Anti-inflammatory Effect of Pomelo Peel and Its Bioactive Coumarins

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Bioactive Constituents, Metabolites, and Functions

Anti-inflammatory effect of Pomelo peel and its bioactive coumarins Yun-Li Zhao, Xiong-Wu Yang, Bai-Fen Wu, Jian-Hua Shang, Ya-Ping Liu, Dai Zhi, and Xiao-Dong Luo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02511 • Publication Date (Web): 18 Jul 2019 Downloaded from pubs.acs.org on July 19, 2019

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Journal of Agricultural and Food Chemistry

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Anti-inflammatory effect of Pomelo peel and its bioactive coumarins

2 3

Yun-Li Zhao†,§‖, Xiong-Wu Yang§‖, Bai-Fen Wu‡, Jian-Hua Shang§, Ya-Ping Liu§, Zhi-Dai†, Xiao-Dong Luo†,§,*

4

†

Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education

5

and Yunnan Province, School of Chemical Science and Technology, Yunnan

6

University, Kunming 650091, People’s Republic of China

7

§

Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China

8 9 10

State Key Laboratory of Phytochemistry and Plant Resources in West China,

‡

Yunnan University of Chinese Medicine, Yunnan Province, Kunming 650500, P. R. China

11

__________________________________________________

12

* Corresponding author. Tel.: +86 871 65223177; fax: +86 871 65220227.

13 14

E-mail address: [email protected] (X.-D. Luo) 1 These

authors contributed equally.

ACS Paragon Plus Environment


15

ABSTRACT: Citrus grandis (L.) Osbeck is a popular fruit cultivated around the

16

world and its peels are sometimes used for the treatment of cough, abdominal pain

17

and indigestion in China. However, the peel is discarded after fruits consumption in

18

most cases and its chemical constituents and biological activities haven’t been

19

validated before. The present study focused on evaluation of the chemical and

20

pharmacological profile of coumarins from peels of C. grandis against inflammation.

21

The extracts and phytochemicals from peels of C. grandis were prepared and

22

anti-inflammatory activities were carried out in vivo and in vitro, including inhibiting

23

xylene-induced ear edema, carrageenan-induced paw edema in mice, and the

24

production of inflammatory cytokines (IL-1, PGE2, TNF-) in lipopolysaccharide

25

(LPS) induced RAW 264.7 cells. Results indicated that methanolic extract (ME),

26

ethyl acetate fraction (EAC) and four major coumarins (compounds 7, 8, 13, and 16)

27

inhibited swelling induced by xylene and carrageenan respectively in vivo.

28

Furthermore, 18 coumarins inhibited inflammatory factors secretion in macrophages

29

primed by LPS, in which compounds 4, 6, 7, 10, 17 showed the most pronounced

30

change, which were comparable to dexamethasone (DXM). In summary, peel of C.

31

grandis showed an anti-inflammatory effect, and coumarins compounds were

32

responsible for the regulating inflammatory mediators and cytokines, which might

33

provide a novel nutritional strategy for inflammatory diseases.

34

KEYWORDS: C. grandis; coumarins; anti-inflammatory effect; lipopolysaccharide


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INTRODUCTION

36

Pomelo (Citrus grandis (L.) Osbeck), belonging to the Rutaceae family, is one of

37

the most important fruit crops grown in the world 1. Owing to the refreshing texture,

38

aromatic smell, soft juicy pulp and a pleasant sugar to acid ratio, pomelo fruits and

39

fruit juice are becoming a popular fruit and beverage of the daily life in many

40

countries 2. Pomelo has a history of over 3000 years and distributed pervasively in

41

south region of China, not only a fruit but also used in folk medicine against

42

hyperlipemia 3, cardiovascular disease 4, nervous system disease 5, peroxidation

43

damage 2, and even ameliorate fatigue, loss of energy, lack of vitality, bruising,

44

wounds, acne or osteoarthritis 6. Pomelo peels, the immature or nearly mature dry

45

outer skin of C. grandis, are the main “Citris Grandis Exocarpium” of pomelo fruits

46

recorded in the Chinese Pharmacopoeia accounting for one third or even two fifths of

47

grapefruits, which have long been used as herbal remedies to promote blood

48

circulation and remove blood stasis in diseases caused by blood stagnation 7. It not

49

only contains vitamins, water, minerals, but also many physiological active

50

ingredients, such as flavonoids, coumarins, volatile oils and limonoids constituents

51

8-10.

52

this fruits are consumed, which not only constitutes a possible environmental

53

pollution but also leads to waste of natural resources. If the more bioactivities of

54

pomelo peels could be identified, this agricultural waste can be recycled and reused,

55

thereby increasing the commercial value of it.

However, pomelo peels are commonly discarded as daily waste materials after



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Coumarin-Benzopyrone, a lactone of cis-O-hydroxycinnamic acid, is the basic

57

nucleus of coumarin compounds and one of main chemical constituents in pomelo

58

peels, which possess various bioactivities. For example, prenylated coumarins have

59

been reported for the antitumor activities

60

treatment for skin disease for a long time 13, 14. Even more activities have been found

61

as antimicrobial 15, antiviral 16, antioxidant 17, antiarrhythmic 18, anticoagulation 19. To

62

evaluate anti-inflammatory activity of pomelo peel related to the folk use,

63

phytochemical and pharmacological investigations were carried out. As a result, 18

64

coumarins,

65

5-(6-Hydroxy-7-methoxy-3,8-dimethyl-2E-2-octenyloxy)psoralen

66

5-(6-Hydroxy-3,7-dimethyl-2E,7-octadienyloxy)psoralen

67

8-(6-Hydroxy-7-methoxy-3,7-dimethyl-2E-octenyloxy)psoralen

68

8-(6,7-Dihydroxy-3,7-dimethyl-2E-octenyloxy)psoralen (6)

69

Marmin (8)

70

(9)

71

Umbelliferone

72

7-Methoxy-8-(2-formyl-2-methylpropyl)coumarin (14)

73

Meranzin hydrate (16)

74

All the structures were determined by comparing their experimental data with those

75

described in the literature. Furthermore, some of them were responsible for as

76

anti-inflammatory agents both in vitro and in vivo.

77

MATERIALS AND METHODS

Bergamottin

26,

27,

11, 12,

furanocoumarins was used as the

20,

(1)

Bergaptol

(2)

21,

(3)

22, 23,

(4)

24,

24,

(5) Auraptene (7)

25,

7-[6-Hydroxy-7-methoxy-3,7-dimethyl-(2E)-2-octenyloxy]coumarine 7-(6-Hydroxy-3,7-dimethyl-2E,7-octadienyloxy)coumarin (11)

33,

29,

Auraptenol

Toddanone (17)

34,

(12)

30, 31,

Isoauraptene

(13)

Yuehgesin B (15)

Omphalocarpin (18)


(10)28,

35

30

32,

were isolated.

