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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 vasodil-
atation, 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 antinoci-
ceptive 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
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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. The combined effluent was were obtained with a punch (a diameter of 8 mm) and weighed. 144
89 adjusted to pH 7 with a solution of sodium hydroxide and evap- Weight-increase-rate of the right ear over the left one indicated 145
90 orated using a rotary evaporator to give a non-alkaloids fraction the vasodilatation. 146
91 (NAF). The NAF was then suspended in distilled water and suc-
92 cessively partitioned with petroleum ether, ethyl acetate (EtOAc) Acetic acid-induced vascular permeability 147
93 and n-butanol (BtOH) to yield EtOAc (18.25 g) and BtOH (77.33 g) The acetic acid-induced increased vascular permeability in mice 148
94 fractions, as well as an H2O residue. was carried out using the reported technique (Whittle, 1964). 149
Briefly, grouping and administration were the same as mentioned 150
95 Estimation of the total phenolic, flavonoid and saponin content in ear vasodilatation assay. 60 min after via gavage, 10 mL/kg body 151
weight of 2% Evans blue in normal saline solution to the tail vein 152
96 The total amount of phenolic and flavonoid contents were then applied 20 mL/kg body weight of 0.6% (v/v) acetic acid imme- 153
97 determined in EtOAc and BtOH fractions using the Folin–Ciocalteu diately (i.p.). 20 min after the administration of acetic acid, the 154
98 colorimetric method and aluminum chloride colorimetric assay, mice were sacrificed by cervical dislocation. The peritoneal cavity 155
99 respectively. The percentage of total phenolic content expressed as was rinsed by 10 mL of saline. The washing solutions were col- 156
×
100 gallic acid equivalents for EtOAc and BtOH fractions were 15.08% lected in centrifuge tube and centrifuged at 1000 g for 5 min. 157
101 and 3.05%, respectively. But there have no flavonoid in NAF. In addi- The supernatant was measured at 590 nm by spectrometry and 158
102 tion, the saponins were measured by vanillin–perchloric acid assay. the absorbance of Evans blue in the exudates under 590 nm rep- 159
103 The saponin contents expressed as oleanolic acid equivalents were resented the capillary permeability. 160
104 9.37 and 3.83% respectively.
Carrageenan-induced paw edema 161
105 Drugs and chemicals The paw edema was induced according to Winter et al. (1962). 162
Animal of each sex were divided into seven groups of seven each. 163
106 The following drugs and chemicals were used: acetic acid, They were pretreated orally with the vehicle (0.5%, w/v sodium car- 164
107 dimethylbenzene and formaldehyde solution were purchased from boxyl methyl cellulose, control group), EtOAc (500 and 250 mg/kg), 165
108 Damao Chemical Company (Tianjin, China). Carrageenan (BR; no. BtOH (500 and 250 mg/kg), H2O (500 mg/kg) and indomethacin 166
109 Q1555) was from Shanghai Wanjiang Biological Technology Co., (10 mg/kg, p.o.). After 60 min, edema was induced with the injec- 167
110 Ltd. Indomethacin (no. A130602) was obtained from Shanxi Yun- tion of 0.1 mL of 3% (w/v) freshly prepared carrageenan suspension 168
111 peng Pharmaceutical Co., Ltd. Evans blue (no. E2129) was purchased in saline into the right hind paw of each rat. The inflammation was 169
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
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15
170 quantified by measuring the volume (mL) displaced by the paw
171 using a plethysmometer (Beijing Zhongshidichuang Science and
172 Technology Development Co., Ltd, model YLS-7C, China) at 0, 1, 2, 3,
173 4, 5 h after carrageenan injection. The edema volume was expressed
174 by the variation in volume between each time (1, 2, 3, 4 and 5 h) 10
175 and basal time (0 h). * * 176 Antinociceptive activity *
Edema (mg) 5
177 Acetic acid-induced writhing response
178 Mice were used according to the method previously reported
179 (Koster et al., 1959). The doses and the routes of administration
180 were provided in ear vasodilatation assay. 60 min after the via gav- 0
181 age, the responses to an intraperitoneal injection of acetic acid
182 solution, manifesting as a contraction of the abdominal muscles Control
183 stretching of hind limbs, were counted cumulatively after 5 min Indometacin O (500 mg/kg)O (250 mg/kg)
H 2 H 2
184 of stimulus over a period of 20 min. Nociception was induced by BtoH (500 mg/kg)BtoH (250 mg/kg)EtoAc (500 mg/kg)EtoAc (250 mg/kg)
185 intraperitoneal injection of 0.6% acetic acid solution at the dose of
Fig. 1. Effects of oral pretreatment with BtOH (500 and 250 mg/kg), EtOAc (500 and
186 20 mL/kg body weight.
