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Bioactivity-guided fractionation for anti-inflammatory and analgesic properties and constituents of Xanthium strumarium L.

Abstract

The aim of this study was to fractionate an extract of Xanthium strumarium L. (EXS) and to investigate the anti-inflammatory and analgesic properties of the extract and its fractions. The ethanol extract of X. strumarium (EXS) was fractionated on the basis of polarity. Among the different fractions, the n-butanol fraction showed the highest anti-inflammatory activity in the croton-oil-induced ear edema test and furthermore reduced the number of writhings induced by acetic acid in mice in a dose-dependent manner. This indicates that the n-butanol fraction of X. strumarium possesses potent analgesic effects which are likely to be mediated by its anti-inflammatory activity. Bioassay-guided fractionation of EXS led to the isolation and identification of ten caffeoylquinic acids and three heterocyclics by HPLC-DAD-[MS.sup.n] from the active n-butanol fraction, implying that the active compounds are polar in nature. The isolated caffeoylquinic acids could partially explain the antinociceptive effect of X. strumarium polar extract.

[c] 2007 Elsevier GmbH. All rights reserved.

Keywords: Xanthium strumarium; Anti-inflammatory; Analgesic; Croton-oil-induced ear edema; Acetic-acid-induced writhing; Bioassay-guided isolation

Introduction

The genus Xanthium (family Compositae) is represented by 25 species in the world, of which 3 species and one variety are found in China (Chen, 1979). Historically, Xanthium species have been used as traditional herbal medicines in oriental countries. Xanthium strumarium L., is the principle species found abundantly throughout China and used in traditional Chinese medicine to treat nasal sinusitis, headache, urticaria and arthritis (Pharmacopoeia PR China I, 2000). It has also been reported to possess curative effects against chronic bronchitis, chronic rhinitis, allergic rhinitis, lumbago and other ailments (Zhu, 1998). In countries other than China, the genus Xanthium is also used as a diuretic and an emetic, and is reported to be effective against prostate diseases (Cumanda et al., 1991). In previous chemical studies on X. strumarium, carboxyatractyloside, xanthanol, isoxanthanol, hydroquinone, alkaloids, caffeyolquinic acids, and thiazinedione were identified (Ma et al., 1998; Cumanda et al., 1991; China Herbal, 1999). There are, however, no experimental reports on the screening of bioactive fractions and components from this plant. The present study focuses on the evaluation of anti-inflammatory and analgesic activities of a fruit extract of X. strumarium and aims to establish the most active part(s) of the plant.

Materials and methods

Chemicals and medicines

Indomethacin was supplied by the Jiangsu Yabang Pharma Group, Norm: 2.5 mg/tablet. Croton oil, Tween 80, acetic acid, ethanol, petroleum (60-90[degrees]C), chloroform, ethyl acetate and n-butanol were purchased from Sinopharm Chemical Reagent Co. Ltd., China. HPLC-grade acetonitrile and formic acid (FA) were purchased from J.T. Baker (Baker Mallinckrodt, Phillipsburg, NJ, USA). HPLC-grade water was prepared using a Millipore (Bedford, MA, USA) Milli-Q purification system.

Apparatus

The chromatographic separation was performed on an Angilent 1100 HPLC system (Angilent 1100, Boblingen, Germany), equipped with a quaternary pump, an autosampler, a degasser, an automatic thermostatic column compartment, a DAD detector and a computer using a Chemstation software program for analysis of the HPLC data. A Zorbax SB C-18 reversed-phase column (150 x 3.0 mm, 3 [micro]m) together with a Phenomenex C-18 guard column (10 x 4.6 mm, 5 [micro]m) were used with column temperature set at 30[degrees]C.

The mobile phase consisted of methanol (eluent A) and 0.1% aqueous formic acid (eluent B); The ratios of A:B were as follows: 0 min, 10:90; 10min, 20:80; 30min, 40:60; 50min, 40:60; 60min, 10:90 (v/v, respectively). The mobile phase was degassed automatically using the electronic degasser system and the flow rate was 0.2 ml/min, with the detector wavelength set at 327 nm.

MS analysis was performed using an Agilent Series 1100 LCMSD (Agilent technologies Inc., Palo Alto, CA, USA) equipped with the Agilent LCMSD ChemStation software. Normal operating conditions were as follows: varying fragmentor voltages 50-100V were used, the sample was introduced through a syringe infusion pump; heated nitrogen (300[degrees]C) was used as a countercurrent drying gas.

