Inhibition of COXs and 5-LOX and activation of PPARs by Australian Clematis species (Ranunculaceae)

Journal of Ethnopharmacology 104 (2006) 138–143

Inhibition of COXs and 5-LOX and activation of PPARs by Australian Clematis species (Ranunculaceae) Rachel W. Li a,∗ , G. David Lin a , David N. Leach b , Peter G. Waterman b , Stephen P. Myers a a

Australian Centre for Complementary Medicine Education and Research, A Joint Venture of the University of Queensland and Southern Cross University, P.O. Box 157, Lismore, NSW 2480, Australia b Centre for Phytochemistry and Pharmacology, Southern Cross University, P.O. Box 157, Lismore, NSW 2480, Australia Received 9 March 2005; received in revised form 10 August 2005; accepted 26 August 2005 Available online 3 October 2005

Abstract The species of Clematis (Ranunculaceae) have been traditionally used for inflammatory conditions by indigenous Australians. We have previously reported that the ethanol extract of Clematis pickeringii inhibited COX-1. In this study, we examined the ethanol extracts and fractions of three Clematis species, Clematis pickeringii, Clematis glycinoides and Clematis microphylla, on cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX). We further examined the activating effects on the protein expression of peroxisome proliferator-activated receptor alpha (PPAR␣) and gamma (PPAR␥) in HepG2 cells. The ethanol extracts of three Clematis species inhibited the activities of COX-1, COX-2 and 5-LOX in the different extents. The stem extract of Clematis pickeringii showed the highest inhibitory activities among the three species on COX-1, COX-2 and 5-LOX with the IC50 values of 73.5, 101.2 and 29.3 ␮g/mL. One of its fractions also significantly elevated PPAR␣ expression by 173, 280 and 435% and PPAR␥ expression by 140, 228 and 296% at 4, 8 and 16 ␮g/mL, respectively. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Anti-inflammatory activity; Cyclooxygenase-1, -2; 5-Lipoxygenase; PPAR␣; PPAR␥

1. Introduction The Australian continent is one of the vast repositories of medicinal plants that have been used in traditional medicine. The Australian aboriginal people are estimated to have inhabited the Australian continent for at least 40,000 years (Semple et al., 1998). Experimentation with plants and passage of knowledge from one generation to the next resulted in the development of a large body of knowledge about plants to use for foods, implements, medicine and narcotics, and the methods of plant Abbreviations: ACN, acetonitrile; COX-1, cyclooxygenase-1; COX-2, cyclooxygenase-2; EtAc, ethyl acetate; 5-HETE, 5-hydroxyeicosatetraenoic acid; HepG2, human Caucasian hepatocyte carcinoma; HPLC, highperformance liquid chromatography; 5-LOX, 5-lipoxygenase; NSAIDs, nonsteroidal anti-inflammatory drugs; PPAR␣, peroxisome proliferator-activated receptor alpha; PPAR␥, peroxisome proliferator-activated receptor gamma; PGE2 , prostaglandin E2; PGD2 , prostaglandin D2 ∗ Corresponding author. Present address: Tropical Plant and Soil Sciences, CTAHR/University of Hawaii, 112 St. John Hall, 3190 Maile Way, Honolulu, HI 96822, USA. Tel.: +1 808 956 7940; fax: +1 808 956 3894. E-mail address: [email protected] (R.W. Li). 0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.08.061

reparation (Webb, 1973). Traditional medical practices still play an important role in some areas of Australia today. A number of studies have recently revealed that some of the plants used by indigenous Australians produce valuable active compounds (Hungerford et al., 1998; Wickens and Pennacchio, 2002; Li et al., 2004). Three Australian Clematis species (Ranunculaceae), Clematis glycinoides DC., Clematis microphylla DC. and Clematis pickeringii A. Gray, have been used by traditional medical practitioners in the treatment of inflammatory conditions such as pain, rheumatism, common colds, headache and infections (Cribb and Cribb, 1981; Lassak and McCarthy, 1997). However, earlier use of these plant materials was based on subjective experience and no experimental data are available as to their efficacy and/or mechanisms of action. We have previously shown that the ethanol extract of Clematis pickeringii stem inhibited cyclooxygenase-1 (Li et al., 2003). In this study, we used a panel of enzyme assays to test whether the activities of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) would be inhibited by the ethanol extracts of three Australian Clematis species and then we

