Metabolism of Phenolic Compounds in LPS-stimulated Raw264. 7

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Bioactive Constituents, Metabolites, and Functions

Metabolism of Phenolic Compounds in LPS-stimulated Raw264.7 Cells Can Impact Their Anti-inflammatory efficacy: Indication of Hesperetin Yong Ma, Yu He, Taijun Yin, Haoqing Chen, Song Gao, and Ming Hu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04464 • Publication Date (Web): 24 May 2018 Downloaded from http://pubs.acs.org on May 24, 2018

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Journal of Agricultural and Food Chemistry

Metabolism of Phenolic Compounds in LPS-stimulated Raw264.7 Cells Can Impact Their Anti-inflammatory efficacy: Indication of Hesperetin Yong Ma*,1,3, Yu He1, Taijun Yin1, Haoqing Chen2, Song Gao1, Ming Hu*,1 1

Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy,

University of Houston, 1441 Moursund St., Houston, TX, 77030 2

Department of Chemistry, College of Natural Sciences and Mathematics, University of

Houston, 4800 Calhoun Rd., Houston 77004 3

Current address: 343 Oyster Point Blvd #200, South San Francisco, CA 94080

1 ACS Paragon Plus Environment


Key words: Natural phenolic compounds, Raw264.7, Hesperetin, Degradation, Metabolism, In vitro

Abstract Raw264.7 is a murine macrophage-like cell line commonly used to study the antiinflammatory efficacy of natural compounds. However, the impacts of long-time incubation on the tested compounds are often inappropriately ignored. Among 77 natural phenolic compounds (mainly flavonoids), only 36 remain more than 70% after a 15-hour incubation in cell culture medium at 37 °C. Interestingly, for those compounds with a relatively good chemical stability, the presence of Raw264.7 cells could accelerate their disappearance in the medium, indicating that cellular metabolism occurred. As a representative phenolic, hesperetin was found to be efficiently metabolized by Raw264.7 cells and the metabolite was identified as a glucuronide in the further investigation. The glucuronidation activity is constitutive in this cell line. At certain concentration levels of hesperetin, the ability of hesperetin to inhibit PGD2 production in LPS-induced Raw264.7 cells was significantly enhanced by introducing β-glucuronidase which can hydrolyze hesperetin glucuronide into the incubation medium. The results indicate that glucuronidation and excretion of hesperetin can significantly impact its bioactivity in Raw264.7 cells.


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1

Introduction

2

The natural phenols and polyphenols including phenolic acids, lignans, stilbenoids, and

3

flavonoids are widely distributed in nature as the secondary metabolic products of plants

4

or fungus.1 These phytochemicals are also important dietary constituents in cereals, fruits,

5

vegetables, herbals, and mushrooms. They are receiving a lot of attention from

6

researchers and general population, due to their potential benefits to human health.2-4

7

Since early last century, numerous in vitro and in vivo studies have been conducted to

8

demonstrate the impacts of digesting flavonoids and other natural phenols on human

9

health.5-8 The functions of natural phenols include, but are not limited to anti-cancer9, 10,

10

anti-inflammation11-13,

11

cardiovascular protection18, 19.

12

When the pharmacological effects of food constituents are studied in vitro, cell culture

13

models are extensively used by researchers. The cultured cells provide a platform to

14

either screen the natural product pool for lead compounds or study the underlying

15

mechanism of action. For example, the Abelson murine leukemia virus transformed

16

mouse macrophage cell line Raw264.7 is commonly employed to investigate the anti-

17

inflammatory efficacies of natural compounds, as well as their other potential functions

18

in the immune cells.20 A search using the combination of the keywords Raw264.7 and

19

flavonoid retrieves more than 400 publications from PubMed in May, 2018. Like

20

macrophages, upon lipopolysaccharides (LPS) induction, the activation of Raw264.7

21

cells results in dramatic changes in signaling pathways and gene expression.21-25 Most

22

prominently,

23

inflammatory nitric oxide synthase (iNOS) will lead to the production of prostaglandin

the

anti-oxidation14,

induced

15

,

overexpression

anti-aging16,

of

anti-bacteria11,

cyclooxygenase-2


17

,

(COX-2)

and

and


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24

D2 (PGD2) and nitric oxide (NO), respectively, as two important inflammatory

25

biomarkers.25-28

26

To assess the anti-inflammatory effects of natural compounds, Raw264.7 cells are usually

27

incubated with the compound and LPS simultaneously. The incubation time is usually

28

longer than 10 hours, and sometimes it can be as long as 24 hours because it takes several

29

hours for LPS to induce the expression and function of COX-2 and iNOS.29-38 For

30

example, a number of flavonoids including kaempferol, baicalein, quercetin, luteolin,

31

genistein and hesperetin were incubated with Raw264.7 cells for longer than 12 hours in

32

a few studies previously.29,

33

biotransformation of the tested compound were not characterized in most studies which

34

employed Raw264.7 cells for long-time incubations. Indeed, it is already well-known that

35

some phenolic compounds rapidly decompose in the aqueous solutions.39,

36

phenolic compounds can be excellent substrates of metabolic enzymes, especially phase

37

II

38

biotransformation by the Raw264.7 cells may alter the concentration of a compound in

39

the medium and its efficacy as well. When the necessary characterization of compound

40

stability (either chemical or metabolic) is inappropriately ignored in the experiment

41

design, it may not only lead to biased observations of compound bioactivity in Raw264.7

42

cells, but also increase the difficulty in extrapolating the in vitro results to in vivo. In this

43

study, the chemical stability and potential cellular metabolism is studied under the

44

Raw264.7 cell incubation conditions for a broad panel of phenolic compounds (mainly

45

flavonoids). The biotransformation of hesperetin, a flavonoid with the anti-inflammatory

46

activity which has been reported in this cell line previously,29, 42, 43 is further investigated

drug

metabolizing

31, 35, 36

enzymes.41

However, the potential degradation and/or

Thus,

chemical


degradation

and

40

Also,

potential

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47

as a representative compound to illustrate how the metabolism of hesperetin impacts its

48

efficacy to inhibit PGD2 production.

