Identification and Quantification of Phytochemical Composition and

Lizziane de Francisco , Diana Pinto , Hélen Rosseto , Lucas Toledo , Rafaela Santos , Flávia Tobaldini-Valério , Terezinha Svidzinski , Marcos Brus...
0 downloads 0 Views 886KB Size
Subscriber access provided by FORDHAM UNIVERSITY

Article

Identification and quantification of phytochemical composition, and anti-inflammatory and radical scavenging properties of methanolic extracts of Chinese propolis Haiming Shi, Haisha Yang, Xiaowei Zhang, and Liangli Lucy Yu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf3042775 • Publication Date (Web): 23 Nov 2012 Downloaded from http://pubs.acs.org on November 26, 2012

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

Journal of Agricultural and Food Chemistry

Identification and quantification of phytochemical composition, and anti-inflammatory and radical scavenging properties of methanolic extracts of Chinese propolis Haiming Shi,†,‡ Haisha Yang,† Xiaowei Zhang,† and Liangli (Lucy) Yu*,†,§ †

Institute of Food and Nutraceutical Science, Key Lab of Urban Agriculture (South),

Bor S. Luh Food Safety Research Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China; ‡College of Chemistry & Material Engineering, Wenzhou University, Zhejiang, Wenzhou 325027, China; §

Department of Nutrition and Food Science, University of Maryland, College Park,

MD 20742, United States

*Corresponding author (Tel: 86-21-34207961; Fax: 86-21-34205758; E-mail: [email protected])

1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

1

ABSTRACT

2

Fifteen propolis samples collected from different regions of China were investigated

3

and compared for their phytochemical composition, and anti-inflammatory and

4

radical scavenging properties. Eleven compounds including caffeic, p-coumaric,

5

ferulic, isoferulic, 3, 4-dimethylcaffeic acids, pinobanksin, chrysin, pinocembrin,

6

galangin, pinobanksin 3-acetate and caffeic acid phenylethyl ester were quantified

7

for the fifteen propolis samples using UHPLC method, while thirty-eight compounds

8

were identified by UPLC/Q-TOF-MS. The fifteen propolis samples significantly

9

differed in their total phenolic and total flavonoid contents, as well as their

10

phytochemical profiles. The methanol extracts of propolis also showed significant

11

anti-inflammatory effects in LPS-stimulated RAW 264.7 mouse macrophage cells at

12

10 µg propolis extract/mL concentration. Additionally, the propolis samples differed

13

in their DPPH, ABTS cation, hydroxyl and peroxide radical scavenging capacities

14

and ferric reducing abilities. The results from this study may be used to improve the

15

commercial production and consumption of Chinese propolis products.

16

KEYWORDS: propolis, phenolic, flavonoid, anti-inflammation, radical scavenging,

17

methanolic extract, chemical constituent

Page 2 of 38

18

2

ACS Paragon Plus Environment

Page 3 of 38

Journal of Agricultural and Food Chemistry

19 20

INTRODUCTION Propolis, a resinous and adhesive natural substance produced by honeybees, has

21

been used in functional foods and folk medicines for several centuries. Previous

22

studies have shown that propolis may possess several health benefits, including their

23

antioxidant, anticancer, anti-inflammatory, antibacterial, antiviral, antifungal and

24

immunomodulatory properties.1-4 The health benefits were mainly attributed to the

25

flavonoids and phenolic acids, the two major classes of phytochemicals in propolis. In

26

the past ten years, propolis has attracted more and more attentions and has been

27

extensively used in functional foods and nutritional supplemental products.5, 6

28

It is well accepted that the chemical constituents and health properties of propolis

29

greatly depend on several ecological factors, including geographical region, plant

30

source, season and method of harvesting.7-10 For instance, Hamasaka et al.11 reported

31

that the fourteen propolis samples collected in different locations of Japan differed in

32

their chemical compositions and antioxidant activities in 2004. Additionally, Chinese

33

propolis samples were shown to be rich in phenolics, including phenolic acids and

