Anti-inflammatory effects and mechanisms of Fatsia polycarpa
Hayata and its constituents
Hsueh-Ling Chenga, Nurkholisab, Shi-Yie Chengc, Yan-Ting Lua, Shen-Da Huanga,
Xiao-Wen Wanga, Yu-Liang Liua, Chang-Hung Choud*
a Department of Biological Science and Technology,National Pingtung University of Science
and Technology, No. 1, Shuehfu Rd., Neipu, Pingtung 91201, Taiwan
b Department of Agricultural Product Technology, Brawijaya University, Jalan Veteran
Malang 65145, Indonesia
c Department of Life Sciences, National University of Kaohsiung, No.700, Kaohsiung
University Road, Nan-Tzu District, Kaohsiung 811, Taiwan
d Graduate Institute of Ecology and Evolutionary Biology, China Medical University, 91,
Hsueh-Shih Road, Taichung, 40402, Taiwan
* Corresponding author: Chang-Hung Chou, Professor
Tel.: 886-4-2205366 ext1633; fax: 886-4-22071500. E-mail: [email protected] Address: Graduate Institute of Ecology and Evolutionary Biology, China Medical
Abstract
Fatsia polycarpa is a plant endemic to Taiwan and an herbal medicine for treating several inflammation-related diseases, but its biological function needs scientific support. Thus, the anti-inflammatory effects and mechanisms of the methanolic crude extract of F. polycarpa (MCE) and its feature constituents, i.e. brassicasterol (a phytosterol), and triterpenoids 3-hydroxyolean-11,13(18)-dien-28-oic acid (HODA), 3α-hydroxyolean-11-en-28,13β-olide (HOEO), fatsicarpain D and fatsicarpain F, was investigated. Consequently, MCE and HOEO, but not brassicasterol, dose-dependently inhibited lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase and cyclooxygenase-2 in RAW 264.7
macrophage line, whereas HODA, fatsicarpain D and fatsicarpain F were toxic to RAW 264.7 cells. MCE and HOEO also suppressed LPS-induced production of nitric oxide, prostaglandin E2 and interleukine-1β. Furthermore, MCE and HOEO interfered with
LPS-promoted activation of the inhibitor kappa B kinase (IKK)/nuclear factor-B (NF-B) pathway, and that of the mitogen-activated protein kinases (MAPKs) extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38. In animal tests, MCE and HOEO effectively ameliorated 12-O-tetradecanoylphorobol-13 acetate (TPA)-induced ear edema of mice. Thus, MCE of F. polycarpa exhibited an obvious anti-inflammatory activity
in vivo and in vitro, which likely involved the inhibition of the IKK/NF-қB pathway and the MAPKs. Triterpenoids such as HOEO may contribute to this activity.
1. Introduction
There are three known species of plants in the genus of Fatsia (belonging to the Araliaceae family), i.e. Fatsia polycarpa Hayata, an evergreen shrub endemic to Taiwan, Fatsia japonica and Fatsia oligocarpela, which are originated from Japan and Bonin Island, respectively [1]. Fatsia plants have been used as an herbal medicine in Taiwan and in Japan in treating diseases such as coughing, ankylosing spondyloarthritis, osteoarthritis,
rheumatism, rheumatoid arthritis and tendinitis, and in improving blood circulation [1, 2]. However, these therapeutic activities of the plants have seldom been scientifically
investigated, and the bioactive constituents from these plants are not clear.
Previously, the constituents in the methanolic extract of leaves and twigs of Fatsia polycarpa were characterized [1]. The extract was rich in phytosterols in which brassicasterol was a major one (unpublished data). Brassicasterol is a common phytosterol present in many plants and was suggested to reduce blood cholesterol levels together with other phytosterols [3]. Triterpenoids is another type of compounds rich in the crude extract of F. polycarpa [1, 4, 5]. Seven novel structures (named fatsicarpains A-G) and two known ones
[3-hydroxyolean-11,13(18)-dien-28-oic acid (HODA); 3α-hydroxyolean-11-en-28,13β-olide (HOEO)] belonging to oleanane-type triterpenoids were isolated and identified from the extract [1]. Among these triterpenes, HODA, HOEO, fatsicarpain D and fatsicarpain F are in larger amounts. Whether these molecules play roles in the medical functions of Fatsia
polycarpa deserves to be investigated.
