Triglycerides constituted of short and medium chain fatty acids and dicarboxylic acids in momordica charantia as well as capric acid inhibit PGE2 production in RAW264.7 macrophages

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Triglycerides constituted of short and medium chain fatty acids and

dicarboxylic acids in Momordica charantia, as well as capric acid, inhibit PGE

2

production in RAW264.7 macrophages

Wen-Huey Wu

a

, Bi-Yu Lin

a

, Yueh-Hsiung Kuo

b,c,1

, Ching-jang Huang

d,*,1

a_{Graduate Program of Nutrition, Department of Human Development and Family Studies, National Taiwan Normal University, 162, Sec. 1, Hoping E. Rd., Taipei 106, Taiwan} b_{Tsuzuki Institute for Traditional Medicine, College of Pharmacy, China Medical University, 91, Hsueh-Shih Rd., Taichung 404, Taiwan}

c

Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan

d

Department of Biochemical Science and Technology and Institute of Microbiology and Biochemistry, National Taiwan University, 1, Sec. 4, Roosevelt Rd., Taipei 106, Taiwan

a r t i c l e

i n f o

Article history:

Received 2 November 2008

Received in revised form 1 March 2009 Accepted 1 April 2009 Keywords: Momordica charantia Bitter gourd Macrophages Prostaglandin E2 Capric acid

a b s t r a c t

This study was aimed to identify compounds in bitter gourd (Momordica charantia) ethyl acetate extract (EAE), which inhibit lipopolysaccharide-induced prostaglandin E2(PGE2) production in RAW264.7 cells. Bitter gourd EAE was partitioned between n-hexane and methanol/H2O (90/10). The hexane fraction was further separated by repeated silica gel chromatographies, and a reverse phase (RP) C18 chromatog-raphy. Fraction RP-10 showed the highest inhibition effect on PGE2production (Max inhibition = 96%, IC50= 2.3lg/ml) and was identiﬁed to be triglycerides constituted of short and medium chain fatty acids by1_{H NMR, IR and H–HCOSY, and dicarboxylic acids by GC/MS. Fatty acids with 3–20 carbons were tested} for the inhibitory activity, and capric acid exhibited the highest effect (Max inhibition = 99%, IC50= 6.5lM). In conclusion, triglycerides composed of short and medium chain fatty acids and dicarbox-ylic acids in bitter gourd inhibit PGE2production, and capric acid is the most potent inhibitor among the fatty acids.

1. Introduction

Prostaglandin E2 (PGE2) is an important pro-inﬂammatory

mediator in many chronic inﬂammatory conditions, including rheumatoid arthritis (Robinson, McGuire, & Levine, 1975), inﬂam-matory bowel diseases (Subbaramaiah et al., 2004), cancer (Sinha, Clements, Fulton, & Ostrand-Rosenberg, 2007), atherosclerosis (Gómez-Hernández et al., 2006), atherosclerotic plague rupture (Cipollone et al., 2004) and age-related degeneration (Wu & Meydani, 2004). In response to intrinsic cytokines (Arias-Negrete, Keller, & Chadee, 1995), or extrinsic factors, such as lipopolysac-charide (LPS) (Rhee & Hwang, 2000), macrophages release large amounts of PGE2. PGE2 also stimulates the production of

proin-ﬂammatory cytokines (Williams & Shacter, 1997). Inhibition of the production of PGE2has been used to ameliorate inﬂammatory

symptoms and suppress related diseases (Guadagni et al., 2007). Bitter gourd (Momordica charantia) is a very common oriental vegetable in tropical and subtropical areas. It is traditionally re-garded as a ‘‘cooling” or ‘‘ﬁre-reducing” food. In our previous study, that characterised ‘‘heating” and ‘‘cooling” foods by their effects on

PGE2 production in LPS-stimulated RAW264.7 macrophages, we

found that ethyl acetate extract (EAE) of bitter gourd inhibited PGE2production (Huang & Wu, 2002). This study was thus aimed

to further investigate the active components in bitter gourd EAE that inhibit PGE2production.

