Antinociceptive activity and chemical composition of constituents from Caragana microphylla seeds

Antinociceptive activity and chemical composition of constituents from Caragana microphylla seeds

ARTICLE IN PRESS Phytomedicine 14 (2007) 143–146 www.elsevier.de/phymed Antinociceptive activity and chemical composition of constituents from Carag...

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ARTICLE IN PRESS

Phytomedicine 14 (2007) 143–146 www.elsevier.de/phymed

Antinociceptive activity and chemical composition of constituents from Caragana microphylla seeds Y. Huoa,b, C. Guoc, Q.-Y. Zhanga, W.-S. Chenb, H.-C. Zhenga, K. Rahmand, L.-P. Qina, a

Department of Pharmacognosy, School of Pharmacy, Second Military Medical University, Shanghai 200433, PR China Department of Pharmacy, Changzheng Hospital, Shanghai 200003, PR China c Department of Pharmacy, Sixth People’s Hospital, Shanghai Jiao Tong University, Shanghai 200233, PR China d Faculty of Science, Liverpool John Moores University, Liverpool L3 3AF, UK b

Abstract This investigation was undertaken to ascertain the antinociceptive activity of Caragana microphylla Lam. seeds and isolate and characterize the constituents. Antinociceptive activity was screened using acetic acid-induced abdominal constriction in ICR mice. The 75% ethanol extract and some fractions showed analgesic activity, but the antinociceptive activity of chloroform fraction was the strongest and was more productive than other fractions. Seven compounds were isolated from it and identified as: (1) machaeric acid, (2) b-sitosterol, (3) stigmasterol, (4) pratol, (5) dehydrocavidine, (6) formononetin and (7) sucrose. Caragana microphylla Lam. seeds showed analgesic activity, with the chloroform fraction showing the strongest analgesic activity among the fractions. r 2006 Elsevier GmbH. All rights reserved. Keywords: Caragana microphylla Lam.; Antinociceptive activity; Triterpenes; Alkaloid; Flavone; Isoflavone

Introduction Caragana microphylla Lam. (Leguminosae) is widely distributed in northern and western China. Its roots and seeds have been used in folk medicine as treatments for swollen and painful throat (Shanghai Scientific & Technical Press, 1985). Literature reports indicate that this plant has some bioactivities, including antinociceptive, anti-inflammatory and anticancer activities (Liu HX et al., 2004). Recently, our research showed that the 75% ethanolic extract and some fractions of the seeds of the plant exhibited analgesic activities in vivo (Jin ZN et al., 1994). These bioactivities prompted us to continue to investigate its chemical components. In the present Corresponding author. Fax: +86 2125070394.

E-mail addresses: [email protected], [email protected] (L.-P. Qin). 0944-7113/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2006.03.008

work, we studied the antinociceptive activity of an ethanolic extract and its fractions on acetic acid-induced abdominal constriction in ICR mice and obtained seven compounds: (1) machaeric acid, (2) b-sitosterol, (3) stigmasterol, (4) pratol, (5) dehydrocavidine, (6) formononetin and (7) sucrose, from the chloroform fraction.

Experimental Medicines and animals Medicines: Indomethacin (Xiaoyantong), supplied by the Jiangsu Yabang Pharma Group, Norm:2.5 mg/p. Tween 80, acetic acid, ethanol, petroleum(60–901), CHCl3, EtOAc and n-BuOH were purchased from

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Sinopharm Chemical Reagent Co., Ltd., China. All the chemicals and reagents used were of the highest grade of purity. Animals: ICR species mice, 18–22 g each, half male and half female, were supplied by Shanghai SLAC Laboratory Animal Co., Ltd. Animals were raised at constant room temperature (251, relative humidity 70–75%). For each experiment, the mice were divided randomly into groups of 10 per cage. 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).

Preparation of plant extract and fractions The seeds of Caragana microphylla Lam. were collected from the middle of Inner Mongolia Autonomous Region, People’s Republic of China, in July, 2004. The specimens were identified by Prof. Zheng HanChen, School of Pharmacy, Second Military Medical University of China, where voucher specimens have been deposited. The seeds of Caragana microphylla Lam. (25 kg) were ground and extracted exhaustively using 75% EtOH. The solvent was evaporated in vacuo, and the extract was dissolved in water and partitioned successively with petroleum (60–901), CHCl3, EtOAc and n-BuOH, yielding Caragana fractions (CF), CF1, CF2, CF3, CF4, respectively, and an aqueous residue (CF5). The CF, CF4 and CF5 were dissolved in water, and CF1,

CF2 and CF3 were dissolved in water with 1.25% Tween 80 for the antinociceptive tests.

Methods and results Acetic-acid-induced abdominal constriction Groups of animals were separated and food and water removed 1 h before treatment. Animals were pre-treated orally (p.o.) with CF or its fractions (5 g/kg body wt) 60 min before acetic acid injection. The abdominal constrictions induced by intraperitoneal (i.p.) injection of 0.7% acetic acid at a dose of 10 ml/kg were carried out according to Koster et al. (1959). As positive control for antinociception, a group of mice was treated with Indomethacin at 5 mg/kg, body wt, p.o. (Indo group), 30 min before the noxious stimulus. The nociception control group received an equal volume of vehicle (water with 1.25% Tween 80). Control experiments did not show altered response due to oral administration of the vehicle at the concentration used. After challenge, pairs of mice were separated in boxes and the number of writhes was noted for 15 min beginning 5 min after acetic acid injection (Loro et al., 1999). Antinociceptive activity was detected as a reduction in the number of abdominal constrictions exhibited by treated mice as compared to the nociception control group, and was expressed as the percent of pain inhibition. We found the CF, CF1 and CF2 significantly inhibited the frequency of acetic-acid-induced abdominal

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50

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40 ** ** 30

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0 Control

Indo

CF

CF1

CF2

CF3

CF4

CF5

Fig. 1. Effect of p.o. administration of CF, its fractions and Indomethacin on writhing induced in groups of mice by an intraperitoneal injection of 0.7% acetic acid. Data are expressed as mean7s.e.m. po0:05,   po0:01.