Fatsia plants exhibit pleiotropic therapeutic activities as described above. However, several of these activities, such as the treatment of ankylosing spondyloarthritis,
osteoarthritis, rheumatoid arthritis, tendinitis etc., are related to anti-inflammation. Thus, in this study, the anti-inflammatory effects of the methanolic crude extract of F. polycarpa (MCE), and its major or feature constituents, including brassicasterol, HODA, HOEO, fatsicarpain D and fatsicarpain F (structures shown in Figure 1), were characterized.
2. Materials and Methods
2.1. Chemicals
Specific antibodies against phosphorylated IKK-IKK-, total IKK-, total IKK-, phosphorylated IB-, total IB-, phosphorylated JNK, total JNK, phosphorylated ERK, total ERK, phosphorylated p38, total p38, p65 and lamin B were purchased from Cell Signaling Technology (Beverly, MA, USA); antibodies against inducible nitric oxide synthase and cyclooxygenase-2 from BD Biosciences (Franklin Lakes, CA, USA); an actin-specific antibody from Chemicon (Temecula, CA, USA); all secondary antibodies from Santa Cruz Biotechnologies (Santa Cruz, CA, USA); fetal bovine serum (FBS) from Invitrogen (San Diego, CA, USA); cell culture media, lipopolysaccharide (LPS) derived from E. coli
055:B5, NaF, sodium orthovanadate, sodium pyrophosphate, indomethacin, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), and 12-O-tetradecanoylphorobol-13
acetate (TPA) from Sigma-Aldrich (St. Louis, MO, USA) at reagent grade or cell-culture grade. MCE, HODA, HOEO, fatsicarpain D and fatsicarpain F were prepared, or isolated and identified as described previously [1]. MCE was dissolved in DMSO (dimethylsulfoxide) in a concentration of 50 mg/ml as a stock solution; HODA, HOEO, fatsicarpain D and
fatsicarpain F were dissolved in DMSO to a concentration of 10 mM as the stock solutions.
2.2. Isolation and Identification of brassicasterol
The air-dried and powdered leaves and twigs of F. polycarpa (7.1 kg) were extracted with methanol (MeOH) for three days at room temperature (three times), and the combined extracts were concentrated in vacuum (under 35 °C). The resulting dark green gum was suspended in H2O and extracted sequentially with CH2Cl2, ethyl acetate (EtOAc), and n-butanol (n-BuOH) (saturated with H2O). The CH2Cl2 extract (100 g) was subjected to column chromatography on silica gel using n-hexane, n-hexaneEtOAc, and EtOAcMeOH mixtures of increasing polarity for elution to furnish 40 fractions. Fraction 20 (7.0 g) eluted with n-hexaneEtOAc (1:10) was fractionated over Sephadex LH-20 (100% acetone) to afford a major phytosterol (580 mg), which was grown by slow evaporation of the mixture CH2Cl2MeOH (1:1) solution at room temperature to give a suitable colorless crystal. The chemical structure of the crystal was confirmed by comparison of its spectroscopic data with those of brassicasterol [6, 7], which has been reported specifically in only one alga, an
unidentified species of the order Sarcinochrysidales (Chrysophyceae). Brassicasterol was dissolved in 100% ethanol in a concentration of 10 mM as a stock solution.
2.3. Animal test
All protocols of animal tests were approved by the Committee for Animal
Experiments of National Pingtung University of Science and Technology in accordance with international guidelines. Experiments were performed using groups of 8 male ICR mice (30-35 g; purchased from BioLASCO, Taipei, Taiwan) that were fed with a regular laboratory rodent diet and housed under a 12-hour light–dark cycle. TPA-induced ear edema of mice was performed according to previously published methods with modifications [8, 9]. In the experiment for MCE, TPA (2.5 μg/ear dissolved in 20 μl of acetone) was topically applied to both surfaces of both ears of mice, yet in the group of TPA control, TPA was applied to only the right ear. In the group of normal control, 20 μl of acetone was applied to both ears. Four hours later MCE or indomethacin (both dissolved in acetone) was applied to the right ear and vehicle to the left ear, whereas in normal control and TPA control, an equal amount of vehicle was applied to both ears. The thickness of ears was measured before and at 4 h, 16 h and 24 h after TPA treatment using a dial thickness gauge (Peacock, Ozaki, Tokyo, Japan). The experiment for HOEO was performed similarly using a different batch of mice, but each group contained 6 mice, the dosage of TPA was 3.0 μg/ear, and HOEO or indomethacin was
applied to the ears at 1 h (instead of 4 h) after TPA treatment.
