Botanical Studies (2012) 53: 431-438.
biochemistry
The antinociceptive and anti-inflammatory activities of Petasites formosanus Kitamura extract
Shyr-Yi LIN1'2, Hsueh-Lian TENG3, Sung-Hui TSENG2, Lih-Geeng CHEN4, Ching-Chiung
WANG,5,6,*
1Department of Primary Care Medicine, Taipei Medical University Hospital, Taipei, Taiwan
2Department of General Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan
3Graduate Institute of Pharmacognosy Science, Taipei Medical University, Taipei, Taiwan
4Department of Microbiology, Immunology and Biopharmaceuticals, College of Life Sciences, National Chiayi University,Chiayi, Taiwan
5School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan
6Orthopedics Research Center, Taipei Medical University Hospital, Taipei, Taiwan
(Received January 10, 2012; Accepted May 30, 2012)
abstract.
Petasites formosanus Kitamura (Compositae) is native to Taiwan and is used in folk medicine
to treat hypertension and asthma. Aqueous methanolic (50%) extracts of leaves of P. formosanus (Leaves-MeOH extract) showed the strongest inhibitory effect against NO production by lipopolysaccharide (LPS)-induced RAW 264.7 cells with an IC50of 22.85 μg/mL. In an in vi~vo assay, 200 mg/kg of the extract also significantly decreased the acetic acid-induced writhing response, increased the hot-plate latency, and significantly sup­pressed carrageenan-induced paw edema. The principle anti-inflammatory constituents of the leaf extract were isolated by column chromatography combined with bioassay-guided fractionation, and 4 compounds, isopeta-sin, S-isopetasin, S-petasin, and caffeic acid methyl ester, were obtained. S-Ispopetasin and S-petasin more significantly inhibited NO production, inducible nitric oxide synthase, and cyclooxygenase-2 expression in a dose-dependent manner in LPS-induced RAW 264.7 cells, and S-ispopetasin showed stronger potency than S-petasin. S-Isopetasin also showed stronger reductions in the acetic acid-induced writhing response and car-rageenan-induced paw edema than S-petasin. Taken together, the results indicate that P. formosanus possesses both anti-inflammatory and anti-nociceptive activities, and S-isopetasin was the major constituent mediating those activities and may be used as a lead for new anti-inflammatory drug development.
Keywords:
Anti-inflammation; Antinociceptive; Compositae; Petasites formosanus Kitamura; S-isopetasin; S-
petasin.
introduction
Lin et al., 2008). Supporting evidence for the traditional beneficial claims of P. formosanus in hypertensive man­agement was also reported. Administration of S-petasin and S-isopetasin dose-dependently reduced the heart rate and blood pressure in anesthetized rats. Mechanistic stud­ies suggested that these two sesquiterpenes have calcium channel-blocking actions in vascular smooth muscle cells (Wang et al., 2001, 2002, 2004). The traditional usage also suggests that this plant may have anti-inflammatory and analgesic activities (Lin et al., 2004). Indeed, in other parts of the world, other species of Petasites are used to treat inflammatory disorders (Thomet et al., 2001; Panthong et al., 2003; Fiebich et al., 2005).
Petasites formosanus Kitamura is a Petasites species of the Asteraceae (Compositae) indigenous to Taiwan. Inter­est in this plant arose because in Taiwanese folk medicine, the plant is used to treat asthma and hypertension (Lin et al., 1998). A study by Lin et al. first isolated several com­pounds, including S-petasin and S-isopetasin, from the aer­ial parts of P. formosanus (Lin et al., 1998). A later study demonstrated significant relaxant effects of S-petasin and S-isopetasin in the isolated guinea pig trachea pretreated with a contractile agent (Ko et al., 2000). The antispas-modic and tracheal relaxation may be due to antimuscarin-ic activity of the two compounds, supporting the folkloric usage of P. formosanus to treat asthma (Ko et al., 2001;
Usually the entire plant of P. formosanus is used indig­enously. However, the part of the plant that is most useful has not been explored, and the active ingredients of P. for­mosanus for anti-inflammation and anti-nociception are as yet unknown. The purpose of this study was to investigate the ability of P. formosanus extracts and constituents to in-

