J Basic Clin Physiol Pharmacol 2014; aop
Asie Shojaii, Manijeh Motevalian* and Nazanin Rahnama
Evaluation of anti-inflammatory and analgesic activities and the phytochemical study of Astragalus arbusculinus gum in animal models Abstract
DOI 10.1515/jbcpp-2014-0092 Received August 16, 2014; accepted November 4, 2014
Background: The importance of inflammatory diseases and side effects of conventional drugs necessitate the finding of new anti-inflammatory agents from natural sources. In this study, for the first time, the anti-inflammatory and analgesic effects of the aqueous extract of Astragalus arbusculinus gum were evaluated in animal models. Methods: Thirty-five male Wistar rats were divided into five groups and pretreated with different doses of A. arbusculinus gum extract before the injection of formalin. Paw edema was measured by a plethysmometer at time 0 and after 8 days and compared to controls. The analgesic effect of the extract was evaluated using the hot-plate test in 42 male albino mice. Results: The extract of A. arbusculinus gum decreased the rat paw edema in a dose-dependent manner. The effect on inflammation of the highest dose of extract was comparable to sodium salicylate. Astragalus arbusculinus gum extract at doses of 300 and 1000 mg/kg showed analgesic effects comparable to sodium salicylate and morphine, respectively. A preliminary phytochemical study and the determination of the total phenolic content of the gum extract were performed for the first time. Conclusions: The aqueous extract of A. arbusculinus gum reduced the inflammation and pain in a dose-dependent manner and is a good candidate for further studies of safety and efficacy. The clarification of active components of the plant is necessary. Keywords: analgesic; anti-inflammatory; arbusculinus; gum; rat paw edema.
Astragalus
*Corresponding author: Manijeh Motevalian, Department of Pharmacology, School of Medicine and Razi Institute for Drug Research, Iran University of Medical Sciences, Tehran, Iran, E-mail: [email protected], [email protected] Asie Shojaii: Research Institute for Islamic and Complementary Medicine, Iran University of Medical Sciences, Tehran, Iran Nazanin Rahnama: Department of Pharmacology, School of Medicine and Razi Institute for Drug Research, Iran University of Medical Sciences, Tehran, Iran
Introduction Inflammation is the response of living tissues to a variety of hostile agents, including infectious organisms, toxic chemical substances, physical injury, or tumor growth leading to the local accumulation of plasma fluid and blood cells [1]. Drugs used to treat mild to severe pain, such as nonsteroidal anti-inflammatory drugs (NSAIDs) and opiates, show some adverse reactions; gastrointestinal disturbances, renal damages, respiratory depression, and possibly drug dependence may be induced by the prolonged use of these drugs [2, 3]. During the last decade, there has been an increasing interest in traditional herbal products [4]. Some medicinal herbs in traditional medicine in Iran have been recommended for inflammatory and painful conditions [5, 6]. Astragalus is a widely distributed flowering plant, with an estimated 2500–3000 species mostly found in Central Asia [7]. Previous studies have shown its immunomodulatory, antiviral, and antibacterial activities. Astragalus has been effective against cardiovascular diseases, nephritis, and male infertility and as an adjunct cancer therapy [8, 9]. Its gum (called anzaroot) has been used as an analgesic and anti-inflammatory agent and in orthopedics in Iranian traditional medicine [6]. To the best of our knowledge, there are no reports about the analgesic and anti-inflammatory effects of this plant. Therefore, in the present study, we evaluated the anti-inflammatory and analgesic effects of the aqueous extract of A. arbusculinus gum using the formalin test and hot-plate test in animal models.
Materials and methods Plant materials Preparation of the gum extract: Astragalus arbusculinus (anzaroot) gum was purchased from a local herbal medicine shop in Tehran,
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2 Shojaii et al.: Anti-inflammatory and analgesic activities of A. arbusculinus gum Iran, and its authenticity was approved by Mr. Kamali Nejad, a member of the School of Pharmacy, Shaheed Beheshti University, Tehran, Iran (No. 8006). The gum was extracted with distilled water by maceration for three times, and the aqueous extract was dried with freeze dryer (Operon Co., South Korea). Phytochemical screening: The phytochemical investigation of the aqueous extract of A. arbusculinus gum was carried out using standard methods and tests [10, 11]. Total phenolic content (TPC): The TPC in the gum extract of A. arbusculinus was determined according to the Folin-Ciocalteu method with minor modifications [12]. For the preparation of the calibration curve, 1 mL aliquots of 75, 100, 150, and 200 mg/mL hydroethanolic/gallic acid solutions (50:50) were mixed with 5 mL Folin-Ciocalteu reagent (Merck, Germany) (diluted 10-fold) and 4 mL (75 mg/mL) sodium carbonate (Exir, Tehran, Iran). The absorption was read after 30 min at 765 nm and the calibration curve was drawn. One milliliter of the gum extract (10 mg/mL) was mixed with the same reagents as described above, and the absorption was measured for the determination of plant phenolics. All determinations were performed in triplicate. The total content of phenolic compounds in A. arbusculinus gum extract was expressed as the mean ± SD of milligram of gallic acid equivalents (GAE) per milligram of extract.