34

flavonoids, and had strong antioxidant activities measured as reducing power,

35

β-carotene bleaching inhibition, and scavenging ability against DPPH and ABTS

36

cation radicals.12, 13 Recently, our group isolated five new glycerol esters and

37

tentatively identified twelve minor constituents using UPLC-Q-TOF-MS from Wuhan

38

propolis.14 All the five isolated compounds showed significant anti-inflammatory

39

activities on interleukin (IL)-1β, IL-6 and cyclooxygenase (COX)-2 mRNA

40

expressions. To date, there is little report on hydroxyl (HO•) or peroxide anion (O2•-) 3

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 4 of 38

41

radical scavenging capacity of propolis. Also noted was that few UHPLC analysis has

42

been performed to quantitatively characterize the detailed chemical composition of

43

propolis, though the previous studies generally employed HPLC to quantify

44

flavonoids and phenolic compounds by comparing the retention time and UV spectra

45

with the standard compounds.

46

In the present study, fifteen Chinese propolis were evaluated for their total phenolic

47

contents (TPC), total flavonoid contents (TFC), potential anti-inflammatory effects,

48

scavenging capability against DPPH•, ABTS•+, HO• and O2•-, and ferric reducing

49

ability. The anti-inflammatory effects were measured as their ability in suppressing

50

IL-1β, IL-6 and COX-2 mRNA expressions in the LPS stimulated RAW 264.7 mouse

51

macrophages. In addition, a rapid and effective UHPLC analysis method was

52

developed and applied to quantify the major compounds in the Chinese propolis

53

samples. Finally, the chemical profiles of Chinese propolis from different origins were

54

compared basing on the UPLC/Q-TOF-MS analysis. The results advanced our

55

understanding of the different chemical composition among propolis, and to promote

56

its better use in health food and dietary supplement.

57

MATERIALS AND METHODS

58

Materials. Fifteen propolis samples were collected from various locations in

59

China (Figure S1 in the supporting information) and stored at -20 °C before use. Iron

60

(III) chloride, fluorescein (FL), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic

61

acid (trolox), and 2,2-diphenyl-1-picryhydrazyl radical (DPPH•), gallic acid,

62

1,3,5-tri(2-pyridyl)-2,4,6-triazine (TPTZ), DMSO and 2-peproponal were purchased 4

ACS Paragon Plus Environment

Page 5 of 38

Journal of Agricultural and Food Chemistry

63

from Sigma-Aldrich (St. Louis, MO, USA). Reference compounds: caffeic acid,

64

p-coumaric acid, ferulic acid, isoferulic acid, 3, 4-dimethylcaffeic acid, caffeic acid 1,

65

1-dimethylallyl ester and caffeic acid phenylethyl ester were purchased from

66

Sigma-Aldrich (St. Louis, MO, USA); pinocembrin was obtained from Shanghai

67

ANPEL Scientific Instrument Co., Ltd. (Shanghai, China); quercetin, apigenin,

68

isorhamnetin, chrysin and galangin were obtained from Shanghai R&D Centre for

69

Standardization of Chinese Medicines; pinobanksin, caffeic acid isopent-3-enyl ester,

70

caffeic acid 2-methyl-2-butenyl ester, pinobanksin 3-acetate, p-coumaric acid benzyl

71

ester, caffeic acid cinnamyl ester and chrysin-7-methyl-ether were isolated from

72

propolis in our laboratory. The purities of isolated compounds were all above 98%

73

by HPLC analysis. The chemical structures of all these compounds as standards were

74

also confirmed by 1H NMR and HR-MS. Folin-Ciocalteu (FC) reagent was

75

purchased from Ambrosia Pharmaceuticals (Shanghai, China). 2, 2'-Azobis

76

(2-amidinopropane) dihydrochloride (AAPH) was purchased from J&K Scientific

77

(Beijing, China). Thirty percent H2O2 regent, analytical grade acetone, methanol,

78

ethyl ether, ethyl acetate, petroleum ether, sodium hydroxide, sodium nitrite, sodium

79

dihydrogen phosphate and disodium hydrogen phosphate were purchased from

80

Sinopharm (Beijing, China). Aluminum nitrate was obtained from Aladdin

81

(Shanghai, China). HPLC-grade formic acid, methanol and acetonitrile were

82

purchased from Merck (Darmstadt, Hesse-Darmstadt Germany). RAW 264.7 mouse

83

macrophage was purchased from Chinese Academy of Sciences (Shanghai, China).