2. Materials and methods 2.1. Materials

Wild bitter gourds (M. charantia L., strain Hualian No. 4), were kindly provided by Mr. Chong-Ho Chuan of Hualien District Agri-cultural Research & Extension Station.1_{H NMR and H–HCOSY}

spec-tra were obtained on Bruker AM-300 and Bruker-500 instruments, respectively. An HP 6890 series gas chromatograph (GC) equipped with a 30 m 0.25 mm 0.25

l

m HP-5 MS column was used for the analysis of fatty acids. Mass spectral analysis was done on a JEOL JMS-HX300 mass spectrometer. Thin-layer chromatography (TLC) was performed on silica gel 60F254 TLC plates (Merck, Darmstadt, Germany). Silica gel (230–400

l

m) (Macherey-Nagel, Germany) and reverse phase C18 were used for column chroma-tography. Infrared spectra were obtained on a Bio-Rad FTS-40 FT-IR. The RAW 264.7 macrophage cell line (CCRC60001, originally from the American Type Culture Collection; designation, TIB-71)

*Corresponding author. Tel.: +886 2 2362 1301. E-mail address:cjjhuang@ntu.edu.tw(C.-j. Huang).

1 _{Equal contribution to this article.}

Contents lists available atScienceDirect

Food Chemistry

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was obtained from the cell bank of the Food Industry Research and Development Institute, Hsin Chu, Taiwan. Dulbecco’s minimal essential medium (DMEM) and fetal bovine serum (FBS) were pur-chased from Gibco (Gaithersburg, MD, USA). LPS (Escherichia coli, serotype 026: B6) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Fatty acids, propionic acid (3:0), butyric acid (4:0), caproic acid (6:0), caprylic acid (8:0), capric acid (10:0), lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0) and dicarboxylic acids, octanedioic acid (suberic acid), nonanedioic acid (azelainic acid) and decanedioic acid (sebacic acid), were purchased from Sig-ma–Aldrich; oleic acid (18:1), linoleic acid (18:2n6), arachidonic acid (20:4n6) and conjugated linoleic acid (CLA) were purchased from Cayman (Ann Arbor, MI, USA); medium chain triglycerides (MCT) containing 67% caprylic acid (8:0), and 23% capric acid (10:0) were purchased from Mead Johnson (Evansville, IL, USA). Conjugated linolenic acid (CLN) was isolated and puriﬁed from bit-ter gourd in our laboratory, previously (Chuang et al., 2006); it was composed of 77% 9c, 11t, 13t-CLN, 11% stearic acid and 12% pal-mitic acid, based on the mass spectral data.

2.2. Preparation of water-soluble and ethyl acetate-soluble fractions of bitter gourd

Fresh whole bitter guard was homogenised and filtered through cotton cloth. The juice was centrifuged and the supernatant was lyophilised to obtain the water extract (WE). Lyophilised whole bitter guard was extracted with ethyl acetate (EA) for 48 h. The extract was filtered through Whatman no. 1 filter paper, followed by removing solvent in a rotary vacuum evaporator (Buchi, Essen, Germany), and ethyl acetate extract (EAE) was obtained. WE and EAE were tested for their effects on LPS-stimulated PGE2

produc-tion in RAW264.7 cells.

2.3. Fractionation of bitter gourd ethyl acetate extract (EAE) in large quantity

One hundred and thirteen kilograms of bitter gourd were lyoph-ilised to produce 7.8 kg of powder. The powder was extracted with EA twice (2 80 l) at room temperature for two days. The pooled extracts were evaporated in a rotary evaporator to remove the sol-vent and yielded 250 g of EAE. EAE was suspended in 1 l of n-hex-ane, and partitioned with 1 l of methanol (MeOH)/H2O (90/10)

three times. The resulting two fractions were evaporated to yield 234 g of n-hexane extract and 6 g of MeOH/H2O (90/10) extract.

Hexane extract was subsequently chromatographed over silica gel with an EA/hexane gradient solvent system and ﬁnally an EA/ MeOH (90/10) solvent. The eluted fractions showing similar thin-layer chromatography (TLC) and1_{H NMR patterns were collected,}

evaporated and weighed; the evaporated crude compounds were screened for their inhibition effects upon LPS-stimulated PGE2

pro-duction in the RAW264.7 macrophages.