2.4. Cell culture and Western blot analysis
RAW 264.7 cells (ATCC number AT TIB-71) were cultured in DMEM (Dulbecco’s modified Eagle’s medium) containing 10% fetal bovine serum. FL83B cells (ATCC number CRL-2390) were cultured as previously described [10]. Both cells were incubated at 37˚C in a humidified incubator supplied with 5% CO2.
RAW 264.7 cells were seeded at a density of 1.5 - 2.0 x 106/35-mm plate, grown for 16
h, and treated with MCE or the natural products for 3 h. LPS was then added to the medium (final concentration 100 ng/ml) for 16 h. Cells were washed with cold PBS (phosphate-buffered saline, pH 7.4) twice and submerged in lysis buffer [1x Cell Culture Lysis Reagent (Promega, Madison, WI, USA) containing 1 mM of phenylmethylsulfonyl fluoride, 1g/ml of pepstatin, 1 g/ml of leupeptin, and 1 g/ml of aprotinin]. For the detection of
phosphorylated proteins, the lysis buffer also contained 10 mM of NaF, 1 mM of sodium orthovanadate and 10 mM of sodium pyrophosphate. Cells were scraped off the plate on ice, and the suspension was centrifuged at 14,000×g for 15 min at 4ºC. The supernatant was collected and the protein concentration analyzed using Bradford assay reagent (Bio-Rad, Hercules, CA, USA). Equal amounts of proteins were sampled and subjected to
scanned by a gel documentation and image analysis system (Syngene, Frederick, MD, USA), and band intensities were analyzed using the program supplied with the system.
Alternatively, after hybridization with a horseradish peroxidase (HRP)-conjugated secondary antibody, followed by incubation with “Western HRP substrate” (LuminataTM Classico,
Millipore, Temecula, CA, USA), immunoreactive bands were detected using a biospectrum imaging system (UVP Biospectrum, UVP, LLC, Upland, CA, USA) and band intensities analyzed by the supplied software.
2.5. Cytotoxicity assay
Cytotoxicity was assayed using MTT. RAW 264.7 (3 x 105 cell/well) or FL83B cells
(2 x 105 cell/well) were cultured in a 96-well plate. Cells were washed twice with PBS,
treated with the compounds in the indicated concentrations in serum-free medium for 24 h (HODA) or 19 h (other compounds). Cells were then washed twice with PBS and incubated in 30 l of 5 mg/ml MTT solution at 37 °C in the dark for 1 h. Subsequently, DMSO (100 l) was added to each well for 10 minutes, withdrawn to another 96-well plate, and the
absorbance at 570 nm determined using a microplate reader (Molecular Devices, Sunnyvale, CA, USA). Experiments were performed in triplicate. Average ratio ± standard deviation of viable cells was calculated against the control (cells treated by the solvent).
Total cellular RNA was purified using Trizol (Invitrogen) according to the manufacturer’s instruction. The quality of RNA was checked by agarose gel electrophoresis. Sequences of the forward and reverse PCR primers of interleukin-1IL-1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as published previously [12].RT-PCR was performed using the SuperScriptTM One-Step RT-PCR System (Invitrogen) kit with the following formulation: 2 μg of total RNA, 0.4 M of forward primer and reverse primer, respectively, 12.5 μl of 2× reaction mix, 1 μl of SuperScript RT/Platinum Taq. The final volume was adjusted to 25 l with sterilized distilled water. It was then subjected to PCR amplification in a thermal cycler by the following program:48°C, 45 min (for cDNA synthesis); one cycle of 95°C, 2 min, 35 cycles of 95°C, 45 s, 60°C, 45 s, 72°C, 1 min, one cycle of 72°C, 5 min.The RT-PCR products were separated electrophoretically on 1% agarose gels and visualized by ethidium bromide staining. The resulting images were recorded and band intensities quantified as described previously [12]. Meanwhile, the RT-PCR products were purified and the sequences confirmed by DNA sequencing (Mission Biotech, Taipei, Taiwan).