84

DMEM, fetal bovine serum and 1×PBS were purchased from Gibco (Life 5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

85

Technologies, Carlsbad, CA, USA). TRIzol reagent was obtained from Invitrogen

86

(Life Technologies, Carlsbad, CA, USA). Lipopolysaccharide (LPS) from E. Coli

87

0111: B4 was obtained from Millipore (Millipore, Billerica, MA, USA). IScriptTM

88

Advanced cDNA Synthesis kit was purchased from Bio-Rad (Bio-Rad Laboratories,

89

Hercules, CA, USA), while AB Power SYBR Green PCR Master Mix was purchase

90

from ABI (Applied Biosystems, Carlsbad, CA, USA). Ultrapure water was prepared

91

by a Millipore ultra-Genetic polishing system with < 5 ppb TOC and resistivity of

92

18.2 mΩ (Millipore, Billerica, MA, USA) and was used for all experiments.

93

Sample Preparation. The frozen propolis samples were powdered using a mill.

94

Approximately one gram of propolis was extracted by 10 mL pure methanol at room

95

temperature. After the mixtures were sonicated (320 W, 40 KHz) for 2 h, the extracts

96

were collected. The extracts were kept at 4 °C in the refrigerator until further

97

analysis. The average methanolic extract of propolis yield of 15 samples was 75.5 ±

98

6.2%. For the quantitative and qualitative analyses, an accurately weighed mass of

99

the propolis powder (1.0 g) was transferred into a 50 mL volumetric flask adjusted

100

with methanol and sonicated for 30 min. The supernatant of sample solution was

101

filtered through a 0.22 µm GHP membrane for UPLC/Q-TOF-MS analysis and

102

UHPLC quantification.

103

Page 6 of 38

Total Phenolic Contents (TPC). The TPC of each methanol extract was

104

measured according to a laboratory procedure described previously.15 Briefly, the

105

reaction mixture consisted of 50 µL of sample extracts, 250 µL Folin-Ciocalteu

6

ACS Paragon Plus Environment

Page 7 of 38

Journal of Agricultural and Food Chemistry

106

regent, 750 µL of 20% sodium carbonate and 3 mL ultrapure water. Gallic acid was

107

used as the standard. Absorbance was read at 765 nm after 2 h of reaction at ambient

108

temperature. The results were reported as mg gallic acid equivalent (GAE) per gram

109

of propolis.

110

Total Flavonoid Contents (TFC). Total flavonoid was determined using an

111

aluminum colorimetric method described previously.16 In brief, 150 µL propolis

112

extracts was mixed with 1.5 mL 5% sodium nitrite, and 1 mL 10% aluminum nitrate

113

was added after 6 min. Then 4 mL of 4% sodium hydroxide was added into the

114

mixture. The absorbance was read at 502 nm using after 15 min of reaction at

115

ambient temperature. The results were reported as mg quercetin equivalent (QE) per

116

gram propolis.

117

Identification of Chemical Constituents by UPLC-Q-TOF-MS Analysis.

118

Propolis samples were analyzed for their chemical profiles by Waters Xevo G2

119

Q-TOF mass spectrometer (Milford, MA, USA). UPLC was performed at 40 °C

120

using an Acquity UPLC BEH C18 column (100 mm × 2.1 mm i.d.; 1.7 µm; Waters,

121

Milford, MA, USA), equipped with an Acquity UPLC VanGuard precolumn (5 mm

122

× 2.1 mm i.d.; 1.7 µm; Waters). The elution gradient (eluent A, 0.1% formic acid;

123

eluent B, acetonitrile) was: 20% B for 1.3 min, 20-30% B in 0.9 min, 30-40% B in

124

6.6 min, 40-60% B in 3.3 min, 60-90% B in 3.4 min, and 90% B for 3.2 min. The

125

flow rate was 0.5 mL min-1 and the injection volume was 1.5 µL. MS conditions

126

were: capillary voltages for negative and positive ion modes were 2.8 kV and 3.0 kV;

7

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

127

sampling cone voltages for negative and positive ion modes were 55.0 V and 43.0 V;

128

source temperature 100 °C; desolvation temperature 300 °C; desolvation gas flow

129

500.0 L/h; cone gas flow 50.0 L/h; scan range m/z 80-1000; scan time 0.3 s; and

130

inter-scan time 0.02 s. Data were collected and analyzed with Waters MassLynx v4.1

131

software.