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78

Plant materials. The peels of C. grandis were collected in October 2017 in

79

Kunming, Yunnan Province, People’s Republic of China, and identified by Dr.

80

Ya-Ping Liu, State Key Laboratory of Phytochemistry and Plant Resources in West

81

China, Kunming Institute of Botany, Chinese Academy of Sciences. A voucher

82

specimen (No. Luo. 20171021) has been deposited in the State Key Laboratory of

83

Phytochemistry and Plant Resources in West China, Kunming Institute of Botany.

84

Extracts and coumarins fractions preparation. The fresh peels of C. grandis

85

were (20.7 kg) cut to pieces and extracted with 90% methanol (MeOH) three times

86

(each time for 65 L, 1 day) at room temperature. The solvent was evaporated in vacuo

87

to afford a residue (608 g) as methanol extract, which was suspended in hot water,

88

and then the solid were removed by filtering the solution. The filtrate was partitioned

89

with EtOAc to afford EtOAc fraction (coumarins fractions). Then the sample was

90

kept cooling and standing for a period of time and the solid matter were removed by

91

filtering the sample. The filtrate was partitioned with ethyl acetate three times (each

92

time for 10 L, 1 day) and after evaporation of the solvent in reduced pressure, ethyl

93

acetate fraction (coumarins fractions) (32 g) was obtained.

94

Isolation and purification of coumarins. The EtOAc fraction was subjected to

95

chromatography column on silica gel eluted with CHCl3-MeOH (from 100:1 to 4:1,

96

v/v) to afford 6 fractions (Fr.Ⅰ - Fr.Ⅵ). Compounds (Figure 1) were obtained from

97

fractions Fr.Ⅰ - Fr.Ⅵ by silica gel, RP-C18, Sephadex LH-20 and semipreparative

98

HPLC. Fraction Fr.Ⅰ was purified by Sephadex LH-20 eluting with 100% CH3OH to



99

afford three fractions (Fr.Ⅰ-1 to Fr.Ⅰ-3), then the fraction Fr.Ⅰ-3 was further

100

purified over silica gel column eluting with PE/EtOAc (8:1, v/v) and compounds 1

101

(28.4 mg), 2 (10.2 mg) and the residue were obtained, then the residue was purified

102

by silica gel column and eluted by PE/Acetone (10:1, v/v) to afford compound 7 (608

103

mg). Fraction Fr.Ⅱ was separated by silica gel column eluting with PE/EtOAc (8:1,

104

v/v) to provide compound 5 (5 mg) and fractions Fr.Ⅱ-1 and Fr.Ⅱ-2. Fraction

105

Fr.Ⅱ-1 then was purified by Sephadex LH-20 eluting with 100% CH3OH and was

106

further prepared by HPLC using the mobile phase of CH3CN/H2O (68:32, v/v) at a

107

flow rate of 2.5 mL/min to give compound 12 (40 mg, tR 18 min). Fraction Fr.Ⅱ-2

108

was separated by silica gel column eluting with PE/Acetone (10:1, v/v) to afford

109

compound 17 (10.1 mg). Fraction Fr.Ⅲ afforded three fractions Fr.Ⅲ-1, Fr.Ⅲ-2 and

110

Fr.Ⅲ-3 by silica gel column and eluted by CHCl3/EtOAc (10:1, v/v). Compounds 3

111

(27.6 mg) and 4 (5.0 mg) were obtained from Fr.Ⅲ-2 by HPLC eluting with

112

CH3CN/H2O (65:35, v/v) at a flow rate of 2.5 mL/min. Fraction Fr.Ⅲ-3 was separated

113

by silica gel column eluting with CHCl3/EtOAc (3:1, v/v) to provide compound 13

114

(290 mg) and 14 (5.8 mg). Fraction Fr. Ⅳ was separated by Sephadex LH-20 eluting

115

with 100% CH3OH to afford two fractions (Fr. Ⅳ-1 and Fr. Ⅳ-2). After a period of

116

time, fraction Fr. Ⅳ-1 precipitated crystallization and then the crystal was identified

117

to compounds 8 (350 mg). Compounds 15 (9.7 mg, tR 21 min) and 18 (1.3 mg, tR 23

118

min) were obtained from fraction Fr. Ⅳ-2 by HPLC using the mobile phase of

119

CH3CN/H2O (55:45, v/v) at a flow rate of 2.5 mL/min. Fraction Fr.Ⅴ was separated

120

by silica gel column and eluted with CH3Cl/Acetone (3:1, v/v) to provide three


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121

fractions (Fr.Ⅴ-1 to Fr.Ⅴ-3). Fraction Fr.Ⅴ-1 was purified by HPLC using the

122

mobile phase of CH3CN/H2O (65:35, v/v) at a flow rate of 2.5 mL/min was gave

123

compounds 6 (14.7 mg, tR 19 min) and 9 (9.2 mg, tR 21 min). Otherwise, compound

124

16 (1.01 g, tR 33 min) was obtained from Fr.Ⅴ-2 by HPLC using the mobile phase of

125

CH3CN/H2O (55:45, v/v) at a flow rate of 2.5 mL/min. Fraction Fr.Ⅵ was separated

126

by RP-C18 eluting with CH3OH/H2O (from 10:90 to 100:0, v/v) to yield three fractions

127

(Fr.Ⅵ-1 to Fr.Ⅵ-3). Compounds 10 (4.0 mg, tR 20.5 min) and 11 (6.1 mg, tR 23 min)

128

were obtained from fraction Fr.Ⅵ-2 by HPLC using the mobile phase of CH3CN/H2O

129

(65:35, v/v) at a flow rate of 2.5 mL/min. The properties of the compounds were

130

presented below (the entire data set is contained in the supporting information).

131

Bergamottin (1). The molecular formula was obtained as C21H22O4 and the

132

separation gave of 28.4 mg white powder. The 1H NMR(400MHz, CDCl3) and EIMS

133

were consistent with previously published data

134

supported by

135

C-9), 143.4 (d, C-4), 142.3 (s, C-3'), 131.9 (s, C-7'), 128.6 (s, C-5), 123.6 (d, C-6'),

136

118.4 (d, C-2'), 113.2 (d, C-6), 112.9 (d, C-3), 112.4 (s, C-10), 101.6 (d, C-8), 65.4 (t,

137

C-1'), 39.5 (t, C-4'), 26.2 (d, C-5'), 25.6 (q, C-8'), 17.7 (q, C-9'), 16.7 (q, C-10').