Plant material

The ripe fruits of X. strumarium L., Xanthii Fructus, were harvested from a local research farm in Sunqiao town, Shanghai, China. A voucher specimen of the plant was deposited at the Herbarium of the Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai, China under the acquisition number CE20031107.

Preparation of the ethanol extract and the fractions

Dried fruits of X. strumarium (20 kg) were ground and extracted with 75% aqueous ethanol at room temperature. The solvent was evaporated under vacuum to afford 1100g crude extract (yield, 5.5%). This extract (EXS) was then suspended in water and partitioned successively with petroleum ether, chloroform, ethyl acetate and aqua-saturated n-butanol. Each fraction was evaporated in vacuo to yield the residues of petroleum ether 55 g (5.0%), chloroform 60 g (5.4%), ethyl acetate 60.5g (5.5%), n-butanol 200g (18.2%) and aqueous 725 g (65.9%), respectively.

For pharmacological studies, EXS and the fractions were suspended in a 1% aqueous solution of Tween-80. When the extracts were administered topically in the croton-oil test, they were dissolved in acetone. The doses employed are expressed as mg of the dried extract per kg body weight.

Animals

Studies were carried out in accordance with current guidelines for the care of laboratory animals and ethical guidelines for the investigation of experimental pain in conscious animals (Zimmermann, 1983). Male ICR mice (approximately 25 g each) were housed in temperature-controlled rooms (22-23[degrees]C) until use. Food and water were supplied ad libitum and each experimental group contained 10 animals.

Evaluation of anti-inflammatory activity: croton oil-induced ear edema

This evaluation was performed essentially as described by Tubaro et al. (1985). Mice were anesthetized with ether and 20 [micro]l of an acetone solution containing 0.4 [micro]g croton oil and appropriate amounts of test samples were applied to the inner surface of the right ear of each mouse. The left ear remained untreated. Control animals received only the irritant, while indomethacin (300 [micro]g per ear) served as the reference. The animals were sacrificed by cervical dislocation 5 h later and a plug (7 mm in diameter) was removed from both the treated ear and the untreated ear. The difference in weight between the two plugs was taken as a measure of edematous response. The percentage of protection was calculated using the following ratio: (control mean-treated mean) x 100/control mean.

Evaluation of analgesic activity: acetic acid-induced writhing test

Analgesic activities of the ethanol extract (400mg/kg body wt., p.o.) and its five fractions were studied by the reduction of acetic acid-induced writhing in the mice (Witkin et al., 1961). After 30min administration, the animals received 10 ml/kg body wt. acetic acid (0.7%; i.p.). The number of abdominal contractions and stretching with a jerk of the hind limb were counted for 15 min after administering acetic acid. The percentage of protection was calculated using the following ratio: (control mean--treated mean) x 100/control mean.

Chemical analysis

From the n-butanol fraction, the following were isolated and identified as described by Han et al. (2006): 5 phenolic acids, namely: chlorogenic acid, cynarin, 1,5-O-dicaffeoylquinic acid, 1,4-O-dicaffeoyl-quinic acid, 1,3,5-O-tricaffeoylquinic acid and 3 heterocyclics. By the application of HPLC-DAD-[MS.sup.n] another 5 phenolics were determined from the n-butanol fraction (Fig. 1) according to the methods described by Clifford et al. (2005) and Tolonen et al. (2002). The 3 heterocyclics are absent from the chromatogram because of their relatively low content.

Statistical analyses

The data were analyzed for statistical significance using Student's t-test. P-values less than 0.05 were considered to be significant.

Results and discussion

Croton-oil-induced ear edema

The topical anti-inflammatory activities of EXS and its subfractions were evaluated as inhibition of croton-oil-induced ear edema in mice. The percent inhibition edema (Table 1) of EXS was significant (p<0.05) at the dose tested (33.72% at 1.0mg/ear), in comparison with the control. The n-butanol fraction exhibited significant anti-inflammatory activity between 0.5, 0.75 and 1.0mg/ear, in a dose-dependent manner. At 1.0mg/ear dose, the anti-inflammatory activity of the n-butanol fraction was comparable to that of indomethacin at a dose of 0.3mg/ear (54.77% vs. 54.25%). In contrast, the chloroform fraction showed a mild anti-edema effect at the 1.0mg/ear dose, while the petroleum ether and other fractions showed fewer or no effects at this dose (Table 1). As a positive control, indomethacin (0.3 mg/ear) significantly inhibited the ear edema by 54.25%. The results demonstrate the topical anti-inflammatory properties of X. strumarium fruits and they may justify the use of this plant for the treatment of inflammatory diseases in Chinese, folk and herbal medicine.