250 mg/kg), H2O (500 and 250 mg/kg) or indomethacin (10 mg/kg) on DMB-induced
ear vasodilatation. The result of ear edema represents mean ± SD (n = 8). Differences
187 Formalin test
between the groups were determined by an ANOVA followed by Dunnett’s test.
188 Formalin-induced nociception was induced in mice according *p < 0.05, **p < 0.01 when compared to the control group.
189 to a previously described procedure (Hunskaar et al., 1985). A vol-
190 ume of 20 L of a 2.5% formalin solution in saline was injected
Anti-inflammatory effects on DMB-induced ear vasodilatation 226
191 intraplantarly (i.pl.) in the plantar surface of the right hind paw
192 to mice. After formalin injection, the mice were individually placed
Treatment with BtOH (500 and 250 mg/kg) 1 h before applied 227
193 in a glass cylinder of 22 cm diameter and were observed from 0
DMB in mice ears significantly (Fig. 1; p < 0.05) inhibited the edema 228
194 to 5 min (neurogenic pain response) and 15–30 min (inflamma-
formation when compared to the control group. Dose-dependent 229
195 tory pain response). The time spent licking the injected paw was
manner was observed at BtOH groups. BtOH group at the dose 230
196 recorded with a chronometer for both phases and considered as
of 500 mg/kg could remarkably decrease the vasodilatation with 231
197 indicative of nociception. The animals were pretreated with oral
inhibition rate over 50%. 232
198 doses of vehicle, indomethacin, EtOAc (500 and 250 mg/kg), BtOH
199 (500 and 250 mg/kg) and H2O (500 and 250 mg/kg) 60 min before
Anti-inflammatory effects on acetic acid-induced vascular 233
200 formalin administration.
permeability enhancement 234
201 Hot plate test
Fig. 2 showed acid-induced vascular permeability enhancement 235
202 Mice were subjected to the hot plate test (MacDonald et al.,
was significantly (p < 0.05, compared to control group) inhibited 236
203 1946; Sahley and Berntson, 1979). A glass cylinder was placed on a
in mice pretreated orally with BtOH fraction and EtOAc fraction 237
204 hot-plate with adjustable temperature. The temperature of the hot-
◦ (500 mg/kg, p.o.), with an inhibition of 32.13 and 27.30%, respec- 238
205 plate was then regulated to 55 ± 1 C. The female mice with baseline
tively. The standard drug reference indomethacin resulted in a 239
206 latencies of more than 20 s were eliminated from the study and the
significant reduction (39.46%) of the control number. 240
207 cut-off time of 40 s was fixed to avoid damage to the paws. Each
208 mouse was placed in the glass on the hot-plate in order to obtain
3
209 the animal’s response to heat-induced nociceptive pain stimulus
210 (linking of the hindpaws, jumping or shaking). The reaction times
211 obtained at 0, 30, 60, 90 and 120 min prior to via gavage of vehicle
212 and drugs. 2
213 Statistical analysis ** *
**
214 The results were analyzed using a statistical program SPSS
215 Statistics, version 17.0. One-way ANOVA followed by Dunnett’s 1
216 test was used for determining the statistically significant differ-
Absorbance of evance blue Absorbance of evance
217 ences between the values of various experimental groups. Data are
218 expressed as means ± SD and p < 0.05 was considered statistically 219 significant. 0
220 Results Control
Indometacin O (500 mg/kg)O (250 mg/kg)
2 2
221 Acute toxicity studies H H
BtoH (500 mg/kg)BtoH (250 mg/kg)EtoAc (500 mg/kg)EtoAc (250 mg/kg)
222 The EtOAc and BtOH fractions from NAF at various dose levels Fig. 2. Effects of oral pretreatment with BtOH (500 and 250 mg/kg), EtOAc (500 and
250 mg/kg), H2O (500 and 250 mg/kg) or indomethacin (10 mg/kg) on the capillary
223 did not show any mortality, any morbid symptoms or deleterious
permeability under acetic acid challenge. The absorbance of Evans Blue in the leak-
224 effects even at the highest dose (40 g/kg, orally). The LD50 value by
age under 590 nm represented indicated the capillary permeability. Date represents
225 oral route could not be determined as no lethality was observed up
mean ± SD (n = 8). Differences between the groups were determined by an ANOVA
to 40 g/kg in mice. followed by Dunnett’s test. *p < 0.05, **p < 0.01 when compared to the control group.
