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Protective role of flavonoids and lipophilic compounds from Jatropha platyphylla on the suppression of Lipopolysaccharide (LPS)-induced inflammation in macrophage cells. Dulce L Ambriz-Perez, Woo Young Bang, Vimal Nair, Miguel A Angulo Escalante, Luis Cisneros-Zevallos, and J. Basilio Heredia J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05534 • Publication Date (Web): 12 Feb 2016 Downloaded from http://pubs.acs.org on February 14, 2016

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Journal of Agricultural and Food Chemistry

Protective role of flavonoids and lipophilic compounds from Jatropha platyphylla on the suppression of Lipopolysaccharide (LPS)-induced inflammation in macrophage cells.

Dulce L. Ambriz-Pérez¥, Woo Young Bang‡,φ, Vimal K. Nair‡, Miguel A. AnguloEscalante¥, Luis Cisneros-Zevallos‡,†,*, J. Basilio Heredia¥,*.

¥

Centro de Investigación en Alimentación y Desarrollo A.C., Carretera a Eldorado

km 5.5 Col. Campo el Diez C.P. 80110, Culiacán, Sinaloa, México. ‡

Department of Horticultural Sciences, †Department of Nutrition and Food Science,

Texas A&M University, College Station, Texas 77843-2133, USA.

*

Corresponding authors.

Luis Cisneros-Zevallos Phone: 979-8453244, Fax: 979-8450627, e-mail: [email protected] J. Basilio Heredia Phone: +52 1 6671 890101; Tel.: +52 1 6677 605536. e-mail: [email protected]

φ

Present address: National Institute of Biological Resources, Environmental

Research Complex, Incheon 404-708, Korea. 1

1 ACS Paragon Plus Environment


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ABSTRACT

3

Seventeen polyphenols (e.g, Apigenin, genistein and luteolin glycosydes) and 11

4

lipophilic compounds (e.g., fatty acids, sterols, terpenes) were detected by LC-

5

MS/MS-ESI and GC-MS, respectively, in Jatropha platyphylla. Extracts from pulp,

6

kernel and leaves and fractions were studied in order to know their effect on some

7

pro-inflammatory mediators. Phenolic and lipophilic extracts showed significant

8

inhibitory effects on ROS, NO production while not affecting mitochondrial activity

9

nor superoxide generation rate in lipopolysaccharide (LPS)-induced inflammation

10

in RAW 264.7 macrophage cells. In addition, NO production was also diminished

11

by lipophilic leaf fractions F1 and F2 with the latter fraction showing a greater effect

12

and composed mainly of sterols and terpene. Furthermore, total extracts showed

13

non-selective inhibitions against cyclooxygenases COX-1 and COX-2 activity. All

14

together, these results suggest that Jatropha platyphylla extracts have potential in

15

treating inflammatory diseases and their activity is mediated by flavonoids and

16

lipophilic compounds.

17

18

KEYWORDS. Jatropha platyphylla, flavonoid glycosides, fatty acids, sterols,

19

terpene, inflammation, ROS and NO pro-inflammatory mediators, macrophages.


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INTRODUCTION

21

The inflammatory process is a natural response as a critical protective reaction to

22

injury or pathogens, and produce a great accumulation of immune cells at the

23

damaged site. Initially the cells recognize the antigen by specific receptors,

24

recognize the pathogen and activate the macrophages, which proliferate and

25

secrete inflammatory mediators to the extracellular medium, such as reactive

26

oxygen species (ROS), nitric oxide (NO), prostaglandins (synthetized by

27

cyclooxygenase COX 1 and 2), and cytokins; these mediators amplify the

28

inflammatory response, unfortunately they have also the potential to generate

29

damage to the peripheral tissues1,2. Currently, some inflammatory mediators have

30

been associated to important pathologies including neurodegenerative diseases,

31

cardiovascular disorders, atherosclerosis and cancer, which were not related

32

before with inflammation3,4.

33

It has long been known that ROS perform essential roles in immune responses to

34

pathogens, however, it has been implicated in inflammatory diseases as well 5.

35

ROS activate metabolic pathways such as the NF-κB mediated pathway, this

36

activation leads to the production of various proinflammatory mediators that play an

37

important role in the inflammatory response like cytokines, prostaglandins, iNOS

38

and COX6. Therefore, appropriate inhibition of proinflammatory mediators

39

represent a therapeutic target to reduce inflammation7. Because of this, the search

40

for new anti-inflammatory agents from medicinal plant resources is intensifying.

41

In this sense, in folklore medicine the leaves, stems, roots and seeds of different

42

species from the genus Jatropha, have taken popularity as anti-inflammatory and



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8-11

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antioxidant agents

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the genus Jatropha, including phenolics like gadain, gossypidien, isogadain,

45

isovitexin,

46

dimethoxyphenyl)-naphthalene dihidroprasantalin, apigenin 7-O-neohespredoside,

47

ferulic acid, quercetin, vicinin-II, isoorientin, luteolin, among others9. In addition

48

there are reports of presence of lipophilic compounds, including terpenoids like

49

jatrophone12, fatty acids like linoleic and oleic acid13, sterols like lanosterol14, β-

50

sitosterol15 and ς-sitosterol16, and others. In México there are endemic nontoxic

51

species of Jatropha, one of them is Jatropha platyphylla, this species grows in the

52

Pacific coast, from the state of Sinaloa to Michoacán, where it is traditionally

53

consumed by the local people, as roasted seeds17 or pulp marmalades; although J.