*Corresponding author: E-mail: crystal@tmu.edu.tw; Tel: 886-2-27361661 ext. 6161; Fax: 886-2-27329368.
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hibit inflammatory and nociceptive responses. The results provide scientific evidence for the clinical use of P. formo-sanus in Taiwanese folk medicine.
and 3 (142.9 mg, 0.0150%) (Figure 2).
The n-hexane-EtOAc ( 1: 1) eluate was chromato-graphed over a silica gel column (2.5 cm i.d. x 40 cm) eluted with n-hexane-EtOAc (5: 2) to yield caffeic acid methyl ester (4) (43.3 mg, 0.0046%) (Figure 2).
materials and methods
Plant material
Isopetasin (1) ESI-MS (m/z): 317 [M+H]+. 1H-NMR (500MHz, CDCy 5: 1.01 (3H, d, J =6.8Hz, H-14), 1.99 (3H, dd, J =7.0, 1.1Hz, H-4'), 1.05 (3H, s, H-15), 1.50 (1H, m, H-2a), 1.71 (1H, m, H-4),1.86 (3H, s, H-12), 1.89 (3H, s, H-5'), 2.10 (3H, d, J =1.7 Hz, H-13), 2.19 (1H, m, H-6a), 2.24 (1H, m, H-2b), 2.36 (1H, m, H-1a), 2.48 (1H, m, H-1b), 2.93 (1H, d, J =13.7Hz, 6b), 4.93 (1H, td, J =4.3, 11.1Hz, H-3), 5.78 (1H, d, J =1.58Hz, H-9), 6.07 (1H, m, H-3'). 13C-NMR (126MHz, CDCy 5: 10.8 (C-14), 15.7 (C-4'), 17.1 (C-15), 20.6 (C-5'), 22.1 (C-12), 30.1 (C-1), 31.7 (C-2), 41.2 (C-6), 42.2 (C-5), 46.2 (C-4), 73.3 (C-3), 126.7 (C-9), 127.1 (C-7), 128.0 (C-2'), 137.9 (C-3'), 143.4 (C-11), 165.1 (C-10), 167.7 (C-1'), 191.6 (C-8).
Petasites formosanus was identified and cultivated by Dr. Din-Lin Chang of the Taiwan Seed Improvement and Propagation Station, Council of Agriculture, Taichung, Taiwan for 1 year and was harvested in April 2006. Vouch­er specimens (PF-006) were deposited in the School of Pharmacy, College of Pharmacy, Taipei Medical Univer­sity, Taipei, Taiwan. After harvest, leaves of P. formosanus were dried in a 40°C oven before being used.
Drug and Chemicals
Dimethyl sulfoxide (DMSO), MTT [3-(4, 5-dimeth-ylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide], trypan blue, lipopolysaccharide (LPS) (E. coli serotype 0127-8B), carrageenan, indomethacin, tramadol and other chemicals were purchased from Sigma Chemical (St. Louis, MO, USA). Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), antibiotics, L-glutamine, and trypsin-EDTA were purchased from Gibco BRL (Grand Island, NY, USA). Western blotting was performed using an antibody specific to mouse iNOS (sc-650), anti-COX-2 (sc-1745), anti-GAPDH (sc-32233), anti-rabbit IgG-AP (sc-2007), and anti-mouse IgG-AP (sc-2008), anti-goat IgG-AP (sc-2022) which was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Column chromatography was carried out on silica gel (Merck). All solvents used for column chromatography were of analytical grade.
S-Isopetasin (2) ESI-MS (m/z): 335 [M+H]+. 1H-NMR (500MHz, CDCy 5: 0.99 (3H, d, J =6.7Hz, H-14), 1.03 (3H, s, H-15), 1.49 (1H, qd, J =1.4, 14.3 Hz, H-2a), 1.66 (1H, m, H-4), 1.85 (1H, s, H-13), 2.09 (3H, d, J=1.04Hz, H-12), 2.18 (1H, brd, J =13.7 Hz, H-6a), 2.22 (1H, m, H-2b), 2.34 (1H, td, J =4.0, 15.0Hz, H-1a), 2.41 (3H, s, S-CH3), 2.45 (1H, td, J=3.8, 14.5 Hz, H-1b), 2.92 (1H, m, H-6b), 4.93 (1H, td, J =4.3, 12.7Hz, H-3), 5.84 (1H, d, J =10.1Hz, H-2'), 5.77 (1H, s, H-9), 7.08 (3H, d, J = 10.1Hz, H-3'). 13C-NMR (126MHz, CDCy 5: 10.7 (C-14), 17.1 (C-15), 19.3 (S-CH3), 22.1 (C-13), 22.6 (C-12), 30.1 (C-1), 31.8 (C-2), 41.2 (C-6), 42.2 (C-5), 46.2 (C-4), 73.3 (C-3), 113.0 (C-2'), 126.7 (C-9), 127.2 (C-7), 143.3 (C-11): 152.4 (C-3'), 165.2 (C-10), 166.3 (C-1'), 191.6 (C-8).
