Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Antinociceptive and anti-inflammatory activities of Schefflera octophylla extracts Yanfen Chen a, Shuhong Tao a, Fanling Zeng a, Luwei Xie b, Zhibin Shen a,n a b
School of Traditional Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, PR China School of Pharmacy, Harbin University of Commerce, Harbin 150000, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 3 March 2015 Received in revised form 25 April 2015 Accepted 26 April 2015
Ethnopharmacological relevance: Schefflera octophylla (Lour.) Harms, a traditional Chinese herb mainly distributed in Southeast Asia, is extensively prescribed to alleviate pain and treat rheumatoid arthritis (RA), influenza, throat swelling and pain, etc. In this paper, the antinociceptive and anti-inflammatory activities of the ethanol extract and its five different polar fractions of this plant were evaluated. Furthermore, the anti-rheumatoid arthritis activity of the ethanol extract and its active fraction (CHCl3 fraction) were evaluated. And the chemical constituents of the CHCl3 active fraction displayed significant antinociceptive and anti-inflammatory activities were investigated. Materials and methods: Antinociceptive and anti-inflammatory activities were investigated by hot plate test, acetic acid-induced abdominal writhing test and formalin test, xylene-induced ear edema test. The anti-rheumatoid arthritis activity was evaluated through the model of adjuvant-induced arthritis (AA) in rats, paw swelling, pain response, arthritis index and histopathological changes of ankle, the levels of TNF-α, IL-1β, IL-6 and rheumatoid factor (RF) of rats were detected. The chemical constituents of the CHCl3 fraction were isolated using chromatographic techniques. Their structures were elucidated by spectroscopic data analysis. Results: The results showed that the ethanol extract of S. octophylla has significant dose-dependent antiinflammatory and antinociceptive activities. And its five different polar fractions especially the CHCl3 fraction significantly inhibited the abdominal writhing induced by acetic acid and ear edema induced by xylene, also reduced pain threshold in hot plate test in 120 min and ticking times in formalin test. The ethanol extract of S. octophylla and the CHCl3 fraction demonstrated an anti-RA effect in a dosedependent manner. The levels of TNF-α, IL-1β and IL-6 in ethanol extract (600 mg/kg) and CHCl3 fraction (300 mg/kg) groups were significantly lower than those of the model group. The chemical constituents study of the CHCl3 fraction from S. octophylla led to six triterpenoids which were identified as taraxerone (1), epitaraxerol (2), aleuritolic acid (3), 3-oxofriedelan-28-oic acid (4), 3β,19α -dihydroxy-urs-12-ene24,28-dioic acid (5) and asiatic acid (6). Compounds 1–5 were obtained from this plant for the first time. Conclusion: This study proved the antinociceptive, anti-inflammatory and anti-rheumatoid arthritis activities of S. octophylla. Triterpenoids obtained from its CHCl3 fraction may be responsible for those activities. These results could support the fact that S. octophylla is used traditionally to cure inflammatory and pain diseases. & 2015 Published by Elsevier Ireland Ltd.
Keywords: Schefflera octophylla Antinociception Anti-inflammatory Adjuvant-induced arthritis Triterpenoids
1. Introduction Arthritis is the most common chronic diseases in the world. During all kinds of arthritis, rheumatoid arthritis and osteoarthritis are the two most common types of arthritis. According to reports in the literature (Li et al., 2014), rheumatoid arthritis affects around 0.5–1% of adults in the developed world with between 5 and 50 per 100,000 people newly developing the condition each
n
Corresponding author. Tel.: þ 86 20 39352179; fax: þ86 20 39352174. E-mail address: [email protected] (Z. Shen).
year, while osteoarthritis affects about 3.6% of the global population and nearly 27 million people in the United States. The symptoms of arthritis are very diverse, mainly shown as red, swollen, and dysfunctional joints, with feeling of heat and pain. Thus the conventional medication treatments for arthritis, especially in acute period, are analgesics and non-steroidal antiinflammatory drugs (NSAIDs). However, those drugs have undesirable side effects on gastrointestinal, kidney, bronchus and cardiovascular system (Wallace and Vong, 2008). As a result, researchers all of the world are looking for safer, more effective alternatives to both the traditional analgesics and NSAIDs (WalkerBone, 2003). Furthermore, inflammation is typically a fundamental
http://dx.doi.org/10.1016/j.jep.2015.04.050 0378-8741/& 2015 Published by Elsevier Ireland Ltd.
Please cite this article as: Chen, Y., et al., Antinociceptive and anti-inflammatory activities of Schefflera octophylla extracts. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.04.050i
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and important pathological process in many diseases, researchers focused in recent years on study of antinociceptive and antiinflammatory activities, and medicinal plants derived natural products such as flavonoids, steroids, polyohenols, terpenes, stearic acid which have wide range of pharmacological efficacy but lesser side effects (Shah and Alagawadi, 2011; Shukla et al., 2010). The genus Schefflera (Araliaceae) comprises of 200 species distributed in the tropical and some subtropical areas in the world. There are 38 species and 2 varieties in China. Schefflera octophylla (Lour.) Harms is distributed mainly in Guangxi, Guangdong and Yunnan provinces in China. It is widely used as a folk medicine for the rheumatic pain, traumatic pain, and sore throat, etc (Editorial committee of Chinese Materia Medica, 1999). Previous phytochemical investigations have revealed that triterpenoids were the main components. More than 40 triterpenoids were isolated from this plant, including oleanane type (Sung and Adam, 1991; Maeda et al., 1994), ursane type (Maeda et al., 1994; Sung et al., 1992) and lupane type (Sung et al., 1991; Kitajima et al., 1990; Adam et al., 1982; Lischewski et al., 1984; Kitajima and Tanaka, 1989; Sung and Adam, 1992; Sung et al., 1991), etc. However, in the pharmacological aspects, only anti-virus and anti-oxidant effects of the extract from S. octophylla have been reported previously (Li et al., 2004a, 2004b; Zheng et al., 2009). In Southeast Asia, S. octophylla is extensively prescribed to alleviate pain and treat rheumatoid arthritis (RA). Considering the traditional uses in alleviating pains of this herb, we firstly evaluated the antinociceptive and anti-inflammatory activities of ethanol extract of S. octophylla and its five different polar fractions through three antinociceptive tests and one anti-inflammatory test. For further investigation, we evaluated the anti-rheumatoid arthritis activity through the model of adjuvant-induced arthritis (AA) in rats, arthritis index, paw swelling, pain response and histopathological changes of ankle, the levels of TNF-α, IL-1β, IL-6 and rheumatoid factor (RF) of rats were detected. In order to clarify the main active components of S. octophylla, we also investigated the chemical constituents of the CHCl3 fraction which displayed significant antinociceptive and anti-inflammatory activities.
2. Materials and methods
successively with petroleum ether, chloroform (CHCl3), ethyl acetate (EtOAc) and n-butyl alcohol (n-BuOH) for three times. The volume for the above extracted organic solvent was 2 L each time. Each partition was evaporated to dryness and resulting fractions of petroleum ether, CHCl3, EtOAc and water. These fractions and the ethanol extract of S. octophylla (EES) were evaluated for activity tests. 2.4. Isolation of the CHCl3 fraction The CHCl3 fraction (365 g) was separated by silica gel chromatography (100–200 mesh, petroleum ether-EtOAc 100:0–7:3 and CHCl3– MeOH 9:1–7:3) to yield four fractions (F1–4). F1 was differentiated with silica gel chromatography again (200–300 mesh) using gradient of petroleum ether–EtOAc (97:3–8:2) to afford compound 1 (70 mg). F2 was separated by silica gel chromatography (200–300 mesh, petroleum ether–EtOAc 95:5–7:3) to yield three sub-fractions (F2a– 2c). F2b was purified by Sephadex LH-20 chromatography using MeOH to yield compound 2 (10 mg), 3 (8 mg) and 4 (10 mg). F4 was run with silica gel chromatography again (200–300 mesh, CHCl3– MeOH 9:1–7:3) to generate compound 5 (3 g) and 6 (12 mg). 2.5. Structure identification of compound 1–6 The structures of compound 1–6 were identified by comparing the 1H NMR and 13C NMR spectral data with previous literature reports. 2.6. Biological activity assays 2.6.1. Drugs and reagents Acetic acid (Guangdong Chemical Reagent Factory, China); formaldehyde (Luoyang Chemical Reagent Factory, China); xylene (Guangdong Guanghua Chemical Reagent Co., Ltd., China); indometacin (Southern China Pharmaceutical Group Co., Ltd., China); rotundine (Sichuan Kangfulai Pharmaceutical Group Co., Ltd., China); complete Freund's adjuvants (Sigma, Saint Louis, Missouri, USA); leflunomide (Suzhou Changzheng Xinkai Pharmaceutical Co., Ltd.); CMC-Na (Guangdong Device of Medical Equipment Co., Ltd., China). All other chemicals were of analytical grade.