saline as positive and negative controls, respectively. The paw volume of each rat was measured with a plethysmometer (model 7141; UgoBasile, Comerio Varese, Italy) before formalin injection. Then, the inflammation was induced by an injection of 50 μL of 0.2% formalin solution into the plantar surface subcutaneously. The volume measurements were repeated in 1, 2, and 3 h and at 8 days after formalin injection. The percentage inhibition of edema was calculated by the following formula: Percentage inhibition= [( Vt −V0 ) / V0 ] × 100 where Vt is the rat paw volume after injection and V0 is the rat paw volume before injection. Hot-plate test: To evaluate the antinociceptive effects, 42 male healthy albino mice (25–30 g) were randomly divided into six groups to receive an intraperitoneal injection of A. arbusculinus extract (100, 300, and 1000 mg/kg), sodium salicylate (positive control), normal saline (negative controls), or morphine hydrochloride. The reaction time of animals on the hot plate was recorded at 15, 30, 60, and 120 min after drug administration. The percentage increase in the basal reaction time (index of analgesia) at each time interval was calculated. The basal reaction time was recorded by observing hind paw licking or jump response in animals when placed on a hot plate maintained at a constant temperature (55 ± 1 °C). A cutoff period of 15 s was set to avoid paw injury.
Chemicals
Statistical analysis
Sodium salicylate (Sigma, Munich, Germany) and morphine hydrochloride (Darou Pakhsh, Iran) were used in control groups. Formalin solution (0.2%) was obtained from Sigma (Germany).
The results are presented as mean ± SE. Data analysis was performed using SPSS 19.0 for Windows (SPSS, Inc., Chicago, IL, USA). To compare the test results in all groups, a one-way ANOVA test was used. p < 0.05 was considered statistically significant.
Animals Thirty-five healthy male Wistar rats (weight 180–240 g) and 42 healthy male albino mice (weight 25–30 g) were housed in the Animal Unit of Iran University of Medical Sciences under standard laboratory conditions (temperature 23 ± 2 °C) with 12 h dark and 12 h light cycle. Animals had free access to standard dry pellet diet and tap water ad libitum. All possible steps were taken to avoid animal suffering at each stage of the experiment. All procedures involving animals were conducted according to the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (National Research Council, 2003) and guidelines of the Institutional Animal Ethical Committee (IUMS-AC).
Results The phytochemical study of the gum extract of A. arbusculinus was carried out for the first time in this study. The results showed the presence of saponins, phenolic compounds, glycosides, triterpenoids, alkaloids, and anthraquinone. The TPC of the aqueous extract of A. arbusculinus gum was 27.76 ± 1.38 mg GAE/mg extract.
Anti-inflammatory activity Experimental studies Formalin-induced inflammation: The test was carried out using the method described by Hunskaar and Hole [13]. To evaluate the anti-inflammatory effects of Astragalus gum extract, 35 healthy male Wistar rats were randomly divided into five groups (n = 7). The first three groups were treated with an intraperitoneal injection of A. arbusculinus extract in doses of 100, 300, and 1000 mg/kg. The other two groups received sodium salicylate (300 mg/kg) or normal
The findings of the formalin-induced inflammation have shown a dose-dependent decrease in inflammation produced by formalin in rat paw in different groups in the acute phase of inflammation (Table 1). A decrease in edema was observed in the chronic phase in a dosedependent manner for 8 days, except on day 6. On day 6, the mean paw volume in A. arbusculinus extract of 100, 300, and 1000 mg/kg and normal saline-treated groups
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Shojaii et al.: Anti-inflammatory and analgesic activities of A. arbusculinus gum 3
Table 1 Evaluation of the anti-inflammatory effect of A. arbusculinus extract (acute inflammation).
Volume of rat paw, mm3 before injection
Increase in paw volume, mL after 2 h
3 h, mean ± SE
% Inhibition after 2 h, mean
% Inhibition after 3 h, mean
p-Value
N.S. S.S. Extract 100 Extract 300 Extract 1000
3.014 ± 0.16a 3.2 ± 0.07 3.4 ± 0.12 3.4 ± 0.13 3.3 ± 0.12
2.09 ± 0.1 0.76 ± 0.02 1.9 ± 0.05 1.5 ± 0.02 1 ± 0.02
2.68 ± 0.13 0.93 ± 0.03 2.4 ± 0.07 1.9 ± 0.05 1.2 ± 0.01
30.65 76.25 44.11 55.8 69.7
11.1 70.9 29.4 44.11 63.64
– 0.001 0.007 0.001 0.001
Groups
Data are mean ± SE (n = 7). p < 0.05. N.S., normal saline; S.S., sodium salicylate.
a
were 0.38 ± 0.1, 0.37 ± 0.03, 0.35 ± 0.03, and 0.36 ± 0.03 mL, respectively (p > 0.05). The lowest level of inflammation was in the sodium salicylate group, but the maximum percentage inhibition in the A. arbusculinus groups was observed in the group receiving extract at a dose of 1000 mg/kg, and this was comparable to the established anti-inflammatory drug, sodium salicylate (Table 2). Although the amount of inflammation in the normal saline-treated group increased in the final days, the other groups experienced a decrease in inflammation (Figure 1).