49



50

Materials and Methods

51

Chemicals and reagents. All the tested natural phenolic compounds (purity > 98%) were

52

purchased from INDOFINE Chemical Company (Hillsborough, NJ) unless otherwise

53

stated. PGD2 and PGD2-d4 were purchased from Cayman Chemical (Ann Arbor, MI).

54

LPS, β-glucuronidase, magnesium chloride (MgCl2), D-saccharic acid 1,4-lactone

55

monohydrate, alamethicin, uridine 5'-diphospho-glucuronic acid (UDPGA), testosterone,

56

N-(1-naphthyl)ethylenediamine, sulfanilic acid and sodium nitrite were purchased from

57

Sigma-Aldrich (St. Louis, MO). Phosphate-buffered saline (PBS), Dulbecco's Modified

58

Eagle's Medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco

59

(Gland Island, NY). Acetonitrile and water (mass spectrometer grade) were purchased

60

from EMD (Gibbstown, NJ). All other materials (typically analytical grade or better)

61

were used as received.

62

Cell culture. The Raw264.7 cell line was purchased from the American Type Culture

63

Collection (Manassas, VA). The cells were maintained in DMEM supplemented with 10%

64

FBS at 37 oC and under 5% CO2 in a humidified incubator. The cells were passaged

65

every 2 to 3days with standard aseptic techniques, and no antibiotics were added in the

66

culture medium.

67

Chemical stability of phenolic compounds in DMEM. The stock solutions of the tested

68

compounds were prepared in DMSO. 0.2 mL blank DMEM was spiked with 10 µM

69

tested compounds and then kept at 37° C and under 5% CO2 in a humidified incubator.

70

15 hours later, the sample was mixed with 50 µL acetonitrile and centrifuged (15,000 × g,

71

15 min, 4 ºC) before analysis. The aqueous stability of the tested compound was


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determined by comparing its peak area in DMEM after 15-hour incubation with that in

73

freshly spiked DMEM.

74

Incubation of phenolic compounds with Raw264.7 cells. 5 × 104 cells in 0.5 mL

75

DMEM supplemented with 10% FBS were seeded in each well of a 24-well cell culture

76

plate. 48 hours later, when the cells reached 100% confluence, the old medium was

77

removed and the cells were washed with pre-warmed PBS twice before experiment.

78

To study the metabolic stability of phenolic compounds, Raw264.7 cells were incubated

79

with tested compound (10 µM) in the presence of 0.1 µg/mL LPS and or vehicle in fresh

80

DMEM. The plate was kept at 37° C and under 5% CO2 in a humidified incubator for 15

81

hours before the medium was harvested to analyze PGD2/NO production and compound

82

metabolism.

83

To study the metabolism and anti-inflammation activity of hesperetin, Raw264.7 cells

84

were incubated with 10 µM hesperetin in the presence or absence of 0.1 µg/mL LPS in

85

fresh DMEM (n = 3). The plate was kept at 37° C and under 5% CO2 in a humidified

86

incubator. After 0, 1, 2, 4, 8, 12, 16, 20, 24 hours of incubation, the cell medium was

87

harvested to analyze PGD2/NO production and hesperetin metabolism.

88

For investigating the impact of metabolism on the anti-inflammation activity of

89

hesperetin, Raw264.7 cells were incubated with 0 – 50 µM hesperetin and 0.1 µg/mL

90

LPS in the presence or absence of 10 units/mL β-Glucuronidase in fresh DMEM (n = 3).

91

The plate was kept at 37° C and under 5% CO2 in a humidified incubator for 15 hours

92

before the medium was harvested to analyze PGD2/NO production.



93

Preparation of cell lysate and protein concentration assay. The cell lysate was

94

prepared from Raw264.7 cells which were suspended in 50 mM potassium phosphate

95

buffer (pH 7.4), and sonicated in an ice-cold water bath by Aquasonic 150D sonicator

96

(VWR Scientific, Bristol, CT) for 20 min at the maximum power (135 average watts).

97

Protein concentration of the lysate was determined by a pierce BCA protein assay kit

98

(Rockford, IL), using bovine serum albumin as standards.

99

Glucuronidation of hesperetin by Raw264.7 cell lysate. The glucuronidation rates of

100

hesperetin by Raw264.7 cell lysate were determined as previously reported.44 Briefly, in

101

200 µL 50 mM potassium phosphate buffer (pH 7.4), Raw264.7 cell lysate (final protein

102

concentrations between 0.1 - 1 mg/mL), MgCl2 (0.88 mM), D-saccharic acid 1,4-lactone

103

monohydrate (4.4 mM), alamethicin (0.022 mg/mL), and different concentrations of

104

hesperetin were mixed. The sample was pre-warmed at 37 °C for 5 minutes before

105

UDPGA (final concentration 3.5 mM) was added to start the reaction. After 30 minutes

106

incubation at 37 °C, the reaction was stopped by adding 50 µL acetonitrile containing 100

107

µM testosterone as the internal standard. After centrifugation (15,000 × g, 15 min, 4 ºC),

108

10 µL of supernatant was injected for ultra-performance liquid chromatography (UPLC)-

109

UV analysis.