13C

20.

The identification was further

NMR (100MHz, CDCl3): 162.2 (s, C-2), 161.3 (s, C-7), 155.8 (s,

138

Bergaptol (2). The molecular formula was obtained as C11H6O4 and the separation

139

gave of 10.2 mg white powder. The 1H NMR(400MHz, CDCl3) and EIMS were in

140

agreement with a previous work

141

NMR (100 MHz, CDCl3): 160.4 (s, C-2), 157.1 (s, C-7), 152.7 (s, C-9), 147.9 (s, C-5),

21.

The identification was further supported by 13C



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142

145.0 (d, C-12), 139.8 (d, C-4), 112.5 (s, C-6), 110.9 (d, C-3), 104.7 (d, C-11), 103.7

143

(s, C-10), 91.0 (d, C-8). 5-(7-Hydroxy-8-methoxy-3,8-dimethyl-2-enyloxy)psoralen

144

(3).

It

had

the

145

molecular formula of C22H26O6 and the separation gave of 27.6 mg white powder. The

146

1H

147

The identification was further supported by 13C NMR (100 MHz, CDCl3): 161.3 (s,

148

C-2), 158.1 (s, C-7), 152.6 (s, C-9), 148.9 (s, C-5), 144.9 (d, C-12), 143.1 (s, C-3'),

149

139.6 (d, C-4), 119.1 (d, C-2'), 114.3 (s, C-6), 112.5 (d, C-3), 107.6 (s, C-10), 105.0

150

(d, C-11), 94.2 (d, C-8), 77.3 (s, C-7'), 76.3 (d, C-6'), 69.7 (t, C-1'), 49.19 (q,

151

7'-OCH3), 36.6 (t, C-4'), 29.2 (t, C-5'), 20.7 (q, C-8'), 18.7 (q, C-9'), 16.7 (q, C-10').

NMR(400MHz, CDCl3) and EIMS were the same as those reported in literature 22.

5-(6-Hydroxy-3,7-dimethyl-2E,7-octadienyloxy)psoralen

152

(4).

The

molecular

153

formula was assigned as C21H22O5 and the separation gave of 5 mg white powder. The

154

1H

NMR(400MHz, CDCl3) and EIMS were identical with those reported in literature

155

23.


156

C-2), 158.1 (s, C-7), 152.7 (s, C-9), 148.9 (s, C-5), 147.3 (s, C-7'), 144.9 (d, C-12),

157

142.8 (s, C-3'), 139.6 (d, C-4), 119.1 (d, C-2'), 114.2 (s, C-6), 112.6 (d, C-3), 111.3 (t,

158

C-8'), 107.5 (s, C-10), 105.0 (d, C-11), 94.3 (d, C-8), 75.4 (d, C-6'), 69.7 (t, C-1'),

159

35.5 (t, C-4'), 32.7 (t, C-5'), 17.6 (q, C-9'), 16.7 (q, C-10').

160

8-(6-Hydroxy-7-methoxy-3,7-dimethyl-(2E)-2-octenyloxy)psoralen (5). It gave a

161

molecular formula C22H26O6 and the separation gave of 5 mg white powder. The

162

NMR(400MHz, CDCl3) and EIMS agreed with the result reported before


24.

1H

The

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163

identification was further supported by 13C NMR (100 MHz, CDCl3): 160.3 (s, C-2),

164

148.5 (s, C-7), 148.3 (d, C-12), 145.8 (d, C-4), 143.9 (s, C-9), 143.8 (s, C-3'), 130.8 (s,

165

C-8), 126.1 (s, C-6), 119.4 (d, C-2'), 116.8 (s, C-10), 114.8 (d, C-3), 114.5 (d, C-5),

166

107.5 (s, C-11), 77.2 (s, C-7'), 75.0 (d, C-6'), 69.7 (t, C-1'), 49.1 (q, 7'-OCH3), 36.7 (t,

167

C-4'), 29.5 (t, C-5'), 21.9 (q, C-8'), 19.9 (q, C-9'), 16.7 (q, C-10').

168

8-(6,8-Dihydroxy-3,8-dimethyl-2-octenyloxy)psoralen (6) gave the molecular

169

formula of C21H24O6 and 14.7 mg white powder. The 1H NMR (400MHz, CDCl3) and

170

EIMS agreed with the previous studies 24. The identification was further supported by

171

13C

172

C-4), 143.9 (s, C-9), 142.8 (s, C-3'), 131.5 (s, C-8), 125.9 (s, C-6), 120.1 (d, C-2'),

173

116.5 (s, C-10), 114.7 (d, C-5), 113.4 (d, C-3), 107.8 (s, C-11), 77.7 (d, C-6'), 73.0 (t,

174

C-1'), 70.1 (s, C-7'), 36.4 (t, C-4'), 29.3 (t, C-5'), 26.4 (q, C-8'), 23.1 (q, C-9'), 16.4 (q,

175

C-10').

NMR (100 MHz, CDCl3): 160.7 (s, C-2), 148.7 (s, C-7), 146.7 (d, C-12), 144.5 (d,

176

Auraptene (7) exhibited a molecular formula of C19H22O3 and the separation gave

177

of 608 mg white powder. The 1H NMR (400 MHz, CDCl3) and EIMS were

178

correspond with the report of literatures previously 25. The identification was further

179

supported by 13C NMR (100 MHz, CDCl3): 162.2 (s, C-2), 161.3 (s, C-7), 155.8 (s,

180

C-9), 143.4 (d, C-4), 142.3 (s, C-3'), 131.9 (s, C-7'), 128.6 (d, C-5), 123.6 (d, C-6'),

181

118.4 (d, C-2')，113.2 (d, C-6), 112.9 (d, C-3), 112.4 (s, C-10), 101.6 (d, C-8), 65.4 (t,

182

C-1'), 39.5 (t, C-4'), 26.2 (d, C-5'), 25.6 (q, C-8'), 17.7 (q, C-9'), 16.7 (q, C-10').

183

Marmin (8) possessed a molecular formula of C19H24O5 and the separation gave of



Page 10 of 35

184

350 mg white powder. The 1H NMR (400 MHz, CDCl3) and EIMS agreed well with

185

the observed values in the literature 26. The identification was further supported by 13C

186

NMR (100 MHz, CDCl3): 162.2 (s, C-7), 160.8 (s, C-2), 155.8 (s, C-9), 144.8 (d,

187

C-4), 142.5 (s, C-3'), 129.9 (d, C-5), 118.8 (d, C-2'), 113.3 (d, C-3), 112.8 (d, C-6),

188

112.7 (s, C-10), 101.8 (d, C-8), 77.4 (d, C-6'), 72.0 (s, C-7'), 65.7 (t, C-1'), 36.8 (t,

189

C-4'), 29.4 (t, C-5'), 26.9 (q, C-8'), 24.9(q, C-9'), 17.1(q, C-10').