2.1. General Column chromatography: silica gel (100–200 and 200–300 mesh; Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), Sephadex LH-20 (Amersham Biosciences; Sweden). TLC: precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). NMR Spectra: Bruker AVANCE III 500 spectrometer with Me4Si as internal standard. ESI-TOF-MS: Acquity UPLC-Q-TOF Microinstrument; in m/z. 2.2. Plant material The root bark of S. octophylla was collected in April 2010 from Guangzhou, Guangdong province, PR China. The specimen was identified by Associate Professor Jizhu Liu, School of Traditional Medicine, Guangdong Pharmaceutical University. A voucher specimen has been deposited at the lab 435 of School of Traditional Chinese Medicine, Guangdong Pharmaceutical University. (Accession number: TCMS002). 2.3. Extraction The air-dried and powdered plant materials of S. octophylla (23 kg) were refluxed with 95% ethanol (300 L) for 3 times (2 h per time). After evaporation of the alcohol under reduced pressure, 920 g of viscous residue was obtained with a yield of 4.0%. The ethanol extract (730 g) was suspended in H2O, and extracted
2.6.2. Experimental animals Male or female NIH mice (18–22 g) and male Sprague-Dawley rats (150–180 g) were obtained from the Medical Experimental Animal Center of Guangdong Province (China). They were kept in plastic cages at 2272 1C with free access to pellet food and water. The experiment procedures were conducted in compliance with the National Institutes of Health Guide for care and use of the laboratory animals, and were approved by the Committee of Experimental Animal Administration of the university (Document no. SCXK 2008-0002). All experimental protocols were approved by the Institutional Animal Ethics Committee. 2.6.3. Hot plate test Experiments were carried out according to previously described method (Shinde et al., 1999). During test of antinociceptive and anti-inflammatory activities in mice, EES was administrated to the animals at doses of 300, 600 and 1200 mg/kg (equivalent to 7.5, 15, 30 g/kg of crude medicine), and petroleum ether, CHCl3, EtOAc, n-BuOH, water fractions were administrated at doses of 6 and 600, 32, 55, 100 mg/kg respectively (each dose was equivalent to 30 g/kg of crude medicine). The control group received only vehicle (0.5% CMC-Na, 20 ml/kg), and the reference group received rotundine (0.02 g/kg). Mice were individually placed on the heated plate which maintained at 5570.5 1C after treatment for 60, 90
Please cite this article as: Chen, Y., et al., Antinociceptive and anti-inflammatory activities of Schefflera octophylla extracts. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.04.050i
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67 EES at doses of 150, 300 and 600 mg/kg. The CHCl3 groups of animals 68 were administered CHCl3 fraction at a dose of 75, 150, and 300 mg/kg 69 respectively. Paw swelling, arthritis index, pain response and histo70 pathological changes of ankle were employed to evaluate the anti71 inflammatory activities of drugs. The paw swelling was periodically 72 examined using toe volume measuring instrument. According to the 2.6.4. Acetic acid-induced writhing test 73 degree of joint swelling, we assessed the arthritis index (AI) by Experiments were carried out according to previously described 74 the standard as previously descri bed (Gu and Brandwein, 1998):0, Q2 method (Xu et al., 2014). Mice were treated orally one hour before 75 normal; 1, slight swelling and/or erythema of toes; 2, most of the toes injection of acetic acid with EES or different fractions. The control 76 and foot swelling; 3, paw swelling below the ankle; 4,complete group received only vehicle (0.5% CMC-Na, 20 ml/kg), and the 77 swelling of toes and ankle, inability to bend the ankle or wrist. The reference group received indometacin (0.01 g/kg) respectively. Wri78 pain response score assessment standard also based on the literature thing was induced by an intraperitoneal injection (20 ml/kg) of a 79 (Gu and Brandwein, 1998), according to the leg withdrawal reaction 0.7% acetic acid solution. The numbers of writhing that occurred in 80 in rats, the ankle pain score was 0–5. During the experiment, the 15 min were counted for each animal. 81 hind paw swelling was measured at days 4, 8, 12, 16, 20, 24, 28 after 82 the injection, and the arthritis index, pain score were monitored at 2.6.5. Formalin test 83 days 12, 16, 20, 24, and 28. At the end of the experiments (day 28 Experiments were carried out according to previously described 84 after immunization for AA rats), rats were fasted overnight and method (Hunskaar and Hole, 1987). Mice were orally treated with 85 sacrificed. The hind paws of all the rats were removed, the ankle EES or different fractions. The control group received only vehicle 86 joints were decalcified and embedded, then sections were stained (0.5% CMC-Na, 20 ml/kg), and the reference group received indo87 with haematoxylin and eosin for histopathological observation to metacin (0.01 g/kg). One hour later, 20 ml of 2.5% formalin was 88 find out the degree of chronic inflammation occurred in the joints. injected s.c. into the right hind paw of each mouse. Each mouse was 89 Blood samples were collected into labeled vacuum tubes and procimmediately placed into a glass cylinder to observe the nociceptive 90 essed into serum, then immediately stored at 80 1C in closed. behavior. The time spent in licking the injected paw (an index of 91 Levels of cytokines (IL-1β,IL-6, TNF-α) and RF were determined by the nociception) was measured in the first phase (0–5 min) and second 92 ELISA Kits (Wuhan Huamei Biological Engineering Co. Ltd.,Wuhan, phase (15–30 min) after formalin injection. 93 China) according to the manufacturer's instructions. 94 2.6.6. Xylene-induced ear edema test 95 2.7. Statistical analysis of data The improved method was adopted as previously described 96 (Zhang et al., 2008). Mice were pretreated with EES or different 97 The data were presented as the mean 7SD and analyzed by fractions, indometacin (0.01 g/kg), or the vehicle alone. One hour 98 one-way ANOVA. P values less than 0.05 (P o0.05) were defined as later, each animal received 20 ml of xylene on the anterior and 99 the significant level. posterior surfaces of the right ear lobe, the left ear serving as the 100 control. One hour later, the animals were sacrificed under ether 101 anesthesia, their bilateral ears were removed and weighed pre3. Results and discussion 102 cisely. The degree of ear edema was calculated based on the 103 weight of the left ear without applying xylene. 3.1. Antinociceptive and anti-inflammatory activities of EES 104 105 2.6.7. Adjuvant-induced arthritis in rats The hot plate test, writhing test and the formalin model of 106 Animals like Sprague-Dawley rats were randomly divided into nociception, xylene-induced ear edema are usually used as screen107 nine groups of ten animals each (n¼10). Except the control group, ing tools for the assessment of analgesic or anti-inflammatory 108 rats in other groups were inoculating 0.1 mL of complete Freund's properties of drugs. Therefore, based on clinical application of S. 109 adjuvants (CFA) into the left hind metatarsal rat footpad to develop octophylla, we firstly investigated the antinociceptive and anti110 AA animals. The rats of control group were injected with 0.1 mL inflammatory activities of the ethanol extracts of S. octophylla 111 sterilized physiological saline only. Drug treatment was started in all (EES) and its different polar fractions with these test. 112 groups from the day of arthritis successfully formed and continued In hot plate test, EES showed a significant dose dependent 113 12 d. The control group and the model group received 10 ml/kg of effect. It significantly inhibited pain threshold in 60 (P o0.05), 90, 114 vehicle in which the extract was going to be suspended while the 120 min (P o0.01) at the highest dose of 1200 mg/kg (Fig. 1).The 115 positive control group received leflunomide (6 mg/kg p.o) as the plant extract significantly restrained the writhing response ind116 reference standard. The EES groups of animals were administered uced by intraperitoneal injection of 0.7% acetic acid in mice at all 117 118 70 119 ** ** ** 120 60 121 ** ** ** 50 ** ** 122 * * * 40 123 124 30 125 20 126 10 127 128 0 60 min 0 min 120 min 90 min 129 Hot plate test 130 131 Fig. 1. Antinociceptive effect of EES in the hot plate test. Results are expressed as mean7 SD (n ¼14); *Po 0.05, **Po 0.01 when compared with control group. Con: control; 132 Rot: rotundine; EES: ethanol extract of Schefflera octophylla; (A) petroleum ether fraction; (B) CHCl3 fraction; (C) EtOAc fraction; (D) n-BuOH fraction; (E) water fraction. and 120 min. The time for forepaw licking or jumping was taken as the latency time and the percentage of inhibition was determined. Before the experiment we screened and employed the mice which have the pot responses between 5 s and 30 s.