Active fractions F189–191 (8.3 g) eluted by EA/MeOH (90/10) were combined, dissolved in acetone, subjected to a second silica gel column chromatography, eluted ﬁrst by chloroform/ethanol (6/1), to yield two subfractions, F1–6 (4.3 g) and F8–12 (0.4 g), and then eluted by chloroform/ethanol (5/1) to yield F13–21 (2.8 g). F1–6 and F8–12, but not F13–21, were found to have the PGE2inhibition effect in macrophages. F1–6 (1.7 g) were dissolved

in ethyl acetate, subjected to a third silica gel column chromatog-raphy, and eluted by EA/hexane (60/40). Fractions with similar TLC patterns were combined to yield seven fractions, and tested for the PGE2inhibition effect in macrophages. The three active fractions

F1–6-2 (247 mg), F1–6-3 (237 mg) and F1–6-4 (258 mg) were combined (742 mg) and subjected to a fourth silica gel column chromatography, successively eluted by an EA/hexane gradient, starting at 90% hexane with a 5% increase in EA for every 12

fractions (150 ml/fraction) collected. Fractions with similar TLC patterns were combined to yield 16 fractions. The active fractions p11–13 (220 mg) were combined and subjected to a reverse phase (RP) C18 chromatography, eluted by MeOH/H2O (80/20), MeOH/

H2O (90/10) and 100% MeOH. Fraction RP-10 (19.7 mg), eluted by

100% MeOH, showed the highest PGE2 inhibition effect. RP-10

was analysed by1_{H NMR and H-HCOSY for structural}

identiﬁca-tion. Fraction F1–6 was subjected to GC/MS for fatty acid analysis after saponiﬁcation and methylation by the diazomethane method (Mueller, 1996).

2.4. Cell culture

RAW 264.7 cells were seeded on 96 well plates at a concentra-tion of 5 105_{cells/ml, and incubated for 24 h at 37 °C in 5% CO}

2.

The unattached cells were washed away and the cells were treated with serum-free Dulbecco’s modiﬁed Eagle’s medium (DMEM) containing bitter guard extracts or fatty acids in the present or ab-sence of 100 ng/ml of LPS for 18 h. Then, the medium was collected and analysed for PGE2using a commercial enzyme immunoassay

kit (Cayman, Ann Arbor, Mich., USA). Cell viability and cell num-bers after treatments were assayed using the MTT method ( Deni-zot & Lang, 1986).

2.5. Statistical methods

Data were expressed as means ± SD. Experiments were repeated at least three times in triplicate. The signiﬁcance of difference was analysed by one way analysis of variance (ANOVA) and a Duncan’s multiple rank test, using SPSS software. To calculate the percentage inhibition of LPS-stimulated PGE2production, PGE2production in

the presence of 100 ng/ml of LPS was taken as 0% inhibition and basal PGE2production as 100% inhibition; the percentage

inhibi-tion by a sample at a speciﬁc concentrainhibi-tion was calculated as [1 (PGE2 sample with LPS PGE2 basal)/(PGE2 LPS only PGE2

basal)] 100%. The IC50value, calculated from the curve of

per-centage inhibition versus concentration, is the concentration of sample that results in 50% inhibition.

3. Results

3.1. Culture conditions for measuring PGE2

PGE2production induced by LPS (100 ng/ml) in RAW264.7 was

tested for 0, 12, 18, and 24 h and the maximal production of PGE2

0 10000 20000 30000 40000 50000 60000 70000 80000 0 1 5 10 20 50 100 200 400 LPS (ng/ml) PGE 2 (pg/1 x 10 5 cells) DMEM 10%FBS+DMEM

Fig. 1. PGE2production induced by various concentrations of LPS in RAW264.7 cells

cultured with or without the addition of fetal bovine serum (FBS). The cells were treated with LPS at 0–400 ng/ml with or without 10% FBS for 18 h and medium was collected for PGE2 analysis using an EIA. At least three batches of separate

experiments were carried out with similar results. The values are means ± SD of triplicates in a representative experiment.

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+ LPS IC₅₀ = 14.2 μg/ml Max inhibition = 99.1% 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 0 5 10 50 100 150 200 basal

Bitter gourd EAE (_μg/ml)

PGE 2 (pg/1 x 10 5 cells)

A

a b bc cd d e e e + LPS IC₅₀ = 8.9 _μg/ml Max inhibition = 66.9% 0 2000 4000 6000 8000 10000 12000 14000 16000 18000 0 5 10 50 100 200 basal Bitter gourd WE (_μg/ml) PGE 2 (pg/1 x 10 5 cells)

B

a ab bc bc c c d - LPS 0 200 400 600 800 0 5 25 50 100

Bitter gourd EAE (μg/ml)