Hot-plate analysis The findings of the hot-plate test showed that the A. arbusculinus gum extract possessed a significant antinociceptive effect in comparison to control groups. The results for the gum extracts at concentrations of 300 and 1000 mg/kg were significant and comparable with the effect of sodium salicylate and morphine at 2 h after injection (Table 3; Figure 2). Astragalus arbusculinus extract had no adverse effects on the animals and no animals died of A. arbusculinus extract treatment.
Table 2 Anti-inflammatory effect of A. arbusculinus gum extract on formalin-induced edema in rats (chronic inflammation). Groups
2 days
3 days
4 days
5 days
6 days
7 days
8 days
p-Value
N.S. S.S. Extract 100 Extract 300 Extract 1000
0.24 ± 0.002a 0.19 ± .0.01 0.23 ± 0.01 0.58 ± 0.02 0.53 ± 0.07
0.44 ± 0.002 0.34 ± 0.01 0.46 ± 0.01 0.53 ± 0.02 0.45 ± 0.06
0.32 ± 0.03 0.24 ± 0.02 0.38 ± 0.004 0.47 ± 0.02 0.33 ± 0.04
0.27 ± 0.01 0.26 ± 0.01 0.41 ± 0.01 0.42 ± 0.02 0.27 ± 0.01
0.36 ± 0.01 0.28 ± 0.01 0.38 ± 0.01 0.40 ± 0.02 0.24 ± 0.00
0.43 ± 0.020 0.24 ± 0.010 0.33 ± 0.01 0.39 ± 0.02 0.20 ± 0.01
0.52 ± 0.02 0.23 ± 0.01 0.38 ± 0.01 0.37 ± 0.03 0.16 ± 0.02
0.09 0.0001 0.0001 0.0001 0.0001
Data are mean ± SE (n = 7). p < 0.05. N.S., normal saline; S.S., sodium salicylate.
a
0.6
Normal saline
Mean edema, mL
0.5 Sodium salic.
0.4 0.3
AA 100
0.2
AA 300
0.1 AA 1000
0 Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Day 8
Figure 1 Mean rat paw edema induced by formalin during 8 days in the presence of different concentrations of A. arbusculinus gum extract. N.S., normal saline; S.S., sodium salicylate. p < 0.05.
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4 Shojaii et al.: Anti-inflammatory and analgesic activities of A. arbusculinus gum
Table 3 Mean latency time at 0, 30, 60, and 120 min after Astragalus gum extract and drug injections. Latency time, mean ± SE
Groups N.S. S.S. M.S. Extract 100 Extract 300 Extract 1000
0
30
60
120
6.9 ± 0.35 7.5 ± 0.22 7.8 ± 0.35 7.9 ± 0.81 8.9 ± 0.57 9.4 ± 0.61
6.8 ± 0.31 11.9 ± 0.15 15.5 ± 0.47 8.2 ± 0.4 12.3 ± 0.38 12.5 ± 0.45
6.7 ± 0.32 11.8 ± 0.22 15.28 ± 0.46 9.7 ± 0.18 11.8 ± 0.01 13.5 ± 0.22
7.1 ± 0.31 11.6 ± 0.24 14.7 ± 0.53 10.2 ± 0.11 12.5 ± 0.17 14.9 ± 0.13
M.S., morphine sulfate; N.S., normal saline; S.S., sodium salicylate.