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
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BJP 86 1–6 ARTICLE IN PRESS
4 Y. Zhang et al. / Revista Brasileira de Farmacognosia xxx (2015) xxx–xxx Control Indometacin
BtoH (500 mg/kg) BtoH (250 mg/kg)
EtoAc (500 mg/kg) EtoAc (250 mg/kg)
H2O (500 mg/kg)
0.55
0.50
0.45
0.40 * 0.35 * 0.30 * * * 0.25 * * * * * * Edema volume (mL) 0.20 *
0.15 * * 0.10 * * ** * * 0.05 * 0.00
1 2 3 4 5
Time (h)
Fig. 3. Anti-inflammatory activity of BtOH (500 and 250 mg/kg), EtOAc (500 and 250 mg/kg), H2O (500 mg/kg) or indomethacin (10 mg/kg) in carrageenan induced hind paw
edema. Results were obtained by oral administration of NAF fractions, indomethacin and vehicle. Each value represents the mean ± SD of results from seven rats. Differences
from the control group were determined by ANOVA followed by Dunnett’s test. *p < 0.01.
241 Anti-inflammatory effects on carrageenan-induced paw edema 60
242 Treatment of animals with BtOH fractions (500, 250 mg/kg, p.o.)
243 and EtOAc fraction (500 mg/kg) 1 h before injection of carrageenan *
244 significantly (Fig. 3; p < 0.01) inhibited the edema formation at 40
245 1–5 h when compared to control group. But EtOAc fraction at **
246 dose of 250 mg/kg only significantly decreased the edema at 1 h. **
247 Indomethacin (10 mg/kg, p.o.) also produced a significant inhibi- **
248 tion of the carrageenan-induced paw edema at 1, 2, 3, 4 and 5 h 20
Number of writhes
249 when compared to control (Fig. 3; p < 0.01).
250 Anti-nociceptive activity on acetic acid-induced writhes
0
251 In the acetic acid-induced writhing mice (shown in Fig. 4), EtOAc
Control
252 fractions (250–500 mg/kg, p.o.) evoked a dose-dependent inhibi- Indometacin
O (500 mg/kg)O (250 mg/kg)
253 tion, the inhibitory percentage of 44.67% and 28.11%, respectively. H 2 H 2
BtoH (500 mg/kg)BtoH (250 mg/kg)EtoAc (500 mg/kg)EtoAc (250 mg/kg)
254 All doses of EtOAc fractions significantly (p < 0.01 and p < 0.05)
255 inhibited the frequency induced abdominal constrictions by acetic
Fig. 4. Effects of oral pretreatment with BtOH (500 and 250 mg/kg), EtOAc (500
256 acid when compared to the control group. Treatment with BtOH and 250 mg/kg), H2O (500 and 250 mg/kg) or indomethacin (10 mg/kg) on acetic
acid-induced writhing in mice. Each value represents mean ± SD (n = 8). *p < 0.05,
257 fraction at dose of 500 mg/kg also showed antinociceptive effect in
**p < 0.01 when compared to the control group.
258 comparison with the control (p < 0.01).
