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Anti-inflammatory principles from Heritiera littoralis bark.

ABSTRACT

Compounds from the hexane, dichloromethane and acetone extracts of Heritiera littoralis bark were investigated for their nitric oxide (NO) inhibitory effects using RAW264.7 macrophage cells. The result indicated that ergosterol peroxide (13) exhibited the highest activity against NO release with an [IC.sub.50] value of 2.5 [mu]M, followed by 6-[alpha]-hydroxystigmast-4-en-3-one (11, [IC.sub.50] = 9.5 [micro]M) and stigmast-4-en-3-one (9, [IC.sub.50] = 15.9 [micro]M), whereas other compounds showed moderate and mild effects (25.4- > 100 [micro]M). Ergosterol peroxide (13) and 6-[alpha]-hydroxystigmast-4-en-3-one (11) were also tested against prostaglandin [E.sub.2] (PG[E.sub.2]) and tumor necrosis factor alpha (TNF-[alpha]) releases. It was found that ergosterol peroxide (13) possessed marked activity against PG[E.sub.2] release with an [IC.sub.50] value of 28.7[micro]M. while 6-[alpha]-hydroxystigmast-4-en-3-one(11)was 86.7 [micro]M. However, these two compounds were inactive towards TNF-[alpha] release ([IC.sub.50] > 100[micro]M). The mechanism in transcriptional level of ergosterol peroxide (13) was found to down regulate mRNA expressions of iNOS and COX-2 in dose-dependent manners.

[c] 2010 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Keywords:

NO

Inflammatory mediators

Heritiera littorolis

Sterculiaceae

Ergosterol peroxide

Introduction

Heritiera littoralis Dry., locally known in Thai as Ngon kai thale, is the mangrove that widely distributed in East Africa and Madagascar (Tomlinson 1986). In Thailand, H. littoralis has been found in the eastern and southern parts. This plant is a substantial tree (20-25 m height) and is typically found in the mangrove zones which are upstream and low salinity areas. The bark is grayish, fissured and scaly. In terms of medicinal uses, the Vietnameses use the seeds to treat diarrhea and dysentery by decoction (Bamroongrugsa 1999), whereas the local fishermen in Philippines use the sap as fish poison (Miles et al. 1991).

Since the extract of H. littoralis possessed high NO inhibitory effect (18.8 [mu]g/ml), the compounds from this plant were then isolated and tested for NO, [PGE.sub.2] and TNF-[alpha] inhibitory activities, as well as the mechanism on iNOS and COX-2 mRNA expressions of active compounds using RAW264.7 macrophage cells.

Materials and methods

Reagents

Lipopolysaccharide (LPS, from Escherichia coli), RPMI-1640 medium, 3-(4,5-dimethyl-2-tbiazolyl)-2,5-diphenyl-2H-tetrazo-lium bromide (MTT), L-nitroarginine (l-NA), caffeic acid phenethylester (CAPE), indomethacin and phosphate buffer saline (PBS) were purchased from Sigma Aldrich (Sigma Aldrich, Missouri, USA). Fetal calf serum (FCS) was bought from Gibco (Invitrogen, California, USA). Penicillin-streptomycin was purchased from Invitrogen (Invitrogen, California, USA). 96-Well microplates were obtained from Nunc (Nunc, Birkrod, Denmark). ELISA test kits of [PGE.sub.2] and TNF-a were from R&D systems (R&D systems, Minnesota, USA). Other chemicals were from Sigma Aldrich (Sigma Aldrich, Missouri, USA).

Plant material

H. littoralis bark was collected from Songkhla province, Thailand in November 2004 and was identified by Prof. Puangpen Sirirugsa, Department of Biology, Faculty of Science, Prince of Songkla University. The specimen (No. CD01) was deposited at Prince of Songkla University Herbarium.

Extraction and isolation of compounds from H. littoralis extract

The air-dried bark of H. littoralis (6.0 kg) was extracted with hexane (2 x 301), [CH.sub.2][Cl.sub.2] (2 x 251) and acetone (2 x 251) successively for 5 days at room temperature. The extract was filtered and concentrated under vacuo to give crude extract of hexane (30.7 g), dichloromethane (32.0 g) and acetone fractions (44.0 g), respectively.