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constrictions in mice (po0:01). Inhibition rates were observed for at CF (64.68%) and its fractions CF1 (53.20%), CF2 (56.73%), CF3 (34.00%), CF4 (25.17%), CF5 (27.59%). Indomethacin inhibited the abdominal constrictions by 46.36%. The results are shown in Fig. 1.

Chemical analysis From the chloroform fraction, seven compounds were isolated and identified using NMR data as (1) machaeric acid, (2) b-sitosterol, (3) stigmasterol, (4) pratol, (5) dehydrocavidine, (6) formononetin and (7) sucrose.

Discussion Natural products exhibiting analgesic properties are of great interest for a variety of reasons. The seed of Caragana microphylla Lam. is a rich source of several biological molecules, such as organic molecules produced via the secondary metabolism, including triterpenes, alkaloids, flavones, and isoflavones. Jin et al. (1994) found that the methanol extract of Caragana microphylla Lam. roots showed antinociceptive activity. It has been suggested that there are some analgesic fractions or compounds in this plant. In the present work, we have fractionated the seed of Caragana microphylla Lam. and found the main analgesic fraction. From this effective fraction, we have isolated some interesting compounds, including 1, 4, 5, and 6. The antinociceptive activity of these compounds is under investigation and the findings will be the subject of another paper. The mouse-writhing model is well known for the antinociceptive activity bioassay. It involves different nociceptive mechanisms, such as the sympathetic system (biogenic amines release), cyclooxygenases (COX) and their metabolites (Duarte et al., 1988) and opioid mechanisms (Collier et al., 1968). Acetic acid acts indirectly by inducing the release of endogenous mediators, which stimulate the nociceptive neurons sensitive to NSAIDs (non-steroidal anti-inflammatory drugs) and/or opioids (Collier et al., 1968). Thus, CF and its active fractions can induce antinociception by mechanisms similar to non-narcotics and/or narcotic drugs, perhaps by blocking the receptor or the release of endogenous substances that excite pain nerve endings (Hunskaar and Hole, 1987). NSAIDs such as indomethacin inhibit cyclooxygenases (COX) in peripheral tissues, thereby reducing PGE2 (prostaglandin E2) synthesis and interfering with the mechanism of transduction in primary afferent nociceptors (Dionne et al., 2001).

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The observed central antinociceptive activity reported in this study may be due to the presence of some active constituents, especially the lipophilic fractions. Among the separated compounds, compounds 1, 4, 5, and 6 are of interest. Compound 1 is a triterpene, while compound 5 is an alkaloid, and compounds 4 and 6 are flavonoids. All are more productive in this isolation. Recently, a large number of different kinds of natural alkaloids, triterpenes and flavonoids with antinociceptive and anti-inflammatory activities has been reported (Joao et al., 2000). We suggest that compounds 1, 4, 5, and 6 may be the most effective constituents in this antinociceptive fraction. Further test of the analgesic activity of these compounds is currently being conducted in our laboratory. Caragana microphylla Lam. is a perennial shrub, and a favored plant for restoring vegetation on desertified sandy land (Cao et al., 2000). It can establish and develop the sandy soil water-holding capacity, enhanced organic C and total N accumulation, and decreased pH and bulk density. It can facilitate the colonization and development of herbaceous species by ameliorating stressful environmental conditions (Su and Zhao, 2003). Thus, it has an important role in the restoration of the local ecosystem. Although both its roots and seeds can be used in folk medicine, local people mainly use the roots. If this practice continues, the plant will become extinct in the local area and hence destroy the local environment due to overuse of the root. We therefore decided to investigate its seeds in order to find if there are pharmacological differences between the roots and seeds. According to our data, the seeds showed the same analgesic activity as the roots. Hence, we propose that the seeds should be become the main resource for folk medicine, rather than Caragana’s roots. This practice will protect the plant and the local environment.

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Jin, Z.N., Jin, J.J., Jin, G.Z., 1994. The pharmacology research of Caragana microphylla Lam. J. Med. Sci. Yanbian Univ. 17 (4), 267. Calixto, J.B., Beirith, A., Ferreira, J., et al., 2000. Naturally occurring antinociceptive substances from plants. Phytother. Res. 14, 401–418. Koster, R., Anderson, M., Beer, E.J., 1959. Acetic acid for analgesic screening. Fed. Proc. 18, 412–417. Liu, H.X., Lin, W.H., Yang, J.S., 2004. Research of chemical components and bioactivities of Caragana. Fabr. Chin. Pharm. J. 39 (5), 327–330.

Loro, J.F., del Rio, I., Perez-Santana, L., 1999. Preliminary studies of analgesic and anti-inflammatory properties of Opuntia dillenii aqueous extract. J. Ethnopharmacol. 67, 213–218. Shanghai Scientific & Technical Press, 1985. The Dictionary of Traditional Medicine. Jiangsu New Medical College, China, pp. 392–393. Su, Y.Z., Zhao, H.L., 2003. Soil properties and plant species in an age sequence of Caragana microphylla plantations in the Horqin Sandy Land, North China. Ecol. Eng. (20), 223–235.