PGE 2 (pg/1x10 5 cells)

C

a _a a a a - LPS 0 200 400 600 800 0 5 10 50 100 Bitter gourd WE (_μg/ml) PGE 2 (pg/1 x 10 5 cells)

D

a a a a a

Fig. 2. Effects of ethyl acetate extract (EAE) (A and C) and water extract (WE) (B and D) of bitter gourd on PGE2production in RAW264.7 cells. The cells were treated with

various concentrations of the extracts in the presence (+LPS) (A and B) or absence (-LPS) (C and D) of LPS (100 ng/ml) for 18 h and then medium was collected for PGE2

analysis. The values are means ± SD of triplicates in a representative experiment. Values not sharing the same letter are signiﬁcant different (p < 0.05). Basal: cells incubated with medium only. 0: cells treated with LPS only.

0 2000 4000 6000 8000 10000 12000 14000 16000 18000 20000 F28 F64 F92_F114_F141_F152_F162_F170_F190 0

Fractions from hexane extracts

PGE 2 (pg/1x10 5cells) 25 ug/ml 50 ug/ml 75 ug/ml

A

0 2000 4000 6000 8000 10000 12000 14000 0 0.1 1 10 25 50 75 basal F190 (_μg/ml) PGE 2 (pg/1x 10 5 cells) IC₅₀ = 3.04 _μg/ml Max inhibition = 97.8% c a a

B

0 2000 4000 6000 8000 10000 12000 14000 16000 18000 0 0.1 1 5 10 50 basal F1-6 (_μg/ml) PGE 2 (pg/1 x 10 5 cells) e d cd bc ab a = 7.05 μg/ml Max inhibition = 91.0%

C

0 1000 2000 3000 4000 5000 6000 7000 8000 0 0.1 1 10 25 50 basal RP-10 (μg/ml) PGE 2 (pg/1 x 10 5 cells) IC50 = 2.31 μg/ml Max inhibition = 96.3%

D

IC50

Fig. 3. Effects of different fractionations of bitter gourd EAE after chromatography on PGE2production in LPS-stimulated RAW264.7 cells. Bitter gourd hexane-partitioned

fraction was subjected to a silica gel chromatography and eluted by EA/hexane. F28 from EA/hexane (v/v) 2/98, F64 from 5/95, F92 from 10/90, F114 from 20/80, F141 from 30/70, F152 from 40/60, F162 from 60/40, F170 from 80/20 and F190 from EA/MeOH (90/10) were tested for PGE2suppression effect in LPS-stimulated RAW264.7 cells (A).

Dose–responses of bitter gourd active fractions F190 (B), F1–6 (C) and RP-10 (D) on PGE2suppression. The values are means ± SD of triplicates in a representative experiment.

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was found to occur at 18 h (data not shown). PGE2production

in-duced by LPS at concentrations of 0, 1, 5, 10, 20, 50, 100, 200, 400 ng/ml in RAW264.7, incubated with or without 10% FBS for 18 h, was tested. At LPS concentration of 100 ng/ml, the production of PGE2in cells cultured without FBS reached peak level (Fig. 1)

but, in cells cultured with FBS, PGE2production increased

dramat-ically as the concentration of LPS increased. To avoid over stimula-tion, culture medium without FBS was used during LPS stimulation in the following experiments. The concentrations of extracts used to culture cells were those which did not cause cytotoxicity exam-ined by the MTT assay.

3.2. Inhibition of PGE2production by bitter gourd extracts and the

separated fractions

Both kinds of bitter gourd extracts, WE and EAE, decreased the secretion of PGE2in LPS-stimulated RAW264.7 (Fig. 2A and B), but

the EAE had higher inhibition activity than had WE. Without LPS stimulation, the basal PGE2levels were low and not affected by

either extract (Fig. 2C and D).

After partition of EAE, the hexane-soluble fraction showed high-er inhibitory effect than did the MeOH/H2O (90/10)-soluble

frac-tion (data not shown). The hexane-soluble fracfrac-tion was then separated by a silica gel chromatography. The fraction F190, eluted by EA/MeOH (90/10), showed the highest PGE2inhibitory activity

(Fig. 3A), with maximal inhibition of 97.8% and IC50of 3.04

l

g/ml

(Fig. 3B). Then, fractions F189–191, with similar patterns on TLC,

were pooled and separated by a second silica gel chromatography. The obtained fraction F1–6, eluted by chloroform/ethanol (6/1), had the highest activity (maximal inhibition = 91.0% and IC50= 7.05

l

g/ml) (Fig. 3C). The fraction F1–6 was further

sepa-rated by two successive silica gel chromatographies and ﬁnally a reverse phase chromatography. The fraction RP-10, eluted with methanol, showed the highest PGE2 inhibition effect (maximal

inhibition = 96.3% and IC50= 2.31

l

g/ml) (Fig. 3D).