Discussion and conclusion The A. arbusculinus (Fasciculifolius), Family Papilionaceae, gum (called anzaroot) has been used as an analgesic and anti-inflammatory agent and in orthopedics in Iranian traditional medicine [5, 6]. In this study, the antiinflammatory and analgesic effects of the aqueous extract of A. arbusculinus gum were evaluated using the formalin and hot-plate tests. The findings of formalin-induced inflammation have shown acute and chronic anti-inflammatory activities of the gum extract in a dose-dependent manner. The results of the hot-plate test also revealed a dose-dependent analgesic effect of the gum extract comparable to sodium salicylate and morphine. An inflammatory process is a dynamic complex sequence of highly organized, tightly regulated events,
Normal saline
Sodium salicylate
AA100
ultimately resulting in wound healing or granulation tissue. The inflammation that is initiated by homeostasis and coagulation continues with the recruitment of polymorphonuclear leukocytes and macrophages. These leukocytes, via the activation of the nuclear factor-κB (NF-κB) gene, enhance the release of interleukin (IL)1α, IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) to maintain the inflammatory phase [14, 15]. The synthetic drugs of NSAIDs and corticosteroids suppress leukocyte migration and proinflammatory cytokine release [16]. There are in vivo and in vitro evidence that Astragalus extract has the same immunomodulatory properties. It has been demonstrated that Astragalus extract attenuates the elevated myeloperoxidase (MPO) activity and down-regulates the inducible nitric oxide synthase, P-selectin, and intercellular cell adhesion molecule-1 protein expression [17]. The Astragalus extract also inhibits the production of interferon (IFN)-γ by CD4+ lymphocytes in a similar way to prednisolone [18]. Astragalus extracts, via p38 mitogen-activated protein kinase and NF-κB signaling pathways, inhibits IL-1β and TNF-α secretion in macrophages [19]. In vivo studies have also revealed its anti-inflammatory effects in arthritis via reduced lymphocyte recruitment and TNF-α and IL-1β production and in colitis via the amelioration of elevated colonic MPO activity, reducing overexpressed TNF-α and IL-1β and restoring IL-10 expression; its antiatherosclerotic function via the decreased expression of adhesion molecules; and its antihepatocellular carcinoma effects via increased proapoptotic protein Bax and decreased
AA300
AA1000
Morphine
18 16
Mean analgesia time, s
14 12 10 8 6 4 2 0
0
30 min
60 min
120 min
Figure 2 Mean latency time after treatment by the extract of A. arbusculinus in animal models at 30, 60, and 120 min after injection. Data are mean ± SE (n = 7). *p < 0.05, Compared with normal saline group. †, Compared with positive control group.
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Shojaii et al.: Anti-inflammatory and analgesic activities of A. arbusculinus gum 5
antiapoptotic protein Bcl-2 [20–22]. Our current study is another proof of A. arbusculinus in vivo anti-inflammatory effects in a dose-dependent manner. Inflammatory pain is generated in primary sensory neurons (nociceptors) and transmitted via mediators such as glutamate, substance P, VIP, somatostatin, ATP, and neural growth factor to dorsal horn neurons. Additionally, the release of prostaglandins, bradykinin, and proinflammatory cytokines (IL-1, IL-6, IL-8, and TNF-α) in the inflammatory process produces hyperalgesia [23]. NSAIDs, opioids, and cannabinoids are the main antinociceptive drugs in inflammatory pain processes [24]. Our results showed the antinociceptive effects of the different doses of A. arbusculinus, but its mechanism of action still needs to be described. In addition, previous studies have suggested that primarily central nervous system (CNS)acting drugs are inhibitors of both early (zero time) and late (120 min time) phases of nociception; in other words, they equally suppress inflammatory and neurogenic nociception [25, 26]. Astragalus arbusculinus extract has antinociceptive characteristics in both phases, so it is probably a primarily CNS-acting drug. NSAIDs and steroids are examples of peripherally acting drugs. Our current study has shown that the aqueous extract of A. arbusculinus gum has dose-dependent anti-inflammatory effects in paw edema. In addition, A. arbusculinus gum extract exhibited dose-dependent antinociceptive properties probably through the suppression of both peripheral and central nociceptors. No side effects of A. arbusculinus gum extract were observed during the study. Therefore, A. arbusculinus gum extract might be useful in the treatment of inflammatory diseases. Further studies are needed to investigate this hypothesis. Up to now, different species of Astragalus showed analgesic, antinociceptive, and anti-inflammatory activities in animal models and their active compounds were determined in complementary studies. For example, some fraction and compounds isolated from the roots of Astragalus membranaceus have shown anti-inflammatory activity in an in vitro study [27]. In another study, Astragalus siculus showed anti-inflammatory and analgesic effects in animal models [28]. The anti-inflammatory activity of Astragalus hamosus was determined in a study by Hakim et al. [29]. So far, different compounds were isolated from Astragalus species. Among these compounds, terpenoids (especially saponins) and polysaccharides have shown significant anti-inflammatory and analgesic effects in experimental models [30, 31]. Due to the preliminary phytochemical study of the gum extract of A. arbusculinus (mentioned above), the anti-inflammatory and analgesic