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separated the active fractions via a bioactivity-guided fractionation using high-performance liquid chromatography (HPLC) and preparative HPLC. We also studied some of their cellular actions through enzyme-independent mechanisms. Human Caucasian hepatocyte carcinoma (HepG2) cells were used to determine the expression profile of peroxisome proliferatoractivated receptor alpha (PPAR␣) and gamma (PPAR␥). We used this cell line to test whether protein expression of PPAR␣ and PPAR␥ would be influenced by the extracts or fractions of Clematis species. 2. Materials and methods 2.1. Plant material The stems of Clematis pickeringii, Clematis glycinoides and Clematis microphylla were collected from Western Australia and authenticated by Dr. Elwyn Hegarty of PlantChem, in Australia, where the voucher specimens have been deposited (accession number RL99A056, RL20A042 and RL03A0151, respectively). The dried material (300 g) was powdered and mixed with 5% polyvinylprrolidone (PVP) to remove polyphenols as these may cause an inhibitory result on COX assays (Rogers et al., 2000). The mixture was then percolated with absolute ethanol at room temperature for 48 h (500 mL ×3). The ethanol extracts were combined and evaporated to dryness. Part of the ethanol extract (200 mg) was re-constituted in ethanol and diluted in a series concentration for bioassays. 2.2. Reagents Ovine COX-1, sheep placenta COX-2, human recombinant 5LOX, arachidonic acid, prostaglandin E2 (PGE2 ), prostaglandin D2 (PGD2 ) and 5-hydroxyeicosatetraenoic acid (5-HETE) were purchased from Cayman Chemical (USA). [1-14 C]-arachidonic acid was purchased from Amersham (USA). Antibodies to PPAR␣ and PPAR␥ were purchased from Santa Cruz Biotechnology (Santa Cruz, USA) and all other chemicals were analytical grade obtained from Sigma–Aldrich, Fisher and Ajax Chemicals (Australia). 2.2.1. Bioassay-guided fractionation The extract showing highest inhibition on the enzymes was fractionated using column chromatography (3 g in total) over a silica gel H60 (60 g) column (open column 50 mm × 600 mm) eluting successively with 150 mL of each: (1) hexane, (2) hexane–ethyl acetate (EtAc) (75:25), (3) hexane–EtAc (50:50), (4) hexane–EtAc (25:75), (5) EtAc, (6) EtAc–chloroform (50:50), (7) chloroform, (8) chloroform–methanol (50:50), (9) methanol and (10) methanol–water (50:50). Throughout the separation process, each fraction was monitored by testing its inhibitory activity against COX-1. The fractions showing marked inhibitory activity on COX-1 were analyzed on HPLC (HP 1100 series LC, Hewlett Packard) using an ODS reversed phase C-18 column, 4.0 mm i.d. × 125 mm, 5 ␮m (Hypersil column, Hewlett Packard). The active fractions were further fractionated using a Phenomenex Luna 5 ␮m C18