190

The

molecular

formula

C20H26O5

of

191

7-[6-Hydroxy-7-methoxy-3,7-dimethyl-(2E)-2-octenyloxy]coumarine (9) and the

192

separation gave of 9.2 mg white powder. The 1H NMR (400 MHz, CDCl3) and EIMS

193

were in correlation with the published data 27. The identification was further supported

194

by 13C NMR (100 MHz, CDCl3): 162.1 (s, C-7), 161.3 (s, C-2), 155.9 (s, C-9), 143.5

195

(s, C-4), 142.4 (s, C-3'), 128.7 (d, C-5), 118.5 (d, C-2'), 113.2 (d, C-3), 113.0 (d, C-6),

196

112.4 (s, C-10), 101.6 (d, C-8), 77.0 (s, C-7'), 76.2 (t, C-6'), 65.5 (s, C-1'), 49.1 (q,

197

7'-OCH3), 36.6 (t, C-4'), 29.2 (t, C-5'), 20.7(q, C-8'), 18.8 (q, C-9'), 16.9 (q, C-10').

198

7-(6-Hydroxy-3,7-dimethyl-2E,7-octadienyloxy)coumarin (10) was identified as

199

C19H22O4 and the separation gave of 4.0 mg white powder. The 1H NMR (400 MHz,

200

CDCl3) and EIMS were in agreement with the reported in literature

201

identification was further supported by 13C NMR (100 MHz, CDCl3): 162.1 (s, C-7),

202

161.3 (s, C-2), 156.1 (s, C-9), 147.3 (s, C-7'), 143.4 (s, C-4), 142.1 (s, C-3'), 128.7 (d,

203

C-5), 118.5 (d, C-2'), 113.2 (d, C-3), 113.0 (d, C-6), 112.5 (t, C-8'), 111.3 (s, C-10),

204

101.6 (d, C-8), 75.4 (d, C-6'), 65.4 (s, C-1'), 35.5 (t, C-4'), 32.8 (t, C-5'), 17.6 (q, C-9'),

205

16.8 (q, C-10').


28.

The

Page 11 of 35


Umbelliferone (11) was assigned the molecular formula of C9H6O3 and the

206

1H

207

separation gave of 6.1 mg white powder. The

208

EIMS consistent with literature data

209

13C

210

C-4), 130.2 (d, C-5), 113.6 (d, C-3), 111.9 (d, C-6), 111.7 (s, C-10), 102.6 (d, C-8).

29.

NMR (400 MHz, CDCl3) and

The identification was further supported by


211

Auraptenol (12) exhibited a molecular formula of C15H16O4 and the separation

212

gave of 40 mg white powder. The 1H NMR (400 MHz, CDCl3) and EIMS agree well

213

with the observed values in the literature 30. The identification was further supported

214

by 13C NMR (100 MHz, CDCl3): 161.2 (s, C-2), 160.1 (s, C-7), 153.5 (s, C-9), 147.2

215

(s, C-3'), 143.9 (d, C-4), 127.0 (d, C-5), 115.0 (s, C-8), 112.9 (d, C-3), 112.8 (s, C-10),

216

110.5 (t, C-4'）, 107.4 (d, C-6), 75.2 (d, C-2'), 56.2 (q, 7-OCH3), 29.4 (t, C-1'), 18.0

217

(q, C-5').

218

Isoauraptene (13) had a molecular formula of C15H16O4 and the separation gave of

219

290 mg white powder. The 1H NMR (400 MHz, CDCl3) and EIMS were in agreement

220

with a previous work

221

MHz, CDCl3): 210.8 (s, C-2'), 160.9 (s, C-2), 160.4 (s, C-7), 153.2 (s, C-9), 143.8 (d,

222

C-4), 127.6 (d, C-5), 112.9 (s, C-10), 112.8 (d, C-3), 111.9 (s, C-8), 107.3 (d, C-6),

223

56.1 (q, 7-OCH3), 40.9 (d, C-3'), 34.7 (t, C-1'), 18.4 (q, C-4'), 18.4(q, C-5').

30.


13C

NMR (100

224

7-Methoxy-8-(2-formyl-2-methylpropyl)coumarin (14). The molecular formula

225

was obtained as C15H16O4 and the separation gave of 5.8 mg white powder. The 1H

226

NMR (400 MHz, CDCl3) and EIMS were consistent with previously published data 31.



Page 12 of 35

227


228

-CHO), 160.9 (s, C-2), 160.4 (s, C-7), 153.2 (s, C-9), 143.8 (d, C-4), 127.4 (d, C-5),

229

114.0 (s, C-10), 113.1 (d, C-3), 112.9 (s, C-8), 107.3 (d, C-6), 55.8 (q, 7-OCH3), 47.4

230

(s, C-2'), 30.2 (t, C-1'), 21.5 (q, C-4'), 21.4 (q, C-5').

231

Yuehgesin B (15) exhibited a molecular formula of C16H20O5 and the separation

232

gave of 9.7 mg white powder; 1H NMR (400 MHz, CDCl3) and EIMS were identical

233

with that reported in literature

234


235

C-4), 126.7 (d, C-5), 116.2 (s, C-8), 113.1 (d, C-3), 113.1 (s, C-10), 107.4 (d, C-6),

236

77.2 (s, C-3'), 76.6 (d, C-2'), 56.2 (q, 7-OCH3), 49.4 (q, 3'-OCH3), 25.1 (t, C-1'), 21.0

237

(q, C-4'), 20.3 (q, C-5').

32.


13C

238

Meranzin hydrate (16) gave the molecular formula of C15H18O5 and 1.01 g white

239

powder. The 1H NMR (400 MHz, CDCl3) and EIMS were correspond with the report

240

of literatures previously 33. The identification was further supported by 13C NMR (100

241

MHz, CDCl3): 161.2 (s, C-2), 160.5 (s, C-7), 153.4 (s, C-9), 143.9 (d, C-4), 127.0 (d,

242

C-5), 115.8 (s, C-8), 113.1 (d, C-3), 113.1 (s, C-10), 107.4 (d, C-6), 78.3 (d, C-2'),

243

73.1 (s, C-3'), 56.3 (q, 7-OCH3), 26.1 (q, C-4'), 25.6 (t, C-1'), 24.1 (q, C-5').