effects of the gum may be due to the presence of these compounds. Because no phytochemical study was performed on A. arbusculinus to purify the active compounds, further studies can determine the active principles of this plant, which may responsible for the analgesic and antiinflammatory activities, such as in polysaccharides, saponins, or flavonoids. Acknowledgments: This work was supported by the Research Institute for Islamic and Complementary Medicine, Iran University of Medical Sciences (Grant Number 14513). Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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Asie Shojaii, Manijeh Motevalian* and Nazanin Rahnama
Evaluation of anti-inflammatory and analgesic activities and the phytochemical study of Astragalus arbusculinus gum in animal models Abstract
DOI 10.1515/jbcpp-2014-0092 Received August 16, 2014; accepted November 4, 2014
Background: The importance of inflammatory diseases and side effects of conventional drugs necessitate the finding of new anti-inflammatory agents from natural sources. In this study, for the first time, the anti-inflammatory and analgesic effects of the aqueous extract of Astragalus arbusculinus gum were evaluated in animal models. Methods: Thirty-five male Wistar rats were divided into five groups and pretreated with different doses of A. arbusculinus gum extract before the injection of formalin. Paw edema was measured by a plethysmometer at time 0 and after 8 days and compared to controls. The analgesic effect of the extract was evaluated using the hot-plate test in 42 male albino mice. Results: The extract of A. arbusculinus gum decreased the rat paw edema in a dose-dependent manner. The effect on inflammation of the highest dose of extract was comparable to sodium salicylate. Astragalus arbusculinus gum extract at doses of 300 and 1000 mg/kg showed analgesic effects comparable to sodium salicylate and morphine, respectively. A preliminary phytochemical study and the determination of the total phenolic content of the gum extract were performed for the first time. Conclusions: The aqueous extract of A. arbusculinus gum reduced the inflammation and pain in a dose-dependent manner and is a good candidate for further studies of safety and efficacy. The clarification of active components of the plant is necessary. Keywords: analgesic; anti-inflammatory; arbusculinus; gum; rat paw edema.
Astragalus
*Corresponding author: Manijeh Motevalian, Department of Pharmacology, School of Medicine and Razi Institute for Drug Research, Iran University of Medical Sciences, Tehran, Iran, E-mail: [email protected], [email protected] Asie Shojaii: Research Institute for Islamic and Complementary Medicine, Iran University of Medical Sciences, Tehran, Iran Nazanin Rahnama: Department of Pharmacology, School of Medicine and Razi Institute for Drug Research, Iran University of Medical Sciences, Tehran, Iran
Introduction Inflammation is the response of living tissues to a variety of hostile agents, including infectious organisms, toxic chemical substances, physical injury, or tumor growth leading to the local accumulation of plasma fluid and blood cells [1]. Drugs used to treat mild to severe pain, such as nonsteroidal anti-inflammatory drugs (NSAIDs) and opiates, show some adverse reactions; gastrointestinal disturbances, renal damages, respiratory depression, and possibly drug dependence may be induced by the prolonged use of these drugs [2, 3]. During the last decade, there has been an increasing interest in traditional herbal products [4]. Some medicinal herbs in traditional medicine in Iran have been recommended for inflammatory and painful conditions [5, 6]. Astragalus is a widely distributed flowering plant, with an estimated 2500–3000 species mostly found in Central Asia [7]. Previous studies have shown its immunomodulatory, antiviral, and antibacterial activities. Astragalus has been effective against cardiovascular diseases, nephritis, and male infertility and as an adjunct cancer therapy [8, 9]. Its gum (called anzaroot) has been used as an analgesic and anti-inflammatory agent and in orthopedics in Iranian traditional medicine [6]. To the best of our knowledge, there are no reports about the analgesic and anti-inflammatory effects of this plant. Therefore, in the present study, we evaluated the anti-inflammatory and analgesic effects of the aqueous extract of A. arbusculinus gum using the formalin test and hot-plate test in animal models.
Materials and methods Plant materials Preparation of the gum extract: Astragalus arbusculinus (anzaroot) gum was purchased from a local herbal medicine shop in Tehran,
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2 Shojaii et al.: Anti-inflammatory and analgesic activities of A. arbusculinus gum Iran, and its authenticity was approved by Mr. Kamali Nejad, a member of the School of Pharmacy, Shaheed Beheshti University, Tehran, Iran (No. 8006). The gum was extracted with distilled water by maceration for three times, and the aqueous extract was dried with freeze dryer (Operon Co., South Korea). Phytochemical screening: The phytochemical investigation of the aqueous extract of A. arbusculinus gum was carried out using standard methods and tests [10, 11]. Total phenolic content (TPC): The TPC in the gum extract of A. arbusculinus was determined according to the Folin-Ciocalteu method with minor modifications [12]. For the preparation of the calibration curve, 1 mL aliquots of 75, 100, 150, and 200 mg/mL hydroethanolic/gallic acid solutions (50:50) were mixed with 5 mL Folin-Ciocalteu reagent (Merck, Germany) (diluted 10-fold) and 4 mL (75 mg/mL) sodium carbonate (Exir, Tehran, Iran). The absorption was read after 30 min at 765 nm and the calibration curve was drawn. One milliliter of the gum extract (10 mg/mL) was mixed with the same reagents as described above, and the absorption was measured for the determination of plant phenolics. All determinations were performed in triplicate. The total content of phenolic compounds in A. arbusculinus gum extract was expressed as the mean ± SD of milligram of gallic acid equivalents (GAE) per milligram of extract.