The hexane fraction (30.7 g) was further isolated by quick column chromatography (QCC) using silica gel and eluted with hexane and increasing polarity with [CH.sub.2][Cl.sub.2] and MeOH to obtain 8 fractions (A1-A8). Fraction A2 was further isolated by column chromatography (CC) using silica gel and eluted with hexane to give compounds 1 (936.6 g), 14 (4.5 mg) and 15 (20.2 mg), respectively. Fraction A4 was purified by CC using silica gel and washed with hexane which finally afforded compounds 7 and 8 (mixture, 7.6 mg) and 9 (43.3 mg), respectively. Fraction A6 was further isolated by CC using silica gel and eluted with 90% [CH.sub.2][Cl.sub.2] in hexane to obtain compound 2 (10.4 mg). Fraction A7 was separated by QCC, and Sephadex LH20 to afford compounds 5 (4.6 mg), 10 (13.2 mg) and 11 (9.4 mg), respectively.

Dichloromethane fraction (32.0g) was separated by QCC using silica gel and eluted with hexane and increasing polarity with dichloromethane and methanol, successively to afford seven fractions B1--B7. Fraction B2 was subjected to CC using 15% acetone and hexane to give compound 1 (115.7mg). Fraction B3 was washed with hexane to afford compound 2 (14.0 mg). Fraction B4 was subjected to QCC and eluted with hexane and increasing polarity with ethyl acetate to give seven subfractions. Subfraction B4 was washed with hexane to yield compounds 3 (128.6 mg) and 6 (4.5 mg). Fraction B6 was separated by CC using hexane and increasing polarity with ethyl acetate to afford six subfractions. Subfraction B6 was purified by CC using 10% ethyl acetate in dichloromethane to give compounds 13 (8.3 mg) and 16 (14.4 mg).

Acetone fraction (44.0 g) was purified by QCC using silica gel and washed with gradient eluent of hexane, dichloromethane and methanol to obtain six fractions (C1-C6). Fraction C2 was separated by QCC using 70% dichloromethane in hexane to afford three subfractions. Subfraction C2 was washed with hexane to obtain compounds 3 (33.3 mg) and 4 (6.6 mg). Fraction C3 was purified by QCC using 3% methanol in dichloromethane to obtain three subfractions. Subfraction C3 was rechromatographed on CC using 30% ethyl acetate in hexane as eluent to afford compound 17 (9.4 mg). Fraction C4 was washed with hexane and crystallized with 50% methanol in dichloromethane to give compound 12 (31.5 mg). Fraction C5 was purified by QCC using hexane and increasing polarity with acetone as eluent to afford five sub-fractions. Subfraction C5 was crystallized with 80% methanol in dichloromethane to afford compound 18 (30.0 mg). All these compounds were identified by comparison of their spectroscopic data with those reported in the literatures (Ahad et al. 1991; Arai et al. 1998; Ali et al. 2001; Castola et al. 2002; Cheenpracha 2004; Chu et al. 2005; David et al. 2004; Deachathai 2005; Delia Creca et al. 1990; Elix and Wardlaw 1997; Macias et al. 1994; Martinez et al. 1988; Miles et al. 1991; Moiteiro et al. 2001; Rosecke and Konig 2000; Thongdeeying 2005; Vardamidesa et al. 2003; Yue et al. 2001).