259 Anti-nociceptive activity on formalin test
p.o.) only significantly reduced the inflammatory (75.8%) phase of 269
formalin-induced nociception. 270
260 Fig. 5 shows that both first (neurogenic pain) and second
261 (inflammatory pain) phases of formalin-induced nociception were
262 significantly (p < 0.01, compared to control group) inhibited in mice
Anti-nociceptive activity on hot plate test 271
263 pretreated orally with BtOH fractions (500, 250 mg/kg, p.o.) and
264 EtOAc fraction (500 mg/kg). EtOAC fraction at dose of 250 mg/kg
In the hot plate test, the results presented in Fig. 6 showed 272
265 only significantly (p < 0.05) inhibited the nociception at second
that significant (p < 0.01 and p < 0.05) antinociceptive effect in BtOH 273
266 phase compared to control group. The inhibitions were 50.8 and
fraction and EtOAc fraction groups (500 mg/kg) at 90 and 120 min 274
267 80.3% in BtOH group at the dose of 500 mg/kg, for the first and
after administration when compared to control groups. 275
268 second phases, respectively. In contrast, indomethacin (10 mg/kg,
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
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285
150 1 st phase 2 st phase high molecular weight components such as ions of alkaloids (Yang
and Kong, 2009). The ions of alkaloids were retained in chromato- 286
graphic column, but the non-alkaloids fractions could be washed 287
using deionized water. In our study, no deaths or serious clinical 288
signs in both EtOAc and BtOH fractions at the highest dose of 40 g/kg 289
100 #
(this dose is much higher than the dose of treatment) in acute tox- 290
icity study. Although the evidence of risk in longer use is unknown, 291
## ** ##
but the acute toxic results sufficiently support the fact that BtOH 292
**
** and EtOAc fractions of NAF have no toxicity. 293
Licking time (s) 50
Xylene-induced ear edema in mice is a preliminary and 294
simple acute inflammation model for evaluating potential anti- 295
## ##
inflammatory agents (Cheng et al., 2005). Ear edema may involve 296
inflammatory mediators such as histamine, kinin, fibrinolysin, 297
0 phospholipase A2 and PLA2. These mediators induce edema by pro- 298
moting vasodilation and increasing vascular permeability (Li et al., 299
Control
2011; Xu et al., 2014b). The BtOH fraction of NAF was able to reduce 300
Indometacin
O (500 mg/kg)O (250 mg/kg) inflammation in this model and acetic acid-induced vascular per- 301 2 2
BtoH (500 mg/kg)BtoH (250 mg/kg) H H
EtoAc (500 mg/kg)EtoAc (250 mg/kg) meability enhancement model. These results suggest that BtOH 302
fraction may interfere with the actions of inflammatory mediators 303
Fig. 5. The antinociceptive action of BtOH (500 and 250 mg/kg), EtOAc (500 and
and produce the anti-inflammatory effect. Carrageenan-induced 304
250 mg/kg) and H2O (500 and 250 mg/kg) on both first (0–5 min) and second
paw edema is a largely used test for screening both steroidal 305
(15–30 min) phases of the formalin-induced nociception. Each value represents
mean ± SD (n = 8). *p < 0.05, **p < 0.01 by an ANOVA followed by Dunnett’s test com- anti-inflammatory drugs and NSAID. The inflammatory response 306
# ##
pared to control group at first phase. p < 0.05, p < 0.01 by an ANOVA followed by involves three phases through sequential release of several medi- 307
Dunnett’s test compared to control group at second phase.
ators. The early phase (the first 90 min) involves the release of 308
histamine and serotonin; the second phase (90–150 min) is medi- 309
276 Discussion ated by kinin and the third phase (after 180 min) is mediated by 310
prostaglandin (Di Rosa et al., 1971). The results from this study 311
312
277 The toxicity of alkaloids from A. flavum Hand.-Mazz, Ranuncu- suggest that the BtOH and EtOAc fractions of NAF possibly act by
313
278 laceae, has made us worried, so to obtain nature products with inhibiting the release or action of histamine, serotonin and kinin
314
279 favorable effects and low toxicity from A. flavum become a mean- and prostaglandin of the edema development.