[FIGURE 1 OMITTED]

Acetic acid-induced writhing test

The results of the acetic acid-induced writhing responses in mice which indicate the analgesic activity of EXS are presented in Table 2. It was found that all the extracts and fractions except for the petroleum ether fraction at the dose 400mg/kg body wt. caused a significant inhibition of the writhing responses induced by acetic acid as compared to the control, with values ranging from 24% to 72% protection. At 200mg/kg body wt., EXS and the n-butanol fraction still showed significant inhibition of the writhing responses, with inhibitions of 26.41% and 52.75%, respectively. In contrast, at a dose of 100mg/kg body wt., only the n-butanol fraction showed any significant analgesic effect. Oral administration of the n-butanol fraction at doses of 100, 200 and 400mg/kg body wt. gave rise to inhibitions of 36.44%, 52.75% and 72.33%, respectively, in the number of writhings, indicating that the analgesic activity of n-butanol fraction is dose-dependent. These results provide pharmacological support for the analgesic effect of X. strumarium. These results taken together indicate that EXS possesses strong analgesic activity, with the n-butanol fraction possessing the highest analgesic activity as compared to other fractions.

X. strumarium L. is the principle Xanthium species and the only one indexed in the Chinese Pharmacopoeia (Pharmacopoeia PR China I, 2000). It is found abundantly throughout the world and has been used as a traditional herbal medicine with a long history of use in oriental countries. However, its pharmacological actions have not been well-documented in spite of its increasing use. In the present work, we elucidated the anti-inflammatory effect of EXS using the croton-oil-induced ear edema test. In the aspect of ear plug weight, EXS shows an inhibitory effect on inflammation, which supports its folkloric usage in inflammatory diseases. At present, it is difficult to ascertain whether EXS is able to inhibit iNOS and/or COX-2 enzyme. EXS was found to possess both anti-inflammatory and analgesic activities similar to those displayed by classical anti-inflammatory drugs. Its analgesic activity may be mediated by its anti-inflammatory action. As a step toward identifying the active principle(s) of X. strumarium, EXS was fractionated successively using different organic solvents. Among the fractions, the n-butanol fraction appeared to be the most effective against inflammation and as an analgesic, implying that X. strumarium could contain active anti-inflammatory component(s) with relatively hydrophilic characters.

Bioactivity-guided fractionation of EXS led to the isolation and identification of ten phenolic acids and three heterocyclic compounds, of which two are new natural compounds (Han et al., 2006). The HPLC-DAD-[MS.sup.n] method was successfully used to identify the three types of caffeoylquinic acids (mono-, di- and tri-caffeoylquinic acids). Previous studies showed that these naturally occurring phenolic acids have various pharmacological properties and can be used as cholagogues, stomach stimulants, and immunostimulants, as well as anti-tumor, antioxidant, antibacterial, and antifungal agents (Mishima et al., 2005; Hamauzu et al., 2005; Jiang et al., 2005; Zhang et al., 2001). Santos et al. (2005) reported that two dicaffeoylquinic acids (3,5- and 4,5-O-dicaffeoylquinic acids) but not 3,4,5-0-tricaffeoylquinic acid showed significant analgesic activity in the acetic-acid-induced mouse writhing test at low but not at high doses. Ferreira et al., 2006 obtained 3,5- and 4,5-O-dicaffeoylquinic acids from the antinociceptive fraction of Ipomoea cairica. The isolated caffeoylquinic acids could partially explain the antinociceptive effect of X. strumarium polar extract. However, the bioactivities of the thiazinedione heterocyclics have not been reported previously. Furthermore, the anti-inflammatory activities of the mono-, di- and tri-caffeoylquinic acids and the bioactive effects of the heterocyclics reported here are currently under investigation and the findings will be the subject of a future publication.

Based on these results, it can be suggested that X. strumarium has topical anti-inflammatory and analgesic activities. These observed activities may be associated with the presence of phenolics of known analgesic activity, as well as a probable synergistic effect of the heterocyclics components and other secondary metabolites. Further studies are required to determine the possible mechanism of actions of these compounds and their potential for clinical use needs to be demonstrated in clinical trials.