110

UPLC-UV analysis of phenolic compounds. A 200 µL aliquot of medium sample was

111

added 50 µL acetonitrile and vigorously mixed. After centrifugation (15,000 × g, 15 min,

112

4 ºC), the samples were analyzed using a Waters AcquityTM UPLC equipped with a diode

113

array detector (DAD). The conditions were: column, Waters C18, 1.7µm, 50mm × 2.1mm

114

(Waters, Milford, MA); mobile phase A, 2.5mM ammonium acetate in water (pH 7.4);

115

mobile phase B, 100% acetonitrile; gradient, 0 - 2.0 min, 10% - 20% B, 2.0 - 3.0 min, 20% 8 ACS Paragon Plus Environment

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- 40% B, 3.0 - 3.5 min, 40% - 50% B, 3.5 - 4.0 min, 50% - 90% B, 4.0 - 4.5 min, 90% B,

117

4.5 - 5.0 min, 90% - 10% B; flow rate, 0.45 mL/min; column temperature, 45 oC. The

118

detection wavelengths in DAD were dependent on the maximum UV absorbance of the

119

tested compounds, ranging from 240 to 380 nm.

120

UPLC-MS/MS quantitation of PGD2. For PGD2 quantitation, a 100 µL aliquot of

121

medium sample was spiked with 5 ng/mL PGD2-d4 as the internal standard and then

122

mixed with 50 µL acetonitrile. After centrifugation (15,000 × g, 15 min, 4 ºC), the

123

concentrations of PGD2 were determined by a Waters AcquityTM UPLC coupled with an

124

AB Sciex 5500 triple quadrupole mass spectrometer (MS) equipped with a

125

TurboIonSprayTM source. The UPLC conditions were: column, Waters BEH C18, 1.7µm,

126

100mm × 2.1mm (Waters, Milford, MA, USA); mobile phase A, 0.1% formic acid in

127

water; mobile phase B, 0.1% formic acid in acetonitrile; gradient, 0 - 0.5 min, 5% B, 0.5 -

128

1.0 min, 5% - 37% B, 1.0 - 4.5 min, 37% B, 5.0 - 5.5 min, 37% - 95% B, 5.5 - 6.0 min,

129

95% - 5% B, 6.0 - 7.0 min, 5% B; flow rate, 0.5 mL/min; column temperature, 45 oC. The

130

concentration of PGD2 was determined by using Multiple Reaction Monitoring (MRM)

131

scan type in negative mode. The ion pairs selected for PGD2 and PGD2-d4 were 351/271

132

and 355/275, respectively. The compound dependent parameters were: DP, -65; EP, -10;

133

CE, -24; CXP, -15. The instrument dependent parameters were: ionspray voltage, -4500

134

V; ion source temperature, 650 oC; nebulizer gas, 30 psi; turbo gas, 30 psi; curtain gas, 20

135

psi. The calibration curve was prepared by spiking different concentrations of PGD2

136

standard to DMEM. The quantitation range of PGD2 was 0.5 - 250 ng/mL.

137

Quantitation of NO. The concentrations of NO in cell culture medium were determined

138

by Griess reagent as described previously.45 Before each assay, the Griess Reagent was 9 ACS Paragon Plus Environment


139

freshly made by mixing equal volumes of 0.1% N-(1-naphthyl)ethylenediamine solution

140

in water and 1% sulfanilic acid in 5% phosphoric acid. A 100 µL aliquot of medium

141

sample was mixed with 100 µL Griess Reagent in a 96-well plate and incubated at room

142

temperature for 30 minutes. The plate was read by an ELx800 absorbance microplate

143

reader (BioTek Instruments, Inc., Winooski, VT) at the wavelength of 548 nm. Blank

144

DMEM samples spiked with different concentrations of sodium nitrite were used as the

145

calibration curve in nitrite quantitation. The relationship between spectrometer reading

146

and nitrite concentration was determined by polynomial regression. The calibration range

147

was 2 - 1000 µM.

148


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Results

150

The chemical stability of phenolic compounds in DMEM. The chemical stability in

151

DMEM at 37 oC was assessed for totally 77 phenolic compounds, including 61

152

flavonoids and 16 others. The remaining percentages of these compounds after a 15-hour

153

incubation in DMEM at 37 oC were shown in Table 1, ranging from 0 to 100%. As

154

expected, these compounds exhibited various stabilities in the physicochemical

155

environment of cell culture. Some phenolic compounds were found to be intrinsically

156

instable in the aqueous solutions because their chemical structures are extremely

157

vulnerable under such conditions. For example, flavones with a hydroxyl group on the 3-

158

position, such as 3-hydroxyflavone, 3,5-dihydroxyflavone, quercetin and galangin, were

159

found to be relatively less stable, with 0.0, 0.0, 0.0 and 5.6% left after the incubation,

160

respectively. However, it should be noticed that the compounds instable in DMEM are

161

not necessarily lacking of anti-inflammatory efficacy. For example, 100% of quercetin

162

degraded during the incubation. However, quercetin has been repeatedly reported as a

163

bioactive agent in Raw264.7 cells.33, 35 During the incubation, if the concentration of the

164

tested compound decreases rapidly in the medium, it is supposed that unidentified

165

degradation product(s) will emerge at the same time. It is possible that the degradation

166

products of quercetin are the responsible species accounting for its bioactivities in

167

Raw264.7 cells.