54

platyphylla has been nutritionally characterized17, the nutraceutical potential has

55

not been explored. In the present study we hypothesized that phenolics and

56

lipophilic compounds present in J platyphylla were responsible for the anti-

57

inflammatory properties claimed by folklore use. Thus, to confirm this we

58

characterized the polyphenols and lipophilic compounds present in J. platyphylla

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and their anti-inflammatory mode of action in LPS-induced inflammation

60

macrophage cell models. To our knowledge this is the first report of the chemical

61

characterization and anti-inflammatory properties of Jatropha platyphylla species.

vitexin,

. Several studies have focused in identifying compounds in

2,3-bis-(hydroxymethyl)

-6,7-methylenedioxy-1

-

(3'4'-

62 63

MATERIALS AND METHODS

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Plant material. For the present study, pulp, kernel meal and leaves from wild

65

Jatropha platyphylla trees were used. The fruits were collected at a ripen mature


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green stage in the months of August and September 2012, in Mazatlán, Sinaloa,

67

Mexico (23,5333°N, 106,4667°W, 60 m.a.s.l.). The samples were washed in water

68

chlorine solution (150 ppm), lyophilized and ground for the extraction process. The

69

kernel flour was defatted to obtain a meal by hexane solvent extraction before

70

analysis

71 72

Reagents and Chemicals

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The following chemicals were used in the experiments: 2′,7′-Dichlorofluorescin

74

diacetate (DCFA), Griess reagent, Sodium nitrite solution, Dulbecco’s Modified

75

Eagle’s Medium (DMEM)/low glucose, phenol red-free DMEM/low glucose,

76

penicillin/streptomicyn mixture, DMSO and Fetal Bovine Serum (FBS) were

77

purchased from Sigma (St. Louis, MO). Glucose and sodium bicarbonate were

78

purchased from Acros Organics (Fair Lawn, NJ) and sodium bicarbonate from

79

Mallinckrodt Chemicals (Phillipsburg, NJ), respectively. The CellTiter 96®

80

AQueous Non-Radioactive Cell Proliferation Assay kit was purchased from

81

Promega (Madison, WI). The COX Inhibitor Screening Assay Kit and the DuP-697,

82

a COX-2 inhibitor, were purchased from Cayman Chemical (Ann Arbor, MI). The

83

Macrophages RAW 264.7 (cell line TIB-71™) was acquired from the American

84

Type Culture Collection (ATCC) (Manassas, VA) and LH-20 Sephadex was

85

purchased from GE-healthcare Biosciences (Uppasala).

86 87

Extract preparation.

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The extraction process is presented in Figure 1. To obtain lipophilic extracts a 0.1 g

89

of lyophilized powder of leaf, pulp or kernel of Jatropha platyphylla were extracted 5 ACS Paragon Plus Environment


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in 1 mL hexane and stirred at 8 °C and medium speed for 2 h. The extracts were

91

centrifuged and the hexane supernatants were discarded while the pellets were

92

extracted in 1 mL chloroform and stirred at 8 °C and medium speed for 4 h. After

93

centrifugation at 4000 rpm (2147g), the chloroform supernatants were recovered

94

and concentrated at 45 °C until all of the volatile solvents were evaporated in a

95

Centrivap concentrator connected to a cold trap (Labconco, Kansas City, MO).

96

Finally, the dry extracts were kept in the dark at -4°C until tested. The yields

97

obtained for pulp, leaf and kernel were 10.8, 47.7 and 25.6 mg, respectively.

98 99

For the phenolic extracts, the pellets from the previous extraction were extracted in

100

5 mL methanol/acetone/water (5:4:1) and stirred for 24 h at medium speed at 8 °C.

101

The extracts were sonicated (15 min) and filtered, the solutions were centrifuged at

102

4000 rpm (2147g) and the supernatants were concentrated at 45 °C until all of the

103

volatile solvents were evaporated in a Centrivap concentrator connected to a cold

104

trap (Labconco, Kansas City, MO), after that methanol/water (8:2) was added to

105

the dry extract. Finally, the chlorophyll was removed with hexane and the aqueous

106

phases were evaporated until dryness at 45 °C in a Centrivap concentrator and

107

kept in the dark at -4°C until used18. The yields obtained for dry extracts of pulp,

108

leaf and kernel were 9.8, 4 and 3.8 mg, respectively.

109

To obtain the whole extract from Jatropha platyphylla, chloroform and

110

methanol/water supernatants were mixed and then evaporated until dryness.

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These extracts contained flavonoids and lipophilic compounds, and were used to

112

evaluate the COX-inhibitory activity of the whole extract from Jatrophylla

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platyphylla. 6 ACS Paragon Plus Environment

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All three dry powder samples, lipophilic, phenolic and whole extracts were re-

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suspended in DMSO for further studies of pro-inflammatory mediators.