Page 8 of 38

132

Quantification of Eleven Compounds in Propolis by UHPLC Analysis. The

133

analytical UHPLC system was a Shimadzu LC-30AD series Ultra-High-Performance

134

liquid chromatography equipped with a Shimadzu series diode-array detector

135

(Shimadzu Technologies, Kyoto, Japan). The UHPLC pumps, auto sampler, column

136

oven and diode-array system were monitored and controlled using the Shimadzu

137

LC-solution computer program (Shimadzu, Kyoto, Japan). The individual compound

138

in propolis was quantified according to the absorbance at 280 nm. The quantitative

139

analysis was carried out on an Acquity BEH ODS-C18 column (Waters, Milford, MA,

140

USA) (150 mm × 2.1 mm i.d., 1.7 µm). Temperature of the column oven was set to

141

45 °C. The mobile phases consisted of 0.1% aqueous formic acid (solvent A) and

142

acetonitrile (solvent B). A gradient elution was: start at 20% B; held for 1.2 min;

143

1.2-2.0 min, increase via linear gradient to 30% B; 2.0-8.0 min, increase via linear

144

gradient to 40% B; 8.0-11.0 min, increase via linear gradient to 60% B; 11.0-15.0

145

min, increase via linear gradient to 90% B; and held for 2 min. Flow rate of the

146

mobile phase was 0.5 mL/min, and the injection volume was 1.0 µL. All eleven

147

compounds were quantified against external standards. Quantification was based on

148

peak area. Calibration curves of the standards were made by diluting stock standards 8

ACS Paragon Plus Environment

Page 9 of 38

Journal of Agricultural and Food Chemistry

149

150

in methanol.

Inhibition of IL-1β, IL-6 and COX-2 mRNA Expression in RAW 264.7 Mouse

151

Macrophage Cells. Solvent was removed from known volume of the methanol

152

extract for each propolis sample. The residue was re-dissolved in known amount of

153

DMSO (100 mg/mL) and diluted in the medium for cell treatment. RAW 264.7

154

mouse macrophages were cultured in 6-well plates and reached the confluence of

155

80%. The cells were pretreated with media containing propolis extracts for 24 h at an

156

initial concentration of 0.1 mg of propolis equiv/mL. After pretreatment, LPS was

157

added at an initial concentration of 10 ng/mL, cells were incubated at 37 °C under 5%

158

CO2 for another 4 h. After induction, culture media was discarded and cells were

159

collected to perform total isolation and real-time PCR.17

160

RNA isolation and real-time PCR were performed according to the previously

161

published protocol.18 After LPS induction, cells were washed with 1×PBS, and

162

TRIzol reagent was added for total RNA isolation. IScriptTM Advanced cDNA

163

Synthesis kit was used to reverse transcribe complementary DNA. Real-time PCR

164

was performed on an ABI 7900HT Fast Real-Time PCR System (Applied

165

Biosystems, Foster City, CA, USA) using AB Power SYBR Green PCR Master Mix.

166

Primers used in this study were as follows: IL-1β (Forward:

167

5'-GTTGACGGACCCCAAAAGAT-3', Reverse:

168

5'-CCTCATCCTGGAAGGTCCAC-3'); IL-6 (Forward:

169

5'-CACGGCCTTCCCTACTTCAC-3', Reverse:

9

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 10 of 38

170

5'-TGCAAGTGCATCATCGTTGT-3'); COX-2 (Forward:

171

5'-GGGAGTCTGGAACATTGTGAA-3', Reverse:

172

5'-GCACGTTGATTGTAGGTGGACTGT-3'). The mRNA amounts were normalized

173

to an internal control, GAPDH mRNA (Forward:

174

5'-AGGTGGTCTCCTCTGACTTC-3', Reverse:

175

5'-TACCAGGAAATGAGCTTGAC-3').The following amplification parameters

176

were used for PCR: 50 °C for 2 min, 95 °C for 10 min, and 46 cycles of

177

amplification at 95 °C for 15 s and 60 °C for 1 min.