Pain threshold(sec)
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67 BuOH and water fractions. And during those antinociceptive and 68 anti-inflammatory activities experiments, we also studied the 69 different fractions at the same crude medicine dosage (30 g/kg) 70 to screening the active site. In our work, the results showed that in 71 the writhing test the CHCl3 site and water site all showed obvious 72 effects (Po 0.01), the inhibition rates were 53.61% and 30.85% 73 respectively(Fig. 2). However in the hot plate test, the formalin 74 model of nociception test and xylene-induced ear edema test only 75 the CHCl3 site showed obvious activities (Fig. 1). And in the hot plate test the low percentage inhibition shown by the CHCl3 site in Q3 76 77 this assay suggests that it is not a centrally acting analgesic 78 (Ibrahim et al.,2012). These results indicated the CHCl3 site may 79 be the active site of S. octophylla. 80 81 3.2. Effect of active fractions of S. octophylla on AA rats 82 83 FCA-induced arthritis model in rats is suggested as the most 84 suitable model of chronic and sub-chronic inflammation. The 85 above experiments have been described chloroform site was the 86 active site of anti-inflammatory and antinociceptive effects, in 87 order to study the treatment effect of active site of S. octophylla on 88 rheumatic diseases, by utilizing AA rats animal model, we obs89 erved the effects of EES and CHCl3 fraction at three doses. During 90 the experiment, the arthritic effect and other clinical symptoms 91 such as body weight and pain response were observed in all the 92 animals up to 28 d. After the experiment the proinflammatory 93 cytokines (IL-1β, IL-6, TNF-α) and rheumatoid factor (RF) levels in 94 serum were determined. 95 96 97 3.2.1. Evaluation of antiarthritic activity 98 Rats injected with complete Freund's adjuvant (CFA) will 99 appear with primary lesion and the secondary lesion. Primary 100 lesion is similar to general inflammatory edema, usually local 101 swelling of injection appear after 12 h. The secondary lesion is 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 Fig. 2. Antinociceptive and anti-inflammatory effects of EES in mice. Results are expressed as mean 7 SD (n¼ 14); *Po 0.05, **Po 0.01 when compared with control group. 131 Con: control; Ind: indomethacin; EES: ethanol extract of Schefflera octophylla; (A) petroleum ether fraction; (B) CHCl3 fraction; (C) EtOAc fraction; (D) n-BuOH fraction; 132 (E) water fraction.
doses of 300, 600, 1200 mg/kg (Po 0.01) (Fig. 2), the inhibition rates were 35.45%, 50.33%, 62.14% respectively. The pain model induced by formalin is an international kind of model for study analgesia effect, the pain reaction is divided into two phases, which represent different types of pain; the first phase (the first 5 min) is acute pain caused by direct stimulation of nerve endings; the second phase of (15–30 min) is the delayed pain caused by inflammatory mediators producing and releasing. In this formalin test, EES produced inhibition of analgesic reaction with peak effect in the first phase, but it is not significant compared with control group. However, in the second phase, the middle, highest dose of EES (600, 1200 mg/kg) and indomethacin all inhibited the licking response significantly (P o0.01), the licking times were (54.21 722.57)s, (53.71 718.72)s, (46.50 7 10.38)s respectively, compared with the control group (80.43 7 27.76)s. And the lowest dose group of EES (300 mg/kg) reflected no significant effect(69.647 25.63)s (Fig. 2). The results from this study suggest that the extract of S. octophylla possibly exhibits its effects via inhibiting the release and/or action of inflammatory mediators, which can be used to explain why EES showed better effect on inflammatory pain. The xylene-induced ear edema is used in the screening of anti-acute inflammatory activity (Wang et al., 2011). A significant (P o0.01) inhibition of edema development was produced by EES, the inhibition rates were 42.43%, 46.61%, 47.41% at doses of 300, 600, 1200 mg/kg (Fig. 2), but all lower than that of indomethacin group (78.09%). All these results suggested that EES had significant antinociceptive and antiinflammatory effects, it also showed dose–response relationship. The positive drug (rotundine or indomethacin) all showed significant antinociceptive and anti-inflammatory effects in these experiments. In order to clarify the active ingredient of antinociceptive and anti-inflammatory effects in EES mainly concentrating in which polar parts, we used classical organic solvent extraction and separation methods, obtained petroleum ether, CHCl3, EtOAc, n-
Please cite this article as: Chen, Y., et al., Antinociceptive and anti-inflammatory activities of Schefflera octophylla extracts. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.04.050i
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Fig. 3. Effect of active fractions of Schefflera octophylla on the left hind paw swelling of AA rats. 0.5 0.45 0.4 0.35
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Fig. 4. Effect of active fractions of Schefflera octophylla on right hind paw swelling of AA rats. 3 2.5
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Y. Chen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Fig. 6. Effect of active fractions of Schefflera octophylla on pain response of the right hind paw of AA rats.
paw (the right hind) also significant in 16, 20, 24, 28 d (Po0.05), which symptoms were similar to RA. As those treatment groups, it seemed leflunomide acted better, because its group diminished swelling degree significantly in 20, 24, 28 d with either the left hind or the right hind comparing with model group. 600 mg/kg of EES (Po0.05) and 300 mg/kg of CHCl3 fraction (Po0.01) all diminished swelling degree significantly in 24, 28 d (Figs. 3 and 4) for both the left hind and right hind, the inhibition rate of EES were 11.83%,11.55% on left hind and 13.55%, 15.88% on right hind in 24, 28 d respectively, but the inhibition rate of CHCl3 fraction were 14.58%, 15.21% on left hind and 17.94%, 23.27% on right hind respectively. And 150 mg/kg of CHCl3 fraction also diminished the secondary paw swelling (right hind) significantly in 24, 28 d (Po0.05).The pain response of primary paw (the left hind) and secondary paw (the right hind) showed similar results (Figs. 5 and 6). Furthermore, the arthritis index of each group in 12, 16, 20, 24, 28 d were significantly different (Po0.05) when compared with the control group. Apart from the control group, arthritis index of others all more than 4 from the 12d, which illustrated the AA model was successful established. When compared with the model group, 300 mg/kg and 600 mg/kg of EES diminished arthritis index in 24 d or/and 28 d respectively. However, treatment of 150 mg/kg and 300 mg/kg of CHCl3 fraction all diminished arthritis index in 24 d and 28 d (Table 1). In addition, we also observed that body weight of all groups injected with CFA increased slowly.
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Fig. 5. Effect of active fractions of Schefflera octophylla on pain response of the left hind paw of AA rats.
induced by delayed hypersensitivity (DH), usually contralateral and forelimb paw edema appear after 10 d, and ears, tail show inflammatory nodules and erythema, body weight decreased too. And these rats experiment results showed that when compared with the control group, the swelling degree of primary paw (the left hind) of model group were significant in 4, 8, 12, 16, 20, 24, 28 d all the time (P o0.05), and the swelling degree of secondary
3.2.2. Histopathological analysis of ankle AA is characterized by a rapid onset and progression to polyarticular inflammation, eventually leads to permanent joint malformations. Symmetric joint involvement, lymphocyte infiltration and cartilage, synovial hyerplasia are shared features with human RA (Li et al., 2014). From the histopathological detection results (Figs. 7 and 8), the ankle joint cavity of the control rats were clean, smooth articular surface, complete cartilage layer, rich cartilage cells and bone matrix, and there was no drop of joint capsule synovial epithelium, no inflammatory exudation edema and inflammatory cell infiltration. But in the model group, we discovered most typical pathological changes: multiple defects of articular surface, pyknosis and necrosis in cartilage cells, superficial stained in the matrix, joint capsule hyperplasia with lots of inflammatory cells infiltration and little exudation. Compared with the model group, the degree of pathological changes in those treatment groups appeared slightly, hyperplasia and inflammation of synovial relieved, especially in 300 mg/kg and 600 mg/kg of EES,150 mg/kg and 300 mg/kg of CHCl3 fraction, and leflunomide groups.
Please cite this article as: Chen, Y., et al., Antinociceptive and anti-inflammatory activities of Schefflera octophylla extracts. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.04.050i
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Y. Chen et al. / Journal of Ethnopharmacology ∎ (∎∎∎∎) ∎∎∎–∎∎∎
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Table 1 Effect of active fractions of Schefflera octophylla on arthritis index of AA rats (n¼ 10, x7 S). Group
Control Model Leflunomide EES
B
Dose (g/kg)
– – 0.006 0.15 0.30 0.60 0.075 0.15 0.30
Arthritis index of AA rats 12 d
16 d
20 d
24 d
28 d
0.0 7 0.00 5.8 7 0.79nn 5.8 7 0.79 5.9 7 0.74 5.7 7 0.82 5.9 7 0.88 5.8 7 0.97 5.8 7 0.79 5.7 7 1.06
0.0 7 0.00 7.3 7 1.34nn 6.5 7 1.08 7.2 7 1.23 7.17 1.37 7.0 7 0.82 7.2 7 0.83 7.2 7 0.79 7.17 0.74
0.0 70.00 8.6 71.35nn 7.6 70.97△ 8.5 71.08 8.4 71.17 8.3 70.95 8.1 70.78 8.0 71.05 7.8 71.03
0.0 70.00 8.2 70.92nn 6.8 70.79△△ 7.9 70.74 7.7 71.06 7.2 71.14△ 7.9 71.13 7.2 70.67△ 7.1 70.57△△
0.0 70.00 8.3 70.67nn 6.7 71.06△△ 7.7 70.95 7.4 70.97△ 7.0 70.82△△ 7.9 70.99 7.1 70.93△ 7.0 70.94△△
n
Po 0.05, compared with control group. EES: ethanol extract of S. octophylla; B: CHCl3 fraction. nn
△
Po 0.01 compared with control group. Po 0.05 compared with model group. Po 0.01 compared with model group.
△△
Fig. 7. Histopathological images under Microscopic magnification 10 of ankle; picture (a–i) comes from control, model, leflunomide, 600 mg/kg of EES, 300 mg/kg of EES, 150 mg/kg of EES, 300 mg/kg of CHCl3 fraction, 150 mg/kg of CHCl3 fraction, 75 mg/kg of CHCl3 fraction groups, respectively.