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Fractionation of extracts. Both extracts, methanol/water and chloroform, were

118

spotted in a TLC plate to determine the possible presence of phenolics and/or

119

terpenoids using the ferric chloride (FeCl3) test19 and the Liebermann Burchard20

120

test respectively, confirming visible spots of both phenolics and terpenoids. After

121

the TLC analysis, extracts were fractionated (0.1 g for each extract) by polarity in a

122

size

123

chloroform/methanol (1:1) as mobile phase by collecting 20 mL of each fraction

124

(Figure 2). In phenolic extracts, we did not observe defined fractions. For lipophilic

125

extracts only two different fractions, F1 and F2, were identified in the chloroform

126

leaf extract with yields of 1.9 and 9.5 mg, respectively. Fractions were completely

127

evaporated at 45 °C using a Centrivap (Labconco, Kansas City, MO) and kept in

128

the dark at -4°C until tested.

exclusion

LH-20

sephadex

column

chromatography

using

129 130

Reversed-phase high-performance liquid chromatography. The identification of

131

phenolic compounds was performed by HPLC/MS. Dry powder samples were re-

132

suspended in methanol at 1mg/mL, filtered and 10 µL were injected. The HPLC

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system was a Surveyor (Thermo Scientific, USA) coupled to Surveyor DAD.

134

Separations were performed using a Synergi™ (Phenomenex, Torrance, CA, USA)

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4 µm, Hydro-RP 80 Å, LC, C18 Column 150 x 1 mm. The eluents were

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acetonitrile/methanol (1:1), formic acid (0.5:99.5, vv-1) (phase A) and formic acid–

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water (0.5:99.5, vv-1) (phase B). The applied elution conditions were: 0-5 min, 2% 7 ACS Paragon Plus Environment


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A, 98% B; 5-20 min, 5% A, 95% B; 20-45min, 30% A, 70% B; 45-55 min, 65% A,

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35% B; 55-66 min, 100% A; 66-70 min, 2% A, 98% B. The chromatograms were

140

monitored at 330, 280, 210 nm; and complete spectral data were recorded in the

141

range 200–600 nm.

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Mass spectrometry. Mass spectra were obtained on a MS Finnigan LCQ Deca XP

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Max, Ion trap mass spectrometer coupled at the exit of the diode array detector

144

and equipped with a Z-spray ESI source, and run by Xcalibur version 1.3 software

145

(Thermofinnigan-Surveyor, San José, USA). A flow of 200 µLmin-1 from the DAD

146

eluent was directed to the ESI interface using a flow-splitter. Nitrogen was used as

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desolvation gas, at 275 °C at a flow rate of 60 Lh-1. A potential of 1.5 kV was used

148

on the capillary for negative ion mode. The source block temperature was held at

149

120 °C, a potential of 6.8 V was used on the capillary for positive ion mode. Spray

150

voltage of 4.57 kV and the source block temperature was held at 255◦C

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Gas chromatography/mass spectrometry.

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The identification of lipophilic compounds was performed by GC/MS. Dry powder

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samples were re-suspended in dichloromethane/hexane (50:50) at 1mg/mL,

154

filtered and 10 µL were injected. The GC/MS analyses were performed with an

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Agilent 6890N gas chromatograph (Agilent Technologies, Inc., Santa Clara, CA,

156

USA) connected with an Agilent 5975 mass-selective detector (MSD; electron

157

impact ionization, 70 eV) and equipped with G1701DA GC/ MSD ChemStation

158

software and a non-polar Agilent Technologies Ultra 1 cap. column (25 m × 0.32

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mm i.d., film thickness 0.52 mm). The oven temperature was programmed

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isothermal at 75°C for 4 min, then linearly rising from 75 to 200°C at 5°/min, and


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finally held isothermal at 200° for 1 min; injector temp., 280°C; detector temp.,

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290°C; carrier gas, He (1.5 ml/min); injection volume, 1 ml; split ratio, 1 : 50.

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The peaks obtained were tentatively identified by comparing their mass spectra

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with those included in the NIST-05a and ADAMS libraries and/or reported in the

165

literature21.

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Cell culture and treatment of Jatropha platyphylla extracts. Macrophages were

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grown in the DMEM-low glucose (pH 7.2 – 7.4) including 4 gL-1 glucose, 3.7 gL-1

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sodium bicarbonate, 10% fetal bovine serum (FBS) and antibiotics (100 units/mL

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penicillin and 100 µgmL-1 streptomycin) in a humidified atmosphere with 5% CO2 at

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37°C. Cells were used at a passage of 5 to 9 in this study.

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For treatments of cells with J. platyphylla extracts, the cells were plated at 0.5×105

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cells/well in a 96-well black and clear bottom plates (Costar, Cambridge, MA) for

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MTS test and measurements of NO, ROS and superoxide. The cells were treated

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with the growth medium containing 1 µg/ml LPS for 19 h either with or without the 5

175

h-pre-treatment of J. platyphylla extracts. The re-suspended J. platyphylla extracts

176

in DMSO were dissolved in the growth medium to obtain a final 0.5 % DMSO,

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which was used as a control in all experiments. Finally, cells and medium at 19 h

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after LPS challenge were used for various measurements in this study.

179 180

Tests for cell viability and LPS-induced mitochondrial dehydrogenase

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activity. Macrophages were plated at 0.5×105 cells/well in a 96-well clear bottom

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plates (Costar) and cultured overnight. The cells were stimulated by LPS for 19 h

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either with or without the 5 h-pre-treatment of J. platyphylla extracts as described

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above and were subjected to the tests for cell viability and LPS-induced 9 ACS Paragon Plus Environment


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mitochondrial dehydrogenase activity, which were evaluated in macrophages using

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the MTS assay kit (Promega, Madison, WI), according to the manufacturer’s

187

instructions. The quantity of formazan product was measured at 490 nm and is

188

directly proportional to the mitochondrial dehydrogenase activity.