244

Toddanone (17) possessed a molecular formula of C16H18O5 and the separation

245

gave of 10.1 mg white powder. The 1H NMR (400 MHz, CDCl3) and EIMS agreed

246

well with the observed values in the literature

247

supported by 13C NMR (100 MHz, CDCl3): 211.3 (s, C-2'), 161.3 (s, C-5), 161.2 (s,

34.

The identification was further


Page 13 of 35


248

C-2), 156.2 (s, C-7), 153.9 (s, C-9), 138.9 (d, C-4), 110.8 (d, C-3), 104.1 (s, C-6),

249

103.7 (s, C-10), 90.2 (d, C-8), 56.0 (q, 5-OCH3), 55.9 (q, 7-OCH3), 40.7 (d, C-3'),

250

34.3 (t, C-1'), 18.4 (q, C-4'), 18.4 (q, C-5').

251

The molecular formula C17H22O6 of Omphalocarpin (18), and the separation gave

252

of 1.3 mg white powder. The 1H NMR (400 MHz, CDCl3) and EIMS were in

253

agreement with the published data 34. The identification was further supported by 13C

254

NMR (100 MHz, CDCl3): 161.4 (s, C-2), 161.3 (s, C-7), 155.6 (s, C-5), 154.1 (s,

255

C-9), 138.8 (d, C-4), 110.9 (d, C-3), 108.2 (s, C-8), 103.9 (s, C-10), 90.4 (d, C-6),

256

77.2 (s, C-3'), 76.7 (d, C-2'), 56.2 (q, 7-OCH3), 55.9(q, 5-OCH3), 49.4(q, 3'-OCH3),

257

24.6(t, C-1'), 21.0(q, C-4'), 20.2(q, C-5').

258

Animals. ICR male mice (22-24 g) were purchased from Kunming Medical

259

University (License number SCXK 2015-0002). All animals were housed at room

260

temperature (20-25 °C) and constant humidity (40-70%) under a 12 h light-dark cycle

261

in SPF grade laboratory. Animals were acclimatized to the laboratory environment for

262

3 days and allowed free access to water and a standard diet prior to the experiment.

263

The experiment was conducted in accordance with the revised Animals (Scientific

264

Procedures) Act 1986 in the UK, Directive 2010/63/EU in Europe, Basel Declaration

265

and International Council for Laboratory Animal Science (ICLAS) and local laws and

266

regulations. The experiment was reviewed and approved by the Institutional Animal

267

Care and Use Committee of Kunming Institute of Botany, Chinese Academy of

268

Sciences (SYXK K2018-0005).



269

Xylene-induced acute inflammatory model of mice. The experiment was 36.

270

carried out as a previously described procedure

The male mice were randomly

271

assigned to 15 groups and administrated with methanolic extract (ME), ethyl acetate

272

extract (EAC) and four compounds (7, 8, 13, 16) for three consecutive days except

273

that the positive controls (Aspirin and Dexamethasone) were given once on the day of

274

the experiment. Two dose groups were set for each sample and every group contained

275

12 animals and the dose regimens were set according to the preliminary toxicity

276

experiment. Among them, ME and EAC groups were invented by intragastric

277

administration at the volume of 20 mL/kg except DXM and four compounds groups

278

were treated with intraperitoneal injection at 10 mL/kg. The control group was given

279

the equal volume of normal sodium by gavage. The right auricle of all animals was

280

externally coated with 30 μL xylene 30 minutes later after the last administration and

281

the left auricle served as a control. Mice were sacrificed by inhalation anesthesia 1 h

282

after xylene application. The auricles in the same position and area were harvested

283

and weighted, and the extent of edema was evaluated by the weight difference

284

between the right and the left auricle of the same animal.

285

Carrageenan-induced subacute inflammatory model of mice. The study was

286

carried out following the method reported previously 37. Group and dose settings were

287

the same as xylene-induced acute inflammatory model except the time of

288

administration was prolonged to 5 days. The paw swelling was induced by a

289

subcutaneous injection of 50 μL of 1% (w/v) carrageenan suspension in 0.9% normal

290

saline into the left hind paw 30 minutes after the last oral administration. The same


Page 14 of 35

Page 15 of 35


291

paw volume was measured by a digital vernier caliper before (time 0) and at 4 h after

292

carrageenan injection. The anti-inflammatory activity was calculated using the

293

following formula: Averagevolume difference(control) - Averagevolume difference(test) %inhibition =

294

Averagevolume difference(control)

×100%

295

Measurement of pro-inflammatory cytokines in vitro study. According to the

296

methods from literatures 38, the murine macrophage RAW 264.7 cells were plated in

297

96-well plates at 2×104 cells/well and cultivated in DMEM in a humidified

298

atmosphere with 5% CO2 at 37 °C for 24h. Cells were pretreated with different

299

compounds at the concentrations of 5 μg/mL for 2h and next induced with 1 μg/mL

300

LPS for 24h, with dexamethasone (10 μg/mL) as a positive control. Cell-free

301

supernatant was collected for the quantification of interleukin (IL)-1, prostaglandin

302

E2 (PGE2) and tumor necrosis factor-a (TNF-) by using enzyme-linked immuno

303

sorbent assay (ELISA) kits (Wuhan Huamei Biotechnology, Wuhan, China)

304

according to the manufacturers' protocols. Meantime, tetrazolium bromide reduction

305

(MTT) assay was performed to study the effect of coumarin compounds on RAW

306

264.7 cells growth at the same concentration.

307

Statistical analysis. Results are expressed as the mean ± SEM. Statistical

308

significance was determined using the two tailed Student’s t-test, with **p < 0.01 or

309

*p < 0.05 accepted as significant.

310

RESULTS



311

Compound isolation and structure determination. In the study, methanol

312

extract of pomelo peels was obtained by the way of cold-maceration with methanol.

313

Then the extraction, which was evaporated in vacuo to remove the organic solvents,

314

was suspended in hot water. The solid substance was removed by filtering after

315

sample standing for a while, and then the filtrate was partitioned with ethyl acetate.

316

Ethyl acetate fraction was collected, which was rich in coumarins after removed most

317

flavonoids and glycosides using the above ways. Finally, the structure of 18

318

coummarin compounds was identified by using spectroscopic methods and comparing

319

with literature data, the most structures of coumarins were 6,7-furocoumarins and 7-

320

or 7, 8-substituted coumarins compounds.