Assay for NO inhibitory effect from RAW264.7 cells

Inhibitory effect on NO production by murine macrophage-like RAW264.7 cells was evaluated using a modified method from that previously reported (Banskota et al. 2003). Briefly, the RAW264.7 cell line (purchased from Cell Lines Services) was cultured in RPMI medium supplemented with 0.1% sodium bicarbonate and 2mM glutamine, penicillin G (100 U/ml), streptomycin (100[micro]g/ml) and 10% FCS. The cells were harvested with trypsin-EDTA and diluted to a suspension in a fresh medium. The cells were seeded in 96-well plates with 1 x [10.sup.5] cells/well and allowed to adhere for 1 h at 37 [degrees]C in a humidified atmosphere containing 5% [CO.sub.2] After that the medium was replaced with a fresh medium containing 200 [micro]g/ml of LPS together with the test samples at various concentrations (3-100 p.g/ml for crude extract and 3-100 [micro]/M for pure compounds) and was then incubated for 48 h. NO production was determined by measuring the accumulation of nitrite in the culture supernatant using the Griess reagent. Cytotoxicity was determined using the MTT colorimetric method. Briefly, after 48 h incubation with the test samples, MTT solution (10 [micro]l, 5 mg/ml in PBS) was added to the wells. After 4 h incubation, the medium was removed, and isopropanol containing 0.04 M HCl was then added to dissolve the formazan production in the cells. The optical density of the formazan solution was measured with a microplate reader at 570 nm. The test compounds were considered to be cytotoxic when the optical density of the sample-treated group was less than 80% of that in the control (vehicle-treated) group. L-NA, CAPE and indomethacin were used as positive controls. The stock solution of each test sample was dissolved in DMSO, and the solution was added to the medium RPMI (final DMSO is 1%). Inhibition (%) was calculated using the following equation and [IC.sub.50] values were determined graphically (n = 4):

Inhibition (%) = A-B/A-C x 100

A-C: [NO.sub.2.sup.-] concentration ([micro]M) [A: LPS (+), sample (-); B: LPS (+), sample(+); C: LPS (-), sample (-)].

Inhibitory effects on LPS-induced [PGE.sub.2] and TNF-[alpha] releases from RAW264.7 cells

Briefly, the RAW264.7 cell line was cultured in RPMI medium supplemented with 0.1% sodium bicarbonate and 2 mM glutamine, penicillin G (100 U/ml), streptomycin (100 [micro]g/ml) and 10% FCS. The cells were harvested with trypsin--EDTA and diluted to a suspension in a fresh medium. The cells were seeded in 96-well plates with 1.0 x [10.sup.5] cells/well and allowed to adhere for 1 h at 37[degrees]C in a humidified atmosphere containing 5% [CO.sub.2]. After that the medium was replaced with a fresh medium containing 200 [micro]g/ml of LPS together with the test samples at various concentrations (3-100 [micro]M) and was then incubated for 48 h. The supernatant was transferred into 96 well ELISA plate and then [PGE.sub.2] and TNF-[alpha] concentrations were determined using commercial ELISA kits. The test sample was dissolved in DMSO, and the solution was added to RPMI. The inhibition on [PGE.sub.2] and TNF-[alpha] releases was calculated and [IC.sub.50] values were determined graphically.

Total RNA isolation and RT-PCR

In order to acquire the mechanism of action on cytokine release of ergosterol peroxide (13), the assays for mRNA expression of iNOS and COX-2 were carried out. The total RNA was isolated from RAW264.7 cells and was harvested after 20 h of incubation with samples in various concentrations (3, 10, 30, 100 [micro]M) using the RNeasy Mini Kit (Qiagen Operon Co. Ltd., USA). The total RNA from each sample was used for cDNA synthesis using first strand cDNA synthesis kit (Rever Tra Ace-[alpha], TOYOBO Co., Ltd., Japan), followed by RT-PCR (Rever Tra Dash, TOYOBO Co., Ltd., Japan). The primers for iNOS and COX-2 were used (forward primer for iNOS: 5'-ATCTGGATCAGGAACCTGAA-3' and its reverse primer: 5'-CCTTTTTTGCCCCATAGGAA-3'; forward primer for COX-2: 5'-GGAGAGACTATCAAGATAGTGATC-3' and its reverse primer: 5'-ATGCTCAGTAGACTTTTACAGCTC-3'; forward primer for [beta]-actin (an internal standard): 5'-TGTGATGGTGGGAATGGGTCAG-3' and reverse primer: 5'-TTTGATGTCACGCACGATTTCC-3'.