3.3. Chemical structures of active components

Analysis of RP-10, using1_{H NMR and H–HCOSY, revealed that}

its major components were triglycerides (TGs) constituted of short and medium chain fatty acids. To identify the fatty acid composi-tion, the F1–6 fraction was further saponiﬁed, methylated and ana-lysed using GC/MS. As a result, common fatty acids, and three dicarboxylic acids, octanedioic, nonanedioic and decanedioic acids, were identiﬁed.

3.4. Inhibition of PGE2production by speciﬁc fatty acids

Of all long chain fatty acids tested, none inhibited PGE2

produc-tion in LPS-stimulated RAW264.7, but linoleic and arachidonic acids induced dose-dependent increase in the production of PGE2

(Fig. 4A). In contrast, among short and medium chain fatty acids, capric (10:0) and lauric (12:0) acids induced dose-dependent inhi-bition of PGE2production in LPS-stimulated RAW264.7, and capric

0 2000 4000 6000 8000 10000 12000 14000 16:0 18:0 18:1 18:2 18:3n6 9-c,11-t CLA 10-t,12-c CLA CLN 20:4n6 none Fatty acids PGE 2 (pg/1 x 10 5 cells) 25 μΜ 50 μΜ 100 μΜ

A

0 2000 4000 6000 8000 10000 12000 14000 16000 18000 3:0 4:0 6:0 8:0 10:0 12:0 14:0 16:0 none

Fatty acids

PGE 2 (pg/1 x 10 5 cells) 25 μΜ 50 μΜ 100 μΜ

B

Fig. 4. Effects of different fatty acids on PGE2production in LPS-stimulated RAW264.7 cells. The cells were treated with long chain (A) and short and medium chain fatty acids

(B) in the presence of LPS (100 ng/ml) for 18 h and then medium was collected for PGE2analysis. CLA, conjugated linoleic acid; CLN, 77% 9c, 11t, 13t-conjugated linolenic acid.

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acid showed the highest inhibition activity (Fig. 4B). At a concen-tration of 50

l

low and is likely to occur in the blood stream (Richieri & Kleinfeld, 1995). Capric acid has also been found to be the most effective among six fatty acids in killing Gram-negative bacteria by disrupt-ing the outer membrane (Thormar, Hilmarsson, & Bergsson, 2006). The remaining fatty acids tested, including caprylic acid (8:0), lau-ric acid (12:0), mystic acid (14:0), palmitoleic acid (16:1) and oleic acid (18:1), had either no effect or only a low effect (Thormar et al., 2006). LPS is a major component of the outer membrane of Gram-negative bacteria, contributing greatly to the structural integrity of the bacteria. Capric acid might serve as a permeabiliser that in-duced the release of LPS from the outer membrane of Gram-nega-tive bacteria as lactic acid did (Alakomi et al., 2000), and our observations indicated that it could simultaneously abolish the stimulation of LPS on PGE2production of macrophages.

Only few foods contain signiﬁcant amounts of capric acid, of which bovine milk fat, coconut oil and MCT are major sources. Milk fat contains 2.4% capric acid, but also contains 11% myristic acid and 33% palmitic acid (Odongo et al., 2007). Coconut oil contains 6% capric acid, but also contains 49% lauric acid and 19% myristic acid (Laureles et al., 2002). The co-existence of atherogenic fatty acids may counteract the potential beneﬁt of capric acid in these two oils. MCT oil, on the other hand, has a higher level of capric acid (23%) and is devoid of atherogenic fatty acid. Our study further showed that MCT inhibited the production of PGE2 as well

(Fig. 5D).Kono et al. (2003)compared the effects of MCT and corn oil on endotoxemia in rats, and found that MCT caused much less mortality and liver injury. Kupffer cells, isolated from rats given 0 2000 4000 6000 8000 10000 12000 0 0.1 1 10 25 50 100 basal Capric acid (_μM) PGE 2 (pg/1x10 5 cells) a a b c d e e μ