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150 mm × 21.2 mm preparative column and sub-fractions were collected using a 10% acetonitrile (ACN) to 95% ACN gradient over 20 min at 25 mL/min or a gradient of EtAc/hexane (gradient 5:95 to 95:5 over 45 min) on the normal phase silica gel column. 2.3. Enzyme assays COX-1 and COX-2 assays were performed according to the method described previously (Li et al., 2004). Diluted extract was pre-incubated with COX-1 (or COX-2) before initiating the reaction by the addition of [1-14 C]-arachidonic acid. After incubation, PDE2 and PGD2 were extracted and their radioactivities (c.p.m.) were measured on a liquid scintillation counter. The 5-LOX assay was based on the formation of 5-HETE from arachidonic acid converted by 5-LOX. The diluted extract was incubated with 5-LOX (46 ␮g protein) for 10 min at 24 ◦ C before initiating the enzyme reaction by adding [1-14 C] arachidonic acid (50 nCi). The reaction was terminated after 5 min by acidification with 4 M formic acid. The metabolite, 5-HETE, was separated and its radioactivity was measured in the liquid scintillation counter. Indomethacin was used as a positive control for COX-1, COX-2 and 5-LOX assays. 2.4. Cell culture HepG2 cells were purchased from European Collection of Cell Cultures (Porton Down, Salisbury, UK). To study PPARs activating effect, all experiments were performed when the cells reached 80% confluence. HepG2 cells grown in 75-cm2 flasks were seeded in six-well culture dishes and cultured in color-free and serum-free Dulbecco’s modified Eagle’s medium (DMEM) supplemented with glutamine, penicillin and streptomycin for 12 h before experiments. After a 12-h period of serum deprivation, duplicate wells were exposed to each experimental condition in 4 mL fresh medium for 24 h. The extracts or fractions of Clematis species dissolved in DMSO or ethanol were added in volumes of less than 0.05% of total. Control wells containing the equivalent final concentration of DMSO or ethanol were run with each experiment. 2.5. Immunoblotting analysis After 24 h treatment with or without test agents, HepG2 cells were trypsinized and washed twice with ice-cold PBS. The cells were centrifuged for 5 min at 500 × g at 4 ◦ C. The pellet was suspended in lysis buffer containing protease inhibitor cocktail tablets (Roche Diagnostics) according to the manufacture’s protocols. The cell extract was centrifuged for 10 min at 10,000 × g at 4 ◦ C and the supernatant corresponding to the nuclear extract was collected, aliquoted and stored at −80 ◦ C. The concentration of protein in the extracts was determined using a protein assay kit (Bio-Rad Laboratories, USA). Samples containing 40 ␮g of protein were electrophoresized on 10% SDS-polyacrylamide gels (Minigel system, Bio-Rad) for 1.5 h at 140 V. The separated proteins were then electrophoretically transferred onto nitrocellulose membranes overnight at 4 ◦ C. Non-specific binding sites

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were blocked with 5% skim milk powder diluted in PBS containing 0.05% Tween-20 (PBS-T) at room temperature for 1 h. The membranes were then incubated with primary antibodies (1:500 dilutions of rabbit anti-human PPAR␣ or PPAR␥ polyclonal antibodies) diluted in PBS-T containing 5% skim milk for 4 h at room temperature. The membranes were washed three times with PBS-T and then incubated with peroxidaseconjugated anti-rabbit antibodies diluted in PBS-T containing 5% skim milk (1:6000) at room temperature for 1 h. After a subsequent six washes of 10 min, the bands were visualized with the enhanced chemiluminescence Western blotting detection system (Amersham). Bio-Rad Precision Plus Unstained Standard in combination with Bio-Rad Precision StrepTactin-hrp Conjugate was used for the protein molecular weight standard. For quantitative analysis, bands were detected and evaluated densitometrically by Quantity One 1-D Analysis Software and Gel-Doc system from Bio-Rad. 2.6. Statistical analysis All data are presented as mean ± S.E.M. Statistical analysis was performed using SPSS 11.5. The differences between experimental groups were analyzed by two tailed unpaired Student’s t-test. Differences were considered significant at p < 0.05.