2.7. Analysis of nitric oxide (NO) and prostaglandin E2 (PGE2)
The concentration of NO in the culture medium of cells was analyzed using a kit of Griess reagent according to the manufacturer’s protocol (Promega). That of PGE2 was
analyzed using an ELISA kit according to the protocol suggested by the manufacturer (R&D Systems, Minneapolis, MN, USA).
2.8. Protein extraction from the nuclear and cytoplasmic fractions of cells
RAW 264.7 cells were seeded on 10-mm dishes until 80% confluence and treated with MCE or HOEO for 3 h, followed by treating with LPS for 2 h. Cells were harvested, washed with cold PBS twice, and centrifuged at 500×g for 5 min. Nuclear proteins and cytoplasmic proteins of the cell pellet were then separated and extracted using a nuclear extract kit according to the manufacturer’s instruction (Millipore, Temecula, CA, USA). Protein concentrations in the extracts were quantified using Bradford assay reagent before being subjected to Western blot analysis.
2.9. Statistical analysis
Data were analyzed by one-way analysis of variance (ANOVA) followed by Scheffe's post hoc test. Significance was considered when p < 0.05 and F > 3.5546.
3. Results
3.1. In vivo efficacy of MCE in TPA-induced mouse ear edema
To assess the anti-inflammatory effect of MCE in vivo, a topical inflammation model, TPA-induced mouse ear edema, was used. Both ears of mice were stimulated with TPA to
induce inflammation, and the right ear was treated with MCE (100, 300 or 500 μg/ear) or indomethacin (500 μg/ear, as a reference drug) [13, 14] at 4 h after TPA stimulation, whereas the left ear was remained un-medicated for comparison. As shown in Figure 2A, ear edema was obvious at 4 h, 16 h and 24 h post TPA application without any medication (TPA control), while all three dosages of MCE significantly reduced the edema of the right ear at 16 h and 24 h in a time-dependent manner and in an extent comparable to the effect of indomethacin. When comparing the thickness of the right ear and that of the un-medicated left ear at 24 h post TPA stimulation, the data also revealed that all three dosages of MCE effectively ameliorated the TPA-induced edema (Figure 2B). These results confirmed the anti-inflammatory activity of MCE in vivo. Accordingly, the anti-inflammatory effects of MCE and its characteristic components brassicasterol, HODA, HOEO, fatsicarpain D and fatsicarpain F were further investigated.
3.2. The anti-inflammatory effects of MCE and its feature constituents on macrophage cells
The cytotoxicity of MCE and the compounds were evaluated in RAW 264.7 macrophage cell line and in FL83B normal liver cell line. MCE did not display obvious cytotoxicity in FL83B cells in concentrations of 10 - 400 μg/ml, but did result in reduced cell survival in 400 μg/ml in RAW 264.7 cells (Figure 3A). Brassicasterol did not show obvious cytotoxicity in both cell lines in 10-100 M (Figure 3B). HOEO did not have a significant
toxic effect in FL83B cells in concentrations of 10 - 100 μM, but did result in about 20% inhibition for the growth of RAW 264.7 cells in 100 μM (Figure 3C). HODA, fatsicarpain D and fatsicarpain F on the other hand, obviously suppressed the growth of RAW 264.7 cells in concentrations of 50 M or ≧20 M , but they were not toxic to FL83B cells (Figure 3, D, E, F, respectively). Therefore, the anti-inflammatory effects of MCE, brassicasterol and HOEO were further characterized using RAW 264.7 cells as a model. Those of HODA, fatsicarpain D and fatsicarpain F will be analyzed using other appropriate models.
Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are the major enzymes involved in the synthesis of the inflammatory mediators, NO and prostaglandin E2
(PGE2), respectively, and are common markers of inflammation [12, 15, 16]. Thus, the
dosage-dependent effects of MCE and brassicasterol on the inhibition of LPS-induced
expression of iNOS and COX-2 in RAW 264.7 cells were characterized. These enzymes were highly expressed in LPS-stimulated cells (Figure 4, A and B, lane 2 versus lane 1). MCE obviously inhibited the LPS-elevated expression of iNOS in concentrations of 50-200 μg/ml, and that of COX-2 in concentrations of 100-200 μg/ml (Figure 4A, lanes 4-6 versus lane 2). Accordingly, 100 μg/ml of MCE was considered as an effective dosage for the following experiments. In contrast, brassicasterol had no obvious inhibitory effect on the expression of iNOS and COX-2 at dosages of 10-100 μM (Figure 4B, lanes 3-6 versus lane 2). Thus, brassicasterol was not considered as a potential anti-inflammatory agent. In Figure 4C,
HOEO markedly suppressed LPS-induced expression of iNOS and COX-2 in concentrations of 5-30 μM (lanes 4-7 versus lane 2). Thus, 20 μM of HOEO was used for the subsequent experiments.
Whether MCE and HOEO could suppress LPS-stimulated NO and PGE2 secretion
was also examined. Figure 4D showed that LPS-induced NO secretion (Group 2) was significantly reduced by the treatment of MCE or HOEO (Groups 3, 4). Similarly, MCE or HOEO obviously inhibited LPS-induced production of PGE2 in RAW 264.7 cells (Figure 4E,
Groups 3, 4 versus Group 2). Moreover, both MCE and HOEO suppressed LPS-elevated expression of the mRNA of IL-1 (Figure 4F, lanes 3, 4 versus lane 2). These results further confirmed the anti-inflammatory activities of MCE and HOEO in LPS-stimulated
macrophages.
Thus, the anti-inflammatory effect of HOEO was examined in animal models. As shown in Figure 5A, the application of 300 g/ear of HOEO on the right ear significantly reduced the extent of TPA-induced ear edema in a time-dependent manner, and the effect was similar with that of 500 g/ear of indomethacin. Figure 5 B showed the comparison of the extents of edema between the right ear and the un-medicated left ear on the same group of mice at 24 h post TPA stimulation, it also supported that HOEO effectively hindered TPA-induced edema. These results confirmed the anti-inflammatory activity of HOEO in vivo.
3.3. MCE and HOEO interfere with the activation of the IKK/NF-B pathway and MAPKs
The activation of nuclear factor-B (NF-қB) plays a key role in mediating
inflammatory responses. LPS is known to activate IKK (IB kinase), which in turn catalyzes the phosphorylation of IқB (inhibitor of NF-B), leading to IқB degradation and the release of NF-қB. NF-қB enters the nucleus and activates the expression of pro-inflammatory factors such as iNOS, COX-2, IL-1 etc [17-19].
As revealed in Figure 6 A and B, MCE strongly inhibited LPS-induced
phosphorylation of IKK (A) and IқB (B) (lanes 5, 6) as compared with cells treated with LPS alone (lanes 3, 4). HOEO exhibited a less potent inhibitory effect on the activation of IKK (A, lanes 7, 8 versus lanes 3, 4), yet a substantial suppressive effect on IқB phosphorylation (B, lanes 7, 8 versus lanes 3, 4) was still observed. Therefore, LPS-induced IқB
phosphorylation was obviously inhibited by both MCE and HOEO. Furthermore, Figure 6C showed that the nuclear translocation of p65, a NF-B subunit, was apparently increased by LPS stimulation (lanes 3, 4) as compared to untreated cells (anes 1, 2), whereas MCE (lanes 5, 6) or HOEO (lanes 7, 8) critically suppressed this LPS-induced effect, indicating that MCE and HOEO inhibited the shift of NF-B to the nucleus. Overall, these data suggested that MCE and HOEO inhibited the LPS-mediated activation of IKK/NF-B signaling.
Mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and p38, are also known to mediate the
LPS-induced inflammatory responses [20-23]. Thus, whether MCE and HOEO could inhibit the activation of JNK, ERK and p38 was examined. As shown in Figure 6D, treatment of the cells with MCE (lanes 5, 6) or HOEO (lanes 7, 8) effectively inhibited the LPS-induced phosphorylation of ERK as compared with the LPS control (lanes 3, 4). HOEO also markedly inhibited the phosphorylation of JNK (Figure 6E, lanes 7, 8 versus lanes 3, 4), whereas MCE exhibited a less strong inhibitory action on the activation of JNK (Figure 6E, lanes 5, 6 versus lanes 3, 4). Figure 6F manifested that both MCE (lanes 5, 6) and HOEO (lanes 7, 8)
substantially suppressed the LPS-induced phosphorylation of p38 as compared with the control (lanes 3, 4).