321

Anti-acute inflammation effect. We evaluated the auricle swelling caused by

322

xylene to observe the anti-acute inflammatory effects of C. grandis peels. All the

323

animals in the intraperitoneal injection groups treated with compounds 7, 13, 16 for

324

consecutive 3 days were shown remarkably reduced auricular swelling compared to

325

that in control group (Figure 2, p < 0.05/0.01) with the inhibition ratio of 26.2%

326

(compound 7, 2 mg/kg), 23.5% (compound 7, 1 mg/kg), 19.4% (compound 13, 1

327

mg/kg), 35.6% (compound 13, 0.5 mg/kg), 19.5% (compound 16, 3 mg/kg) and 17.9%

328

(compound 16, 1.5 mg/kg), respectively. Likewise, a similar inhibitory effect was

329

observed after the pretreatment of methanolic extract (ME) and ethyl acetate extract

330

(EAC) accompanied with the rates of change to 43.4% (2 g/kg), 38.9% (1 g/kg), 28.2%

331

(60 mg/kg) and 36.4% (30 mg/kg). The positive control had good suppressing effect

332

on both aspirin and dexamethasone compared to the control (p < 0.01), by 41.6% and


Page 16 of 35

Page 17 of 35


333

51.1% at the dose of 200 mg/kg (aspirin) and 3 mg/kg (dexamethasone). However,

334

compound 8 did not show significant inhibitory effects on xylene-induced ear edema

335

at the dose of 1 mg/kg and 0.5 mg/kg in vivo (p > 0.05).

336

Anti-subacute inflammatory effect. As shown in Figure 3, ME, EAC and four

337

coumarins, orally and intraperitoneally pretreated for 5 successive days before

338

carrageenan injection, could significantly inhibit the carrageenan-induced paw edema,

339

of which the EAC groups showed the most pronounced change in comparison to the

340

control group (p < 0.01), and the inhibitory ratio reached 60.3% and 62.3% at the dose

341

of 60 mg/kg and 30 mg/kg. The next was ME which showed a good effect of

342

anti-inflammation and the inhibiting rates were 49.9% (2 g/kg) and 51.4% (1 g/kg).

343

Besides, four coumarin had obvious inhibitory effect in varying degrees on the

344

carrageenan-induced sub-acute inflammatory model (p < 0.05/0.01), and the ratios of

345

compound 7 with 2 mg/kg and 1 mg/kg were 37.3% and 34.0%, compound 13 with 1

346

mg/kg and 0.5 mg/kg by 31.8% and 38.7%, compound 8 with 1 mg/kg and 0.5 mg/kg

347

by 28.3% and 44.1%, compound 16 with 3 mg/kg and 1.5 mg/kg by 47.6% and 37.8%.

348

Generally speaking, the results indicated that coumarins were the substance basis of

349

the anti-inflammatory action of pomelo peels and effective even at low doses. In

350

addition, prednisolone (10 mg/kg) and dexamethasone (3 mg/kg), the positive control,

351

played a similar role in reducing paw edema induced by carrageenan as well as, and

352

the suppression rate was 79.5% and 81.6%, respectively.



353

Anti-inflammatory effects in vitro. As shown in figure 4, LPS alone

354

significantly increased the IL-1 (Figure 4A, 6.1  0.3 vs. 3.5  0.3 ng/L), PGE2

355

(Figure 4B, 15.5  1.0 vs. 9.2  0.2 ng/L) and TNF- (Figure 4C, 26.2  1.6 vs. 11.4 

356

0.7 ng/L) production respectively compared with that in the control. However, the

357

secretions of pro-inflammatory cytokines were all decreased after the pretreatment of

358

coumarin compounds, of which 4, 6, 7, 10, and 17 compounds showed the most

359

pronounced effect in comparison to the control group (p < 0.05/0.01). Meantime,

360

coumarin compounds 8, 13, 16 showed a decreasing trend on the production of IL-1,

361

PGE2, TNF- at the concentration of 5 μg/mL (IL-1, 4.1  0.8, 4.3  0.6, 4.4  1.0

362

ng/L; PGE2, 11.7  1.0, 12.1  1.0, 11.9  0.9 ng/L; TNF-, 21.3  0.9, 19.5  1.9,

363

21.5  1.0 ng/L) contrast with that observed with LPS alone. Taken together, all of

364

these data indicated that levels of pro-inflammatory cytokines were inhibited by the

365

pretreatment of coumarin compounds. Besides, they had no inhibitory effect on the

366

proliferation of RAW 264.7 cells (Figure 4D), and the the cell survival rate were not

367

obviously difference between control and tested group.

368

DISCUSSION

369

Xylene-induced ear swelling indicates the secretion of inflammatory factors and

370

increased vascular permeability leading to edema, a typical feature of inflammatory

371

reaction, and is the classical animal acute model used for anti-inflammatory activity

372

evaluating 39, 40. The results of xylene-induced edema in mice suggested that ME and

373

EAC could significantly inhibit the formation of edema and reduce the thickness of


Page 18 of 35

Page 19 of 35


374

ear tissues. The therapeutic effects of compound 7, 13, and 16 were preferable among

375

of the high yield of coumarin compounds. Likewise, carrageenan is a strong stimulant

376

of proinflammatory mediators for sub-acute inflammation used in determining

377

effective anti-inflammation drugs 41. The early phase (1 h after carrageenan injection)

378

is mainly mediated by the release of histamine and serotonin; whereas the late phase

379

(the first 2-4 h) is mediated by the release of bradykinin, TNF-, and leukotrienes and

380

is sustained by PGE2 and COX-2

381

drugs target the late phase 43.

42.

Thus, most conventional anti-inflammatory

382

In vivo study showed that orally administered ME and EAC inhibited the

383

carrageenan-induced paw edema at 4 h after the carrageenan subcutaneous injection

384

and had no dose-dependent relationship. The results suggested that EAC exhibited the

385

powerful anti-inflammatory effects by suppressing the release of bradykinin, TNF-

386

and leukotrienes. Coumarins as the major bio-active components in the ethyl acetate

387

extract had various degree of suppressive action in edema. However, it was worth

388

noting that compound 8 could remarkably inhibit paw edema induced by carrageenan

389

in mice, but had no influence on xylene-induced auricular swelling, which implied

390

compound 8 might act as an anti-inflammatory based on its inhibitory effect on the

391

leukocyte migration. In other words, the coexistence of different coumarins enhanced

392

the anti-inflammatory action of EAC and showed synergetic effect with each other.

393

Then, an in vitro assay was selected to support the assumption.