168

The incubation of selected compounds with Raw264.7 cells. To study the cellular

169

biotransformation of phenolic compounds in Raw264.7 cells, 36 phenolic compounds

170

showing a relatively good chemical stability in DMEM were selected for a further

171

investigation in the presence of Raw264.7 cells (Table 1). All of these compounds 11 ACS Paragon Plus Environment


172

remained more than 70% in DMEM after a 15-hour incubation at 37 oC in the absence of

173

Raw264.7 cells. Under the same incubation conditions in the presence of Raw264.7 cells,

174

the recovery of these compounds were determined by comparing the peak areas of parent

175

before and after the incubation. Several flavonoids were observed to have less than 30%

176

left in the medium at the end of incubation, including 7,3'-dihydroxyflavone, 7,2'-

177

dihydroxyflavone, 8-hydroxy-7-methoxyflavone, hesperetin and more. Similar results

178

were also observed for some other phenolic compounds including 4'-hydroxychalcone

179

and 6-hydroxy-7-methoxy-4-phenylcoumarin. Here, the accelerated disappearance of the

180

parent compounds in the presence of Raw264.7 cells is indicating that the cellular

181

metabolism of phenolic compounds is possible during the incubation.

182

Identification of the hesperetin metabolite in Raw264.7 cells. UPLC-UV analysis of

183

cell culture medium revealed that during the incubation of hesperetin with Raw264.7

184

cells, the peak area of hesperetin decreased and a new peak with shorter retention time

185

(Figure 1, A and B) and similar spectrum of UV absorption was observed (Figure 1, D

186

and E). In negative mode MS, Q1 full scan indicated the ionization of hesperetin

187

generated m/z 301, while the ion for the new peak was found to be m/z 477. MS2 scan

188

revealed that after fragmentation induced by collision, a major daughter ion of m/z 477 is

189

m/z 301, which is consistent with the loss of a glucuronic acid moiety (176 amu) (Figure

190

1, F). Meanwhile, after β-glucuronidase treatment, the new peak disappeared in UV

191

profile and hesperetin was recovered (Figure 1, C). Thus, the identity of hesperetin

192

metabolites was confirmed as hesperetin mono-glucuronide, although the conjugation site

193

is still unknown.


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Besides hesperetin, the metabolite identification by mass spectrometry was also

195

conducted for 16 out of 36 compounds which were incubated with the Raw264.7 cells.

196

For all these 16 compounds, the glucuronides (+ 176 amu) of the parent compounds were

197

detected in the medium. In addition, it is interesting to observe that multiple metabolites

198

of 6,7,3'-trihydroxyflavone were detected, including a methylated metabolite (+ 14 amu),

199

a glucuronide (+176 amu), and a methylated glucuronide (+ 190 amu).

200

The kinetics of hesperetin glucuronidation by Raw264.7 cells. The ability of

201

Raw264.7 cells to conjugate hesperetin was confirmed by incubating hesperetin with cell

202

homogenates and the cofactor UDPGA. The conjugation rates of hesperetin were found

203

to be dependent on the substrate concentrations, and the kinetics could be best described

204

by a Michaelis-Menten equation (Figure 2).46 The Michaelis constant Km and the

205

maximum reaction rate Vmax were calculated as 4.6 ± 0.3 µM and 129.9 ± 2.8

206

pmole/min/mg protein, respectively.

207

The time courses of hesperetin metabolism and PGD2/NO production in Raw264.7

208

cells. To further investigate whether metabolism of hesperetin can impact its anti-

209

inflammation efficacy in Raw264.7 cells, the time courses of hesperetin metabolism and

210

PGD2/NO production were determined (Figure 3, A - D). When 10 µM hesperetin was

211

incubated with Raw264.7 cells in the presence of 0.1 µg/mL LPS, PGD2 and NO started

212

to be produced by the cells at 4 hours after the incubation began, indicating that it would

213

take up to 4 hours for LPS to induce the expression of relevant enzymes and the enzymes

214

begin to function after that. PGD2 concentration in the medium burst to a high level and

215

stayed the same in the following 16 hours. In contrast, NO concentration in the medium



216

kept increasing from 4 to 24 hours. Without LPS induction, no PGE2 and NO were

217

produced by Raw264.7 cells.

218

The UPLC-UV analysis of the medium showed that hesperetin was rapidly metabolized

219

to its glucuronide by Raw264.7 cells, regardless of the presence or absence of LPS,

220

indicating that the expression of glucuronidation enzyme is constitutive and not altered

221

by LPS induction. In the presence of LPS, hesperetin concentration in the medium

222

decreased to the lowest at 8 hours, and then started to increase toward the end of

223

incubation. Part of hesperetin glucuronide was converted back to hesperetin, probably

224

due to the expression and excretion of glucuronidase activity from the LPS-stimulated

225

Raw264.7 cells.47 In the incubation without LPS, hesperetin concentration in the medium

226

kept decreasing, reached the trough after 8 hours, and did not show any “rebound” later,

227

because of no glucuronidase activity induction.