References

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Hamauzu, Y., Yasui, H., Inno, T., Kume, C, Omanyuda, M., 2005. Phenolic profile, antioxidant property, and anti-influenza viral activity of Chinese quince (Pseudocydonia sinensis Schneid.), quince (Cydonia oblonga Mill.), and apple (Mains domestica Mill.) fruits. J. Agric. Food Chem. 53 (4), 928-934.

Han, T., Li, H.L., Zhang, Q.Y., Zheng, H.C., Qin, L.P., 2006. New thiazinediones and other comonents from Xanthium strumarium L. Chem. Nat. Comp. 42 (5), 567-570.

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Tolonen, A., Joutsamo, T., Mattlla, S., Kamarainen, T., Jalonen, J., 2002. Identification of isomeric dicaffeoylquinic acids from Eleutherococcus senticosus using HPLC-ESI/TOF/MS and [.sup.1.H]-NMR methods. Phytochem. Anal. 13 (6), 316-328.

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T. Han (a), H.-L. Li (b), Q.-Y. Zhang (a), P. Han (a), H.-C. Zheng (a), K. Rahman (c), L.-P. Qin (a,*)

(a) Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai 200433, China

(b) Department of Natural Medicinal Chemistry, School of Pharmacy, Second Military Medical University, Shanghai 200433, China

(c) Faculty of Science, School of Biomolecular Sciences, Liverpool John Moores University, Byrom Street, Liverpool L3 3AF, UK

*Corresponding author. Tel./fax: +8621 25070394.

E-mail address: 1pqin@smmu.edu.cn (L.-P. Qin).
Table 1. Effects of Xanthium stramarium on ear edema induced by croton
after topical administration in mice

 Dose Weight of edema (mg),
Treatment (mg/ear) mean [+ or -] S.E.M. Inhibition (%)

Control -- 7.65 [+ or -] 0.86
EXS 1.0 5.07 [+ or -] 1.09 (a) 33.72
Petroleum ether 1.0 6.76 [+ or -] 0.72 11.63
 fraction
Chloroform 1.0 5.38 [+ or -] 0.73 (a) 29.67
 fraction
Ethyl acetate 1.0 7.47 [+ or -] 1.21 2.35
 fraction
Aqueous fraction 1.0 7.22 [+ or -] 0.96 5.62
n-Butanol fraction 1.0 3.46 [+ or -] 0.57 (b) 54.77
 0.75 4.87 [+ or -] 1.02 (a) 36.34
 0.5 5.55 [+ or -] 0.68 (a) 27.45
Indomethacin 0.5 3.50 [+ or -] 0.76 (b) 54.25

(a) p < 0.05.
(b) p < 0.01 compared with control.

Table 2. Effects of Xanthium strumarium on acetic acid-induced writhing
responses in mice

 Dose
 (mg/kg Number of writhings,
Treatment body wt.) mean [+ or -] S.E.M. Inhibition (%)

Control -- 38.39 [+ or -] 8.62
EXS 400 20.45 [+ or -] 2.32 (b) 46.73
 200 28.25 [+ or -] 5.78 (a) 26.41
 100 34.36 [+ or -] 6.34 10.50
Petroleum ether 400 37.50 [+ or -] 6.22 2.32
 fraction
Chloroform 400 26.47 [+ or -] 4.63 (a) 31.05
 fraction
 200 32.25 [+ or -] 5.32 15.99
 100 34.58 [+ or -] 3.33 9.92
Ethyl acetate 400 22.89 [+ or -] 3.20 (b) 67.71
 fraction
 200 31.97 [+ or -] 2.89 16.72
 100 33.79 [+ or -] 6.08 11.98
Aqueous fraction 400 28.88 [+ or -] 7.34 (a) 24.77
 200 36.72 [+ or -] 3.88 4.35
 100 38.26 [+ or -] 7.04 0.34
n-Butanol fraction 400 12.56 [+ or -] 1.22 (b) 72.33
 200 18.14 [+ or -] 3.86 (b) 52.75
 100 24.40 [+ or -] 5.61 (a) 36.44
Indomethacin 10 14.22 [+ or -] 1.78 (b) 62.96

(a) p < 0.05.
(b) p < 0.01 compared with control.
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Author:Han, T.; Li, H.-L.; Zhang, Q.-Y.; Han, P.; Zheng, H.-C.; Rahman, K.; Qin, L.-P.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Dec 1, 2007
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