S-Petasin (3) ESI-MS (m/z): 335 [M+H]+. 1H-NMR (500MHz, CDCy 5: 0.96 (3H, d, J =6.8Hz, H-15), 1.23 (3H, s, H-14), 1.50 (1H, m, H-2a), 1.62 (1H, m, H-4), 1.74 (3H, s, H-13), 1.90 (1H, td, J =13.9Hz, H-6a), 2.03 (1H, dd, J =4.4, 13.1Hz, H-6b), 2.24 (1H, m, H-2b), 2.36 (1H,
Extraction and isolation
Dried leaves of P. formosanus (950 g) were refluxed with 50% methanol (10 L x 2) for 1 h. The filtrate was concentrated with a rotary evaporator at 45°C to obtain a syrup-like aqueous methanolic extract (258 g). The aque­ous methanolic extract was suspended in water and parti­tioned with ethyl acetate (EtOAc). The ethyl acetate layer was absorbed by Celite 545 and was chromatographed over a silica gel column (9.6 cm i.d. x 70 cm) with n-hexane— n-hexane-EtOAc (10:1 ― 5:1 ― 1:1 ― 1:5 —1:10)— EtOAc— EtOAc-acetone (10:1)— acetone— MeOH). Among these fractions, n-hexane-EtOAc (10:1, 5:1, and 1:1) eluates were further characterized (Figure 1).
The n-hexane-EtOAc (10:1) eluate was chromato-graphed over a VersaPak silica gel column (40 mm i.d. x 150 mm, Supelco, Bellefonte, PA, USA), developed with n-hexane-EtOAc (5:1). The major compound was moni­tored with thin-layer chromatography (TLC) and recrystal-lized with n-hexane and EtOAc to yield isopetasin (1) (1.9 mg, 0.0002%) (Figure 2).
The n-hexane-EtOAc (5:1) eluate yielded crude S-iso-petasin (2) and S-petasin (3). Each compound was recrys-tallized with n-hexane to yield pure 2 (153.6 mg, 0.0162%)
Figure 1.
Fractionation and isolation procedures from the dried
leaves of P. formosanus.
LIN et al. ― Anti-inflammatory and anti-nociceptive effects of Petasites formosanus
433
m, H-1a), 2.41 (3H, s, S-CH3), 2.52 (1H, m, H-1b), 3.11 (1H, dd, J =4.5, 14.4Hz, H-7), 4.82 (1H, s, H-12a), 4.95 (1H, dd, J =4.4, 15.5, H-3), 4.98 (1H, d, J =1.5 Hz, H-12b), 5.78 (1H, d, J =1.5Hz, H-9), 5.83 (1H, d, J =10.1Hz, H- 2'), 7.09 (1H, d, J = 10.1Hz, H-3'). 13C-NMR (126MHz, CDCy 5: 10.4 (C-15), 17.1 (C-14), 19.3 (S-CH3), 20.0 (C-13), 30.6 (C-1), 31.7 (C-2), 40.0 (C-5), 41.7 (C-6), 47.3 (C-4), 50.3 (C-7), 73.0 (C-3), 112.9 (C-2'), 114.4 (C-12), 124.6 (C-9), 143.3 (C-11), 152.5 (C-3'), 166.3 (C-1'), 166.8 (C-10), 198.5 (C-8).
mice per group). The total number of writhes that occurred within 30 min were observed and counted after the animal was placed in a plastic cage. The writhing response con­sisted of a contraction of the abdominal muscles together with a stretching of the limbs. The anti-nociceptive activ­ity was expressed as a writhing number over a period of 30 min (Ojewole, 2005).
Hot-plate latent pain response test in rats
For thermal hyperalgesia, rats were individually placed in a hot plate instrument (Ugo Basile, Comerio, VA, Italy). Oral treatment with the vehicle, tramadol (10 mg/kg), or P. formosanus (100, 200, or 400 mg/kg) was given 1 or 2 h prior to the hot plate test (3 rats per group). A radiant heat source was applied underneath the glass floor. The time between placement of the rats on the platform and licking of the paws was recorded as the hot-plate latency (Mar-rassini et al., 2010).