showed potent inhibition on 5-LOX with the IC50 values of 79.4 and 60.6 ␮g/mL, respectively. However, neither stem nor leaf extracts of Clematis microphylla inhibited COX-2. Both stem and leaf extracts of Clematis glycinoides showed inhibitory activities against 5-LOX, though they were not as potent as the other two species. Neither stem nor leaf extracts of Clematis glycinoides inhibited COXs. 3.2. Activating activity of Clematis extracts on protein expression of PPARα and PPARγ We evaluated the activating effects of the extracts from the stems of three Clematis species on the protein expression of PPAR␣ and PPAR␥ in HepG2 cells. Consistent with the data reported by Han et al. (2002), the HepG2 cells express both PPAR␣ and PPAR␥ (Fig. 1). The treatment with the stem extract of Clematis pickeringii resulted in 300% increases in PPAR␣ protein expression (Fig. 1A) and 278% increase in PPAR␥ protein expression (Fig. 1B), results which are statistically significant compared to the control (p < 0.001). The stem extract from Clematis macrophylla showed slightly enhancing effects on PPAR␣ (130%) and on PPAR␥ (124%) protein expression, however, these increases were not significant (p = 0.09). The stem extract of Clematis glycinoides activated neither PPAR␣ nor PPAR␥ under the same experimental conditions.

3. Results 3.1. Enzyme assays

3.3. Chromatographic separation of stem extract from Clematis pickeringii

The ethanol extracts of three Clematis species inhibited the activities of COX-1, COX-2 and 5-LOX in vitro in the different extents as showed in Table 1. Indomethacin used as positive control gave the IC50 values of 1.6 ␮M on COX-1, 10 ␮M on COX-2 and 30 ␮M on 5-LOX, respectively. The stem extract of Clematis pickeringii showed the highest inhibition on COX-1, COX-2 and 5-LOX among the extracts tested with the IC50 values of 73.5, 101.2 and 29.3 ␮g/mL, respectively. The leaf extract of Clematis pickeringii also inhibited COX-1 and 5-LOX; however, it showed low COX-2 inhibition (IC50 >200 ␮g/mL). The extracts from stem and leaves of Clematis microphylla inhibited COX-1 (IC50 values 175.2 and 134.1 ␮g/mL) and also

To determine the active fraction of the stem extract of Clematis pickeringii, which gave the highest inhibition on enzyme assays and activation on the PPAR␣ and PPAR␥ protein expressions, a preliminary fractionation was performed on a silica gel column (see Section 2). Ten fractions were collected and designated as F1 to F10. COX-1 and 5-LOX assays were performed and the results are presented in Table 2. Fraction 3 eluted with n-hexane:ethyl acetate (50:50, v/v) exhibited significantly inhibitory effects on COX-1 (80%) and 5-LOX (95%), respectively, at a concentration of 100 ␮g/mL. F7 eluted with 100% chloroform showed about 55 and 46% inhibition on COX-1 and 5-LOX at the concentration of 100 ␮g/mL, respectively, while

Table 1 Anti-inflammatory effects of the ethanol extracts of three Clematis species Sample

Parts extracted

Yielda

IC50 (␮g/mL)b COX-1

COX-2

5-LOX

Clematis glycinoides

Stem Leaf

5.2 2.8

>200 >200

>200 >200

98 ± 2.0 154.0 ± 4.0

Clematis microphylla

Stem Leaf

4.3 2.2

175.2 ± 5.3 134.1 ± 1.1

>200 >200

79.4 ± 1.9 60.6 ± 0.3

Clematis pickeringii

Stem Leaf

4.7 2.9

73.5 ± 1.2 120.5 ± 1.2

101.2 ± 4.3 >200

29.3 ± 2.3 68.4 ± 4.1

Indomethacin

–

–

1.6 ± 0.0c

10.5 ± 0.1c

30.2 ± 0.5c

a b c

Gram of solid extract/100 g of dried plant materials. The values are means ± S.E.M. from six replicates of two independent experiments. IC50 values are determined by regression analysis. ␮M.

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Fig. 2. HPLC chromatograph of F3-4 from the stem extract of Clematis pickeringii. The F3-4 was separated through a normal phase prep-HPLC analysis and 30 fractions were collected. Two sub-fractions of F3-4-16 and F3-4-18 showed potent COX-1 and 5-LOX inhibition.