saline as positive and negative controls, respectively. The paw volume of each rat was measured with a plethysmometer (model 7141; UgoBasile, Comerio Varese, Italy) before formalin injection. Then, the inflammation was induced by an injection of 50 μL of 0.2% formalin solution into the plantar surface subcutaneously. The volume measurements were repeated in 1, 2, and 3 h and at 8 days after formalin injection. The percentage inhibition of edema was calculated by the following formula: Percentage inhibition= [( Vt −V0 ) / V0 ] × 100 where Vt is the rat paw volume after injection and V0 is the rat paw volume before injection. Hot-plate test: To evaluate the antinociceptive effects, 42 male healthy albino mice (25–30 g) were randomly divided into six groups to receive an intraperitoneal injection of A. arbusculinus extract (100, 300, and 1000 mg/kg), sodium salicylate (positive control), normal saline (negative controls), or morphine hydrochloride. The reaction time of animals on the hot plate was recorded at 15, 30, 60, and 120 min after drug administration. The percentage increase in the basal reaction time (index of analgesia) at each time interval was calculated. The basal reaction time was recorded by observing hind paw licking or jump response in animals when placed on a hot plate maintained at a constant temperature (55 ± 1 °C). A cutoff period of 15 s was set to avoid paw injury.
Chemicals
Statistical analysis
Sodium salicylate (Sigma, Munich, Germany) and morphine hydrochloride (Darou Pakhsh, Iran) were used in control groups. Formalin solution (0.2%) was obtained from Sigma (Germany).
The results are presented as mean ± SE. Data analysis was performed using SPSS 19.0 for Windows (SPSS, Inc., Chicago, IL, USA). To compare the test results in all groups, a one-way ANOVA test was used. p < 0.05 was considered statistically significant.
Animals Thirty-five healthy male Wistar rats (weight 180–240 g) and 42 healthy male albino mice (weight 25–30 g) were housed in the Animal Unit of Iran University of Medical Sciences under standard laboratory conditions (temperature 23 ± 2 °C) with 12 h dark and 12 h light cycle. Animals had free access to standard dry pellet diet and tap water ad libitum. All possible steps were taken to avoid animal suffering at each stage of the experiment. All procedures involving animals were conducted according to the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (National Research Council, 2003) and guidelines of the Institutional Animal Ethical Committee (IUMS-AC).
Results The phytochemical study of the gum extract of A. arbusculinus was carried out for the first time in this study. The results showed the presence of saponins, phenolic compounds, glycosides, triterpenoids, alkaloids, and anthraquinone. The TPC of the aqueous extract of A. arbusculinus gum was 27.76 ± 1.38 mg GAE/mg extract.
Anti-inflammatory activity Experimental studies Formalin-induced inflammation: The test was carried out using the method described by Hunskaar and Hole [13]. To evaluate the anti-inflammatory effects of Astragalus gum extract, 35 healthy male Wistar rats were randomly divided into five groups (n = 7). The first three groups were treated with an intraperitoneal injection of A. arbusculinus extract in doses of 100, 300, and 1000 mg/kg. The other two groups received sodium salicylate (300 mg/kg) or normal
The findings of the formalin-induced inflammation have shown a dose-dependent decrease in inflammation produced by formalin in rat paw in different groups in the acute phase of inflammation (Table 1). A decrease in edema was observed in the chronic phase in a dosedependent manner for 8 days, except on day 6. On day 6, the mean paw volume in A. arbusculinus extract of 100, 300, and 1000 mg/kg and normal saline-treated groups
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Shojaii et al.: Anti-inflammatory and analgesic activities of A. arbusculinus gum 3
Table 1 Evaluation of the anti-inflammatory effect of A. arbusculinus extract (acute inflammation).
Volume of rat paw, mm3 before injection
Increase in paw volume, mL after 2 h
3 h, mean ± SE
% Inhibition after 2 h, mean
% Inhibition after 3 h, mean
p-Value
N.S. S.S. Extract 100 Extract 300 Extract 1000
3.014 ± 0.16a 3.2 ± 0.07 3.4 ± 0.12 3.4 ± 0.13 3.3 ± 0.12
2.09 ± 0.1 0.76 ± 0.02 1.9 ± 0.05 1.5 ± 0.02 1 ± 0.02
2.68 ± 0.13 0.93 ± 0.03 2.4 ± 0.07 1.9 ± 0.05 1.2 ± 0.01
30.65 76.25 44.11 55.8 69.7
11.1 70.9 29.4 44.11 63.64
– 0.001 0.007 0.001 0.001
Groups
Data are mean ± SE (n = 7). p < 0.05. N.S., normal saline; S.S., sodium salicylate.
a
were 0.38 ± 0.1, 0.37 ± 0.03, 0.35 ± 0.03, and 0.36 ± 0.03 mL, respectively (p > 0.05). The lowest level of inflammation was in the sodium salicylate group, but the maximum percentage inhibition in the A. arbusculinus groups was observed in the group receiving extract at a dose of 1000 mg/kg, and this was comparable to the established anti-inflammatory drug, sodium salicylate (Table 2). Although the amount of inflammation in the normal saline-treated group increased in the final days, the other groups experienced a decrease in inflammation (Figure 1).