228

The effects of β-glucuronidase on anti-inflammatory efficacy of hesperetin. To

229

investigate the impact of rapid glucuronidation on the anti-inflammatory efficacy of

230

hesperetin in Raw264.7 cells, β-glucuronidase (10 units/mL) was introduced into the

231

incubation medium. During the incubation, hesperetin glucuronide was formed inside the

232

cells, excreted out, and then promptly hydrolyzed to hesperetin if β-glucuronidase was

233

present in the medium. The inhibitory effects of hesperetin on the production of PGD2

234

and NO were assessed and compared before and after the introduction of β-glucuronidase

235

(Fig.4). Interestingly, with respect to PGD2 production, β-glucuronidase significantly

236

enhanced the inhibitory effects of hesperetin at the concentrations of 6.25, 12.5 and 25

237

µM (p = 0.013, 0.041, and 0.012, respectively). However, at lower or higher hesperetin

238

concentrations, no significant impacts of β-glucuronidase on PGD2 production were 14 ACS Paragon Plus Environment

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observed. Meanwhile, as high as 50 µM hesperetin showed very limited inhibitory effects

240

on NO production in LPS-induced Raw264.7 cells, showing that hesperetin is much less

241

potent in inhibiting NO production. This is probably due to the impotent nature of

242

hesperetin to inhibit NO production in Raw264.7 cells, which is consistent with the result

243

in previous studies.29 The co-incubation with β-glucuronidase didn’t make any

244

improvement, which means that the metabolism of hesperetin is not the major reason for

245

the lack of efficacy in NO inhibition.

246



247

Discussion

248

In the current study, the results show that chemical stability and biotransformation during

249

incubation of phenolic compounds can be important determinants of their bioactivity in

250

Raw264.7 cells, a commonly used cell model to study anti-inflammatory efficacy of

251

natural compounds. The chemical stability of natural compounds in the aqueous solutions

252

depends on their intrinsic properties and the adopted incubation conditions as well.

253

Among all the phenolic compounds investigated in these study, some exhibit a decent

254

stability in physiological conditions (37 °C and pH 7.4), while others suffer a poor

255

stability and drastic degradation. In some previous studies, it has already been indicated

256

that the aqueous stability of some phenolic compounds may be an issue, especially at pH >

257

7.39 At pH 7 and 25 °C, quercetin was found to have a half-life of about 1 hour in an

258

aqueous solution, while under the same conditions only less than 10% of myricetin was

259

left after 1 hour incubation.40 Zhu et al. reported the instability of catechins, a subclass of

260

flavonoids present in tea green leaves, under incubation conditions which were very

261

similar to those in the current study.48 They found that more than 75% of total catechins

262

was degraded within a first half hour when incubated in Krebs-Ringer bicarbonate buffer

263

(pH = 7.4) at 37 °C. The stability issue of phenolic compounds brings uncertainty to the

264

bioactivity of phenolic compounds. It can be expected that the bioactivities of some

265

phenolic compounds can be impaired by their poor aqueous stability. It is also possible

266

that the observed bioactivity of certain tested compound in Raw264.7 cells actually

267

comes from its degradation product(s). In the in vitro studies, a comprehensive

268

understanding of compound stability under the incubation conditions may lead to the


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discovery of favorable degradation products as the new bioactive species, which is of

270

great value when exploiting the health benefits of natural compounds.

271

The existence of metabolic enzymes in the in vitro tools makes biotransformation of

272

incubated compounds possible, as exemplified by Raw264.7 cells. It should be noticed

273

that certain metabolic enzyme activities have been detected in murine macrophages,

274

including monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT).49, 50

275

Kawai et al. reported uridine 5'-diphospho-glucuronosyltransferase (UGT) activity in

276

Raw264.7 cell line.47 In this study, the major metabolites of phenolic compound observed

277

in the Raw264.7 cells were glucuronides. Taking hesperetin as an example, it was found

278

to be with a relatively good chemical stability but underwent extensive metabolism by

279

Raw264.7 cells. The major metabolite was identified as glucuronide. The time course and

280

concentration course indicated that Raw264.7 cells efficiently conjugate hesperetin to

281

glucuronide. It can be expected that hesperetin glucuronide is too hydrophilic to penetrate

282

the cell membrane, thus certain transporter(s) may help facilitate its excretion from the

283

cells.51 Glucuronidation and excretion of hesperetin can decrease its intracellular

284

concentrations in Raw264.7 cells, which will eventually weaken its bioactivity. In the

285

moderate concentration range between 6.25 and 25 µM, the metabolism of hesperetin by

286

Raw264.7 cells significantly altered its inhibitory effects on PGD2 production, and the

287

presence of β-glucuronidase in the medium enhanced the bioactivity of hesperetin

288

because formed hesperetin glucuronide was converted back to the parent. Meanwhile, no

289

effects were observed at either lower or higher hesperetin concentrations. At

290

concentrations of 3.12 µM or lower, hesperetin itself showed a limited potency in

291

inhibiting PGD2 production, and the addition of β-glucuronidase didn’t help very much. 17 ACS Paragon Plus Environment


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At a higher hesperetin concentration (50 µM), Raw264.7 cells were not efficient enough

293

to conjugate all the hesperetin in the medium, and thus a 100% inhibition of PGD2

294

production was achieved, even without the introduction of β-glucuronidase into the

295

medium. Given these observations, the potency of hesperetin to inhibit PGD2 production

296

in Raw264.7 cells could be determined by at least two aspects: intrinsic efficacy and

297

glucuronidation rate. The interactions between these two aspects lead to the result that

298

metabolism can significantly impact the ability of hesperetin to inhibit PGD2 production

299

at certain concentrations. Here, our study has clearly indicated that the bioactivities of

300

phenolic compounds can be impacted by their chemical and metabolic stabilities in

301

Raw264.7 cells.