394

Lipopolysaccharide (LPS), also known as lipoglycans and endotoxins, is a

395

component of Gram-negative bacterial cell walls. It plays an important role as an



Page 20 of 35

396

inflammation inducer in macrophages and trigger the activation of nuclear factor-B

397

(NF- B)

398

proinflammatory cytokines such as TNF-, IL-6, and IL-1, as well as various

399

pro-inflammatory mediators, such as NO and PGE2 by iNOS and COX-2 during the

400

inflammatory response

401

acute and chronic inflammatory diseases. Therefore, inhibition of a pro-inflammatory

402

mediator is important to curtail an inflammatory disorder

403

macrophage has been used as an effective cellular model to study anti-inflammatory

404

activities and mechanisms in vitro

405

leukocytic pyrogen, is a member of the interleukin 1 family of cytokines and a major

406

pro-inflammatory cytokine that initiates and enhances the inflammatory response,

407

which also is involved in a variety of cellular activities, including cell proliferation,

408

differentiation, and apoptosis 48, 49. In addition, tumor necrosis factor alpha (TNF-) is

409

a cell signaling protein involved in systemic inflammation and one of the cytokines

410

that make up the acute phase reaction, which can activate the NF-B pathway and

411

stimulate the release of other pro-inflammatory cytokines such IL-1 and IL-6

412

Furthermore, metabolites, prostaglandin E2 (PGE2), one of the most abundant

413

prostaglandins produced in the body and an important mediator of many biological

414

functions, such as regulation of immune responses, blood pressure, gastrointestinal

415

integrity, and fertility

416

inflammation as chemokines for increasing the macrophage population, leading to an

417

exacerbated inflammation: fever, redness, swelling and pain

44.

In response to LPS, macrophages induce the expression of

45, 46.

51.

The uncontrolled secretion of these mediators causes

39.

47,

and LPS-stimulated

Interleukin 1 beta (IL-1) also known as

50.

In inflammation, PGE2 participates in the initiation of


52.

Then, the

Page 21 of 35


418

anti-inflammatory properties of coumarins (compounds 7, 8, 13, 16) were further

419

explored in vitro.

420

The effects of coumarin compounds on the extracellular release of IL-1, PGE2

421

and TNF- were investigated in LPS-stimulated RAW264.7 cells. Results showed

422

that LPS markedly increased IL-1, PGE2 and TNF- production, and pretreatment

423

with coumarin compounds significantly inhibited IL-1, PGE2 and TNF- production

424

at a concentration of 5 μg/mL and without cytotoxicity. The result revealed that

425

coumarins were the major components in this fraction. In-vitro pharmacology study,

426

results indicated that production of inflammatory factors in LPS-induced

427

inflammation was decreased in coumarins treated cells, which demonstrated by in

428

vivo anti-inflammatory assay. This study suggests the possibility of pomelo peels to

429

be used for the treatment of inflammatory diseases.

430

Comparison of un-substituent compounds 2 and substituent 1 and 3, compound 4

431

with weak-polar chain substituent at C-5 might increase its anti-inflammatory

432

significantly. Compound 6 possessing polar ten carbons substituent at C-8 showed

433

more anti-inflammatory effect than less polar C-8 substituent group. To the contrary,

434

no polar substituent group at C-7 of coumarin, such as compound 7, indicated much

435

potential bioactivity than other analogs (8, 9, 10, 11). Furthermore, weak-polar five

436

carbons side chain connected at C-6 seem to be the better C-C conjunction position

437

for such coumarins by comparing compounds 17 with 12-16, 18.

438 439

In summary, 18 coumarins were isolated from EtOAc fraction of pomelo peels extracts. Pomelo peels, which account for one third of the fruit


53,

are the waste


440

materials in pomelo fruits industries and daily consumption. A large number of

441

pomelo peels are thrown away as waste every year. It causes a great loss of resources

442

54.

443

first time, which supported the rationality of folk medicine. Meanwhile, the results in

444

vivo showed that the anti-inflammatory effects of pomelo peels were mainly

445

composed of EtOAc fraction, and coumarins were the substance basis that is

446

responsible for the activity of pomelo peel. Furthermore, various coumarins had been

447

observed to be anti-inflammatory through suppressing the secretion of inflammatory

448

cytokines such as IL-1, PGE2 and TNF- induced by LPS in RAW 264.7 cells.

449

AUTHOR INFORMATION

450

Corresponding author

451

*Phone: +86-871-65223177. E-mail: [email protected]

452

Present address

453

*Xiao-Dong Luo: Key Laboratory of Medicinal Chemistry for Natural Resource,

454

Ministry of Education and Yunnan Province, School of Chemical Science and

455

Technology, Yunnan University, Kunming 650091, People’s Republic of China

456

Funding

457

This study was supported by National Key Research and Development Program of

458

China (2017YFC1704007)

459

Notes

460 461

The present study revealed the anti-inflammatory function of pomelo peel for the

There has been no competing financial interest for this work. Acknowledgements


Page 22 of 35

Page 23 of 35


462

The authors are grateful to the National Key Research and Development

463

Program of China (2017YFC1704007), and the “Ten Thousand Plan”, a National

464

High-Level Talents Special Support Plan for partial financial support. And we thank

465

Dr. Ying-Ying He for polishing the article.

466

ABBREVIATIONS USED

467

ME, methanolic extract; EAC, ethyl acetate fraction; LPS, lipopolysaccharide; IL-1 ,

468

interleukin 1 beta; PGE2, prostaglandin E2; TNF-, tumor-necrosis factor (alpha);

469

PRE, prednisone acetate; DXM, dexamethasone; ASP, aspirin



470

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R. G.; Farnsworth, N. R.; Hhs, F.; Pezzuto, J. M., Antimutagenic constituents of Casimiroa edulis

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with potential cancer chemopreventive activity. J Agr Food Chem 1998, 46, 3509-3516.

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25. Rashid, M. A.; Gray, A. I.; Waterman, P. G.; Armstrong, J. A., Coumarins from Phebalium


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26. Wilzer, K. A.; Fronczek, F. R.; Urbatsch, L. E.; Fischer, N. H., Coumarins from Aster

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praealtus. Phytochemistry 1989, 28, 1729-1735.

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27. Masuda, T.; Muroya, Y.; Nakatani, N., Coumarin Constituents of the Juice Oil from Citrus

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hassaku and Their Spasmolytic Activity. Biosci Biotech Bioch 1992, 56, 1257-1260.

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29. Timonen, J. M.; Nieminen, R. M.; Sareila, O.; Goulas, A.; Moilanen, L. J.; Haukka, M.;

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of novel 7-hydroxycoumarin derivatives. Eur J Med Chem 2011, 46, 3845-3850.

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30. Macías, F.; M Massanet, G.; Rodríguez-Luis, F.; Salvá, J.,

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III-Simple coumarins. Magn Reson Chem 1989, 27, 892-894.

