Anti-Inflammatory and Antinociceptive Activities of Non

G Model BJP 86 1–6 ARTICLE IN PRESS Revista Brasileira de Farmacognosia xxx (2015) xxx–xxx www.sbfgnosia.org.br/revista Original Article 1 Anti-inflammatory and antinociceptive activities of non-alkaloids 2 fractions from Aconitum flavum in vivo a,1 a,1 a a a a,b,∗ 3 Q1 Yuanbin Zhang , Zhiheng Shu , Lei Yin , Ling Ma , Xinfang Wang , Xueyan Fu a 4 School of Pharmacy, Ningxia Medical University, Yinchuan, China b 5 Ningxia Engineering and Technology Research Center for Modernization of Hui Medicine, Yinchuan, China 6 a r a b s t r a c t t i c 7 l e i n f o 8 9 Article history: Aconitum flavum Hand.-Mazz., Ranunculaceae, has been used for the treatment of rheumatism, trau- 10 Received 21 September 2014 matic injury in folk and clinical medicine, but the alkaloids has high toxicity. This study was designed 11 Accepted 28 November 2014 to investigate the acute toxicity, anti-inflammatory and antinociceptive activities of non-alkaloids frac- 12 Available online xxx tions from A. flavum in rodents. The anti-inflammatory activity was evaluated by inflammatory models 13 of dimethylbenzene-induced ear vasodilatation and acetic acid-induced capillary permeability enhance- 14 Keywords: ment test in mice and carrageenan-induced paw edema in rats whereas the antinociceptive activity was 15 Aconitum flavum evaluated using acetic acid-induced writhes, hot plate test and formalin test in mice. The result showed 16 Anti-inflammatory activity that the LD50 value of BtOH and EtOAc fractions could not be determined as no lethality was observed up 17 Antinociceptive activity 18 Non-alkaloids to 40 g/kg (p.o.) in mice. BtOH fraction significantly decreased the dimethylbenzene-induced ear vasodilatation, carrageenan-induced paw edema and acetic acid-induced capillary permeability. EtOAc fraction only significantly attenuated paw edema and capillary permeability at the dose of 500 mg/kg. In antinociceptive test, BtOH and EtOAc fractions significantly reduced the writhing number evoked by acetic acid injection and the licking time in both phases of the formalin test. Meanwhile BtOH and EtOAc fractions had significant effect on hot plate test after 90 min. Our data indicate that the BtOH and EtOAc fractions of NAF are no toxicity. BtOH and EtOAc fractions not only inhibit inflammatory and peripheral inflammatory pain but also have central antinociceptive effect. © 2014 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. All rights reserved. 19 Introduction safety between therapeutic and toxic doses of these herbs. Lots of 32 researches showed that the effects and severe toxicity of Aconitum 33 20 The Aconitum consisting of over 300 species are widely dis- are mainly attributed to alkaloids (Chan, 2012; Singhuber et al., 34 21 tributed in Asia, Europe and North America. Most of them grow 2009). Aconitine, mesaconitine, hypaconitine and other alkaloids 35 22 among high altitudes in the northern hemisphere. There are 200 have potent cardiotoxins and neurotoxins found in all parts of the 36 23 species grow in China such as Aconitum kusnezoffii Reichb., Aconi- Aconitum species, especially in the tubers and roots. Patients with 37 24 tum carmichaelithe Debx. and Aconitum flavum Hand. –Mazz. (Xiao, Aconitum poisoning often present with a combination of cardiovas- 38 25 2006). In traditional Chinese medicine, Aconitum have similar cular, neurological, gastrointestinal and other signs and symptoms. 39 26 pharmacological actions and are commonly applied for various The estimated lethal dose is 2 mg of aconitine, 5 mL of aconite 40 27 inflammatory diseases, such as rheumatic fever, painful joints, tincture (herbal medicinal wine) and 1 g of the raw aconite plant 41 28 gastroenteritis, diarrhea, edema, bronchial asthma and various (Chan, 2012; Qin et al., 2012). Facing this dangerous situation, the 42 29 tumors (Qin et al., 2012; Singhuber et al., 2009). However, with effects of non-alkaloids fraction are worth to explore, although the 43 30 the increasing popularity, Aconitum poisoning can occur in many alkaloids of Aconitum species were been seen as important compo- 44 31 parts of the world. Because the highly toxic and narrow margin of nents in disease treatment. Modern Chinese medicine theory has 45 been suggested that the chemical constituents of traditional Chi- 46 nese medicine are complex and many different kinds of chemical 47 compositions may treat the diseases in similar way by additive and 48 ∗ Q2 Corresponding author at: School of Pharmacy, Ningxia Medical University, synergy effect (Tu et al., 2012; Xu et al., 2014a). This theory urged 49 Yinchuan, China. us to explore the effects of non-alkaloids fraction from Aconitum. 50 E-mail: [email protected] (X. Fu). 1 A. flavum Hand.