Caffeic acid methyl ester (4) ESI-MS (m/z): 195 [M+H]+. 1H-NMR (500 MHz, CD3OD) 5: 3.74 (3H, s, H-10), 6.25 (1H, d, J =16.0Hz, H-8), 6.77 (1H, d, J =8.2Hz, H-3), 6.93 (1H, dd, J =16.0, 8.2 Hz, H-2), 7.02 (1H, d, J =1.8Hz, H-6), 7.53 (1H, d, J =16.0Hz, H-7). 13C-NMR (126MHz, CD3OD) 5: 52.0 (C-10), 114.8 (C-8), 115.1 (C-2), 116.5 (C-5), 122.9 (C-6), 127.7 (C-1), 146.8 (C-3), 146.9 (C-7), 149.6 (C-4), 169.8 (C-9).
Animals
Carrageenan-induced paw edema in mice
Adult male Wistar rats weighing about 250 10 g and mice weighing about 25 2 g were purchased from the BioLASCO Taiwan Co., Ltd. and kept on a 12:12-h, day-night cycle. Animals were maintained in polycarbonate cages at 21 2°C and provided food and water ad libitum. All experimental procedures involving animals followed the ethical regulations of Taipei Medical University.
Edema in the left hind paw of mice was induced by an injection of 50 μl of 1% (w/v) carrageenan from Sigma (St. Louis, MO) in saline into the subplantar region. P. formosanus at different doses (100~400 mg/kg) and in­domethacin (100 mg/kg, as a reference substance) were given orally 1 h before the injection. The control group was given the vehicle (0.1 ml/10 g). Another group of mice received a subplantar injection of 0.9% saline and vehicle, and was designated the blank group. Each group consisted of six animals. The paw volume of the animals was measured 1 h before and 1~6 h after the injection us­ing a plyethysometer (Ugo Basile, Comerio, VA, Italy) (Tseng et al., 2006).
Acetic acid-induced writhing test in mice
Acetic acid (0.6%, 0.1 ml) was injected into the peri­toneal cavity of a mouse. Oral treatment with the vehicle, indomethacin (100 mg/kg), or P. formosanus (100~400 mg/kg) was given 1 h prior to an acetic acid injection (6
Figure 2.
The chemical structures of isopetasin (1), S-isopetasin (2), S-petasin (3), and caffeic acid methyl ester (4) isolated from the
dried leaves of P. formosanus.
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In vitro anti-inflammatory assay
Table 1.
Effects of the 50 % methanolic extracts of different
The anti-inflammatory activity was presented in terms of inhibited production of nitric oxide (NO), inducible NO synthase (iNOS), and inducible cyclooxygenase (COX)-2 by LPS-stimulated RAW 264.7 cells. NO was measured as nitrite production in the medium after 24 h of incuba­tion with or without the extracts and/or LPS (500 ng/ml). Briefly, nitrate in the medium was converted to nitrite and measured spectrophotometrically after the Griess reaction (Tseng et al., 2006). Expressions of iNOS and COX-2 by LPS-stimulated RAW 264.7 cells were investigated by a Western blot analysis. RAW 264.7 cells exposed to ex­tracts for 24 h were collected into tubes and then washed with phosphate-buffered saline (PBS). Protein samples were prepared according to our previous paper (Tseng et al., 2006). Total protein (25 μg) was used for the Western blot analysis. Proteins were transferred to nitrocellulose membranes. Membranes were probed using antibodies specific to COX-2, iNOS, and GAPDH and visualized us­ing a BCIP/NBT kit from Gibco BRL (Grand Island, NY, USA) according to the manufacturer's instructions.
amounts of P. formosanus parts on NO production and cell vi­ability of LPS-treated RAW 264.7 cells.