The solution for cDNA synthesis consisted of RNA solution 11 [micro]l, 5x RT buffer 4[micro]l, dNTP mixture (10mM) 2 [micro]l, RNase inhibitor (10U/[micro]l) 1 [micro]l, Oligo(dT)20 1 [micro]l and Rever Tra Ace (reverese transcriptase enzyme) 1 [micro]l for a 20 [micro]l reaction. The condition for cDNA synthesis was as follow; 42[degrees]C for 20 min, 99[degrees]C for 5 min and 4[degrees]C for 5 min. After that, 1/10 times (2 [micro]l) of cDNA product was used further for PCR. The PCR mixture consisted of RT reaction mixture (cDNA product) 2 [micro]l; sterilized water 85 [micro]l, 10x PCR buffer 10 [micro]l, forward primer (10 pmol/[micro]l) 1 [micro]l, reverse primer (10 pmol/[micro]l) 1 [micro]l and KOD Dash (polymerase enzyme)1 [micro]l for final volume of 100 [micro]l. The condition for PCR was as follow; denaturation at 94[degrees]C for 1 min, 98[degrees]C for 30s, 55[degrees]C for 30s and 74 C for 1 min (30 cycles). The PCR products were analyzed in 1.2% agarose gel electrophoresis and visualized by SYBR safe staining and UV irradiation under a wavelength of 312 nm.

Statistical analysis

For statistical analysis, the values are expressed as mean [+ or -] S.E.M. of four determinations. The [IC.sub.50] values were calculated using the microsoft excel programme. The statistical significance was calculated by one-way analysis of variance (ANOVA), followed by Dunnett's test.

Results and discussion

Compounds (Fig. 1) from the hexane, dichloromethane and acetone extracts of H. littoralis bark were investigated for their nitric oxide (NO) inhibitory effects using RAW264.7 macrophage cells. The result indicated that ergosterol peroxide (13) exhibited the highest activity against NO release with an [IC.sub.50] value of 2.5 [micro]M, followed by 6-[alpha]-hydroxystigmast-4-en-3-one (11, [IC.sub.50] = 9.5 [micro]M) and stigmast-4-en-3-one (9, [IC.sub.50 = 15.9 [micro]M), whereas other compounds showed moderate and mild effects (25.4 to > 100 [micro]M) (Table 1). The effect of ergosterol peroxide (13) against NO release was higher than that of CAPE, an NF-kB inhibitor ([IC.sub.50] = 5.6 [micro]M), indomethacin, a non-steroidal anti-inflammatory drug (NSAID, [IC.sub.50] = 25 [micro]M) and L-NA, a NO synthase inhibitor ([IC.sub.50 = 61.8 [micro]M). Ergosterol peroxide (13) and 6-[alpha]-hydroxystigmast-4-en-3-one (11) were also tested against prostaglandin [E.sub.2] ([PGE.sub.2]) and tumor necrosis factor alpha (TNF-[alpha]) releases. It was found that ergosterol peroxide (13) possessed marked activity against [PGE.sub.2] release with an [IC.sub.50] value of 28.7 [micro]M, while 6-[alpha]-hydroxystigmast-4-en-3-one (11) was 86.7 [micro]M (Table 2). However, these two compounds were inactive towards TNF-[alpha] release ([IC.sub.50] > 100 [micro]M). Compound 13 was also examined for its anti-inflammatory mechanism against mRNA expressions. The mechanism in transcriptional level of ergosterol peroxide (13) was found to down regulate mRNA expressions of iNOS and COX-2 in dose-dependent manners (Fig. 2). Ergosterol peroxide (13), a steroidal derivative, has been reported to show marked anti-cancer activity against MCF-7 human breast cancer cells (Ioannou et al. 2009). However, the inhibitory effect of this compound on inflammatory mediators including NO, [PGE.sub.2] and TNF-[alpha] releases have not yet been studied.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]
Table 1

NO inhibitory activity of compounds 1-16 from Heritiera littolaris
bark.

Compound                              % inhibition at various
                                      concentrations ([mu]M) 0