302

The Raw264.7 cell line is derived from murine macrophages, and it is a good model to

303

study the anti-inflammation properties of natural compounds.22 The cell culture models

304

including Raw264.7 are very commonly used tools to investigate the bioactivities of

305

natural compounds in vitro. A wide range of phenolic natural compounds can inhibit the

306

prostaglandin production in Raw264.7 or other cell models at different concentration

307

levels. In Table 2, some previous reports are summarized and listed with the IC50 values

308

and the time lengths of incubation. Nowadays, as more and more evidence has been

309

accumulated to show the bioactivities of natural compounds in vitro, one of the

310

researchers’ primary concerns is whether and how the results from these in vitro studies

311

can be extrapolated to in vivo. Extensive efforts have been made to study the in vivo

312

efficacies of natural compounds in animals or human. It is already well known that the

313

efficacy of a natural compound in vivo is determined by not only its intrinsic bioactivity

314

but also its pharmaceutical characteristics in absorption, distribution, metabolism and 18 ACS Paragon Plus Environment

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Page 19 of 39


315

excretion (ADME).52, 53 For example, the major challenge for a flavonoid to be bioactive

316

in vivo can be its low oral bioavailability.54 Also, because phenolic compounds can be

317

excellent substrates for metabolic enzymes, especially UGTs and sulfotransferases

318

(SULTs), the most abundant species in circulation are usually their conjugates, not the

319

parents.55, 56 In addition, the interactions between the flavonoids and metabolic enzymes,

320

transporters and colonic microflora in the gastrointestinal tract also contribute to the

321

complexity in their in vivo disposition.57-59

322

It should be emphasized that, indeed, just like in the human body, ADME behaviors of

323

compounds also occur in cell culture models and play an important role in determining

324

their in vitro efficacies. However, the degradation, metabolism and excretion of natural

325

compounds are usually underinvestigated, especially in long-time incubations with cell

326

cultures. In Table 2, most studies provide in-depth insights into the effects the tested

327

compounds exert on the cells, but ignore what occurs to the compounds themselves in the

328

incubation systems. Thus, the information we get from an in vitro study may be actually

329

incomplete if the potential transformation of compounds in the incubation is unknown.

330

As for the current study, hesperetin glucuronidation that is observed in Raw264.7 cells

331

occurs extensively in vivo in human intestine or liver,60 and it may also occur in vivo in

332

human macrophages since UGT activity has been reported in the human

333

monocyte/macrophage THP-1 cell line, as well as mature macrophages derived from

334

monocytes in human blood.61, 62 However, for a specific compound, the ADME pathways

335

observed in an in vitro model do not necessarily represent the actual ones which are

336

functioning in vivo. Whether the in vitro degradation, biotransformation, and excretion of

337

compounds resemble that in vivo should be incorporated into the reseachers’ 19 ACS Paragon Plus Environment


338

considerations when predicting the translational value of results from a certain in vitro

339

study. A thorough understanding of both in vitro and in vivo ADME pathways may help

340

the researchers interpret the significance of in vitro observations and explain any

341

potential divergence between in vitro and in vivo studies concerning the bioactivity of a

342

natural compound.

343

In summary, in the long-time incubation with Raw264.7 cells, phenolic natural

344

compounds can undergo extensive degradation and/or cellular metabolism. The

345

glucuronidation of hesperetin impacts its efficacy in inhibiting the production of

346

prostaglandins in LPS-induced Raw264.7 cells. By considering stability and cellular

347

metabolism (as was done in this paper) of natural compounds, factors governing the

348

values of an in vitro study are likely to be fully appreciated and as such may result in

349

improved prediction of their in vivo effects. To improve translational potential, not only

350

the in vivo studies but also the in vitro ones should be more mechanistic to determine

351

ADME factors that affect the efficacy of a natural compound. We believe that such an

352

approach will help bridge the gap between in vitro and in vivo studies and thus better

353

exploit the health benefits of natural compounds including flavonoids and other

354

polyphenols.

355


Page 20 of 39

Page 21 of 39


356

Abbreviations

357

LPS, lipopolysaccharides; COX-2, cyclooxygenase-2; iNOS, inflammatory nitric oxide

358

synthase; PGD2, prostaglandin D2; NO, nitric oxide; MgCl2, magnesium chloride;

359

UDPGA, uridine 5'-diphospho-glucuronic acid; PBS, phosphate-buffered saline; DMEM,

360

Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; UPLC, ultra-performance

361

liquid chromatography; DAD, diode array detector; MS, mass spectrometer; MRM,

362

multiple reaction monitoring; MAO, monoamine oxidase; COMT, catechol-O-

363

methyltransferase;

364

absorption,

365

Acknowledgments

366

The authors thank those who provided help to our experiments in both Colleges of

367

Pharmacy and Natural Sciences and Mathematics at University of Houston.

UGT,

distribution,

uridine

5'-diphospho-glucuronosyltransferase;

metabolism

and

excretion;

368

369


SULT,

ADME,

sulfotransferase.


370

References

371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414

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Author information


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600

Corresponding Authors

601

*

602

*

603

Funding

604

The work was supported by a NIH NIGMS grant, GM070737.

605

Notes

606

The authors declare no competing financial interest.

Telephone: (832)274-8158. E-mail: [email protected]. Telephone: (713)382-6446. E-mail: [email protected].

607



Figure captions Figure 1. Metabolism of hesperetin by Raw264.7 cells and identification of metabolites. UPLC-UV profile of medium containing hesperetin (A) before incubation, (B) after incubation, and (C) after incubation and β-glucuronidase hydrolysis; UV absorbance wavelength of (D) hesperetin and (E) hesperetin glucuronide; and (F) MS2 scan spectrum of hesperetin glucuronide.