Inhibited NO effects

50% MeOH
extract of:

Inhibition        IC50
Cytotoxicity
index (%)a
(%)a        (μg/mL)

Leaves

70.02 ± 3.77

22.85

4.09 ± 3.75
Flowers
33.61 ± 5.12
-
0.51 ± 0.74
Stems
3.09 2.72
-
1.81 ± 2.16
Roots
10.31 ± 0.93
-
0.00 ± 0.00

aThe concentration of tested sample was 200 μg/mL. There were three independent experiments. Results are expressed as Mean ± SD.
Table 2.
Inhibitory effect of Leaves-MeOH extract on heat-
induced foot licking episodes in rats.



Time (h)


0

1

2

Control

7.54 ± 0.42

7.74 ± 0.99

7.32 ± 0.79
Tramal (10 mg/kg)
7.94 ± 0.87
10.29 ± 0.32*
9.39 ± 0.30*
Leaves-MeOH extract (mg/kg)
100
7.41 ± 0.42
9.30 ± 0.58
8.52 ± 0.91
200
7.39 ± 0.79
10.50 ± 0.33*
10.44 ± 0.77*
400
8.52 ± 0.53
10.75 ± 1.62*
10.58 ± 0.94*
Data analysis
Each experiment was performed at least in triplicate. Results are expressed as Mean SD. One way analysis of variance (ANOVA, SPSS 12.0) was used to analyze the data of the animal model. Results were considered statisti­cally significant atp < 0.05.
results

Leaves-MeOH extract was aqueous methanolic leaf extract of P. formosanus. Results are expressed as MeanSD for the foot licking time (second)
*Compared with control group,p < 0.05.
Antinociceptive and anti-inflammatory activities of extracts from P formosanus
After being dried, the different parts (flowers, leaves, stems, and roots) of P. formosanus were separately extract­ed with 50% methanol, and their anti-inflammatory activities were evaluated using LPS-induced RAW 264.7 cells. Aqueous methanolic leaf extract of P. formosanus (leaf extract) showed the most potent inhibitory activity against NO production (Table 1). Therefore, the anti-inflammatory and analgesic activities of the Leaves-MeOH extract were further investigated.
Table 3.
IC50 values of different fractions and natural com-
pounds obtained from the aqueous methanolic leaf extract of P. formosanus on NO production in LPS-induced RAW 264.7 cells.

Fraction

IC50 (μg/mL)

EtOAc layer

12.68
H2O layer
> 100
A
10.07
B
9.10
C
11.14
D
12.80
E
38.90
F
34.34

Natural compounds

μg/mL (μM)

S-isopetasin

1.46 (4.24)
S-petasin
3.54 (10.60)
Caffeic acid methyl ester
8.08 (41.43)