3.2.3. Estimation of cytokines and rheumatoid factor in serum Inflammatory mediators are a group of biologically active chemicals involved in inflammation, which also related to the occurrence and development of adjuvant arthritis. TNF-α is a kind of important inflammatory medium, IL-1 is an important cytokine in the articular cartilage destruction of RA, and the activity of IL-6 is closely related with degree of RA activity, which also can
promote the synthesis of rheumatoid factor(RF). RF is a kind of autoantibody presenting in the serum of RA patients, the continuing action of RA disorders on the lesion is one of the main causes of erosion progression of RA lesions. The results showed the levels of TNF-α, IL-1β and IL-6 in 600 mg/kg of EES and 300 mg/kg of CHCl3 fraction groups were significantly lower than those of model group (Table 2), but the two drugs had no distinct action on RF.
Please cite this article as: Chen, Y., et al., Antinociceptive and anti-inflammatory activities of Schefflera octophylla extracts. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.04.050i
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Fig. 8. Histopathological images under Microscopic magnification 40 of ankle; picture (j–r) comes from control, model, leflunomide, 600 mg/kg of EES, 300 mg/kg of EES, 150 mg/kg of EES, 300 mg/kg of CHCl3 fraction, 150 mg/kg of CHCl3 fraction, 75 mg/kg of CHCl3 fraction groups, respectively.
Table 2 Effect of active fractions of Schefflera octophylla on the level of TNF-α, IL-1β, IL-6 and RF in serum(n¼10, x 7 S). Group
TNF-α (pg/ml)
IL-1β (pg/ml)
IL-6 (pg/ml)
RF (mIU/ml)
Control Model Leflunomide EES
10.707 3.94 21.56 7 3.20nn 12.50 7 3.19△△ 18.79 7 2.36 17.90 7 2.92 17.54 7 3.59△ 18.447 4.54 18.177 3.90 16.65 7 3.24△
129.007 50.99 311.50 7 55.48nn 152.75 7 39.62△△ 260.43 7 95.29 246.147 90.13 230.43 7 93.71△ 280.43 7 81.12 231.86 7 73.42△ 213.29 7 79.34△
12.187 0.76 18.777 1.64nn 14.217 2.23△△ 17.89 7 1.87 17.23 7 1.92 16.747 1.48△ 18.08 7 1.10 17.32 7 1.51 16.55 7 1.56△
442.06 766.99 838.94 7112.48nn 688.31 7112.02 946.75 7212.22 782.37 7221.86 731.75 7196.65 869.61 7221.56 716.04 7242.84 696.04 7177.99
B
n
P o0.05 compared with control group. nn
Po 0.01 compared with control group. Po 0.05 compared with model group. △△ Po 0.01 compared with model group. △
150 mg/kg of CHCl3 fraction group also significantly lowered level of IL-1β. And all those results proved EES and CHCl3 fraction had the effects of anti-rheumatoid arthritis, and also further proved CHCl3 fraction is effective parts of EES. Leflunomide is a novel drug for the treatment of active rheumatoid arthritis in adults, which not only acts as isoxazoles immune inhibitor, but also has shown anti-inflammatory effect in vivo and in vitro experiments. Leflunomide
has many common adverse reactions such as anorexia, abdominal pain, diarrhea, nausea, vomiting, gastritis and enteritis and other gastrointestinal symptoms and liver function damage, etc. In this study, leflunomide diminished paw swelling, arthritis index, pain response and histopathological changes significantly, and also significantly reduced the levels of TNF-α, IL-1β and IL-6 in serum, but showed no obvious effect on the RF too (Table 2). Although RF is a kind
Please cite this article as: Chen, Y., et al., Antinociceptive and anti-inflammatory activities of Schefflera octophylla extracts. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.04.050i
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of autoantibody which is routinely used as a factor in patients with RA to evaluate disease activity and severity. But in clinical practice, it occurs frequently that RF values do not decrease according to clinical improvement in RA patients (Xiao et al., 2008). 3.3. Structure identification Compounds 1–6 were all known pentacyclic triterpenoids. Their structures were identified by comparing the 1H NMR and 13 C NMR spectral data with those reported (Fan et al., 2011; Wang et al., 2011; Yang et al., 2007; Wang et al., 2006; Chen et al., 2010; Dong et al., 2011) as taraxerone (1), epitaraxerol (2), aleuritolic acid (3), 3-oxofriedelan-28-oic acid (4), 3β,19α -dihydroxy-urs-12ene-24,28-dioic acid (5) and asiatic acid (6). Compounds 1–5 were obtained from S. octophylla for the first time. Triterpenoids are a kind of widely distributed natural compounds with various bioactivities. And anti-inflammatory activity is one of the most important. Gautam and Jachak (2009) had reviewed the developments in anti-inflammatory natural products, and gave statements on anti-inflammatory activities and mechanism of different pentacycle triperpenoids in detail. Previous phytochemical investigations indicated that triterpenoids were the major and important constituents of the genus Schefflera (Araliaceae). Among those 199 compounds isolated from this genus, over 70% were triterpenoids (Wang et al., 2013). In the present study, compounds 1–6 were isolated from the antiinflammatory CHCl3 fraction, which may support that triterpenoids are the important active constituents of S. octophylla. 4. Conclusion The present study showed that S. octophylla extracts possessed significant antinociceptive and anti-inflammatory activities in a dose-dependent manner. We also demonstrated that the CHCl3 fraction was the active site. AA rats experiment proved that S. octophylla also possessed anti-rheumatoid arthritis activity, it could diminish the hind paw swelling, arthritis index, pain degree, and ankle pathological changes distinctly. This strong efficacy may be associated with the down-regulation of proinflammatory cytokines (TNF-α, IL-1β and IL-6). Furthermore, we studied the acute toxicity of EES, when the dosage was 100 times than the usual amount of adult, then still no mouse died and no toxicity reaction was seen. We also tested the maximum amount of administration of EES, the mice maximum dosage was more than 3400 times of the clinical adult oral dose. Further research is warranted to separate and purify chemical composition of chloroform parts, and to study the mechanism of anti-inflammatory and analgesic with the chemical elements or compounds. Acknowledgments The authors thank for the National Natural Science Foundation of China for financial supporting this study (No. 81302894). References Adam, G., Lischewski, M., Phiet, H.V., Preiss, A., Schmidt, J., Sung, T.V., 1982. Natural products from Vietnamese plants. Part 6. 3-Hydroxy-lup-20(29)-ene-23,28dioic acid from Schefflera octophylla. Phytochemistry 21, 1385–1387.