Figure 2. The kinetics of hesperetin glucuronidation by Raw264.7 cell homogenates. The relationship between substrate concentration and glucuronidation rates were fit by a Michaelis-Menten equation.

Figure 3. The time courses of PGD2/NO production and hesperetin glucuronidation by Raw264.7 cells. The contents of (A) PGD2 and (B) NO in the medium with or without LPS induction at different time points; the concentrations of hesperetin and hesperetin glucuronide in medium (C) with or (D) without LPS at different points.

Figure 4. The effects of β-glucuronidase on the efficacy of hesperetin at various concentrations to inhibit (A) PGD2 and (B) NO production in Raw264.7 cells.


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In incubation with Raw264.7 cells

#

Compound Name

Chemical stability (%)

1

Glycetin

2

Stability with cells (%)

Inhibition of PGD2 production (%)

Inhibition of NO production (%)

Metabolite(s) identified

101.3

86.1

57.2

0.0

ND

Chrysin

100.6

32.2

91.1

61.5

3

6,7,3'-Trihydroxyflavone

98.9

0.0

92.1

48.7

4

4-Hydroxy-6-methylcoumarin

96.7

51.7

46.8

7.2

Glucuronide Methylated metabolite; Glucuronide; Methylated glucuronide ND

5

Ferulic acid

96.6

68.0

80.8

15.8

ND

6

Equol

96.5

83.2

83.3

7.9

ND

7

7,2'-Dihydroxyflavone

96.4

25.1

22.7

0.0

Glucuronide

8

Daidzein

95.7

84.3

78.1

18.8

ND

9

7-hydroxyflavone

95.0

70.2

93.3

18.3

ND

10

7-Hydroxy-6-methoxyisoflavone

94.1

68.5

66.6

17.8

ND

11

4'-Hydroxychalcone

92.4

1.9

80.1

20.9

ND

12

7-Hydroxyflavanone

92.1

69.2

32.0

6.7

ND

13


91.9

24.0

35.1

21.5

Glucuronide

14

7-Hydroxy-2'-methoxyflavone

91.2

82.8

91.9

0.0

ND

15

Genistein

90.8

73.7

82.3

34.2

Glucuronide

16

Scopoletin

90.6

44.3

23.7

5.4

Glucuronide

17


89.3

76.4

83.8

12.9

ND

18

Formononetin

87.9

59.0

82.6

22.4

ND

19

Wogonin

87.6

63.7

100.0

0.0

ND

20

Apigenin

86.3

44.9

71.7

49.0

Glucuronide

21

8-Hydroxy-7-methoxyflavone

85.8

5.6

24.1

18.1

Glucuronide

22


84.9

58.0

50.7

14.3

Glucuronide

23

7-Hydroxy-5-methyflavone

84.0

80.0

64.5

21.5

ND

24

4'-hydroxyflavone

84.0

76.7

96.9

18.8

ND

25

7-Hydroxy-2-chromanone

83.6

86.9

48.5

10.2

ND

26

Hesperetin

82.3

14.1

38.0

3.1

Glucuronide

27

7-Hydroxy-4'-methoxyflavone

81.9

69.9

76.9

17.1

ND

28

3'-hydroxyflavone

81.7

43.4

35.9

10.8

Glucuronide

29

10-Hydroxywarfarin

81.0

72.4

78.6

3.6

ND

30

1-Naphthol

77.4

0.0

92.0

2.3

Glucuronide

31

77.4

36.8

11.3

24.4

Glucuronide

77.3

65.5

34.9

7.8

ND

33

Naringenin 5,7-Dihydroxy-3'4'5'trifmethoxyflavone 4'-hydroxy-3'-methoxyflavanone

76.3

10.2

70.2

19.5

Glucuronide

34

3,4'-Dimethoxy-3',5,7-

74.2

0.0

24.8

18.5

Glucuronide

32



Page 30 of 39

trihydroxyflavone 35

37

4-Hydroxy-7-methoxyflavone 6-Hydroxy-7-methoxy-4phenylcoumarin 6-hydroxyflavone

38


69.6

ND

ND

ND

ND

39

Diosmetin

69.1

ND

ND

ND

ND

40

5,7-Dihydroxyflavone

67.3

ND

ND

ND

ND

41


67.0

ND

ND

ND

ND

42


66.4

ND

ND

ND

ND

43

2'-hydroxyflavone

64.0

ND

ND

ND

ND

44

Datiscetin

63.4

ND

ND

ND

ND

45

6-Methoxyluteolin

61.8

ND

ND

ND

ND

36

70.6

52.9

40.8

21.9

Glucuronide

70.1

28.3

87.5

7.6

Glucuronide

69.7

ND

ND

ND

ND

46

Eugenol

59.8

ND

ND

ND

ND

47

Prunetin

59.8

ND

ND

ND

ND

48

3'4'-Dihydroxyflavone

55.9

ND

ND

ND

ND

49

Chrysoeriol

55.9

ND

ND

ND

ND

50

2-Hydroxychalcone

51.5

ND

ND

ND

ND

51


46.7

ND

ND

ND

ND

52


38.3

ND

ND

ND

ND

53

32.5

ND

ND

ND

ND

32.1

ND

ND

ND

ND

29.4

ND

ND

ND

ND

21.3

ND

ND

ND

ND

20.2

ND

ND

ND

ND

58

3,7-Dihydroxyflavone 3,7-Dihydroxy-3'4'dimethoxyflavone 5,4'-Dihydroxy-7methoxyflavone 5-hydroxyflavone 3,4-Diphenyl-7hydroxycoumarin Luteolin