There were three independent experiments.
Oral administration of the Leaves-MeOH extracts (200 or 400 mg/kg) to rats 1 or 2 h prior to a hot plate test caused a significant delay in the production of the first foot-licking episode induced by radiant heat (Table 2). The Leaves-MeOH extract dose-dependently and significantly inhibited iNOS expression in LPS-induced RAW 264.7 cells (Figure 3A). The in vivo anti-inflammatory effect of the Leaves-MeOH extract was then examined with a carrageenan-induced acute inflammatory model in ICR mice. Oral administration of the Leaves-MeOH extract 1 h before the injection exerted a significant dose-dependent inhibitory effect on the development of paw swelling (Fig-ure 3B). Oral administration of the Leaves-MeOH extracts (100, 200, or 400 mg/kg) to mice 1 h before the injection significantly reduced the number of writhing episodes in-
LIN et al. ― Anti-inflammatory and anti-nociceptive effects of Petasites formosanus
435
duced by acetic acid compared to the control group (Figure 3C). In according the antinociceptive and anti-inflammato­ry screening test, we suggested the Leaves-MeOH extracts contains bioactive components.
Bioactive sesquiterpenes isolated from leaves of P. formosanus
The Leaves-MeOH extract of P. formosanus was parti­tioned with EtOAc and distilled H2O and chromatographed as described. Among these fractions, n-hexane, n-hexane-EtOAc (10: 1, 5: 1, and 1: 1), acetone, and methanol elutes were further characterized. The inhibitory effect of each fraction (A~F) on NO production in LPS-induced RAW 264.7 cells was evaluated with IC50 values shown in Table
Figure 4.
Dose-dependent inhibitory effects of S-isopetasin
and S-petasin on iNOS and COX-2 production by LPS-induced RAW 264.7 cells. There were three independent experiments.
3. The NO inhibitory effect of the EtOAc layer was stron­ger than that of the H2O layer, and the A to D fractions isolated from the EtOAc layer also showed strong efficacy. Further isolation of fractions A, B, and D yielded 1.9 mg isopetasin (1) from fraction A (yield, 0.0002%); 153.6 mg of S-isopetasin (2) and 142.9 mg of S-petasin (3) from fraction B (yields, 0.0162% and 0.0150%, respectively); and 43.3 mg of caffeic acid methyl ester (4) from fraction D (yield, 0.0046%). However, the yield from fraction C was very little, and the amount of isopetasin also was in­sufficient for a bio-analysis.
Antinociceptive and anti-inflammatory activities of S-isopetasin and S-petasin
The inhibitory effects of S-isopetasin, S-petasin, and caffeic acid methyl against NO were measured in LPS-induced RAW 264.7 cells. S-isopetasin and S-petasin showed the most potent NO inhibitory effect, with respec­tive IC50 values of 1.46 and 3.54 μg/mL (Table 3). Both compounds also significantly inhibited iNOS and COX-2 expressions in LPS-induced RAW 264.7 cells (Figure 4), and S-ispopetasin was stronger than S-petasin in a dose-dependent manner. Both compounds also significantly reduced the number of writhing episodes induced by acetic acid compared to the control (Figure 5). However, S-petasin compared to S-ispopetasin at 1.25 mg/kg that S-ispopetasin was a significant anti-nociceptive effect, but not S-petasin (Figure 5). Moreover, only S-isopetasin
Figure 3.
Inhibitory effects of aqueous methanolic leaf extract
of P. formosanus (Leaves-MeOH extract). (A) Analysis iNOS expression in LPS-induced RAW 264.7 cells, there were three independent experiments; (B) The assay was carrageenan-induced paw edema in mice and per one group were 6 mice; (C) The assay was acetic-acid induce writhing response in mice and per one group were 6 mice. Results are expressed as Mean±SD. *p < 0.05, **p < 0.005.
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Botanical Studies, Vol. 53, 2012
Figure 5.
Inhibitory effects of S-isopetasin and S-petasin on the acetic acid-induced abdominal writhing test in mice. Each group was