63 Chen, Q.L., Li, H.L., Peng, J.N., 2010. Chemical constituents from Ilex hainanensis. Chin. J. Spectrosc. Lab. 27, 131–134. 64 Dong, J.J., Wang, D.C., Li, Y.S., Deng, X.M., 2011. Chemical constituents of Rabdosia Q5 65 japonica var. glaucocalyx. Lishizhen Med. Mater. Medica Res. 22, 2346–2348. 66 Editorial committee of Chinese Materia Medica, 1999. Chinese Materia Medica, first ed.5. Shanghai Science and Technology Press, Shanghai, pp. 859–867. 67 Fan, Q., Zhang, Y., Zhou, X.L., Ge Sang, S.L., 2011. Study on chemical constituents of 68 Codonopsis nervoa Chipp Nannf. Chin. Pharm. J. 46, 256–259. 69 Gautam, G., Jachak, S.M., 2009. Recent developments in anti-inflammatory natural 70 products. Med. Res. Rev. 29, 767–820. Gu, W.Z., Brandwein, S.R., 1998. Inhibition of typeII collagen induced arthritis in rats 71 by triptolide. Int. Immunopharmacol. 20, 389–400. 72 Hunskaar, S., Hole, K., 1987. The formalin test in mice: dissociation between 73 inflammatory and non-inflammatory pain. Pain 1, 103–114. 74 Kitajima, J., Tanaka, Y., 1989. Two new triterpenoid glycosides from the leaves of Schefflera octophylla. Chem. Pharm. Bull. 37, 2727–2730. 75 Kitajima, J., Shindo, M., Tanako, Y., 1990. Two new triterpenoid sulfates from the 76 leaves of Schefflera octophylla. Chem. Pharm. Bull. 38, 714–716. 77 Li, Y.L., Ooi Vincent, E.C., 2004a. Antiviral activities of medicinal herbs traditionally used in southern mainland China. Phytother. Res. 18, 718–722. 78 Li, Y.L., Paul, P.H., Ooi Vincent, E.C., 2004b. Antiviral terpenoids from the Chinese Q6 79 medicinal plant Schefflera octophylla. Antivir. Res. 62 A78-A78. 80 Lischewski, M., Ty, Ph.D., Schmidt, J., Preiss, A., Phiet, H.V., Adam, G., 1984. Natural 81 products from Vietnamese plants. Part 8. 3,11-Dihydroxylup-20(29)-ene-23,28dioic acid from Schefflera octophylla. Phytochemistry 23, 1695–1697. 82 Li, G.P., Fu, Y.Q., Zheng, J.S.H., Li, D., 2014. Anti-inflammatory activity and mechan83 ism of a lipid extract from hard-shelled mussel (mytilus coruscus) on chronic 84 arthritis in rats. Mar. Drugs.. 12, 568–588. 85 Maeda, C., Ohtani, K., Kasai, R., Yamasaki, K., Duc, N.M., Nham, N.T., Cu, N.K.Q., 1994. Oleanane and ursane glycosides from Schefflera octophylla. Phytochemistry 37, 86 1131–1137. 87 Sung, T.V., Lavaud, C., Porzel, A., Steglich, W., Adam, G., 1992. Triterpenoids and 88 their glycosides from the bark of Schefflera octophylla. Phytochemistry 31, 89 227–231. Sung, T.V., Steglich, W., Adam, G., 1991. Triterpene glycosides from Schefflera 90 octophylla. Phytochemistry 30, 2349–2356. 91 Sung, T.V., Adam, G., 1992. An acetylated bidesmosidic saponin from Schefflera 92 octophylla. J. Nat. Prod. 55, 503–505. Sung, T.V., Peter-Katalinic, J., Adam, G., 1991. A bidesmosidic triterpenoid saponin 93 from Scheflerra octophylla. Phytochemistry 30, 3717–3720. 94 Shah, A.S., Alagawadi, K.R., 2011. Anti-inflammatory,analgesic and antipyretic 95 properties of Thespesia populnea Soland ex. Correa seed extracts and its 96 fractions in animal models. J. Ethnopharmacol. 137, 1504–1509. Shukla, S., Mehta, A., Mehta, P., Vyas, S.P., Shukla, S., Bajpai, V.K., 2010. Studies on 97 anti-inflammatory, antipyretic and analgesic properties of Caesalpinia bondu98 cella F.seed oil in experimental animal models. Food Chem. Toxicol. 48, 61–64. 99 Shinde, U.A., Phadke, A.S., Nair, A.M., Mungantiwar, A.A., Dikshit, V.J., Saraf, M.N., 100 1999. Studies on the anti-inflammatory and analgesic activity of Cedrus deodara (Roxb.) Loud. wood oil. J. Ethnopharmacol. 65, 21–27. 101 Walker-Bone, K., 2003. ‘Natural remedies’ in the treatment of osteoarthritis. Drugs 102 Aging 20, 517–526. 103 Wang, Q.H., Li, Z.Y., Shen, Y., Lin, H.W., Shu, W., Zhou, J.B., 2006. Studies on 104 triterpenoids from Potentilla chinensis. China J. Chin. Mater. 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Contents lists available at ScienceDirect
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Antinociceptive and anti-inflammatory activities of Schefflera octophylla extracts Yanfen Chen a, Shuhong Tao a, Fanling Zeng a, Luwei Xie b, Zhibin Shen a,n a b
School of Traditional Medicine, Guangdong Pharmaceutical University, Guangzhou 510006, PR China School of Pharmacy, Harbin University of Commerce, Harbin 150000, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 3 March 2015 Received in revised form 25 April 2015 Accepted 26 April 2015
Ethnopharmacological relevance: Schefflera octophylla (Lour.) Harms, a traditional Chinese herb mainly distributed in Southeast Asia, is extensively prescribed to alleviate pain and treat rheumatoid arthritis (RA), influenza, throat swelling and pain, etc. In this paper, the antinociceptive and anti-inflammatory activities of the ethanol extract and its five different polar fractions of this plant were evaluated. Furthermore, the anti-rheumatoid arthritis activity of the ethanol extract and its active fraction (CHCl3 fraction) were evaluated. And the chemical constituents of the CHCl3 active fraction displayed significant antinociceptive and anti-inflammatory activities were investigated. Materials and methods: Antinociceptive and anti-inflammatory activities were investigated by hot plate test, acetic acid-induced abdominal writhing test and formalin test, xylene-induced ear edema test. The anti-rheumatoid arthritis activity was evaluated through the model of adjuvant-induced arthritis (AA) in rats, paw swelling, pain response, arthritis index and histopathological changes of ankle, the levels of TNF-α, IL-1β, IL-6 and rheumatoid factor (RF) of rats were detected. The chemical constituents of the CHCl3 fraction were isolated using chromatographic techniques. Their structures were elucidated by spectroscopic data analysis. Results: The results showed that the ethanol extract of S. octophylla has significant dose-dependent antiinflammatory and antinociceptive activities. And its five different polar fractions especially the CHCl3 fraction significantly inhibited the abdominal writhing induced by acetic acid and ear edema induced by xylene, also reduced pain threshold in hot plate test in 120 min and ticking times in formalin test. The ethanol extract of S. octophylla and the CHCl3 fraction demonstrated an anti-RA effect in a dosedependent manner. The levels of TNF-α, IL-1β and IL-6 in ethanol extract (600 mg/kg) and CHCl3 fraction (300 mg/kg) groups were significantly lower than those of the model group. The chemical constituents study of the CHCl3 fraction from S. octophylla led to six triterpenoids which were identified as taraxerone (1), epitaraxerol (2), aleuritolic acid (3), 3-oxofriedelan-28-oic acid (4), 3β,19α -dihydroxy-urs-12-ene24,28-dioic acid (5) and asiatic acid (6). Compounds 1–5 were obtained from this plant for the first time. Conclusion: This study proved the antinociceptive, anti-inflammatory and anti-rheumatoid arthritis activities of S. octophylla. Triterpenoids obtained from its CHCl3 fraction may be responsible for those activities. These results could support the fact that S. octophylla is used traditionally to cure inflammatory and pain diseases. & 2015 Published by Elsevier Ireland Ltd.
Keywords: Schefflera octophylla Antinociception Anti-inflammatory Adjuvant-induced arthritis Triterpenoids
1. Introduction Arthritis is the most common chronic diseases in the world. During all kinds of arthritis, rheumatoid arthritis and osteoarthritis are the two most common types of arthritis. According to reports in the literature (Li et al., 2014), rheumatoid arthritis affects around 0.5–1% of adults in the developed world with between 5 and 50 per 100,000 people newly developing the condition each
n
Corresponding author. Tel.: þ 86 20 39352179; fax: þ86 20 39352174. E-mail address: [email protected] (Z. Shen).
year, while osteoarthritis affects about 3.6% of the global population and nearly 27 million people in the United States. The symptoms of arthritis are very diverse, mainly shown as red, swollen, and dysfunctional joints, with feeling of heat and pain. Thus the conventional medication treatments for arthritis, especially in acute period, are analgesics and non-steroidal antiinflammatory drugs (NSAIDs). However, those drugs have undesirable side effects on gastrointestinal, kidney, bronchus and cardiovascular system (Wallace and Vong, 2008). As a result, researchers all of the world are looking for safer, more effective alternatives to both the traditional analgesics and NSAIDs (WalkerBone, 2003). Furthermore, inflammation is typically a fundamental
http://dx.doi.org/10.1016/j.jep.2015.04.050 0378-8741/& 2015 Published by Elsevier Ireland Ltd.
Please cite this article as: Chen, Y., et al., Antinociceptive and anti-inflammatory activities of Schefflera octophylla extracts. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.04.050i
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and important pathological process in many diseases, researchers focused in recent years on study of antinociceptive and antiinflammatory activities, and medicinal plants derived natural products such as flavonoids, steroids, polyohenols, terpenes, stearic acid which have wide range of pharmacological efficacy but lesser side effects (Shah and Alagawadi, 2011; Shukla et al., 2010). The genus Schefflera (Araliaceae) comprises of 200 species distributed in the tropical and some subtropical areas in the world. There are 38 species and 2 varieties in China. Schefflera octophylla (Lour.) Harms is distributed mainly in Guangxi, Guangdong and Yunnan provinces in China. It is widely used as a folk medicine for the rheumatic pain, traumatic pain, and sore throat, etc (Editorial committee of Chinese Materia Medica, 1999). Previous phytochemical investigations have revealed that triterpenoids were the main components. More than 40 triterpenoids were isolated from this plant, including oleanane type (Sung and Adam, 1991; Maeda et al., 1994), ursane type (Maeda et al., 1994; Sung et al., 1992) and lupane type (Sung et al., 1991; Kitajima et al., 1990; Adam et al., 1982; Lischewski et al., 1984; Kitajima and Tanaka, 1989; Sung and Adam, 1992; Sung et al., 1991), etc. However, in the pharmacological aspects, only anti-virus and anti-oxidant effects of the extract from S. octophylla have been reported previously (Li et al., 2004a, 2004b; Zheng et al., 2009). In Southeast Asia, S. octophylla is extensively prescribed to alleviate pain and treat rheumatoid arthritis (RA). Considering the traditional uses in alleviating pains of this herb, we firstly evaluated the antinociceptive and anti-inflammatory activities of ethanol extract of S. octophylla and its five different polar fractions through three antinociceptive tests and one anti-inflammatory test. For further investigation, we evaluated the anti-rheumatoid arthritis activity through the model of adjuvant-induced arthritis (AA) in rats, arthritis index, paw swelling, pain response and histopathological changes of ankle, the levels of TNF-α, IL-1β, IL-6 and rheumatoid factor (RF) of rats were detected. In order to clarify the main active components of S. octophylla, we also investigated the chemical constituents of the CHCl3 fraction which displayed significant antinociceptive and anti-inflammatory activities.