other fractions produced low or no inhibitory effect under the assay conditions. Fraction 3 was further subjected to preparative HPLC-guided by COX-1 inhibitory screening. This fraction afforded a highly active sub-fraction, F3-4, with 81% inhibition at a concentration of 100 ␮g/mL (data not shown). F3-4 was further subjected to a normal phase preparative HPLC and separated into 30 sub-fractions. Two active sub-fractions determined from COX1 assay were designated as F3-4-16 and F3-4-18 (Fig. 2). Inhibitory activities on COX-1 observed from F3-4-16 and F3-4-18 were 80 and 71%, respectively, at a concentration of 100 ␮g/mL (Table 2). F3-4-16 demonstrated a potent 5-LOX inhibition (87%) while the F3-4-18 produced 56% inhibition on 5-LOX. Fig. 1. Effects of extracts of Clematis species on the protein expression of PPAR␣ and PPAR␥. The HepG2 cells were exposed to the Clematis extracts (60 ␮g/mL) for 24 h and proteins were separated by electrophoresis and Western blotted using antisera against PPAR␣ in (A) and PPAR␥ in (B). Lanes 1, 2, 3 and 4 represent Clematis macrophylla, Clematis glycinoides, Clematis pickeringii and DMSO, respectively. The corresponding bar charts present the relative expression of PPAR␣ protein (top) and PPAR␥ protein (bottom) as percentage increase of expression by the presence of test samples compared to vehicle control. Data present the mean ± S.E.M. from three independent experiments. * p < 0.01 compared to control.

Table 2 Inhibition of COX-1 and 5-LOX by the fractions of Clematis pickeringii stem extract separated via column chromatography Fractions

Eluting solvent and dried fractions

% of inhibitiona COX-1

5-LOX

F1 F2 F3 F4 F5 F6 F7 F8 F9 F10

Hexane, 10 mg Hexane:EtAc (75:25), 50 mg Hexane: EtAc (50:50), 23 mg Hexane: EtAc (25:75), 12 mg EtAc, 5 mg EtAc:chloroform (50:50), 6 mg Chloroform, 39 mg Chloroform:methanol (50:50), 20 mg Methanol, 34 mg Methanol:water (50:50), 15 mg

0.2 ± 0.1 49.1 ± 4.4 80.0 ± 5.1 9.3 ± 1.4 nd 17.8 ± 3.1 55.1 ± 3.3 13.2 ± 4.1 4.3 ± 0.1 6.9 ± 0.1

ndb 10.1 ± 0.3 95.1 ± 9.3 45.3 ± 1.0 nd 19.2 ± 1.1 46.2 ± 2.1 6.5 ± 0.3 nd 12.3 ± 0.5

F3 subfractions

F3-4-16

80.8 ± 1.2

86.5 ± 5.9

F3-4-18

71.3 ± 3.4

56.1 ± 2.5

The values are means ± S.E.M. from six replicates of two independent experiments. Final concentration of each fraction was 100 ␮g/mL. b nd, not detectable. a

3.4. Dose-dependent effects of active fraction on PPARα and PPARγ To investigate the effects of F3-4-16 on the expression of PPAR␣ and PPAR␥, we then examined PPAR␣ and PPAR␥ protein levels by Western blotting in HepG2 cells. HepG2 cells were incubated with 0, 4, 8 and 16 ␮g/mL for 24 h. Western blotting analysis showed that this fraction elevated both PPAR␣ and PPAR␥ protein levels in a concentration-dependent manner (Fig. 3A and B). PPAR␣ protein expression was increased by 173, 280 and 435% in response to the presence of F3-4-16 at 4, 8 and 16 ␮g/mL, respectively (Fig. 3C). These increases were statistically significant when compared to the vehicle controls (p < 0.05, 0.001 and 0.001, respectively). Similarly, this fraction activated PPAR␥ protein expression by 140, 228 and 296% at 4, 8 and 16 ␮g/mL, respectively. Excepting the concentration of 4 ␮g/mL (p = 0.08), the protein expression was statistically significant (p < 0.01 and 0.001, respectively). 4. Discussion The species Clematis pickeringii, Clematis glycinoides and Clematis microphylla, have been used traditionally in the treatment of inflammatory conditions. In addition, the mechanism remains unclear. In this study, we demonstrated the effects of extracts of three Clematis species on the inhibition of COX-1, COX-2 and 5-LOX and on the activation of protein expression of PPAR␣ and PPAR␥. We also showed that the active fraction of Clematis pickeringii F3-4-16 inhibited COX-1 and 5-LOX and activated the protein expressions of PPAR␣ and PPAR␥ on