189 190

Detection of extracellular nitric oxide (NO) and intracellular reactive oxygen

191

species (ROS) production. Macrophages were plated at 0.5×105 cells/well in a

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96-well black and clear bottom plates (Costar) and cultured overnight. The cells

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were stimulated by LPS for 19 h either with or without the 5 h-pre-treatment of J.

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platyphylla extracts as described above. Finally, cells and medium were used for

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the ROS and NO detections, respectively.

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First, the nitric oxide (NO) production was assessed the accumulation of nitrite

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(NO2−) in the medium using a colorimetric reaction with the Griess reagent. Briefly,

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50 µl of cell culture supernatants were obtained at 19 after LPS treatment and 100

199

mM sodium nitrite solution was diluted with nanopure water for the preparation of

200

standards from 10 to 100 µM. Subsequently, the cell supernatants and standards

201

were mixed with an equal (1:1) volume of Griess reagent and finally absorbance

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was measured at 540 nm using a 96-well microplate reader (Synergy HT, Bio-Tek

203

Instruments, Inc., Winooski, VT). The results were reported as nitrite concentration

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(µM) in the supernatant.

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Second, the intracellular ROS production was measured by 2′,7′-Dichlorofluorescin

206

diacetate (DCFA). Briefly, the cell culture medium was removed by aspiration at

207

times indicated in this study after LPS challenge, and subsequently cells were

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exposed to 10 µM DCFA in the phenol red/FBS-free DMEM for 30 min, then 10 ACS Paragon Plus Environment

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washed twice with the phenol red/FBS-free DMEM. Finally, fluorescence was read

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immediately at wavelengths of 485 nm for excitation and 528 nm for emission on a

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96-well microplate reader (Synergy HT, Bio-Tek Instruments, Inc., Winooski, VT).

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The results were reported as percentage of relative ROS level.

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Measurement of superoxide production in macrophage cells. The LPS-

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induced superoxide production in macrophage cells was measured by the

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cytochrome c reduction assay as described previously22. Briefly, RAW264.7 cells

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(0.5×105 cells/well in 96-well culture plates) were pretreated with J. platyphylla

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extracts or 10 µM DPI, a NOX inhibitor, for 5 h and then stimulated with LPS (1

219

µg/ml) for 19 h. Subsequently, the cell culture medium in each well was changed to

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the Phenol Red-free medium including 0.45 mg/ml cytochrome c. Finally, the

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superoxide generation rate (pmole/105 cells x min) was calculated by the

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superoxide-induced reduction rate of ferricytochrome c to ferrocytochrome c

223

(extinction coefficient = 28.0 mM-1cm-1), monitored spectrophotometrically at 550

224

nm every 2 min for 1 h. Medium with cytochrome c served as a blank.

225 226

COX inhibitory assay. The COX inhibitory assay was carried out using the COX

227

Inhibitor Screening Assay Kit following the manufacturer's instructions (Cayman

228

Chemical, Ann Arbor, MI). Briefly, heme and COX enzyme (COX-1 or COX-2) was

229

added to test tubes containing COX reaction buffer. The mixture was vortex and

230

exposed to either J. platyphylla total extracts (containing flavonoids and lipophilic

231

compounds), the DuP-697, a COX-2 inhibitor, or DMSO (control) for 10 min at 37

232

°C. This was followed by the addition of arachidonic acid with further incubation for 11 ACS Paragon Plus Environment


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2 min. Hydrochloric acid (1 M) was added to stop the COX reaction followed by the

234

addition of stannous chloride (SnCl2) solution. This assay measures PGF2α derived

235

from SnCl2 reduction of PGH2 produced in the COX reaction, through an enzyme

236

immunoassay kit.

237 238

Statistical analysis. The data were analyzed using one-way analysis of variance

239

(ANOVA) followed by Tukey-HSD test, using the software JMP pro v10.0. Results

240

are expressed as means ± standard errors (SE). Different letters show significant

241

differences (p < 0.05).

242 243

RESULTS AND DISCUSSION

244

Identification of compounds in Jatropha platyphylla extracts and fractions.

245

The phenolic extracts of leaves, pulp and kernel of Jatropha platyphylla were

246

analyzed by HPLC/MS2 in positive and negative ion mode. The flavonoid

247

glycosides were analyzed to determine if these were C-glycosylated or O-

248

glycosylated. According to Plazonić et al. (2009)23, the carbon-carbon bond of C-

249

glycosyl flavonoids is resistant to rupture and the sugar unit can be observed,

250

meanwhile, the fragmentation pathway of O-glycosylated flavonoids starts with the

251

cleavage of the glycosidic bonds and elimination of the sugar moieties with charge

252

retention on aglycone. Under the HPLC/MS2 conditions used, all the compounds

253

analyzed had an intense signal corresponding to the pseudo-molecular ions

254

[M+H]+ and/or [M-H]-. Also lower signals were observed from [M+18]+ water

255

adducts and [M+23]+ sodium adducts24. Although the aglycone and the glycane

256

were all identified, the accurate structure of the flavonoid glycosides with the 12 ACS Paragon Plus Environment

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specific identity and the site of connection of monosaccharides should be

258

confirmed with NMR spectroscopy. In the present study the analyzed extracts

259

showed different phenolic profiles, overall, 17 phenolic compounds were detected;

260

most of them were apigenin, genistein and luteolin glycosides. Apigenin and

261

luteolin glycosides have been found in other Jatropha species, such as J. curcas25,

262

J. multifida26 and J. gossypiifolia27. Although isoflavone genistein is characteristic to

263

soybean, it was found in leaves of J. curcas

264

ions and MS2 data of the phenolic compounds detected in the extracts are listed in

265

table 1.