Hot-plate analysis The findings of the hot-plate test showed that the A. arbusculinus gum extract possessed a significant antinociceptive effect in comparison to control groups. The results for the gum extracts at concentrations of 300 and 1000 mg/kg were significant and comparable with the effect of sodium salicylate and morphine at 2 h after injection (Table 3; Figure 2). Astragalus arbusculinus extract had no adverse effects on the animals and no animals died of A. arbusculinus extract treatment.
Table 2 Anti-inflammatory effect of A. arbusculinus gum extract on formalin-induced edema in rats (chronic inflammation). Groups
2 days
3 days
4 days
5 days
6 days
7 days
8 days
p-Value
N.S. S.S. Extract 100 Extract 300 Extract 1000
0.24 ± 0.002a 0.19 ± .0.01 0.23 ± 0.01 0.58 ± 0.02 0.53 ± 0.07
0.44 ± 0.002 0.34 ± 0.01 0.46 ± 0.01 0.53 ± 0.02 0.45 ± 0.06
0.32 ± 0.03 0.24 ± 0.02 0.38 ± 0.004 0.47 ± 0.02 0.33 ± 0.04
0.27 ± 0.01 0.26 ± 0.01 0.41 ± 0.01 0.42 ± 0.02 0.27 ± 0.01
0.36 ± 0.01 0.28 ± 0.01 0.38 ± 0.01 0.40 ± 0.02 0.24 ± 0.00
0.43 ± 0.020 0.24 ± 0.010 0.33 ± 0.01 0.39 ± 0.02 0.20 ± 0.01
0.52 ± 0.02 0.23 ± 0.01 0.38 ± 0.01 0.37 ± 0.03 0.16 ± 0.02
0.09 0.0001 0.0001 0.0001 0.0001
Data are mean ± SE (n = 7). p < 0.05. N.S., normal saline; S.S., sodium salicylate.
a
0.6
Normal saline
Mean edema, mL
0.5 Sodium salic.
0.4 0.3
AA 100
0.2
AA 300
0.1 AA 1000
0 Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Day 8
Figure 1 Mean rat paw edema induced by formalin during 8 days in the presence of different concentrations of A. arbusculinus gum extract. N.S., normal saline; S.S., sodium salicylate. p < 0.05.
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4 Shojaii et al.: Anti-inflammatory and analgesic activities of A. arbusculinus gum
Table 3 Mean latency time at 0, 30, 60, and 120 min after Astragalus gum extract and drug injections. Latency time, mean ± SE
Groups N.S. S.S. M.S. Extract 100 Extract 300 Extract 1000
0
30
60
120
6.9 ± 0.35 7.5 ± 0.22 7.8 ± 0.35 7.9 ± 0.81 8.9 ± 0.57 9.4 ± 0.61
6.8 ± 0.31 11.9 ± 0.15 15.5 ± 0.47 8.2 ± 0.4 12.3 ± 0.38 12.5 ± 0.45
6.7 ± 0.32 11.8 ± 0.22 15.28 ± 0.46 9.7 ± 0.18 11.8 ± 0.01 13.5 ± 0.22
7.1 ± 0.31 11.6 ± 0.24 14.7 ± 0.53 10.2 ± 0.11 12.5 ± 0.17 14.9 ± 0.13
M.S., morphine sulfate; N.S., normal saline; S.S., sodium salicylate.