-Mazz. from the family Ranunculaceae, is widely 51 These authors equally contributed to this manuscript; both should be considered used in western China (Shanxi, Ningxia, Gansu, Inner Mongolia, 52 as first author. http://dx.doi.org/10.1016/j.bjp.2014.11.013 0102-695X/© 2014 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. All rights reserved. Please cite this article in press as: Zhang, Y., et al. Anti-inflammatory and antinociceptive activities of non-alkaloids fractions from Aconitum flavum in vivo. Revista Brasileira de Farmacognosia (2015), http://dx.doi.org/10.1016/j.bjp.2014.11.013 G Model BJP 86 1–6 ARTICLE IN PRESS 2 Y. Zhang et al. / Revista Brasileira de Farmacognosia xxx (2015) xxx–xxx 53 Qinghai and Tibet). It has been used for the treatment of rheuma- from Sigma Co., Ltd. The fractions of NAF were suspended in water 112 54 tism, traumatic injury and toothache in folk and clinical medicine with 0.5% (w/v) sodium carboxyl methyl cellulose (Na-CMC). 113 55 (Ting et al., 2008). To date, Chinese modern research about A. flavum 56 is mainly concentrated in pharmacological functions and quality Animal preparation 114 57 control of alkaloids and different processed products (the toxicity 58 of these products was reduced by decomposing the diester diter- The animals used in this study, including ICR mice (18–22 g; 115 59 pene alkaloids to the less toxic monoester diterpene alkaloids). License No.: SYXK (NING) 2011-0001) and Sprague-Dawley rats 116 60 However, there has been little research on anti-inflammatory and (180–220 g; License No.: SCXK (NING) 2012-0001) were purchased 117 61 antinociceptive activity of non-alkaloids fractions from A. flavum. from the Experimental Animal Center of Ningxia Medical Univer- 118 62 The aim of the present study is to evaluate the anti-inflammatory sity (Ningxia, China). They were maintained in standard laboratory 119 63 and analgesic effects of non-alkaloids fractions from A. flavum in cages, in moderate humidity (50 ± 5%), at constant temperature 120 ◦ ± 64 standard rodent models of inflammation and pain. Additionally, we (22 1 C) in a 12-h light–dark cycle room. All animals had free 121 65 also studied acute oral toxicity of the non-alkaloids fractions. These access to food and water during the experimental period. The 122 66 studies will show a direction of finding low toxic compounds from experiment protocol was approved by the Ethics Committee of 123 67 Aconitum. Ningxia Medical University, Ningxia (Ethics approval: 2013-146). 124 68 Materials and methods Toxicity study 125 69 Herbal preparation and extraction The acute toxicity study of the non-alkaloids fractions was per- 126 formed according to OECD guidelines (2011). The EtOAc and BtOH 127 70 The roots of A. flavum Hand.-Mazz., Ranunculaceae, were pur- fractions was suspended in water with 0.5% (w/v) sodium carboxyl 128 71 chased from an Herbal Medicinal Materials Company of Mingde methyl cellulose (Na-CMC) in the dose of 120, 1200, 6000, 20,000 129 72 Pharmaceutical Co., Ltd (Ningxia, China). The Plant is growing in and 40,000 mg/kg body weight were orally administered to over- 130 73 Liupan mountain, Longde, Guyuan, Ningxia, China. The roots were night-fasted, healthy mice (n = 8) and the animals were observed 131 74 harvest in August to September. The plant sample was authen- continuously for 24 h for mortality. 132 75 ticated by Lin Dong at Pharmacognosy Department, College of 76 Pharmacy, Ningxia Medical University, and a voucher specimen Anti-inflammatory activity 133 77 was deposited in the same unit (Herbarium number: 20130924). 78 Sliced dried roots (3.7 kg) were extracted three times with 70% Dimethylbenzene (DMB)-induced ear vasodilatation assay 134 79 ethanol (mass ratio of solid to liquid was 1:10). The extract was The test was carried out according to the previously described 135 80 then combined and evaporated to dryness under reduced pres- method (Carlson et al., 1985). Mice of either sex were ran- 136 81 sure, which yielded 925 g crude extract. The crude extract was domly divided in eight groups (n = 8 per group). Control 137 82 dissolved in 20% (v/v) ethanol aqueous (2 L), and then diluted with group, indomethacin (10 mg/kg, positive group), EtOAc (500 138 83 a solution (5000 L) of hydrochloric acid 1% in 20% ethanol aqueous. and 250 mg/kg), BtOH (500 and 250 mg/kg) and H2O (500 and 139 84 The 001 × 7 strong acid polystyrene cation exchange resin (Anhui 250 mg/kg) were administered via oral gavage. 60 min after via gav- 140 ␮ 85 Sanxing Resin Technology Co., Ltd., China) were selected for fur- age of test samples, the mice were topical applied 30 L DMB to 141 86 ther work. The sample solution was absorbed in column with a both inner and outer surface of right ear. Mice were sacrificed by 142 87 20 mL/min flow rate. Then column was washed with deionized cervical dislocation 30 min later then the ear biopsies of both ears 143 88 water until the effluent was colorless.

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