266

The lipophilic extracts of leaves, pulp and kernel of Jatropha platyphylla were

267

analyzed by GC/MS. Eleven compounds within different structural type were found,

268

including four fatty acids, two sterols, two alcohols, one aromatic acid, one terpene

269

and one hydrocarbon, these compounds are listed in table 2. Pentadecanoic acid

270

was observed in both kernel and leaf extracts while oleic acid was found in the pulp

271

extract, this fatty acid has been reported in J. gossypifolia extract before13.

272

Similarly, lanosterol and ς-sitosterol found in leaf extract has been reported

273

previously in other Jatropha species, such as J. tanjorensis14, J. curcas16 and J.

274

gossypifolia13, 16. Furthermore the obtained leaf fractions F1 and F2 were analyzed

275

individually, and five additional compounds were obtained, probably because of an

276

increased concentration in fractions of minor compounds (Table 3). In the present

277

study we found presence of terpenes phytol in fractions F1 and F2 and squalene in

278

F2, which have been reported previously in leaves from other Jatropha species as

279

J. curcas29, J. mutabilis30 and J. maheswarii31.

28

. The retention times (Rt), molecular



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Some of the compounds found in lipophilic extracts have shown interesting

281

biological activities, for example, it has been reported that oleate from oleic acid32,

282

lanosterol33, phytol34 and squalene35 have anti-inflammatory activity and ς-sitosterol

283

has radical scavenging activity36.

284

Cell viability and LPS-induced mitochondrial activity. Prior to evaluating

285

whether J. platyphylla extracts showed anti-inflammatory activity, we examined its

286

effect on cell viability in RAW 264.7 macrophages and found that both, phenolic

287

and lipophilic extracts, did not affect cell viability at concentrations up to 400 µgmL-

288

1

289

(Figures 3A, B). We used two different controls, the first one without LPS pre-

290

treatment, and the second one with LPS pre-treatment; the calculated cell viability

291

(%) was based on the control without LPS treatment (Figure 3). Furthermore, LPS

292

induced the increase of mitochondrial dehydrogenase activity at 24 h (Figures 3 A,

293

B), which likely contributed partially to the ROS increase observed (Figures 4 A, B).

294

The LPS-induced mitochondrial activity was not suppressed by increasing levels of

295

the extracts.

296

reduction assay in LPS challenged cells is due to an increase in NADH levels

297

which can also be associated to higher glycolysis activity besides mitochondria

298

activity, both of which are a response associated to the Warburg effect in

299

inflammation

300

did not alter this effect (Figures 3 A, B). Similarly, when leaf fractions F1 and F2

301

were obtained, these fractions in concentration range of 50 – 200 and 94 - 375

302

µgmL-1, respectively, did not affect cell viability nor the Warburg effect (Figure 3 C).

for polyphenols and in a range of 100 – 400 µgmL-1 for lipophilic compounds

It is important to state that the increase observed in the MTS

37

. Thus, the applied extract doses of phenolic and lipophilc extracts


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In the following experiments, J. platyphylla extracts were studied at concentrations

304

described above.

305 306

Effect of J. platyphylla extracts on LPS-induced ROS production.

307

We examined the effects of J. platyphylla extracts on macrophage ROS production

308

using a H2DCFDA probe associated to hydrogen peroxide levels. Results showed

309

that phenolic extracts decreased ROS production in a dose-dependent manner

310

(200 – 400 µg/mL) for both pulp and kernel phenolic extracts, while leaf phenolic

311

extracts, showed large suppression of ROS production to similar levels for both

312

concentrations used (Figure 4A). It is known that phenolic compounds exhibit a

313

range of biological activities in vitro, including antioxidant and anti-inflammatory

314

effects38, the criteria to establish the antioxidant capacity of these compounds is

315

based on several structural characteristics that include the presence of o-

316

dihydroxyl substituents in the B-ring; a double bond between positions 2 and 3; and

317

hydroxyl groups in positions 3 and 539, as in most of phenolic compounds found in

318

J. platyphylla extracts. However, the mode of action of phenolic compounds may

319

not merely exert their effects as free radical scavengers, but may also modulate

320

cellular-signaling processes during inflammation40.

321

In the case of lipophilic extracts, pulp and leaf extracts dramatically diminished

322

ROS production in a dose-dependent way (200 – 400 µg/mL) while kernel extracts

323

showed a partial ROS production decrease only at a concentration of 400 µg/mL

324

(Figure 4B).

325

We also evaluated the LPS-induced superoxide production (Figure 4). The J.

326

platyphylla leaf, pulp and kernel phenolic and lipophilic extracts, did not show any 15 ACS Paragon Plus Environment


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effect on the kinetics of superoxide production, which suggests that these extracts

328

are not affecting NADPH oxidase activity (Figures 4C, D). When DPI is used as

329

inhibitor of NADPH oxidase, the activity was comparable to controls without LPS

330

(data not shown). Among the ROS produced by the cell, superoxide (O2−) is

331

converted by super oxide dismutase into hydrogen peroxide, which is affected by

332

the J. platyphylla phenolic and lipophilic extracts in the present study (Figures 4A,

333

B).