2. Materials and methods
successively with petroleum ether, chloroform (CHCl3), ethyl acetate (EtOAc) and n-butyl alcohol (n-BuOH) for three times. The volume for the above extracted organic solvent was 2 L each time. Each partition was evaporated to dryness and resulting fractions of petroleum ether, CHCl3, EtOAc and water. These fractions and the ethanol extract of S. octophylla (EES) were evaluated for activity tests. 2.4. Isolation of the CHCl3 fraction The CHCl3 fraction (365 g) was separated by silica gel chromatography (100–200 mesh, petroleum ether-EtOAc 100:0–7:3 and CHCl3– MeOH 9:1–7:3) to yield four fractions (F1–4). F1 was differentiated with silica gel chromatography again (200–300 mesh) using gradient of petroleum ether–EtOAc (97:3–8:2) to afford compound 1 (70 mg). F2 was separated by silica gel chromatography (200–300 mesh, petroleum ether–EtOAc 95:5–7:3) to yield three sub-fractions (F2a– 2c). F2b was purified by Sephadex LH-20 chromatography using MeOH to yield compound 2 (10 mg), 3 (8 mg) and 4 (10 mg). F4 was run with silica gel chromatography again (200–300 mesh, CHCl3– MeOH 9:1–7:3) to generate compound 5 (3 g) and 6 (12 mg). 2.5. Structure identification of compound 1–6 The structures of compound 1–6 were identified by comparing the 1H NMR and 13C NMR spectral data with previous literature reports. 2.6. Biological activity assays 2.6.1. Drugs and reagents Acetic acid (Guangdong Chemical Reagent Factory, China); formaldehyde (Luoyang Chemical Reagent Factory, China); xylene (Guangdong Guanghua Chemical Reagent Co., Ltd., China); indometacin (Southern China Pharmaceutical Group Co., Ltd., China); rotundine (Sichuan Kangfulai Pharmaceutical Group Co., Ltd., China); complete Freund's adjuvants (Sigma, Saint Louis, Missouri, USA); leflunomide (Suzhou Changzheng Xinkai Pharmaceutical Co., Ltd.); CMC-Na (Guangdong Device of Medical Equipment Co., Ltd., China). All other chemicals were of analytical grade.
2.1. General Column chromatography: silica gel (100–200 and 200–300 mesh; Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), Sephadex LH-20 (Amersham Biosciences; Sweden). TLC: precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China). NMR Spectra: Bruker AVANCE III 500 spectrometer with Me4Si as internal standard. ESI-TOF-MS: Acquity UPLC-Q-TOF Microinstrument; in m/z. 2.2. Plant material The root bark of S. octophylla was collected in April 2010 from Guangzhou, Guangdong province, PR China. The specimen was identified by Associate Professor Jizhu Liu, School of Traditional Medicine, Guangdong Pharmaceutical University. A voucher specimen has been deposited at the lab 435 of School of Traditional Chinese Medicine, Guangdong Pharmaceutical University. (Accession number: TCMS002). 2.3. Extraction The air-dried and powdered plant materials of S. octophylla (23 kg) were refluxed with 95% ethanol (300 L) for 3 times (2 h per time). After evaporation of the alcohol under reduced pressure, 920 g of viscous residue was obtained with a yield of 4.0%. The ethanol extract (730 g) was suspended in H2O, and extracted
2.6.2. Experimental animals Male or female NIH mice (18–22 g) and male Sprague-Dawley rats (150–180 g) were obtained from the Medical Experimental Animal Center of Guangdong Province (China). They were kept in plastic cages at 2272 1C with free access to pellet food and water. The experiment procedures were conducted in compliance with the National Institutes of Health Guide for care and use of the laboratory animals, and were approved by the Committee of Experimental Animal Administration of the university (Document no. SCXK 2008-0002). All experimental protocols were approved by the Institutional Animal Ethics Committee. 2.6.3. Hot plate test Experiments were carried out according to previously described method (Shinde et al., 1999). During test of antinociceptive and anti-inflammatory activities in mice, EES was administrated to the animals at doses of 300, 600 and 1200 mg/kg (equivalent to 7.5, 15, 30 g/kg of crude medicine), and petroleum ether, CHCl3, EtOAc, n-BuOH, water fractions were administrated at doses of 6 and 600, 32, 55, 100 mg/kg respectively (each dose was equivalent to 30 g/kg of crude medicine). The control group received only vehicle (0.5% CMC-Na, 20 ml/kg), and the reference group received rotundine (0.02 g/kg). Mice were individually placed on the heated plate which maintained at 5570.5 1C after treatment for 60, 90
Please cite this article as: Chen, Y., et al., Antinociceptive and anti-inflammatory activities of Schefflera octophylla extracts. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.04.050i
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67 EES at doses of 150, 300 and 600 mg/kg. The CHCl3 groups of animals 68 were administered CHCl3 fraction at a dose of 75, 150, and 300 mg/kg 69 respectively. Paw swelling, arthritis index, pain response and histo70 pathological changes of ankle were employed to evaluate the anti71 inflammatory activities of drugs. The paw swelling was periodically 72 examined using toe volume measuring instrument. According to the 2.6.4. Acetic acid-induced writhing test 73 degree of joint swelling, we assessed the arthritis index (AI) by Experiments were carried out according to previously described 74 the standard as previously descri bed (Gu and Brandwein, 1998):0, Q2 method (Xu et al., 2014). Mice were treated orally one hour before 75 normal; 1, slight swelling and/or erythema of toes; 2, most of the toes injection of acetic acid with EES or different fractions. The control 76 and foot swelling; 3, paw swelling below the ankle; 4,complete group received only vehicle (0.5% CMC-Na, 20 ml/kg), and the 77 swelling of toes and ankle, inability to bend the ankle or wrist. The reference group received indometacin (0.01 g/kg) respectively. Wri78 pain response score assessment standard also based on the literature thing was induced by an intraperitoneal injection (20 ml/kg) of a 79 (Gu and Brandwein, 1998), according to the leg withdrawal reaction 0.7% acetic acid solution. The numbers of writhing that occurred in 80 in rats, the ankle pain score was 0–5. During the experiment, the 15 min were counted for each animal. 81 hind paw swelling was measured at days 4, 8, 12, 16, 20, 24, 28 after 82 the injection, and the arthritis index, pain score were monitored at 2.6.5. Formalin test 83 days 12, 16, 20, 24, and 28. At the end of the experiments (day 28 Experiments were carried out according to previously described 84 after immunization for AA rats), rats were fasted overnight and method (Hunskaar and Hole, 1987). Mice were orally treated with 85 sacrificed. The hind paws of all the rats were removed, the ankle EES or different fractions. The control group received only vehicle 86 joints were decalcified and embedded, then sections were stained (0.5% CMC-Na, 20 ml/kg), and the reference group received indo87 with haematoxylin and eosin for histopathological observation to metacin (0.01 g/kg). One hour later, 20 ml of 2.5% formalin was 88 find out the degree of chronic inflammation occurred in the joints. injected s.c. into the right hind paw of each mouse. Each mouse was 89 Blood samples were collected into labeled vacuum tubes and procimmediately placed into a glass cylinder to observe the nociceptive 90 essed into serum, then immediately stored at 80 1C in closed. behavior. The time spent in licking the injected paw (an index of 91 Levels of cytokines (IL-1β,IL-6, TNF-α) and RF were determined by the nociception) was measured in the first phase (0–5 min) and second 92 ELISA Kits (Wuhan Huamei Biological Engineering Co. Ltd.,Wuhan, phase (15–30 min) after formalin injection. 93 China) according to the manufacturer's instructions. 94 2.6.6. Xylene-induced ear edema test 95 2.7. Statistical analysis of data The improved method was adopted as previously described 96 (Zhang et al., 2008). Mice were pretreated with EES or different 97 The data were presented as the mean 7SD and analyzed by fractions, indometacin (0.01 g/kg), or the vehicle alone. One hour 98 one-way ANOVA. P values less than 0.05 (P o0.05) were defined as later, each animal received 20 ml of xylene on the anterior and 99 the significant level. posterior surfaces of the right ear lobe, the left ear serving as the 100 control. One hour later, the animals were sacrificed under ether 101 anesthesia, their bilateral ears were removed and weighed pre3. Results and discussion 102 cisely. The degree of ear edema was calculated based on the 103 weight of the left ear without applying xylene. 3.1. Antinociceptive and anti-inflammatory activities of EES 104 105 2.6.7. Adjuvant-induced arthritis in rats The hot plate test, writhing test and the formalin model of 106 Animals like Sprague-Dawley rats were randomly divided into nociception, xylene-induced ear edema are usually used as screen107 nine groups of ten animals each (n¼10). Except the control group, ing tools for the assessment of analgesic or anti-inflammatory 108 rats in other groups were inoculating 0.1 mL of complete Freund's properties of drugs. Therefore, based on clinical application of S. 109 adjuvants (CFA) into the left hind metatarsal rat footpad to develop octophylla, we firstly investigated the antinociceptive and anti110 AA animals. The rats of control group were injected with 0.1 mL inflammatory activities of the ethanol extracts of S. octophylla 111 sterilized physiological saline only. Drug treatment was started in all (EES) and its different polar fractions with these test. 112 groups from the day of arthritis successfully formed and continued In hot plate test, EES showed a significant dose dependent 113 12 d. The control group and the model group received 10 ml/kg of effect. It significantly inhibited pain threshold in 60 (P o0.05), 90, 114 vehicle in which the extract was going to be suspended while the 120 min (P o0.01) at the highest dose of 1200 mg/kg (Fig. 1).The 115 positive control group received leflunomide (6 mg/kg p.o) as the plant extract significantly restrained the writhing response ind116 reference standard. The EES groups of animals were administered uced by intraperitoneal injection of 0.7% acetic acid in mice at all 117 118 70 119 ** ** ** 120 60 121 ** ** ** 50 ** ** 122 * * * 40 123 124 30 125 20 126 10 127 128 0 60 min 0 min 120 min 90 min 129 Hot plate test 130 131 Fig. 1. Antinociceptive effect of EES in the hot plate test. Results are expressed as mean7 SD (n ¼14); *Po 0.05, **Po 0.01 when compared with control group. Con: control; 132 Rot: rotundine; EES: ethanol extract of Schefflera octophylla; (A) petroleum ether fraction; (B) CHCl3 fraction; (C) EtOAc fraction; (D) n-BuOH fraction; (E) water fraction. and 120 min. The time for forepaw licking or jumping was taken as the latency time and the percentage of inhibition was determined. Before the experiment we screened and employed the mice which have the pot responses between 5 s and 30 s.
