y.; Li, K. S.; Xie, T. G. Hypotensive action of the extract of kudingchadongqingye (Ilex kudingcha). Chinese Tradit. Herb Drugs 1995, 26, 250-252.

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(6) Nishimura, K.; Fukuda, T.; Miyase, T.; Noguchi, H.; Chen, X. M. Activity-guided isolation

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of triterpenoid acyl CoA cholesteryl acyl transferase (ACAT) inhibitors from Ilex kudincha. J.

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(7) Hirano, T.; Gotoh, M.; Oka, K. Natural flavonoids and lignans are potent cytostatic agents against human leukemic HL-60 cells. Life Sci. 1994, 55, 1061-1069.

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Macrococculus pomiferus and their inhibitory activities against cyclooxygenases-1 and -2.

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Phytochemistry 2004, 65, 2861-2866.

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(9) Yildirim, A.; Mavi, A.; Kara, A. Determination of antioxidant and antimicrobial activities of Rumex crispus L. extracts. J. Agric. Food Chem. 2001, 49, 4083-4089. (10) Benzie, I. F. F.; Strain, J. J. The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": The FRAP assay. Anal. Biochem. 1996, 239, 70-76.

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(11) Villaño, D.; Fernández-Pachón, M. S.; Moyá, M. L.; Troncoso, A. M.; García-Parrilla, M. C.

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Radical scavenging ability of polyphenolic compounds towards DPPH free radical. Talanta

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(12) Dávalos, A.; Gómez-Cordovés, C.; Batolomé, B. Extending applicability of the oxygen

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radical absorbance capacity (ORAC-fluorescein) assay. J. Agric. Food Chem. 2004, 52,

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48-54.

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(13) Huang, N.; Rizshsky, L.; Hauck, C.; Nikolau, B. J.; Murphy, P. A.; Birt, D. F. Identification

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of anti-inflammatory constituents in Hypericum perforatum and Hypericum gentianoides

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extracts using RAW 264.7 mouse macrophages. Phytochemistry 2011, 72, 2015-2023.

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(14) Yang, L.; Wu, D. F.; Luo, K. W.; Wu, S. H.; Wu, P. Andrographolide enhances

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5-fluorouracil-induced apoptosis via caspase-8-dependent mitochondrial pathway involving

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p53 participation in hepatocellular carcinoma (SMMC-7721) cells. Cancer Lett. 2009, 276,

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(15) Mosmann, T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55-63.

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(16) Franchi, G. C.; Moraes, C. S.; Toreti, V. C.; Daugsch, A.; Nowill, A. E.; Park, Y. K.

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Comparison of effects of the ethanolic extracts of Brazilian Propolis on human heukemic

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cells as assessed with the MTT assay. Evid-based Compl. Alt. 2012, 2012, 918956-918962.

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(17) Green, S. J.; Meltzer, M. S.; Hibbs, J. B. Jr; Nacy, C. A. Activated macrophages destroy

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intracellular Leishmania major amastigotes by an L-Arginine-dependent killing mechanism. J.

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Immunol. 1990, 144, 278-283.

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(18) Ma, L. S.; Chen, H. X.; Dong, P.; Lu, X. M. Anti-inflammatory and anticancer activities of

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extracts and compounds from the mushroom Inonotus obliquus. Food Chem. 2013, 139,

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503-508. 14


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(19) Shi, S. Y.; Zhao, Y.; Zhou, H. G.; Zhang, Y. P.; Jiang, X. Y.; Huang, K. L. Identification of

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antioxidants

from

Taraxacum

mongolicum

by

high-performance

liquid

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chromatography-diode array detection-radical-scavenging detection-electrospray ionization

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mass spectrometry and nuclear magnetic resonance experiments. J. Chromatogr. A 2008,

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1209, 145-152.

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(20) Gordo, J.; Maximo, P.; Cabrita, E.; Lourenco, A.; Oliva, A.; Almeida, J.; Filipe, M.; Cruz, P.;

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Barcia, R.; Santos, M.; Cruz, H. Thymus mastichina: Chemical Constituents and their

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Anti-cancer Activity. Nat. Prod. Commun. 2012, 7, 1491-1494.

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(21) Dai, X. P.; Huang, Q.; Zhou, B. T.; Gong, Z. C.; Liu, Z. Q.; Shi, S. Y. Preparative isolation

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and purification of seven main antioxidants from Eucommia ulmoides Oliv. (Du-zhong)

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leaves using HSCCC guided by DPPH-HPLC experiment. Food Chem. 2013, 139, 563-570.

417 418 419 420 421 422

(22) Jiang, H. L.; Xu, L. Z.; Yang, X. D.; Zhang, D.; Yang, S. L.; Zou, Z. M. Quinic acid esters from herba of Siphonostegia chinensis. China J. Chinese materia medica 2002, 27, 923-926. (23) Mao, Q.; Cao, D.; Jia, X. S. Studies on the chemical constituents of Lonicera Magranthoides Hand. -Msaa. Acta Pharm. Sinica 1993, 28, 271-281. (24) Halliwell, B.; Aeschbach, R.; Loliger, J.; Aruoma, O.I. The characterization of antioxidants. Food Chem. Toxicol. 1995, 33, 601-617.