334 335

Effect of J. platyphylla extracts on LPS-induced NO production. The effect of

336

J. platyphylla phenolic and lipophilic extracts on macrophages NO production was

337

examined (Figure 5). Phenolic extracts (200 – 400 µgmL-1) and lipophilic extracts

338

(100 – 400 µgmL-1) decreased NO levels in a dose-dependent manner (Figures

339

5A, B). Kernel and leaves extracts at 400 µgmL-1 exhibited the largest reduction in

340

NO production, 85 and 63%, respectively; similar to J. curcas methanolic extracts

341

(80% methanol) of fruit and leaves at 1000 ugmL-1, which have exhibited a

342

reduction in NO production of ~75% and ~85%, respectively41. Phenolic extracts of

343

J. platyphylla contain apigenin and luteolin glycosides, which according to other

344

studies luteolin and apigenin strongly inhibit LPS-induced nitrite production in a

345

dose-dependent way, mainly due to the suppression of inducible NO synthase42.

346

J. platyphylla pulp lipophilic extract contain oleic acid, which has been reported as

347

a concentration-dependent NO synthase inhibitor43. Similarly, lanosterol, present in

348

J. platyphylla leaf lipophilic extract, showed around 15% reduction in NO

349

production in a previous study44. Furthermore, leaf fractions F1 and F2 were

350

evaluated; and both showed inhibition on NO levels in a dose-dependent way 16 ACS Paragon Plus Environment

Page 17 of 38


351

(Figure 5C), with a larger effect in macrophages treated with F2; probably due to

352

the presence of sterols ς-Sitosterol and lanosterol, and the triterpene squalene.

353

According to Cárdeno et al. (2015)35, squalene decreases intracellular levels of

354

nitrites and inhibits iNOS activity in LPS-stimulated macrophages.

355

The NO is involved on regulation of inflammatory transcription factors, like NF-κB,

356

among others45. In addtion, NO and superoxide anion can form peroxynitrite

357

(ONOO-) which mediates in the cytotoxic effects of NO, including DNA damage,

358

LDL oxidation, isoprostane formation, tyrosine nitration, inhibition of aconitase and

359

mitochondrial respiration46. Studies showing importance of nitric oxide in

360

inflammation may indicate that agents, which modulate nitric oxide production and

361

bioavailability, could be successfully used in the management of inflammatory

362

diseases46.

363 364

Cyclooxygenase (COX) inhibitor screening assay. As shown in Figure 6, the

365

evaluated whole extracts from Jatropha platyphylla showed inhibitory activity

366

against COX-2 and COX-1, indicating a decrease in prostaglandin production with

367

subsequent anti-inflammatory effect. Whole extracts of pulp, kernels and leaf

368

(which included a mixture of polyphenols and lipophilic compounds) showed

369

inhibition of COX-2 activity in a range of ~64 to 88% (Figure 6A), while inhibition of

370

COX-1 activity was in a range of ~42 to 98% (Figure 6B). J. platytphylla COX

371

inhibition values are higher than those reported for J. zeyheri, which exhibited

372

COX-1 and 2 inhibition of 17 and 25%, respectively47. In addition, the J. platytphylla

373

COX inhibition values are higher than those reported for the anti-inflammatory



Page 18 of 38

374

agent indomethacin™, commonly used as a positive control in COX-2 studies (54–

375

70% inhibition)47. Previous studies have demonstrated that individual constituents

376

of J. platyphylla extracts such as luteolin and apigenin are efficacious in COX-2

377

inhibition48. Srivastava et al. 200949 proved chamomile is COX-2 selective due to

378

the main constituent apigenin, which works by a mechanism of action similar to

379

that attributed to non-steroidal anti-inflammatory COX-2 selective drugs, such as

380

sulindac.

381

In general, the results in the present study confirm that J. platyphylla extracts are

382

non-selective to COX-1 and COX-2 and possibly work similar to aspirin by

383

irreversibly inhibiting both forms of cyclooxygenases and making them different

384

from other non-steroidal anti-inflammatory drugs, NSAIDs (e.g. ibuprofen), which

385

are reversible inhibitors and selective to COX-2. Very likely J. platyphylla extracts

386

have the potential to reduce prostaglandins formation (COX-2) as well as blocking

387

the formation of thromboxane A2 in platelets with an inhibitory effect on platelet

388

aggregation (COX-1).

389 390

Hypothetical model explaining the effect of Jatropha platyphylla phenolic

391

and lipophilic extracts on pro-inflammatory mediator production. The present

392

study generated new information that allowed the formulation of a hypothetical

393

model explaining the role of J. platyphylla flavonoids (e.g., apigenin, genistein and

394

luteolin glycosides) and lipophilic compounds (e.g., fatty acids, sterols, terpene) as

395

inhibitors of LPS-induced production of pro-inflammatory mediators in macrophage

396

cells (Figure 7). Since J. platyphylla bioactive compounds do not affect cell viability


Page 19 of 38


397

nor the LPS-induced mitochondrial activity, these compounds are not cytotoxic nor

398

alter the Warburg effect37.