4. Discussion
This study scientifically proved that the crude extract of F. polycarpa is a potential anti-inflammatory agent. Animal tests confirmed the anti-anti-inflammatory effect of MCE in vivo. Topical application of MCE for 12-20 h effectively ameliorated acute skin inflammation on mice ear. When compared with indomethacin, a medicine for inflammation, MCE showed a similar or even stronger effect in reducing ear edema of mice. Meanwhile, in RAW 264.7 macrophage line, MCE obviously inhibited LPS-stimulated expression or production of iNOS, COX-2, NO, PGE2, and IL-1β. Overall, these results support the traditional use of F.
MCE obviously reduced the phosphorylation of IKK and IқB, and the nuclear translocation of NF-B. These suggested that MCE inhibited the IKK/NF-қB pathway. MAPKs are another group of signaling pathways regulating inflammation. MCE critically suppressed LPS-induced activation of ERK and p38, and had a milder inhibitory effect on that of JNK. Overall, these results suggest that MCE interferes with the IKK/NF-қB pathway and the MAPK pathways, which likely underlies, at least in part, the mechanism of its anti-inflammatory activity.
MCE was rich in triterpenoids as well as phytosterols. It is rational to speculate that these components may contribute to the anti-inflammatory function of F. polycarpa. Thus, brassicasterol and a few major or feature triterpenes of MCE were characterized.
Consequently, brassicasterol did not exhibit an obvious anti-inflammatory activity, implying that brassicasterol may not contribute to the anti-inflammatory effect of MCE. Consistently, brassicasterol is known to be a phytosterol commonly present in many plants and foods [24-26], including those not known to possess an anti-inflammatory activity. Thus, it is
reasonable that brassicasterol does not contain an anti-inflammatory effect.
On the other hand, HOEO displayed a significant anti-inflammatory activity both in vivo and in vitro. HOEO obviously diminished TPA-induced ear edema in mice, and
inhibited the expression of pro-inflammatory markers in LPS-stimulated RAW 264.7 cells, including iNOS, COX-2, IL-1β, NO and PGE2. Furthermore, in concert with the action of
MCE, HOEO interfered with the activation of the IKK/NF-қB pathway, and that of MAPKs ERK, JNK and p38. Thus, HOEO likely represents one of the constituents contributing to the anti-inflammatory function of F. polycarpa. There are very likely other components in MCE that also play roles in the anti-inflammatory effect of F. polycarpa, and deserve to be
explored.
Interestingly, HODA, fatsicarpain D and fatsicarpain F showed obvious inhibitory effects on the survival of RAW 264.7 cells, but were not toxic to the normal liver line FL83B cell. The molecular mechanism is not clear. However, promoting the death of macrophages without hurting other somatic cells could also be an effect of anti-inflammation. Thus, the actions, immunomodulatory effects and molecular mechanisms of these molecules should be further investigated.
5. Conclusions
Evidence presented in this study suggests that the crude extract from the leaves and twigs of Fatsia polycarpa is a potent anti-inflammatory agent both in vitro and in vivo. The anti-inflammatory activity of MCE is probably not due to brassicasterol, a major phytosterol present in the extract, but is likely contributed by other components in the extract, such as triterpenoids like HOEO. The molecular mechanism underlying the effects of MCE and HOEO probably involves the inhibition of the IKK/NF-қB pathway and the MAPKs. More
studies should be conducted to continue exploring the effective constituents in the extract of F. polycarpa.
Acknowledgments
The authors are grateful to the National Science Council of Taiwan for the grant support to H. L. Cheng (NSC 101-2621-B-020-002), and to C. H. Chou (NSC 101-2621-B-039-002).
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Figure Captions
FIGURE 1: Structures of brassicasterol, HODA, HOEO, fatsicarpain D and fatsicarpain F.