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(25) Pietta, P.G. Flavonoids as antioxidants. J. Nat. Prod. 2000, 63, 1035-1042.

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(26) Riboli, E.; Norat, T. Epidemiological evidence of the protective effects of fruits and

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vegetables on cancer risk. Am. J. Clin. Nutr. 2003, 78, 559-569.

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(27) Li, L.; Xu, L. J.; Ma, G. Z.; Dong, Y. M.; Peng, Y.; Xiao, P. G. The large-leaved Kudingcha

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(Ilex latifolia Thunb and Ilex kudingcha C.J. Tseng): a traditional Chinese tea with plentiful

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secondary metabolites and potential biological activities. J. Nat. Med. 2013, 67, 425-437.

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(28) Hu, W.; Shen, T.; Wang, M. H. Cell cycle arrest and apoptosis induced by

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methyl 3,5-dicaffeoyl quinate in human colon cancer cells: Involvement of the PI3K/Akt and

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MAP kinase pathways. Chem. Biol. Interact. 2011, 194, 48-57.

432

(29) Chu, Y. F.; Wise, M. L.; Gulvady, A. A.; Chang, T.; Kendra, D. F.; van Klinken, B. J. W.;

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Shi, Y. H.; O’Shea, M. In vitro antioxidant capacity and anti-inflammatory activity of seven

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common oats. Food Chem. 2013, 139, 426-431.

435

(30) Yang, J. H.; Li, S. C.; Xie, C. F.; Ye, H. Y.; Tang, H.; Chen, L. J.; Peng, A. H. 15



Anti-inflammatory activity of ethyl acetate fraction of the seeds of Brucea Javanica. J.

437

Ethnopharmacol. 2013, 147, 442-446.

438

(31) Pinheiro, M. M. G.; Fernandes, S. B. O.; Fingolo, C. E.; Boylan, F.; Fernandes, P. D.

439

Anti-inflammatory activity of ethanol extract and fractions from Couroupita guianensis

440

Aublet leaves. J. Ethnopharmacol. 2013, 146, 324-330.

441

(32) Shi, H. T.; Dong, L.; Dang, X.Y.; Liu, Y. P.; Jiang, J.; Wang, Y.; Lu, X. L.; Guo, X. Y.

442

Effect of chlorogenic acid on LPS-induced proinflammatory signaling in hepatic stellate cells.

443

Inflamm. Res. 2013, 62, 581-587.

16


Page 16 of 23

Page 17 of 23


I. latifolia Extraction with 95% ethanol 95% ethanol extract Fractional extraction, be partitioned into four fractions

Petroleum ether fration Ethyl acetate fraction

n-butanol fraction

Water fraction

Silica gel column chromatography, elution with chloroform-methanol

100:0

98:2

ODS colum chromatography, elution with methanol-water

95:5

90:10

80:20

Sephadex LH-20 column chromatography, elution with methanol Compound 3

Sephadex LH-20 column chromatography, elution with methanol

98:2

95:5

90:10

Semi-preparative HPLC

30% methanol 60% methanol 90% methanol 100% methanol

Compound 1

0:100

Silica gel column chromatography, elution with chloroform-methanol

99:1

444

50:50

Natural crystal Compound 2

Compound 4

Compound 5

445

Figure 1. The extraction and isolation procedure of compounds from the 95% ethanol extract of I.

446

latifolia.

447

17



Page 18 of 23

448 CH3 H3C

O OH

CH

C CH

CH3

CH2 O

CH3

H

CH3

O H

HO

H

CH3

HO H3C

CH3

OH

B A O OH

O

O

HO

COOH OH

O

O O

HO

OH

HO

C O

OH

O OH

OH

OH

HO

D C O OH

O

O

O

O

O OH

OH

HO OH

OH

E 449 450

Figure 2. Compounds and their chemical structures isolated from I. latifolia. (A) ethyl caffeate; (B)

451

ursolic acid; (C) chlorogenic acid; (D) 3,4-di-O-caffeoylquinic acid methyl ester; (E)

452

3,5-di-O-caffeoylquinic acid methyl ester.