399

Both flavonoids and lipophilic compounds from J. platyphylla inhibit ROS

400

production, and as ROS participates as secondary messengers activating the NF-

401

κB metabolic pathway, this inhibition has an important role on the production of

402

various pro-inflammatory mediators6. Since neither flavonoids nor lipophilic

403

compounds of J. platyphylla extracts inhibit superoxide production kinetics, there is

404

no inhibitory effects on NADPH oxidase, suggesting that ROS decrease levels

405

could be due to direct scavenging or an induced increase activity of antioxidant

406

enzymes. This ROS inhibition is strongly associated to the NO decrease observed

407

in macrophages. Furthermore, leaf fractions F1 and F2 also decreased NO levels,

408

with F2 showing a larger impact due likely to the presence of sterols ς-Sitosterol

409

and lanosterol, and the triterpene squalene. In general, total extracts (mixtures of

410

flavonoids and lipophilic compounds) inhibited both COX-1 and COX-2 activity and

411

prostaglandin production showing no selectivity and suggesting irreversible binding

412

to cyclooxygenases.

413

All of these effects on pro-inflammatory mediators mean that J. platyphylla

414

flavonoids and lipophilic compounds presented anti-inflammatory capacity in

415

macrophage cells. Further investigations should be oriented in identifying the

416

specific molecular targets of these compounds from J. platyphylla against

417

inflammation and studies in vivo to determine dose levels needed for future clinical

418

studies.

419

ACKNOWLEDGMENT



Page 20 of 38

420

This work was supported by the Consejo Nacional de Ciencia y Tecnología

421

(CONACYT) from Mexico through a graduate study fund for author DL Ambriz

422

(grant #264084). We also thank Elisa Schreckinger (TAMU) for technical support.

423


Page 21 of 38


Figure captions Figure 1. Extraction process to obtain lipophilic, phenolic and whole extracts from pulp, kernel and leaves of Jatropha platyphylla. Figure 2. Fractionation process of Jatropha platyphylla leaf lipophilic extracts to obtain two fractions identified as F1 and F2. Figure 3. The effect of Jatropha platyphylla extracts and fractions on LPS-induced mitochondrial activity. Macrophage RAW 264.7 cells were pre-incubated 5 h with extracts of pulp, kernel and leaf from Jatropha platyphylla and leaf fractions and stimulated for 19 h with LPS (1 µg/mL) and compared with controls without or with LPS. Cell viability were assessed for phenolic extracts at 400 µg/mL (A), lipophilic extracts at 100 – 400 µg/mL (B) and lipophilic leaf fractions F1 and F2 at 50 – 200 and 94 – 375 µg/mL (C) Data, obtained from four biological repeats, are shown as mean ± SE values. Different letters show significant differences (p < 0.05) by oneway analysis of variance followed by Tukey-HSD test. Figure 4. The effect of Jatropha platyphylla extracts on LPS-induced reactive oxygen species (ROS) and superoxide generation rate. Macrophage RAW 264.7 cells were pre-incubated 5 h with extracts of pulp, kernel and leaf from Jatropha platyphylla and stimulated for 19 h with LPS (1 µg/mL) and compared with controls without or with LPS. Intracellular ROS was assessed for phenolic extracts at 200 400 µg/mL (A), lipophilic extracts at 200 – 400 µg/mL (B), while superoxide generation was assessed for phenolic extracts at 400 µg/mL(C) and lipophilic extracts at 400 µg/mL (D). Data, obtained from four biological repeats, are shown



Page 22 of 38

as mean ± SE values. Different letters show significant differences (p < 0.05) by one-way analysis of variance followed by Tukey-HSD test. Figure 5. The effect of Jatropha platyphylla extracts and fractions on LPS-induced nitric oxide (NO) production. Macrophage RAW 264.7 cells were pre-incubated 5 h with extracts of pulp, kernel and leaf from Jatropha platyphylla and leaf fractions and stimulated for 19 h with LPS (1 µg/mL) and compared with controls with or without LPS. NO production was assessed for phenolic extracts at 20 – 400 µg/mL (A), lipophilic extracts at 100 – 400 µg/mL (B) and lipophilic leaf fractions F1 and F2 at

50 – 200 and 94 – 375 µg/mL (C) Data, obtained from four biological

repeats, are shown as mean ± SE values. Different letters show significant differences (p < 0.05) by one-way analysis of variance followed by Tukey-HSD test. Figure 6. The effect of whole Jatropha platyphylla extracts on cyclooxygenase activity. Inhibitory effect of pulp, kernel and leaf extracts from Jatropha platyphylla at concentrations of 200 µg/mL were assessed on COX-2 (A) and COX-1 (B) activity by measuring PGF2α (ng/mL) production. Inhibitor DuP-607 (DUP) was used as a positive control for COX-2. The COX inhibitory assay was evaluated using the COX Inhibitor Screening Assay Kit following the manufacturer’s instructions (Cayman Chemical, Ann Arbor, MI, USA). Data obtained from three biological repeats are shown as mean ±SD values. Different letters indicate significant differences by ANOVA/Tukey-HSD (p< 0.05). Figure 7. Proposed mechanistic model of the anti-inflammatory role of flavonoids and lipophilic compounds from Jatropha platyphylla extracts in the TLR pathway.