A

C

0 2000 4000 6000 8000 10000 12000 0 1 10 25 50 100 200 basal Octanedioic acid _μ a a abc bcd d

B

ab cd 0 2000 4000 6000 8000 10000 12000 0 1 10 25 50 100 200 basal Nonanedioic acid (_μM) PGE 2 (pg/1 x 10 5 cells) a a a ab _{ab ab} b c 0 2000 4000 6000 8000 10000 12000 0 0.1 1 10 25 basal MCT (_μg/ml) PGE 2 (pg/1x10 5 cells) b c c d

D

c a

Fig. 5. Dose–response of capric acid (A), octanedioic acid (B), nonanedioic acid (C) and MCT (D) on PGE2suppression in LPS-stimulated RAW264.7 cells. The cells were treated

with various concentrations of fatty acids or MCT in the presence of LPS (100 ng/ml) for 18 h and then medium was collected for PGE2analysis. The values are means ± SD of

triplicates in a representative experiment. Values not sharing the same letter are signiﬁcant different (p < 0.05). Basal: cells incubated with medium only. 0: cells treated with 100 ng/ml LPS only.

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MCT, showed reduced LPS-stimulated TNF

a

production, and CD14 expression (Kono et al., 2003), which is a cell membrane receptor of LPS (Lu, Yeh, & Ohashi, 2008).

The LPS-sensing machinery consists, primarily, of LPS-binding protein (LBP), CD14, and toll-like receptor 4 (TLR4), a signal-trans-ducing integral membrane protein (Lu et al., 2008). Upon binding of LPS to cells, the sensing machinery then triggers intracellular signalling that leads to the expression of genes encoding proin-ﬂammatory cytokines or enzymes producing inproin-ﬂammatory mole-cules through the transcription factor nuclear factor

j

B (NF

j

B). The PGE2production of LPS-stimulated macrophage is known to

be mediated by inducing COX-2 expression through the LPS-sens-ing machinery pathway (Lee & Hwang, 2006). The COX-2 expres-sion and activity is considered the primary limiting factor for PG production in activated macrophages.

It has been demonstrated that saturated fatty acids, and espe-cially lauric acid, which is the predominant fatty acid acylated in lipid A of LPS, could induce NF

j

B activation and expression of COX-2 in macrophages (Lee, Sohn, Rhee, & Hwang, 2001). The acyl-ation and types of acylated fatty acids of lipid A determine the bio-logical activities of LPS (Lee & Hwang, 2006). Conversely, unsaturated fatty acids inhibit lauric acid- or LPS-induced COX-2 expression (Lee et al., 2001). However, our results showed capric acid to be highly effective, and lauric acid (12:0) to a smaller ex-tent, in inhibiting PGE2 production, while linoleic acid (18:2n6)

and arachidonic acid (20:4n6) enhanced PGE2production. We also

found that the co-treatment with LPS and capric acid did not alter COX-2 protein expression in RAW 264.7 cells compared to treat-ment with LPS alone (data not shown). The discrepancy in the ef-fect of saturated fatty acids on LPS-stimulated macrophages between our andLee et al.’s (2003)studies might be partly ex-plained by the differences in fatty acids tested (capric acid vs lauric acid) and experimental conditions (simultaneous treatment with fatty acids and LPS vs treatment with fatty acids prior to LPS) since lauric acid alone could have stimulated COX-2 expression (Lee et al., 2001). Besides the role of COX-2 expression, the activity of COX-2 and the availability of arachidonic acid, which is derived from the hydrolysis of phospholipids catalysed by phospholipase A2, also affect the biosynthesis of PGE2.The effects of capric acid

on COX-2 activity and phospholipase A2expression and activity

need further investigation.

In conclusion, the triglycerides composed of short and medium chain saturated fatty acids and dicarboxylic acids are the active components in bitter gourd EAE contributing to the inhibition of PGE2 production, and capric acid is the most potent inhibitor

among various fatty acids. Results reported here should prompt studies in other food products with similar triglyceride or fatty acid proﬁles. However, the potential beneﬁts of the bioactive com-pounds in vegetables will need to be demonstrated in human trials. Acknowledgement

This study was funded by a Grant, NSC 95-2317-B-003-001, from the National Science Council of the Republic of China, Taiwan. References

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