Pain threshold(sec)
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Please cite this article as: Chen, Y., et al., Antinociceptive and anti-inflammatory activities of Schefflera octophylla extracts. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.04.050i
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67 BuOH and water fractions. And during those antinociceptive and 68 anti-inflammatory activities experiments, we also studied the 69 different fractions at the same crude medicine dosage (30 g/kg) 70 to screening the active site. In our work, the results showed that in 71 the writhing test the CHCl3 site and water site all showed obvious 72 effects (Po 0.01), the inhibition rates were 53.61% and 30.85% 73 respectively(Fig. 2). However in the hot plate test, the formalin 74 model of nociception test and xylene-induced ear edema test only 75 the CHCl3 site showed obvious activities (Fig. 1). And in the hot plate test the low percentage inhibition shown by the CHCl3 site in Q3 76 77 this assay suggests that it is not a centrally acting analgesic 78 (Ibrahim et al.,2012). These results indicated the CHCl3 site may 79 be the active site of S. octophylla. 80 81 3.2. Effect of active fractions of S. octophylla on AA rats 82 83 FCA-induced arthritis model in rats is suggested as the most 84 suitable model of chronic and sub-chronic inflammation. The 85 above experiments have been described chloroform site was the 86 active site of anti-inflammatory and antinociceptive effects, in 87 order to study the treatment effect of active site of S. octophylla on 88 rheumatic diseases, by utilizing AA rats animal model, we obs89 erved the effects of EES and CHCl3 fraction at three doses. During 90 the experiment, the arthritic effect and other clinical symptoms 91 such as body weight and pain response were observed in all the 92 animals up to 28 d. After the experiment the proinflammatory 93 cytokines (IL-1β, IL-6, TNF-α) and rheumatoid factor (RF) levels in 94 serum were determined. 95 96 97 3.2.1. Evaluation of antiarthritic activity 98 Rats injected with complete Freund's adjuvant (CFA) will 99 appear with primary lesion and the secondary lesion. Primary 100 lesion is similar to general inflammatory edema, usually local 101 swelling of injection appear after 12 h. The secondary lesion is 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 Fig. 2. Antinociceptive and anti-inflammatory effects of EES in mice. Results are expressed as mean 7 SD (n¼ 14); *Po 0.05, **Po 0.01 when compared with control group. 131 Con: control; Ind: indomethacin; EES: ethanol extract of Schefflera octophylla; (A) petroleum ether fraction; (B) CHCl3 fraction; (C) EtOAc fraction; (D) n-BuOH fraction; 132 (E) water fraction.
doses of 300, 600, 1200 mg/kg (Po 0.01) (Fig. 2), the inhibition rates were 35.45%, 50.33%, 62.14% respectively. The pain model induced by formalin is an international kind of model for study analgesia effect, the pain reaction is divided into two phases, which represent different types of pain; the first phase (the first 5 min) is acute pain caused by direct stimulation of nerve endings; the second phase of (15–30 min) is the delayed pain caused by inflammatory mediators producing and releasing. In this formalin test, EES produced inhibition of analgesic reaction with peak effect in the first phase, but it is not significant compared with control group. However, in the second phase, the middle, highest dose of EES (600, 1200 mg/kg) and indomethacin all inhibited the licking response significantly (P o0.01), the licking times were (54.21 722.57)s, (53.71 718.72)s, (46.50 7 10.38)s respectively, compared with the control group (80.43 7 27.76)s. And the lowest dose group of EES (300 mg/kg) reflected no significant effect(69.647 25.63)s (Fig. 2). The results from this study suggest that the extract of S. octophylla possibly exhibits its effects via inhibiting the release and/or action of inflammatory mediators, which can be used to explain why EES showed better effect on inflammatory pain. The xylene-induced ear edema is used in the screening of anti-acute inflammatory activity (Wang et al., 2011). A significant (P o0.01) inhibition of edema development was produced by EES, the inhibition rates were 42.43%, 46.61%, 47.41% at doses of 300, 600, 1200 mg/kg (Fig. 2), but all lower than that of indomethacin group (78.09%). All these results suggested that EES had significant antinociceptive and antiinflammatory effects, it also showed dose–response relationship. The positive drug (rotundine or indomethacin) all showed significant antinociceptive and anti-inflammatory effects in these experiments. In order to clarify the active ingredient of antinociceptive and anti-inflammatory effects in EES mainly concentrating in which polar parts, we used classical organic solvent extraction and separation methods, obtained petroleum ether, CHCl3, EtOAc, n-
Please cite this article as: Chen, Y., et al., Antinociceptive and anti-inflammatory activities of Schefflera octophylla extracts. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.04.050i
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Fig. 3. Effect of active fractions of Schefflera octophylla on the left hind paw swelling of AA rats. 0.5 0.45 0.4 0.35
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Fig. 4. Effect of active fractions of Schefflera octophylla on right hind paw swelling of AA rats. 3 2.5
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Fig. 6. Effect of active fractions of Schefflera octophylla on pain response of the right hind paw of AA rats.
paw (the right hind) also significant in 16, 20, 24, 28 d (Po0.05), which symptoms were similar to RA. As those treatment groups, it seemed leflunomide acted better, because its group diminished swelling degree significantly in 20, 24, 28 d with either the left hind or the right hind comparing with model group. 600 mg/kg of EES (Po0.05) and 300 mg/kg of CHCl3 fraction (Po0.01) all diminished swelling degree significantly in 24, 28 d (Figs. 3 and 4) for both the left hind and right hind, the inhibition rate of EES were 11.83%,11.55% on left hind and 13.55%, 15.88% on right hind in 24, 28 d respectively, but the inhibition rate of CHCl3 fraction were 14.58%, 15.21% on left hind and 17.94%, 23.27% on right hind respectively. And 150 mg/kg of CHCl3 fraction also diminished the secondary paw swelling (right hind) significantly in 24, 28 d (Po0.05).The pain response of primary paw (the left hind) and secondary paw (the right hind) showed similar results (Figs. 5 and 6). Furthermore, the arthritis index of each group in 12, 16, 20, 24, 28 d were significantly different (Po0.05) when compared with the control group. Apart from the control group, arthritis index of others all more than 4 from the 12d, which illustrated the AA model was successful established. When compared with the model group, 300 mg/kg and 600 mg/kg of EES diminished arthritis index in 24 d or/and 28 d respectively. However, treatment of 150 mg/kg and 300 mg/kg of CHCl3 fraction all diminished arthritis index in 24 d and 28 d (Table 1). In addition, we also observed that body weight of all groups injected with CFA increased slowly.
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Fig. 5. Effect of active fractions of Schefflera octophylla on pain response of the left hind paw of AA rats.
induced by delayed hypersensitivity (DH), usually contralateral and forelimb paw edema appear after 10 d, and ears, tail show inflammatory nodules and erythema, body weight decreased too. And these rats experiment results showed that when compared with the control group, the swelling degree of primary paw (the left hind) of model group were significant in 4, 8, 12, 16, 20, 24, 28 d all the time (P o0.05), and the swelling degree of secondary
3.2.2. Histopathological analysis of ankle AA is characterized by a rapid onset and progression to polyarticular inflammation, eventually leads to permanent joint malformations. Symmetric joint involvement, lymphocyte infiltration and cartilage, synovial hyerplasia are shared features with human RA (Li et al., 2014). From the histopathological detection results (Figs. 7 and 8), the ankle joint cavity of the control rats were clean, smooth articular surface, complete cartilage layer, rich cartilage cells and bone matrix, and there was no drop of joint capsule synovial epithelium, no inflammatory exudation edema and inflammatory cell infiltration. But in the model group, we discovered most typical pathological changes: multiple defects of articular surface, pyknosis and necrosis in cartilage cells, superficial stained in the matrix, joint capsule hyperplasia with lots of inflammatory cells infiltration and little exudation. Compared with the model group, the degree of pathological changes in those treatment groups appeared slightly, hyperplasia and inflammation of synovial relieved, especially in 300 mg/kg and 600 mg/kg of EES,150 mg/kg and 300 mg/kg of CHCl3 fraction, and leflunomide groups.