Page 23 of 38


Binding of LPS to TLR4 receptor triggers generation of ROS from NADPH oxidase and mitochondria. ROS-mediated redox activates the nuclear translocation of NFκB. The NF-κB activation is ROS-dependent and mediates iNOS expression as well as cyclooxygenase. Both COX-1 and COX-2 activities mediate the production of prostaglandins. J. platyphylla polyphenol (flavonoids) and lipophilic extracts (sterols, terpene, fatty acids) exert anti-inflammatory properties by reducing both, the generation of ROS and NO in LPS-induced macrophage RAW 264.7 cells. These compounds do not affect LPS induced mitochondrial activity (no amelioration of the Warburg effect) and do not affect NADPH oxidase activity (superoxide generation rate). In addition, J. platyphylla extracts (mixture of flavonoids and lipophilic compounds) inhibit both COX-1 and COX-2 activity through irreversible binding. Furthermore, lipophilic leaf fractions F1 and F2 inhibit NO generation, suggesting that both sterols and terpenes are most active among the lipophilic compounds.



424

Page 24 of 38

Table 1. Identification of phenolic compounds from Jatropha platyphylla extracts by HPLC/MS2. Rt (min)

Molecular ions + [M-H] [M+H]

Fragment ions 2 MS (m/z) [M-H]

19.40 20.78 21.29 22.54 23.04 24.51 25.21 26.01 30.57

435.98 593.00 447.23 563.31 447.16 431.00 431.25 444.88 312.00

437.04 594.95 565.14 449.07 433.13 433.09 -

327, 357, 401 311, 431, 473, 504, 564, 593 327, 357, 399, 447 353, 365, 383, 443, 473, 563 327, 357, 378, 387, 399, 429 311, 312, 323, 353, 431 311, 341, 371, 383, 413 227, 271, 315 244

Phloridzin Apigenin 8 -C-glucoside rhamnoside(50) Luteolin 6-C-glucoside(51) Apigenin O-pentosyl 8-C-hexosyl , Luteolin 6-O-glucoside(50) (51) , Apigenin 7-O-glucoside(50) (51) Genistein 7-O-glucoside(50) Luteolin 8-C-glucoside(50) Unknown

20.95 22.54 24.53 25.22

593.26 563.34 431.27 431.27

595.10 565.04 433.10 433.06

311, 431, 473, 504, 564, 593 353, 383, 426, 443, 473, 503, 532, 563 283, 311, 341, 342, 413, 431 294, 311, 323, 341, 372, 382, 413, 431

Apigenin8 -C-glucoside rhamnoside(50) Apigenin O-pentosyl 8-C-hexosyl(51) Apigenin 7-O-glucoside(50) Genistein 7-O-glucoside(50)

16.50 19.80 21.43 24.37 24.37 25.46 26.78 28.27 31.97 32.97

337.21 435.74 430.89 374.98 711.22 579.07 593.23 442.69 485.03 564.77

338.81 436.87 712.28 581.01 595.04 -

163, 164, 173 294 167 329 257, 284, 459, 579, 711 431 256, 268, 285, 42, 534 435 321, 417 323, 473

3-O-p-coumaroylquinic acid(52) Unknown Apigenin 7-O-glucoside(50) Unknown Luteolin 6,8-di-C-glucoside succinate Apigenin 7-O-neohesperoside(53) Apigenin 8-C-glucoside Unknown Unknown Apigenin O-pentosyl 8-C-hexosyl(51)

Proposed compound

Pulp

Leaf

Kernel

425 426

*Bold numbers correspond to the most intense fragment.

427


Page 25 of 38

428


Table 2. Identification of lipophilic compounds from Jatropha platyphylla extracts by GC/MS. Retention time

MW

Identification

Type of compound

11.85 13.60 14.7

282 242 224

Oleic acid Hexadecanol Hexadecene

Fatty acid Saturated alcohol hydrocarbon

11.40 15.40 17.80 18.29

270 270 426 414

n-Octadecanol Pentadecanoic acid Lanosterol ς-Sitosterol

Alcohol Fatty acid Sterol Sterol

11.92 14.06 15.40 16.18

242 256 284 277

Pentadecanoic acid Palmitic acid Steric acid Benzene propanoic acid

Fatty acid Fatty acid Fatty acid Aromatic acid

18.18

456

Lup-20(29)-en-28-oic acid, 3β-hydroxy

Terpene

Pulp

Leaf

Kernel

429 430



431

Page 26 of 38

Table 3. Identification of lipophilic compounds by GC/MS from fractions of Jatropha platyphylla leaf extract. Retention time F1 10.49 10.89 13.98 F2 10.89 13.98 13.98 17.32 18.53 20.42

MW

Identification

Type of compound

153 170 296

Imidazole2-amino-5-[(2-carboxy), vinyl] Trans-2- undecen-1-ol Phytol

Amino compound Unsaturated Alcoholic Diterpene

170 212

Trans-2- undecen-1-ol 9,9 Dimethoxybicyclo[3.3.1]nona-2,4-dione

Unsaturated Alcoholic Ketone

296 426 414 410

Phytol Lanosterol ς-Sitosterol Squalene

Diterpene Sterol Sterol Triterpene

432


Page 27 of 38


Figure 1

433



434

Page 28 of 38

Figure 2

435


Page 29 of 38

436


Figure 3



Page 30 of 38

Figure 4


Page 31 of 38


Figure 5



Page 32 of 38

Figure 6


Page 33 of 38


Figure 7



Page 34 of 38

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