18


Page 19 of 23


453

Table 1. Total phenolic contents (TPC) and ferric reducing antioxidant power (FRAP), scavenging of the free radical DPPH, oxygen radical absorbance capacity (ORAC) and

454

percent w/w extraction yield of I. latifolia ethanol extract and its four fractions on the basis of freeze-dried starting material. samples

TPC

FRAP

DPPH

ORAC

extraction yield

(mg GAE/g DW)

(µmol TE/g DW)

(IC50, µg/mL)

(µmol TE/g DW)

(w/w, %)

95% ethanol extract

84.4 ± 1.3 c

148.65 ± 0.04 c

28.7 ± 2.1 b

1198 ± 89 c

30.4

petroleum ether fraction

32.5 ± 0.6 d

112.38 ± 0.04 d

not found

139 ± 5 e

3.7

ethyl acetate fraction

125.7 ± 7.8 a

265.05 ± 1.5 a

16.3 ± 3.3 c

2367 ± 108 a

15.2

n-butanol fraction

93.2 ± 6.2 b

175.65 ± 0.09 b

29.5 ± 1.5 b

1921 ± 23 b

5.1

water fraction

87.9 ± 4.1 c

139.22 ± 0.04 c

47.7 ± 2.6 a

652 ± 36 d

5.9

455

Data expressed as means ± standard deviation. Averages followed by the different lower case letters in different columns differ significantly by the one-way analysis of

456

variance-Duncan’s multiple range test (p≤0.05). GAE = gallic acid equivalent; TE = Trolox equivalent; DW = dry weight.

19



A

E

I

B

Page 20 of 23

C

D

F

G

H

J

K

L

457

Figure 3. Morphological changes on the growth and survival of Hela cells as examined by phase-contrast microscopy (magnification, 200×). (A) control group or

458

blank group, (B) 95% EtOH (50 µg/ml) (C) petroleum ether fraction (50 µg/ml), (D) ethyl acetate fraction (50 µg/ml), (E) n-butanol fraction (50 µg/ml), (F) water

459

fraction (50 µg/ml), (G) ethyl caffeate (10 µM), (H) ursolic acid (10 µM), (I) chlorogenic acid (10 µM), (J) 4-CAME (10 µM), (K) 5-CAME (10 µM), (L)

460

5-fluorouracil (10 µM).

20


Page 21 of 23


461

PE

60 Inhibition (%)

av

95%EtOH

70

EA

aw

n-BuOH

50

W

40

ax

5-FU

30 20 10

bv

cv

ay az by by ay cy cy bz cx cz by dy

bx cxcx dx dx

bw cwcw

dv ev

dv

dw dw

0 10

20

30

40

50

Concentration (µg/ml)

A

462 50 45 40 Inhibition (%)

av av bv

ethyl caffeate ursolic acid chloric acid 4-CAME 5-CAME 5-FU

35 30

aw bw cw ax

20

bx ay

15 10 5

cv *

25

by azaz cybycy by bzbzbyaz

cx exdx * * *fx

*

dw

dv *

ew *

*

ev

fw

0 2

4

6 8 Concentration (µM)

463

10

B

464

Figure 4. Cell proliferation inhibition rate of samples. (A) 95% ethanol extract (95% EtOH),

465

petroleum ether fraction (PE), ethyl acetate fraction (EA), n-butanol fraction (n-BuOH), and water

466

fraction (W), 5-FU (5-fluorouracil); (B) isolated compounds: ethyl caffeate, ursolic acid,

467

chlorogenic acid, 3,4-di-O-caffeoylquinic acid methyl ester (4-CAME), 3,5-di-O-caffeoylquinic

468

acid methyl ester (5-CAME), 5-FU (5-fluorouracil). Letters a-e refer to significant differences

469

within a fraction and v-z for comparisons across fractions by one-way analysis of

470

variance-Duncan’s multiple range test (p≤0.05); results are means ± standard deviation, which

471

were calculated based on the data from three trials with three replicates per trial.

21



90

95%EtOH PE EA n-BuOH W

80 NO inhibition (%)

70 60

av bv bw

50 40

10

ax

aw

30 20

cv

aw

ay

az bzbz cz

ax by ay cy dy

bxcx

cw

dv dw

ev

ew

dx

0 10

20

30

40

50

Concentration (µg/ml)

A

472

NO inhibition (%)

100

ethyl caffeate ursolic acid

80

chlorogenic acid 4-CAM E

70 60 50 40 30 20 10

ax

5-CAM E

az bz cy cz dy

av bv

aw

90

bw bx cw cx

bxbyay cy

cv cw

dw

dv ev

ev

dw

dx

0 2

4

6

8

Concentration (µM )

473

10

B

474

Figure 5. Inhibitory effect on NO production of total extract, four fractions and isolated

475

compounds from I. latifolia in LPS-induced RAW264.7 macrophages. (A) 95% ethanol extract

476

(95% EtOH), petroleum ether fraction (PE), ethyl acetate fraction (EA), n-butanol fraction

477

(n-BuOH), and water fraction (W); (B) isolated compounds: ethyl caffeate, ursolic acid,

478

chlorogenic acid, 3,4-di-O-caffeoylquinic acid methyl ester (4-CAME), 3,5-di-O-caffeoylquinic

479

acid methyl ester (5-CAME). Letters a-e refer to significant differences within a fraction and v-z

480

for comparisons across fractions by one-way analysis of variance-Duncan’s multiple range test

481

(p≤0.05); results are means ± standard deviation, which were calculated based on the data from

482

three trials with three replicates per trial.

483

22


Page 22 of 23

Page 23 of 23


484

TOC Graphic

485

23


Functional Analyses on Antioxidant, Anti-inflammatory, and

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