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Fig. 3. Dose-dependent effects of F3-4-16 from the stem extract of Clematis pckeringii on PPAR␣ and PPAR␥ expression in HepG2 cells. Cells were incubated for 24 h with increasing concentrations of F3-4-16 at 0, 4, 8 and 16 ␮g/mL as indicated in lane 1, 2, 3 and 4 in (A and B). DMSO was used as a vehicle control. At the end of incubation, the cells were harvested and PPAR␣ and PPAR␥ protein levels determined. The total protein (40 ␮g) was subjected to Western blot analysis. (A) PPAR␣ protein expression; (B) PPAR␥ protein expression; (C) the results are pooled from three experiments each containing two replicates per conditions and are expressed as a percentage of the expression by control wells. * p < 0.05; ** p < 0.001 (compared to control wells).

HepG2 cells in a dose-dependent manner. This finding is novel to the Clematis species. The activities in the crude extracts and fractions of Clematis species support the traditional use of the Clematis species in Australian indigenous medicine. Despite the wide use of non-steroidal anti-inflammatory drugs (NSAIDs) for the treatment of various inflammatory diseases over the last century, their mechanism of action was not fully appreciated until 1971 when Vane identified their molecular target, the COX enzyme (Vane and Botting, 1987). Also, their use is often limited by the side effects in the gastrointestinal tract and the kidney. Two COX isoforms, COX-1 and COX-2 encoded by two different genes, have generated hope of developing a more specific and safe approach for the treatment of inflammatory diseases (Osiri and Moreland, 1999). The isozyme COX-1 is constitutively expressed in most tissues and is involved in the regulation of physiological ‘housekeeping’ functions such as the protection of gastric mucosa and the maintenance of renal perfusion. COX-2 immediately became a drug target after it was identified as an inducible isoform in pathological conditions by inflammatory stimulation. Therefore, it has been suggested that constitutive COX-1 is involved in homoeostatic processes whereas COX-2 plays a major part in the inflammatory reactions. Consequently, it has been hypothesized that selective inhibition of COX-2 might have anti-inflammatory properties of classical NSAIDs, but without altering renal function and without affecting the integrity of the gastric mucosa. However, recent studies have shown that that COX-1 and COX-2 have overlapping actions and that both isoforms are involved in homoeostasis processes. The role of COX-1 is not only physiological, but also it has been noted, in particular, that the anti-inflammatory effects of selective COX-2 inhibitors can-