(l) Friedelin                         0.0 [+ or -] 3.1

(2) 3-[alpha]-Hydroxyfriedelan-2-one  0.0 [+ or -] 4.2

(3) Cerin                             0.0 [+ or -] 4.2

(4) Friedelan-3-one-29-ol             0.0 [+ or -] 3.1

(5) Betulinic acid                    0.0 [+ or -] 3.1

(6) 3-[beta]-O-E-feruloyl oleanolic   0.0 [+ or -] 4.5
acid

(7) [beta]-Sitosterol + (8)           0.0 [+ or -] 6.3
Stigmasterol

(9) Stigmast-4-en-3-one               0.0 [+ or -] 4.5

(10) 6-[beta]-Hydroxy                 0.0 [+ or -] 4.2
stigmast-4-en-3-one

(11) 6-[alpha]-Hydroxy                0.0 [+ or -] 3.8
stigmast-4-en-3-one

(12) [beta]-Sitosterol                0.0 [+ or -] 4.5
glucopyranoside

(13) Ergosterol peroxide              0.0 [+ or -] 4.5

(14) Physcion                         0.0 [+ or -] 1.6

(15) Methyl [beta]-orinol             0.0 [+ or -] 1.6
carboxylate

(16) Vallapin                         0.0 [+ or -] 3.8

(17) 5-Propylresorcinol               0.0 [+ or -] 1.6

(18) (-)-Epicatechin                  0.0 [+ or -] 3.8

L-Nitroarginine (L-NA)                0.0 [+ or -] 9.9

Caffeic acid phenethylester (CAPE)    0.0 [+ or -] 9.9

Indomethacin                          0.0 [+ or -] 3.6

Compound                              3

(l) Friedelin                         -

(2) 3-[alpha]-Hydroxyfriedelan-2-one  -

(3) Cerin                             -

(4) Friedelan-3-one-29-ol             -

(5) Betulinic acid                    -

(6) 3-[beta]-O-E-feruloyl oleanolic   -
acid

(7) [beta]-Sitosterol + (8)           -
Stigmasterol

(9) Stigmast-4-en-3-one               -

(10) 6-[beta]-Hydroxy                 -
stigmast-4-en-3-one

(11) 6-[alpha]-Hydroxy                25.2 [+ or -] 3.1*
stigmast-4-en-3-one

(12) [beta]-Sitosterol                -
glucopyranoside

(13) Ergosterol peroxide              50.2 [+ or -] 2.3**

(14) Physcion                         -

(15) Methyl [beta]-orinol             -
carboxylate

(16) Vallapin                         -

(17) 5-Propylresorcinol               -

(18) (-)-Epicatechin                  -

L-Nitroarginine (L-NA)                11.7 [+ or -] 4.6

Caffeic acid phenethylester (CAPE)    30.7 [+ or -] 3.2

Indomethacin                          14.5 [+ or -] 2.7

Compound                              10

(l) Friedelin                         -

(2) 3-[alpha]-Hydroxyfriedelan-2-one  -

(3) Cerin                             -

(4) Friedelan-3-one-29-ol             9.2 [+ or -] 2.4

(5) Betulinic acid                    -10.3 [+ or -] 1.8

(6) 3-[beta]-O-E-feruloyl oleanolic   15.3 [+ or -] 2.7
acid

(7) [beta]-Sitosterol + (8)           1.0 [+ or -] 7.5
Stigmasterol

(9) Stigmast-4-en-3-one               26.6 [+ or -] 2.6*

(10) 6-[beta]-Hydroxy                 -
stigmast-4-en-3-one

(11) 6-[alpha]-Hydroxy                50.4 [+ or -] 4.0**
stigmast-4-en-3-one

(12) [beta]-Sitosterol                10.2 [+ or -] 2.1
glucopyranoside

(13) Ergosterol peroxide              85.4 [+ or -] 2.2**

(14) Physcion                         -

(15) Methyl [beta]-orinol             -
carboxylate

(16) Vallapin                         19.9 [+ or -] 4.3

(17) 5-Propylresorcinol               -

(18) (-)-Epicatechin                  -

L-Nitroarginine (L-NA)                20.2 [+ or -] 5.9

Caffeic acid phenethylester (CAPE)    68.6 [+ or -] 3.4 (b)**

Indomethacin                          30.2 [+ or -] 1.6**

Compound                              30

(l) Friedelin                         -

(2) 3-[alpha]-Hydroxyfriedelan-2-one  -