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J.; Kuo, D. H.; Wu, T. S., Anti-Inflammatory and Neuroprotective Constituents from the Peels of

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Citrus grandis. Molecules 2017, 22, 967/1-967/11.

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13C

NMR of coumarins.


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35. Wu, T. S.; Liou, M. J.; Kuoh, C. S., Coumarins of the flowers of Murraya paniculata.

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Potent anti-inflammatory and analgesic steroidal alkaloids from Veratrum taliense. J

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Ethnopharmacol 2016, 179, 274-279.

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38. Wang, B.; Yang, Z. F.; Zhao, Y. L.; Liu, Y. P.; Deng, J.; Huang, W. Y.; Li, X. N.; Wang, X.

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H.; Luo, X. D., Anti-Inflammatory Isoquinoline with Bis-seco-aporphine Skeleton from

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Dactylicapnos scandens. Org Lett 2018, 20, 1647-1650.

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Anti-inflammatory effects of 4-o-methyl-benzenesulfonyl benzoxazolone (MBB) in vivo and in

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vitro as a novel NSAIDs lead compound. Pharmacol Rep 2018, 70, 558-564.

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compound K exerts anti-inflammatory and analgesic effects via downregulating COX2.

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Inflammopharmacology 2018.

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Pharm Pharmacol 2011, 63, 1070-1077.

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Page 30 of 35

Page 31 of 35


9'

10'

10'

1'

O 11

7'

5'

3'

5 3

O

7

O

9

8'

OH

11

5

1

9

7

O

7

O

9

O

OH 7'

5' 10'

1

O

7

9'

3' 7'

5'

8'

8'

3

OH

9

O

O

1

OH

9'

O

5

8'

9'

O

7

OH

9

O

O

10'

3 1'

5'

O

3'

7'

O

9

7

1

OH

1

10 7-(6-Hydroxy-3,7-dimethyl-2E,7octadienyloxy)coumarin

9 7-[6-Hydroxy-7-methoxy-3,7-dimethyl -(2E)-2-octenyloxy]coumarine

8 Marmin

O

9

7 Auraptene

3

3'

7

1

5

5'

3

O

3'

1'

7'

5 1'

7'

8'

10'

H3CO

1'

7'

9'

8'

OH

7 9 O O 1 4 5-(6-Hydroxy-3,7-dimethyl2E,7-octadienyloxy)psoralen

5'

6 8-(6,7-Dihydroxy-3,7-dimethyl2E-octenyloxy)psoralen

5

3'

7'

3

10'

9'

OH

10'

10' 5'

5'

O

O

9

O

1'

OCH3

8'

O

9

O

5 8-(6-Hydroxy-7-methoxy-3,7dimethyl-2E-octenyloxy)psoralen

8'

7

3'

5

11

O 3 1 5-(6-Hydroxy-7-methoxy-3,8dimethyl-2E-2-octenyloxy)psoralen

O

7

9''

3' 1'

HO

O

O

8'

OH

3

O

1

O

5

9'

1'

5

11

3

7'

3

O

1 2 Bergaptol

5

5'

3'

3

O

O

1 Bergamottin

11

OCH3

O 11

10'

9'

1'

5 5

5

3

HO

7

O

9

H3CO

O

1

7

O OH

9 1'

1

3 1

H3CO

O

7

O O

9 1'

3'

5'

3

H3CO

1

7

9 1'

O OH

613 614

O

7

9

O

O

H3CO

1

7

O OH

9 1'

1

O

3' 5'

17 Toddanone

OCH3 18 Omphalocarpin

Figure 1. Coumarins from pomelo peels


1

7

O OH

9 1' 3'

3'

14 7-Methoxy-8-(2-formyl2-methylpropyl)coumarin

3 3

OH

16 Meranzin hydrate

1

5

5

3' 5'

O

CHO

OCH3

3'

O H3CO

9

7 1'

13 Isoauraptene

OCH3

1'

O

H3CO

H3CO

5'

12 Auraptenol

5

O

3'

5'

11 Umbelliferone

3

5

3

3

5

5'

OCH3

15 Yuehgesin B

O

O


615 616

Figure 2. Bar graphs representing the level of auricular swelling induced by 30 μL

617

xylene. Animals were treated by intra-gastric administration for consecutive 3 days.

618

Data was shown as means ± SEM values. Statistical differences are represented as */**

619

p < 0.05/0.01 vs. Control group. Experimental results indicated coumarins of pomelo

620

peels could reduce the degree of tumefaction in auricle and showed an effect against

621

acute inflammation.

622

ASP: aspirin;

623

DXM: dexamethasone;

624

ME: methanolic extract;

625

EAC: ethyl acetate fraction.


Page 32 of 35

Page 33 of 35


626 627

Figure 3. Bar graphs representing the level of paw edema induced by the

628

subcutaneous injection of 50 μL of 1% (w/v) carrageenan suspension carrageenan.

629

The degree of swelling of the foot was measured after administration for five days.

630

Data was shown as means ± SEM values. Statistical differences are represented as */**

631

p < 0.05/0.01 vs Control group. Results showed that coumarins of pomelo peels could

632

inhibit the sub-acute inflammatory and were effective even at low doses.

633

PRE: prednisone acetate;

634

DXM: dexamethasone;

635

ME: methanolic extract;

636

EAC: ethyl acetate fraction.



Page 34 of 35

637 638

Figure 4. Effect of coumarin compounds on inflammatory cytokines and cell viability

639

in lipopolysaccharide (LPS) induced RAW 264.7 cells. Data were expressed as the

640

mean ± SEM. Statistics:

641

(dexamethasone) was used as a positive control. A, interleukin 1

642

IL-1   prostaglandin E2 (PGE2). C, tumor-necrosis factor alpha (TNF-). The

643

expression

644

immunosorbent assay. As shown in figure, coumarin analogs 4, 6, 7 and 17 could

645

inhibit production of IL-1β, PGE2, and TNF-α, and they didn’t inhibit the growth of

646

RAW 264.7 cells at the same concentration.

of

##p

< 0.01 vs Control; */**p < 0.05/0.01 vs LPS. DXM

inflammatory

cytokines

was

determined


by

beta 

enzyme-linked

Page 35 of 35

647 648


Table of Contents Graphic:

Anti-inflammatory effect of Pomelo peel and its bioactive coumarins

HO O

O

O

O

H3CO

O O

13 Isoauraptene

O

O

OH

7 Auraptene

8 Marmin

O

H3CO

O OH

O

OH 16 Meranzin hydrate

649


Anti-inflammatory Effect of Pomelo Peel and Its Bioactive Coumarins

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