178

DPPH Radical Scavenging Activity (DPPH). Hydrogen-donating activity was

179

measured using DPPH radicals following a previously reported protocol.19 For each

180

sample, different concentrations ranging from 0.6 to 500 µg/mL were prepared with

181

methanol or 10% DMSO-methanol (v/v). The reaction mixtures in the 96-well plates

182

consisted of sample (100 µL) and DPPH radical (100 µL, 0.2 mM) dissolved in

183

methanol. The absorbance was measured at 517 nm against a blank. The percentage

184

of scavenging activity was calculated as: [1−(A1−A2)/A0]×100%, where A0 is the

185

absorbance of the control and A1 is the absorbance of the sample, A2 is the

186

absorbance of blank that contained sample without DPPH radical. The scavenging

187

activity of the samples was expressed as IC50 value, the concentration required to

188

scavenge 50% of DPPH radicals.

189

ABTS Cation Radical Scavenging Activity (ABTS). The ABTS cation radical

190

scavenging activity assay was carried out via the ABTS cation radical

191

decolorization.19 The samples were prepared in the same procedure as the DPPH 10

ACS Paragon Plus Environment

Page 11 of 38

Journal of Agricultural and Food Chemistry

192

assay. The ABTS cation radical was prepared by reacting 7 mM aqueous solution of

193

ABTS (15 mL) with 140 mM potassium persulphate (264 µL) to obtain a ABTS

194

working reagent with an absorbance of 0.70 ± 0.02 at 734 nm. The reaction mixtures

195

consisted of sample (50 µL) and the ABTS methanol working solution (100 µL). The

196

mixture was kept for 10 min in the dark, and the absorbance was taken at 734 nm

197

against a blank. The scavenging capacity was calculated as: [1−(A1−A2)/A0] × 100%,

198

Where A0 is the absorbance of the control (without sample) and A1 is the absorbance

199

in the presence of the sample, A2 is the absorbance of sample without ABTS working

200

solution. The IC50 value was calculated from the scavenging activities (%) versus

201

concentrations of respective sample curve.

202

Ferric Reducing Ability of Plasma Assay (FRAP). The ability to reduce ferric

203

ions was measured using a modified method described previously.20 Propolis extracts

204

(0.2 mL) was added to 3.8 mL of FRAP regent (10 parts of 300 mM sodium acetate

205

buffer at pH 3.6, 1 part of 10.0 mM TPTZ solution, and 1 part of 20.0 mM FeCl3

206

solution). Absorbance was read at 595 nm after 30 min reaction at 37 °C. The results

207

were reported as millimoles Trolox equivalents (TE) per gram of propolis.

208

Hydroxyl Radical Scavenging Capacity (HOSC). The HOSC values were

209

measured using a previously published laboratory protocol.21 Reaction mixture

210

contained 170 µL of 9.28 × 10-8 M fluorescein, 30 µL of sample, blank or standard,

211

40 µL of 0.1990 M H2O2 and 60 µL of 3.43 M FeCl3. The fluorescence of the

212

reaction mixture was measured every minute for 6 h at ambient temperature, with the

11

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

213

excitation wavelength at 485 nm and emission wavelength at 528 nm. HOSC values

214

were expressed as millimoles of Trolox equivalents (TE) per gram of propolis.

215

Oxygen Radical Absorbance Capability (ORAC). The ORAC values were

216

determined using a Synergy 2 Multi-Mode Microplate Reader (BioTek, Winooski,

217

VT, USA) according to a laboratory protocol described previously.22 Thirty µL of

218

sample, blank or standard solution was added to 225 µL of freshly prepared 81.63

219

nM fluorescence. The mixture was pipetted into a 96-well plate and preheated at

220

37 °C for 20 min. Then 25 µL of 0.36 M AAPH was added to the mixture. The

221

reaction mixture was measured every minute for 2 h, with an excitation wavelength

222

at 485 nm and an emission wavelength at 528 nm. ORAC values were reported as

223

millimoles of TE per gram of propolis.

224

Statistical Analysis. Data were reported as mean ± SD for triplicate

225

determinations. One-way ANOVA and Tukey’s test were employed to identify

226

differences in means. Statistics were analyzed using SPSS for Windows (version rel.

227

10.0.5, 1999, SPSS Inc., Chicago, IL). Statistical significance was declared at P