(3) Cerin                             -

(4) Friedelan-3-one-29-ol             66.8 [+ or -] 4.1**

(5) Betulinic acid                    37.0 [+ or -] 3.1**

(6) 3-[beta]-O-E-feruloyl oleanolic   33.4 [+ or -] 3.6*
acid

(7) [beta]-Sitosterol + (8)           6.0 [+ or -] 4.1
Stigmasterol

(9) Stigmast-4-en-3-one               87.1 [+ or -] 2.6**

(10) 6-[beta]-Hydroxy                 -
stigmast-4-en-3-one

(11) 6-[alpha]-Hydroxy                65.4 [+ or -] 6.5**
stigmast-4-en-3-one

(12) [beta]-Sitosterol                23.5 [+ or -] 2.6
glucopyranoside

(13) Ergosterol peroxide              98.7 [+ or -] 1.2 (b)**

(14) Physcion                         -

(15) Methyl [beta]-orinol             -
carboxylate

(16) Vallapin                         32.4 [+ or -] 4.6

(17) 5-Propylresorcinol               -

(18) (-)-Epicatechin                  -

L-Nitroarginine (L-NA)                34.7 [+ or -] 1.8*

Caffeic acid phenethylester (CAPE)    98.7 [+ or -] 1.2 (b)**

Indomethacin                          47.6 [+ or -] 2.3**

Compound                              100

(l) Friedelin                         47.8 [+ or -] 3.1**

(2) 3-[alpha]-Hydroxyfriedelan-2-one  6.4 [+ or -] 2.5

(3) Cerin                             36.4 [+ or -] 1.9**

(4) Friedelan-3-one-29-ol             97.9 [+ or -] 1.1 (b)**

(5) Betulinic acid                    84.4 [+ or -] 1.9**

(6) 3-[beta]-O-E-feruloyl oleanolic   65.1 [+ or -] 5.6**
acid

(7) [beta]-Sitosterol + (8)           85.8 [+ or -] 1.3**
Stigmasterol

(9) Stigmast-4-en-3-one               94.7 [+ or -] 1.5 (b)**

(10) 6-[beta]-Hydroxy                 36.4 [+ or -] 1.9**
stigmast-4-en-3-one

(11) 6-[alpha]-Hydroxy                89.7 [+ or -] 0.6 (b)**
stigmast-4-en-3-one

(12) [beta]-Sitosterol                58.5 [+ or -] 4.9**
glucopyranoside

(13) Ergosterol peroxide              99.1 [+ or -] 1.0 (b)**

(14) Physcion                         24.6 [+ or -] 1.5**

(15) Methyl [beta]-orinol             23.6 [+ or -] 1.9**
carboxylate

(16) Vallapin                         66.4 [+ or -] 0.5**

(17) 5-Propylresorcinol               22.9 [+ or -] 1.9**

(18) (-)-Epicatechin                  9.7 [+ or -] 1.8

L-Nitroarginine (L-NA)                71.6 [+ or -] 2.6**

Caffeic acid phenethylester (CAPE)    98.9 [+ or -] 2.1 (b)**

Indomethacin                          80.3 [+ or -] 1.5**

Compound                              [IC.sub.50] ([mu]M)

(l) Friedelin                         >100

(2) 3-[alpha]-Hydroxyfriedelan-2-one  >100

(3) Cerin                             >100

(4) Friedelan-3-one-29-ol             25.4

(5) Betulinic acid                    42.5

(6) 3-[beta]-O-E-feruloyl oleanolic   54.1
acid

(7) [beta]-Sitosterol + (8)           64.7
Stigmasterol

(9) Stigmast-4-en-3-one               15.9

(10) 6-[beta]-Hydroxy                 >100
stigmast-4-en-3-one

(11) 6-[alpha]-Hydroxy                9.5
stigmast-4-en-3-one

(12) [beta]-Sitosterol                77.4
glucopyranoside

(13) Ergosterol peroxide              2.5

(14) Physcion                         >100

(15) Methyl [beta]-orinol             >100
carboxylate

(16) Vallapin                         51.9

(17) 5-Propylresorcinol               >100

(18) (-)-Epicatechin                  >100

L-Nitroarginine (L-NA)                61.8

Caffeic acid phenethylester (CAPE)    5.6

Indomethacin                          25.0

(a) Each value represents mean [+ or -] S.E.M. of four determinations.

Statistical significance, *p < 0.05, *p < 0.01.

(b) Cytotoxic effect was observed.