Discussion and conclusion The A. arbusculinus (Fasciculifolius), Family Papilionaceae, gum (called anzaroot) has been used as an analgesic and anti-inflammatory agent and in orthopedics in Iranian traditional medicine [5, 6]. In this study, the antiinflammatory and analgesic effects of the aqueous extract of A. arbusculinus gum were evaluated using the formalin and hot-plate tests. The findings of formalin-induced inflammation have shown acute and chronic anti-inflammatory activities of the gum extract in a dose-dependent manner. The results of the hot-plate test also revealed a dose-dependent analgesic effect of the gum extract comparable to sodium salicylate and morphine. An inflammatory process is a dynamic complex sequence of highly organized, tightly regulated events,
Normal saline
Sodium salicylate
AA100
ultimately resulting in wound healing or granulation tissue. The inflammation that is initiated by homeostasis and coagulation continues with the recruitment of polymorphonuclear leukocytes and macrophages. These leukocytes, via the activation of the nuclear factor-κB (NF-κB) gene, enhance the release of interleukin (IL)1α, IL-1β, IL-6, and tumor necrosis factor-α (TNF-α) to maintain the inflammatory phase [14, 15]. The synthetic drugs of NSAIDs and corticosteroids suppress leukocyte migration and proinflammatory cytokine release [16]. There are in vivo and in vitro evidence that Astragalus extract has the same immunomodulatory properties. It has been demonstrated that Astragalus extract attenuates the elevated myeloperoxidase (MPO) activity and down-regulates the inducible nitric oxide synthase, P-selectin, and intercellular cell adhesion molecule-1 protein expression [17]. The Astragalus extract also inhibits the production of interferon (IFN)-γ by CD4+ lymphocytes in a similar way to prednisolone [18]. Astragalus extracts, via p38 mitogen-activated protein kinase and NF-κB signaling pathways, inhibits IL-1β and TNF-α secretion in macrophages [19]. In vivo studies have also revealed its anti-inflammatory effects in arthritis via reduced lymphocyte recruitment and TNF-α and IL-1β production and in colitis via the amelioration of elevated colonic MPO activity, reducing overexpressed TNF-α and IL-1β and restoring IL-10 expression; its antiatherosclerotic function via the decreased expression of adhesion molecules; and its antihepatocellular carcinoma effects via increased proapoptotic protein Bax and decreased
AA300
AA1000
Morphine
18 16
Mean analgesia time, s
14 12 10 8 6 4 2 0
0
30 min
60 min
120 min
Figure 2 Mean latency time after treatment by the extract of A. arbusculinus in animal models at 30, 60, and 120 min after injection. Data are mean ± SE (n = 7). *p < 0.05, Compared with normal saline group. †, Compared with positive control group.
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Shojaii et al.: Anti-inflammatory and analgesic activities of A. arbusculinus gum 5
antiapoptotic protein Bcl-2 [20–22]. Our current study is another proof of A. arbusculinus in vivo anti-inflammatory effects in a dose-dependent manner. Inflammatory pain is generated in primary sensory neurons (nociceptors) and transmitted via mediators such as glutamate, substance P, VIP, somatostatin, ATP, and neural growth factor to dorsal horn neurons. Additionally, the release of prostaglandins, bradykinin, and proinflammatory cytokines (IL-1, IL-6, IL-8, and TNF-α) in the inflammatory process produces hyperalgesia [23]. NSAIDs, opioids, and cannabinoids are the main antinociceptive drugs in inflammatory pain processes [24]. Our results showed the antinociceptive effects of the different doses of A. arbusculinus, but its mechanism of action still needs to be described. In addition, previous studies have suggested that primarily central nervous system (CNS)acting drugs are inhibitors of both early (zero time) and late (120 min time) phases of nociception; in other words, they equally suppress inflammatory and neurogenic nociception [25, 26]. Astragalus arbusculinus extract has antinociceptive characteristics in both phases, so it is probably a primarily CNS-acting drug. NSAIDs and steroids are examples of peripherally acting drugs. Our current study has shown that the aqueous extract of A. arbusculinus gum has dose-dependent anti-inflammatory effects in paw edema. In addition, A. arbusculinus gum extract exhibited dose-dependent antinociceptive properties probably through the suppression of both peripheral and central nociceptors. No side effects of A. arbusculinus gum extract were observed during the study. Therefore, A. arbusculinus gum extract might be useful in the treatment of inflammatory diseases. Further studies are needed to investigate this hypothesis. Up to now, different species of Astragalus showed analgesic, antinociceptive, and anti-inflammatory activities in animal models and their active compounds were determined in complementary studies. For example, some fraction and compounds isolated from the roots of Astragalus membranaceus have shown anti-inflammatory activity in an in vitro study [27]. In another study, Astragalus siculus showed anti-inflammatory and analgesic effects in animal models [28]. The anti-inflammatory activity of Astragalus hamosus was determined in a study by Hakim et al. [29]. So far, different compounds were isolated from Astragalus species. Among these compounds, terpenoids (especially saponins) and polysaccharides have shown significant anti-inflammatory and analgesic effects in experimental models [30, 31]. Due to the preliminary phytochemical study of the gum extract of A. arbusculinus (mentioned above), the anti-inflammatory and analgesic
effects of the gum may be due to the presence of these compounds. Because no phytochemical study was performed on A. arbusculinus to purify the active compounds, further studies can determine the active principles of this plant, which may responsible for the analgesic and antiinflammatory activities, such as in polysaccharides, saponins, or flavonoids. Acknowledgments: This work was supported by the Research Institute for Islamic and Complementary Medicine, Iran University of Medical Sciences (Grant Number 14513). Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. Research funding: None declared. Employment or leadership: None declared. Honorarium: None declared. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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