6 mice. Results are expressed as Mean±SD. *p < 0.05, **p < 0.005.
exerted significant inhibitory activity on carrageenan-induced paw edema in mice (Figure 6). These results sug­gest that s-isopetasin is the major anti-inflammatory and anti-nociceptive constituent of leaves of P. formosanus.
discussion
The results of our study suggest that the foliar parts of P. formosanus have both anti-inflammatory and anti-no­ciceptive activities, with S-petasin and S-isopetasin being the active compounds for the above-described biological activities. Our data also suggest that both compounds modulate inflammation and nociception at least through their capability to inhibit iNOS and COX-2 expressions. iNOS and COX-2 are well-known mediators of inflamma­tory responses. In addition, an inhibitor of NOS or a natu­ral compound that can modulate NO was shown to affect the nociceptive response to chemically induced nocicep-tion as well (Moore et al., 1991; Perimal et al., 2011). S-Petasin and S-isopetasin exert a calcium channel-blocking action in vascular smooth muscle cells (Wang et al., 2001, 2004). S-Petasin also inhibits calcium channel excitation in neurons (Wu et al., 2003). Blocking calcium channel excitation has the potential to play a valuable role in dis­eases that require modulation of muscle contractions or neuron excitation, such as hypertension, arrhythmia, and chronic pain. In fact, clinical studies showed the ability of calcium channel blockers to lower pain scores when a noxious pressure was applied to a patient's sternum (Del Giaccio and Eblen-Zajjur, 2010). The calcium channel-blocking activity of S-petasin may also contribute to its analgesic effect.
Figure 6.
Inhibitory effects of S-isopetasin and S-petasin on
carrageenan-induced paw swelling in mice. Each group was 6 mice. Results are expressed as MeanSD. *p < 0.05, **p < 0.005.
asthma and hypertension. This study is the first to dem­onstrate potent anti-inflammatory and anti-nociceptive activities of the methanolic extract of P. formosanus leaves. Also, for the first time, the two major sesquiterpene constituents of P. formosanus leaves, S-isopetasin and S-petasin, were shown to possess strong anti-inflammatory and anti-nociceptive abilities. Taken together, the results suggest an interesting therapeutic potential for this species in pain and inflammatory diseases. The results also sug­gest that S-isopetasin is the major constituent mediating the anti-inflammatory and analgesic activities of P. formo-sanus and may be used as a lead compound for new anti-inflammatory drug development.
Studying traditional folk medicine is useful for dis­covering new natural products with potential medicinal properties. In the last century, natural products played a significant role in drug discovery and development pro­cesses (Newman and Cragg, 2007). The results of previous research provided scientific evidence and explained the mechanism of the action of P. formosanus as a remedy for
Acknowledgments.
The authors gratefully acknowledge
the financial support (98TMU-TMUH-11) from Taipei Medical University Hospital, Taipei, Taiwan.
LIN et al. ― Anti-inflammatory and anti-nociceptive effects of Petasites formosanus
437
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Botanical Studies, Vol. 53, 2012
臺灣款冬之抗發炎與止痛主成分
林時宜1,2 鄧學聯3 曾頌惠2 陳立耿4 王靜瓊5,6
1臺北醫學大學附設醫院一般醫學科
2臺北醫學大學醫學院醫學系
3臺北醫學大學藥學院生藥學研究所
4國立嘉義大學生命科學院微生物免疫與生物藥學系
5臺北醫學大學藥學院藥學系
6臺北醫學大學附設醫院骨關節研究中心
臺灣款冬(菊科)是一種臺灣原生種的植物,且為臺灣民間用來治療高血壓與氣喘的草藥。臺灣
款冬的葉子以50%甲醇萃取(葉萃取物)顯示具有極強抑制脂多醣誘導RAW 264.7巨噬細胞引起的一
氧化氮釋放作用,其50%的抑制濃度為22.85 μg/mL 。繼而利用體內試驗檢測其功效,結果顯示管餵老
200 mg/kg萃取物可以有意義的降低醋酸誘導的扭體反應、熱板引起的疼痛刺激及鹿角菜膠誘導的足
掌浮腫。因此利用管柱層析配合生物活性分析追蹤分離臺灣款冬葉萃取物中的抗發炎活性成分,結果得
4個化化合物'分別是isopetasin S-isopetasin S-petasincaffeic acid methyl ester 。其中,S-isopetasin
S-petasin於抑制脂多醣誘導RAW 264.7巨噬細胞試驗中具較強的抑制一氧化氮釋放、誘導型一氧化
氮合成酶及環氧化酶作用,且顯示劑量依存性,而S-isopetasin的作用又比S-petasin強。綜合而言,臺
灣款冬具有抗發炎及止痛作用,其主要的活性成分為S-isopetasin ,且其葉部具發展成抗發炎先驅藥之潛
力。
關鍵詞:
臺灣款冬;菊科;S-isopetasin S-petasin ;止痛;抗發炎作用。