Please cite this article as: Chen, Y., et al., Antinociceptive and anti-inflammatory activities of Schefflera octophylla extracts. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.04.050i
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Table 1 Effect of active fractions of Schefflera octophylla on arthritis index of AA rats (n¼ 10, x7 S). Group
Control Model Leflunomide EES
B
Dose (g/kg)
– – 0.006 0.15 0.30 0.60 0.075 0.15 0.30
Arthritis index of AA rats 12 d
16 d
20 d
24 d
28 d
0.0 7 0.00 5.8 7 0.79nn 5.8 7 0.79 5.9 7 0.74 5.7 7 0.82 5.9 7 0.88 5.8 7 0.97 5.8 7 0.79 5.7 7 1.06
0.0 7 0.00 7.3 7 1.34nn 6.5 7 1.08 7.2 7 1.23 7.17 1.37 7.0 7 0.82 7.2 7 0.83 7.2 7 0.79 7.17 0.74
0.0 70.00 8.6 71.35nn 7.6 70.97△ 8.5 71.08 8.4 71.17 8.3 70.95 8.1 70.78 8.0 71.05 7.8 71.03
0.0 70.00 8.2 70.92nn 6.8 70.79△△ 7.9 70.74 7.7 71.06 7.2 71.14△ 7.9 71.13 7.2 70.67△ 7.1 70.57△△
0.0 70.00 8.3 70.67nn 6.7 71.06△△ 7.7 70.95 7.4 70.97△ 7.0 70.82△△ 7.9 70.99 7.1 70.93△ 7.0 70.94△△
n
Po 0.05, compared with control group. EES: ethanol extract of S. octophylla; B: CHCl3 fraction. nn
△
Po 0.01 compared with control group. Po 0.05 compared with model group. Po 0.01 compared with model group.
△△
Fig. 7. Histopathological images under Microscopic magnification 10 of ankle; picture (a–i) comes from control, model, leflunomide, 600 mg/kg of EES, 300 mg/kg of EES, 150 mg/kg of EES, 300 mg/kg of CHCl3 fraction, 150 mg/kg of CHCl3 fraction, 75 mg/kg of CHCl3 fraction groups, respectively.
3.2.3. Estimation of cytokines and rheumatoid factor in serum Inflammatory mediators are a group of biologically active chemicals involved in inflammation, which also related to the occurrence and development of adjuvant arthritis. TNF-α is a kind of important inflammatory medium, IL-1 is an important cytokine in the articular cartilage destruction of RA, and the activity of IL-6 is closely related with degree of RA activity, which also can
promote the synthesis of rheumatoid factor(RF). RF is a kind of autoantibody presenting in the serum of RA patients, the continuing action of RA disorders on the lesion is one of the main causes of erosion progression of RA lesions. The results showed the levels of TNF-α, IL-1β and IL-6 in 600 mg/kg of EES and 300 mg/kg of CHCl3 fraction groups were significantly lower than those of model group (Table 2), but the two drugs had no distinct action on RF.
Please cite this article as: Chen, Y., et al., Antinociceptive and anti-inflammatory activities of Schefflera octophylla extracts. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.04.050i
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Fig. 8. Histopathological images under Microscopic magnification 40 of ankle; picture (j–r) comes from control, model, leflunomide, 600 mg/kg of EES, 300 mg/kg of EES, 150 mg/kg of EES, 300 mg/kg of CHCl3 fraction, 150 mg/kg of CHCl3 fraction, 75 mg/kg of CHCl3 fraction groups, respectively.
Table 2 Effect of active fractions of Schefflera octophylla on the level of TNF-α, IL-1β, IL-6 and RF in serum(n¼10, x 7 S). Group
TNF-α (pg/ml)
IL-1β (pg/ml)
IL-6 (pg/ml)
RF (mIU/ml)
Control Model Leflunomide EES
10.707 3.94 21.56 7 3.20nn 12.50 7 3.19△△ 18.79 7 2.36 17.90 7 2.92 17.54 7 3.59△ 18.447 4.54 18.177 3.90 16.65 7 3.24△
129.007 50.99 311.50 7 55.48nn 152.75 7 39.62△△ 260.43 7 95.29 246.147 90.13 230.43 7 93.71△ 280.43 7 81.12 231.86 7 73.42△ 213.29 7 79.34△
12.187 0.76 18.777 1.64nn 14.217 2.23△△ 17.89 7 1.87 17.23 7 1.92 16.747 1.48△ 18.08 7 1.10 17.32 7 1.51 16.55 7 1.56△
442.06 766.99 838.94 7112.48nn 688.31 7112.02 946.75 7212.22 782.37 7221.86 731.75 7196.65 869.61 7221.56 716.04 7242.84 696.04 7177.99
B
n
P o0.05 compared with control group. nn
Po 0.01 compared with control group. Po 0.05 compared with model group. △△ Po 0.01 compared with model group. △
150 mg/kg of CHCl3 fraction group also significantly lowered level of IL-1β. And all those results proved EES and CHCl3 fraction had the effects of anti-rheumatoid arthritis, and also further proved CHCl3 fraction is effective parts of EES. Leflunomide is a novel drug for the treatment of active rheumatoid arthritis in adults, which not only acts as isoxazoles immune inhibitor, but also has shown anti-inflammatory effect in vivo and in vitro experiments. Leflunomide
has many common adverse reactions such as anorexia, abdominal pain, diarrhea, nausea, vomiting, gastritis and enteritis and other gastrointestinal symptoms and liver function damage, etc. In this study, leflunomide diminished paw swelling, arthritis index, pain response and histopathological changes significantly, and also significantly reduced the levels of TNF-α, IL-1β and IL-6 in serum, but showed no obvious effect on the RF too (Table 2). Although RF is a kind
Please cite this article as: Chen, Y., et al., Antinociceptive and anti-inflammatory activities of Schefflera octophylla extracts. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.04.050i
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of autoantibody which is routinely used as a factor in patients with RA to evaluate disease activity and severity. But in clinical practice, it occurs frequently that RF values do not decrease according to clinical improvement in RA patients (Xiao et al., 2008). 3.3. Structure identification Compounds 1–6 were all known pentacyclic triterpenoids. Their structures were identified by comparing the 1H NMR and 13 C NMR spectral data with those reported (Fan et al., 2011; Wang et al., 2011; Yang et al., 2007; Wang et al., 2006; Chen et al., 2010; Dong et al., 2011) as taraxerone (1), epitaraxerol (2), aleuritolic acid (3), 3-oxofriedelan-28-oic acid (4), 3β,19α -dihydroxy-urs-12ene-24,28-dioic acid (5) and asiatic acid (6). Compounds 1–5 were obtained from S. octophylla for the first time. Triterpenoids are a kind of widely distributed natural compounds with various bioactivities. And anti-inflammatory activity is one of the most important. Gautam and Jachak (2009) had reviewed the developments in anti-inflammatory natural products, and gave statements on anti-inflammatory activities and mechanism of different pentacycle triperpenoids in detail. Previous phytochemical investigations indicated that triterpenoids were the major and important constituents of the genus Schefflera (Araliaceae). Among those 199 compounds isolated from this genus, over 70% were triterpenoids (Wang et al., 2013). In the present study, compounds 1–6 were isolated from the antiinflammatory CHCl3 fraction, which may support that triterpenoids are the important active constituents of S. octophylla. 4. Conclusion The present study showed that S. octophylla extracts possessed significant antinociceptive and anti-inflammatory activities in a dose-dependent manner. We also demonstrated that the CHCl3 fraction was the active site. AA rats experiment proved that S. octophylla also possessed anti-rheumatoid arthritis activity, it could diminish the hind paw swelling, arthritis index, pain degree, and ankle pathological changes distinctly. This strong efficacy may be associated with the down-regulation of proinflammatory cytokines (TNF-α, IL-1β and IL-6). Furthermore, we studied the acute toxicity of EES, when the dosage was 100 times than the usual amount of adult, then still no mouse died and no toxicity reaction was seen. We also tested the maximum amount of administration of EES, the mice maximum dosage was more than 3400 times of the clinical adult oral dose. Further research is warranted to separate and purify chemical composition of chloroform parts, and to study the mechanism of anti-inflammatory and analgesic with the chemical elements or compounds. Acknowledgments The authors thank for the National Natural Science Foundation of China for financial supporting this study (No. 81302894). References Adam, G., Lischewski, M., Phiet, H.V., Preiss, A., Schmidt, J., Sung, T.V., 1982. Natural products from Vietnamese plants. Part 6. 3-Hydroxy-lup-20(29)-ene-23,28dioic acid from Schefflera octophylla. Phytochemistry 21, 1385–1387.
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Please cite this article as: Chen, Y., et al., Antinociceptive and anti-inflammatory activities of Schefflera octophylla extracts. Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.04.050i
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