16.0

ND

ND

ND

ND

59


14.2

ND

ND

ND

ND

60

Hinokiflavone

6.0

ND

ND

ND

ND

61

Galangin

5.6

ND

ND

ND

ND

62

Bavachinin

5.4

ND

ND

ND

ND

63

Kaempferol

4.7

ND

ND

ND

ND

64

Phloretin

1.7

ND

ND

ND

ND

54 55 56 57

65

Syringetin

0.0

ND

ND

ND

ND

66

3-Hydroxyflavone

0.0

ND

ND

ND

ND

67


0.0

ND

ND

ND

ND

68

Quercetin

0.0

ND

ND

ND

ND

69

Resveratrol

0.0

ND

ND

ND

ND

70

Baicalein

0.0

ND

ND

ND

ND

71

Fisetin

0.0

ND

ND

ND

ND

72


0.0

ND

ND

ND

ND

73


0.0

ND

ND

ND

ND

74

Curcurmin

0.0

ND

ND

ND

ND


Page 31 of 39


75

Bisdemethoxycurcumin

0.0

ND

ND

ND

ND

76

Demethoxycurcurmin

0.0

ND

ND

ND

ND

77

Geraldol

0.0

ND

ND

ND

ND

ND, not determined. Table 1. The Chemical Stability of 77 Natural Phenolic Compounds in DMEM and Their Metabolic Stability and Anti-inflammatory Efficacies in Raw264.7 Cells.



Page 32 of 39

Compounds

Cells

COX-2 induced by

Incubation time (hours)

hesperidin

Raw264.7

200 ng/mL LPS

6.5

10 µM < IC50< 20 µM

63

sophoraflavanone G

Raw264.7

1 µg/mL LPS

24

IC50 ≈ 1 µM

64

apigenin

Raw264.7

50 ng/mL LPS

24

5 µM < IC50 < 15 µM

65

genistein

Raw264.7

50 ng/mL LPS

24

5 µM < IC50 < 15 µM

65

kaempferol

Raw264.7

50 ng/mL LPS

24

5 µM < IC50 < 15 µM

65

amentoflavone

A549

IL-1β/IFNγ/TNFα (20 ng/mL of each)

18

1 µM < IC50 < 10 µM

66

tectorigenin

rat peritoneal macrophages

12-O-tetradecanoylphorbol 13-acetate (TPA, 16.2 nM) or thapsigargin (15.4 nM)

8

IC50 ≈ 3 µM (TPA) or < 3 µM (thapsigargin)

tectoridin

rat peritoneal macrophages

12-O-tetradecanoylphorbol 13-acetate (TPA, 16.2 nM) or thapsigargin (15.4 nM)

8

IC50 ≈ 30 µM (TPA and thapsigargin)

wogonin

Raw264.7

1 µg/mL LPS

24

0.1 µM < IC50 < 0.5 µM

68

oroxylin A

Raw264.7

100 ng/mL LPS

24

IC50 < 5 µg/mL

69

apigenin

J774A.1

1 µg/mL LPS

24

IC50 ≈ 50 µM

70

naringenin

J774A.1

1 µg/mL LPS

24

5 µM < IC50 < 50 µM

70


Potency

Ref

67

67

Page 33 of 39


galangin

J774A.1

1 µg/mL LPS

24

IC50 ≈ 5 µM

70

quercetin

J774A.1

1 µg/mL LPS

24

0.5 µM < IC50 < 5 µM

70

morin

J774A.1

1 µg/mL LPS

24

IC50 > 50 µM

70

silymarin

J774A.1

1 µg/mL LPS

24

5 µM < IC50 < 50 µM

70

wogonin

Raw264.7

1 µg/mL LPS

24

IC50 = 0.3 µM

71

rutin

Raw264.7

50 ng/mL LPS

12

IC50 > 80 µM

72

quercetin

Raw264.7

50 ng/mL LPS

12

IC50 < 40 µM

72

wogonin

Raw264.7

50 ng/mL LPS

12

IC50 < 40 µM

72

rutin

Raw264.7

100 ng/mL LPS

24

IC50 > 40 µM

73

quercetin

Raw264.7

100 ng/mL LPS

24

IC50 > 40 µM

73

quercetin pentaacetate

Raw264.7

100 ng/mL LPS

24

IC50 < 20 µM

73

nobiletin

human synovial fibroblasts

1 ng/mL IL-1α

24

IC50 < 4 µM

74

isoliquiritigenin

Raw264.7

LPS (concentration not indicated)

24

IC50 = 1 µM

75

4,3’,5’trihydroxystilbene

murine lung fibroblast

None (constitutive)

1

IC50 = 2.16 µM

76



Page 34 of 39

4,3’-dihydrox-5’methoxylstilbene


None (constitutive)

1

IC50 = 2.21 µM

76

4-hydrox-3’5’dimethoxylstilbene


None (constitutive)

1

IC50 = 1.29 µM

76

Resveratrol

human mammary epithelial cells

50 ng/mL phorbol ester

4.5

IC50 < 2.5 µM

77

Table 2. Representative Anti-inflammatory Natural Phenolic Compounds that Showed Efficacy in Inhibiting Prostaglandin Production in Raw264.7 or Other Cells.


Page 35 of 39


Figure 1



Figure 2


Page 36 of 39

Page 37 of 39


Figure 3



Figure 4 608


Page 38 of 39

Page 39 of 39


Table of Contents Graphic


Metabolism of Phenolic Compounds in LPS-stimulated Raw264. 7

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