Table 2

Anti-[PGE.sub.2] and TNF-[alpha] production of compounds 11 and 13
from Heritiera littoralis bark.

Compound                              % Inhibition at various
                                      concentrations ([mu]M

                                      0

Against [PGE.sub.2] (11)              0.0 [+ or -] 3.0
6-[alpha]-Hydroxystigmast-4-en-3-one

(13) Ergosterol peroxide              0.0 [+ or -] 3.0

Against TNF-[alpha] (11)              0.0 [+ or -] 2.8
6-[alpha]-Hydroxystigmast-4-en-3-one

(13) Ergosterol peroxide              0.0 [+ or -] 2.8

Compound

                                      3

Against [PGE.sub.2] (11)              10.7 [+ or -] 0.7
6-[alpha]-Hydroxystigmast-4-en-3-one

(13) Ergosterol peroxide              31.6 [+ or -] 1.6

Against TNF-[alpha] (11)              -
6-[alpha]-Hydroxystigmast-4-en-3-one

(13) Ergosterol peroxide              -

Compound

                                      10

Against [PGE.sub.2] (11)              20.4 [+ or -] 1.1
6-[alpha]-Hydroxystigmast-4-en-3-one

(13) Ergosterol peroxide              46.2 [+ or -] 0.2 **

Against TNF-[alpha] (11)              4.1 [+ or -] 1.3
6-[alpha]-Hydroxystigmast-4-en-3-one

(13) Ergosterol peroxide              4.5 [+ or -] 1.2

Compound

                                      30

Against [PGE.sub.2] (11)              36.3 [+ or -] 0.8**
6-[alpha]-Hydroxystigmast-4-en-3-one

(13) Ergosterol peroxide              48.0 [+ or -] 0.3**

Against TNF-[alpha] (11)              3.2 [+ or -] 1.5
6-[alpha]-Hydroxystigmast-4-en-3-one

(13) Ergosterol peroxide              4.1 [+ or -] 0.9

Compound

                                      100

Against [PGE.sub.2] (11)              51.6 [+ or -] 1.5 **
6-[alpha]-Hydroxystigmast-4-en-3-one

(13) Ergosterol peroxide              59.3 [+ or -] 1.0 **

Against TNF-[alpha] (11)              7.7 [+ or -] 1.0
6-[alpha]-Hydroxystigmast-4-en-3-one

(13) Ergosterol peroxide              16.0 [+ or -] 1.5 *

Compound                              [IC.sub.50] ([mu]M)

Against [PGE.sub.2] (11)              86.7
6-[alpha]-Hydroxystigmast-4-en-3-one

(13) Ergosterol peroxide              28.7

Against TNF-[alpha] (11)              >100
6-[alpha]-Hydroxystigmast-4-en-3-one

(13) Ergosterol peroxide              >100

(a) Each value represents mean [+ or -] S.E.M. of four determinations.

Statistical significance, *p < 0.05, *p < 0.01.


The present study may support the use of H. littoralis bark for treatment of inflammatory-related diseases. The antiinflammatory effect of this plant is mainly due to the inhibition on NO and [PGE.sub.2] releases through down regulation of iNOS and COX-2 mRNA expressions.

Acknowledgements

The authors are grateful to the Thailand Research fund (TRF) for financial support through grant number DBG5280008. We also thank the Faculty of Pharmaceutical Sciences for providing laboratory facilities.

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S. Tewtrakul (a), *, P. Tansakul (a), C. Daengrot (b), C. Ponglimanont (b), C. Karalai (b)

(a) Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Prince of Songkla University. Hat-Yai, Songkhla 90112, Thailand

(b) Department of Chemistry, Faculty of Sciences, Prince of Songkla University, Hat-Yai, Songkhla 90112. Thailand

* Corresponding author. Tel.: +66 74 288888; fax: +66 74 428220.

E-mail address: supinyat@yahoo.com (S. Tewtrakul).

0944-7113/$ - see front matter [c] 2010 Elsevier GmbH. All rights reserved.

doi:10.1016/j.phymed.2010.02.011
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Author:Tewtrakul, S.; Tansakul, P.; Daengrot, C.; Ponglimanont, C.; Karalai, C.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9THAI
Date:Sep 1, 2010
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