315
280 ingful research direction. In order to realize this objective, we The analgesic activity of NAF fractions in this study was investi-
316
281 studied the non-alkaloids fractions from this herb. In our study, gated using the abdominal writhing, formalin and hot plate tests in
317
282 001 × 7 strong acid polystyrene cation exchange resin which is mice. The writhing model induced by acetic acid in mice was com-
318
283 a low cross-linking resin and has porous structure was selected monly considered as classical peripheral inflammatory pain animal
319
284 for preparing NAF. This resin may be beneficial to adsorb alkaline model for evaluation of analgestic or anti-inflammatory drugs
Control BtoH (250 mg/kg)
BtoH (500 mg/kg) EtoAc (250 mg/kg) EtoAc (500 mg/kg) H2O (250 mg/kg)
H2O (500 mg/kg)
30 * ** * **
25
20
15 Latency time (s)
10
5 0 30 60 90 120
Time (min)
Fig. 6. Effects of oral pretreatment with BtOH (500 and 250 mg/kg), EtOAc (500 and 250 mg/kg) and H2O (500 and 250 mg/kg) on hot-plate test in mice. Date represents
mean ± SD (n = 8). Differences between the groups were determined by an ANOVA followed by Dunnett’s test. *p < 0.05, **p < 0.01 when compared to the control group.
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
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6 Y. Zhang et al. / Revista Brasileira de Farmacognosia xxx (2015) xxx–xxx
320 (Negus et al., 2006). The peripheral analgesic is due to the liberation and Technology Project of Ningxia Province of China (Project No. 377
321 of several inflammatory mediators such as bradykinin, substance 170). 378
322 P, prostaglandins and cyclo-oxygenases, lipoxygenases, as well as
323 some cytokines such as IL-1, TNF-␣ and IL-8 (Ikeda et al., 2001; References 379
324 Ribeiro and Poole, 2000). The hot plate test has been found suitable
Carlson, R.P., O’Neill-Davis, L., Chang, J., Lewis, A.J., 1985. Modulation of mouse ear 380
325 to evaluate centrally antinociceptive (Hiruma-Lima et al., 2000). In
edema by cyclooxygenase and lipoxygenase inhibitors and other pharmacologic 381
326 order to further clarify the antinociceptive effect of NAF fractions,
agents. Agents Actions 17, 197–204. 382
327 the formalin test was carried out. This model is validly used in anal- Chan, T.Y., 2012. Aconitum alkaloid content and the high toxicity of aconite tincture. 383
Forensic Sci. Int. 222, 1–3. 384
328 gesia research (Tjølsen et al., 1992), it involves two phases (Zakaria
Cheng, W., Li, J., You, T., Hu, C., 2005. Anti-inflammatory and immunomodulatory 385
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352 loids (Shrestha et al., 2006; Wuljitegus et al., 2008). The flavonoids,
Cunha, F.Q., 2000. Involvement of resident macrophages and mast cells in the 416
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354 lyzed by ultraviolet spectrophotometry in our study. No flavonoids
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355 were identified in those two fractions. However the phenolic acids
administration of nicotine in the rat. Psychopharmacology (Berl.) 65, 279–283. 420
356 and saponins gave a 15.08 and 9.73 percent in EtOAc fractions. Shrestha, B.B., Dall’Acqua, S., Gewali, M.B., Jha, P.K., Innocenti, G., 2006. New 421
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357 At the same time, the phenolic acids and saponins percentage of
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430
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366 running the laboratory work. LM contributed to estimation of
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368 YZ wrote manuscript. XW contributed to critical reading of the
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371 All the authors have read the final manuscript and approved
and toxicities scattering effect of pharmacodynamic substances of Traditional 444
372 submission.
Chinese Medicines (TCMs). Some reflections evoked by the study on TCM 445
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373 Conflict of interest
Xu, Q., Wang, Y., Guo, S., Shen, Z., Wang, Y., Yang, L., 2014b. Anti-inflammatory 448
and analgesic activity of aqueous extract of Flos populi. J. Ethnopharmacol. 152, 449
374 The authors have declared that there is no conflict of interest. 540–545. 450
Yang, Y., Kong, D., 2009. The Extraction and Separation Technology of Traditional 451
Chinese Medicine. Chemical Industry Press, Beijing, pp. 249–256. 452
375 Acknowledgements
Zakaria, Z.A., Ghani, Z.D., Nor, R.N., Gopalan, H.K., Sulaiman, M.R., Jais, A.M., 453
Somchit, M.N., Kader, A.A., Ripin, J., 2008. Antinociceptive, anti-inflammatory, 454
and antipyretic properties of an aqueous extract of Dicranopteris linearis leaves 455
376Q4 This work was supported by grants form the National Nature
in experimental animal models. J. Nat. Med. 62, 179–187. 456
Science Foundation of China (Project No. 81102892) and Science
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