FIGURE 2: The anti-inflammatory effect of MCE on TPA-induced ear edema of mice. Mice
were treated with TPA on both ears for 4 h, followed by MCE (100, 300 or 500 μg/ear) or indomethacin (500 μg/ear) administration on the right ear, while the left ear was
un-medicated. In TPA control, TPA was applied to only the right ear; in normal control, an equal amount of vehicle was applied to both ears. Ear thickness was measured before and at 4 h, 16 h and 24 h after TPA treatment. Data represent mean ± SD (N = 8 per group) and were statistically analyzed by One-way ANOVA. A, comparison of the thickness of the right ear at different time points. * P < 0.05 against “TPA 4h” of the same group. B, comparison of the thickness of the right ear and the left ear at 24 h after TPA treatment. * P < 0.05 against the left ear of the same group.
FIGURE 3: Cytotoxicity assays of MCE (A), brassicasterol (B), HOEO (C), HODA (D),
fatsicarpain D (E) and fatsicarpain F (F). RAW 264.7 cells and FL83B cells were treated with the selected compound in the indicated concentrations for 24 h (HODA) or 19 h (other compounds). Relative cell viability was determined against the control (0 g/ml or 0 M of the compound). Data represent mean ± SD of triplicate. *P < 0.05 against the control.
or HOEO. A, B, and C, analysis by Western blot of the expression of iNOS and COX-2 in RAW 264.7 cells. Cells were treated with MCE (A), brassicasterol (B) or HOEO (C) in the indicated concentrations for 3 h (lanes 3-6 or 3-7), followed by stimulation with LPS for 16 h (lanes 2-6 or 2-7). Band intensities relative to lane 1 in each blot were determined after
normalization by the amount of actin, and plotted as the respective histogram. D and E, ELISA analysis for the concentration of NO (D) or PGE2 (E) in the culture medium. Cells
were treated with 100 g/ml of MCE (Group 3) or 20 μM of HOEO (Group 4) for 3 h, followed by stimulation with LPS (Groups 2, 3, 4) for 16 h. Data represent mean ± SD of three independent experiments. * P < 0.05 against Group 2. F, the expression level of IL-1mRNAanalyzed by semi-quantitative RT-PCR. Cells were treated as in D and E. The expression ofGAPDH was assayed simultaneously as the loading control. Fold represents the relative band intensity against lane 1 after normalization by the amount of GAPDH.
FIGURE 5: The anti-inflammatory effect of HOEO on TPA-induced ear edema of mice. Mice
were treated with TPA on both ears for 1 h, followed by HOEO (300 μg/ear) or indomethacin (500 μg/ear) administration on the right ear, while the left ear was treated with vehicle. In TPA control, TPA was applied only to the right ear, and both ears were treated with vehicle at 1 h afterwards. Ear thickness was measured before and at 4 h, 16 h, 20 h and 24 h after TPA treatment. Data represent mean ± SD (N = 6 per group). A, comparison of the thickness
of the right ear at different time points. * P < 0.05 against TPA control of the corresponding time point. B, comparison of the thickness of the right ear and the left ear at 24 h after TPA treatment. * P < 0.05 against the left ear of the same group.
FIGURE 6: Inhibition of LPS-induced activation of the IKK/NF-B pathway and MAPKs by
MCE or HOEO. Western blot analysis for the expression of the indicated proteins in RAW 264.7 cells. Experiments were performed in duplicate. Cells were treated with 100 g/ml of MCE (lanes 5, 6) or 20 μM of HOEO (lanes 7, 8) for 3 h, followed by stimulation with LPS (lanes 3-8) for 1 h or 2 h (C). A, analysis of phosphorylated IKK (P-IKK), total and subunits of the IKK complex (IKK- and IKK-). B, analysis of phosphorylated IB (P-IB) and actin. C, analysis of p65, lamin B and actin in the nuclear and cytosolic fractions of cells, respectively. D, E, and F, analysis of phosphorylated ERK (P-ERK) and total ERK (D), phosphorylated JNK (P-JNK) and total JNK (E), and phosphorylated p38 (P-p38) and total p38 (F), respectively. Band intensities relative to lane 1 in each blot were determined after normalization by the corresponding total protein, actin or lamin B as indicated in the respective histogram.
FIGURE 1