not be seen if the dose is not increased above levels which also inhibit COX-1 activity, suggesting that this latter isoform has a significant role in the synthesis of proinflammatory PGs. Conversely, recent findings have shown that COX-2 is constitutively expressed in some tissues including the brain and kidneys (Davis et al., 1999; Martel-Pelletier et al., 2003). In addition, data have been produced suggesting that COX-1 may be induced at the site of inflammation (Wallace et al., 1998) and, in fact, mice lacking the gene for COX-1 exhibited diminished inflammatory responses in comparison with wild-type controls. In the present study, we found that the stem extract of Clematis pickeringii caused a COX-2 inhibition at a higher concentration than observed on COX-1 and 5-LOX inhibition. The ethanol extracts of Clematis glycinoides stem and leaf inhibited 5-LOX. This was not surprising considering that the vernacular names of this plant are ‘headache vine’ and ‘travellers’ joy’ (Lassak and McCarthy, 1997). Both indigenous Australians and bushman have used the crushed foliage to cure headaches and colds by inhaling the strong and sharp aroma (Webb, 1948, 1959). This traditional method of preparation leads us to consider that the active fractions may contain lipophilic ingredients, an idea needing further investigation. The poultice made from the leaves of Clematis microphylla was used as a counter-irritant (Lassak and McCarthy, 1997). This species also inhibited COX1 and 5-LOX, particularly, the leaf extract demonstrated a higher inhibitory activity than the stem extract. Although all of these extracts and fractions do not present a selective COX-2 inhibition, these data provide supporting evidence for the traditional use of Clematis species in the inflammatory conditions. The 5-LOX pathway generates an important class of inflammatory mediators, such as leukotrienes (LTs), which plays a major part in the inflammatory process. This pathway is upregulated during COX blockade and is thus potentially responsible for undesirable adverse effects. Therefore, dual inhibition of COX and 5-LOX constitute a valuable alternative to NSAIDs and selective COX-2 inhibitors for the treatment of inflammatory diseases. The concentrations of Clematis extracts and its active fractions required to inhibit 5-LOX are lower than that required to inhibit COX-1 and/or COX-2. This provides evidence that active ingredients tend to affect 5-LOX stronger than the COXs. This, again, supports the traditional use of Clematis species. Although many of the functions of PPARs are traditionally associated with the regulation of pathways of lipid metabolism and glucose utilization, the more recent evidence assigns the role of PPARs to cell replication, differentiation and inflammatory responses in different cell types (Gelman et al., 1999). PPAR␣ is highly expressed in the liver where it has initially been thought to regulate mainly genes involved in fatty acid ␤-oxidation. Recent studies have identified additional hepatic functions of PPAR␣. In primary human hepatocytes and hepatoma cells, PPAR␣ activators suppress interleukin (IL)-1 induced C-reactive protein (CRP) and IL-6-induced fibrinogen expression, the major acutephase response (APR) proteins in humans (Kleemann et al., 2003), whose plasma concentrations are elevated not only in acute but also in chronic inflammatory states. We observed that the Clematis pikeringii extract and its active fractions activated the protein expression of PPAR␣ and PPAR␥ on HepG2 cell

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model while those extracts showed to have anti-inflammatory activities through the in vitro enzyme inhibition. This is an interesting result because very few studies report the COX and LOX inhibition and PPARs activation by a medicinal plant product, although the idea that obesity, insulin resistance and diabetes are inflammatory diseases has existed for some time (Hotamisligil et al., 1993). Thus, we may speculate that Clematis pickeringii seems to contain the possible active ingredients acting as natural PPAR␣ and PPAR␥ ligands. We note, however, that we do not have data for a binding assay. As a consequence, we are unable to demonstrate direct interactions between Clematis pickeringii extracts and PPARs. This remains for further studies. Most importantly, a PPARs activation assessment of the Clematis pickeringii extract and its fractions should be conducted on several other cell models, such as macrophages, since PPAR␣ and PPAR␥ play roles in the inflammatory response in different cell types. Follow-up studies should be conducted to determine the influence of the extract of Clematis pickeringii and its fractions on the expression of PPARs induced proteins. The ability of the extract of Clematis pickeringii and its fractions to affect the protein expressions of PPAR␣ and PPAR␥s pathways is being explored currently. In summary, Clematis species have been used continuously as anti-inflammatory agents by indigenous Australians. We provide experimental evidence for their traditional use and basic data for further investigation on the mechanisms of action of these species. Acknowledgments The authors wish to thank Southern Cross University for the grant of the project. We are also grateful to Mr. Don Brushett of Centre for Phytochemistry, Southern Cross University, Australia for technical assistance in preparative HPLC. References Cribb, A.B., Cribb, J.W., 1981. Sydney. In: Wild Medicine in Australia. William Collins, pp. 34–35. Davis, B.J., Lennard, D.E., Lee, C.A., Tiano, H.F., Morham, S.G., Wetsel, W.C., Langenbach, R., 1999. Anovulation in cyclooxygenase-2-deficient mice is restored by prostaglandin E2 and interleukin-1beta. Endocrinology 140, 2685–2695. Gelman, L., Fruchart, J.C., Auwerx, J., 1999. An update on the mechanisms of action of the peroxisome proliferator-activated receptors (PPARs) and their roles in inflammation and cancer. Cellular and Molecular Life Sciences 55, 932–943.

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