Inflammopharmacol (2014) 22:105–114 DOI 10.1007/s10787-013-0196-2

Inflammopharmacology

RESEARCH ARTICLE

Analgesic and antiinflammatory activities of the ethyl acetate fraction of Bidens pilosa (Asteraceae) Aure´lien Fotso Fotso • Frida Longo • Paul De´sire´ Dzeufiet Djomeni • Sime´on Fogue Kouam • Michael Spiteller • Alain Bertrand Dongmo • J. P. Savineau

Received: 20 August 2013 / Accepted: 23 October 2013 / Published online: 16 November 2013 Ó Springer Basel 2013

of Bidens pilosa has both analgesic and antiinflammatory properties. The qualitative analysis of the fraction by the high-performance liquid chromatography (HPLC) fingerprint revealed the presence of two flavonoids, namely quercetin and iso-okanin, known to have antiinflammatory and antinociceptive properties, which could be responsible for the analgesic and antiinflammatory effects observed.

Abstract Bidens pilosa is an Asteraceae widely used in traditional medicine for the treatment of various ailments including pain and inflammation. The present work was undertaken to assess the analgesic and antiinflammatory properties of the ethyl acetate fraction of methylene chloride/methanol (1:1) extract of leaves of Bidens pilosa at the gradual doses of 50, 100 and 200 mg/kg in mice and rats, respectively. The analgesic properties of Bidens pilosa were investigated using the acetic acid writhing, hot plate, capsaicin and formalin-induced pain models. This was followed by a study of the antiinflammatory properties using carrageenan, dextran, histamine and serotonin to induce acute inflammation in rat hind paw. The extract provided a significant (p \ 0.01) reduction in pain induced by all four models of nociception. It also presented significant (p \ 0.05) antiinflammatory activity in all four models of acute inflammation. These results show that the ethyl acetate fraction of methylene chloride/methanol (1:1)

Keywords Bidens pilosa  Analgesic  Antiinflammatory  Flavonoids

A. F. Fotso  F. Longo (&) Laboratory of Animal Physiology, Department of Biological Science, Higher Teachers’ Training College, University of Yaounde´ I, P.O. Box 47, Yaounde´, Cameroon e-mail: [email protected]

M. Spiteller Institute of Environmental Research (INFU) of the Faculty of Chemistry, TU Dortmund, Otto-Hahn-Str. 6, 44221 Dortmund, Germany e-mail: [email protected]

A. F. Fotso e-mail: [email protected] P. D. D. Djomeni (&) Laboratory of Animal Physiology, Department of Animal Biology and Physiology, Faculty of Science, University of Yaounde´ I, P. O. Box 812, Yaounde´, Cameroon e-mail: [email protected] S. F. Kouam Laboratory of Organic Chemistry, Department of Chemistry, Higher Teachers’ Training College, University of Yaounde´ I, P.O. Box 47, Yaounde´, Cameroon e-mail: [email protected]

Introduction Inflammation is the response of living tissues to infections and injuries. The hallmarks of inflammation include pain and swelling (Tracy 2006). The inflammatory response is basically a protective reaction that is deeply associated

A. B. Dongmo Laboratory of Animal Organism Biology, Faculty of Science, University of Douala, P.O. Box 24157, Douala, Cameroon e-mail: [email protected] J. P. Savineau Cardiothoraxic Research Center of Bordeaux (INSERM U 1045), University of Victor Segalen Bordeaux 2, 146, street Le´o Saignat, 33076 Bordeaux cedex, France e-mail: [email protected]

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with tissue repair. Nevertheless, inflammation can be potentially harmful. As reflected by its cardinal signs (redness, pain, heat, swelling and loss of function), inflammation is frequently an extremely dynamic and violent process (Honsoul et al. 2012). Most human diseases have a pain and inflammation component, which leads individuals to seek medical attention (Merskey 1986). As such, analgesic and antiinflammatory drugs are among the most prescribed drugs in clinical practice (Lim and Yap 1999). Despite the progress in the discovery of antiinflammatory and analgesic drugs, the chronic use of these drugs is hampered by their adverse effects such as gastric lesions or tolerance, as seen with NSAIDs and opiate analgesics, respectively. Therefore, it is important to search for potent analgesic and antiinflammatory drugs with fewer adverse effects from plant sources. One such potential plant is Bidens pilosa. B. pilosa is a perennial weed herb used in Cameroonian folk medicine. B. pilosa, either as a whole plant or different parts, has been reported to be useful in the treatment of more than 40 disorders such as inflammation, immunological disorders, digestive disorders, infectious diseases, cancers, metabolic syndrome, wounds and many others (Bartolome et al. 2013). Previous studies have been carried out, for example, on the antiinflammatory effect of leaf aqueous extracts (Masako and Yoshiyuki 2006). In Cameroon, the main use is for the management of inflammation, pain, fever, headaches and stomachaches (Adjanohoun et al. 1996). In our laboratory, different Bidens pilosa leaf extracts have been tested. Their antisecretory and antiulcerogenic properties were demonstrated (Tan et al. 2000). A hypotensive effect has been shown on hypertensive-induced rats (Dimo et al. 2001). Also an antimalarial property was demonstrated (Mbacham et al. 2005). The mechanisms of ocytocic action of aqueous and alcohol extracts on contractile activity of smooth uterine muscle have been tested (Longo et al. 2007; Longo et al. 2008; Longo et al. 2011). In the present study, we investigated the antinociceptive and antiinflammatory properties of the ethyl acetate fraction of methylene chloride/methanol (1:1) extract of the leaves in mice and rats, respectively. By the higher performance liquid chromatography fingerprint, some molecules were determined that could be responsible for the analgesic and antiinflammatory effects.

Materials and methods Preparation of the ethyl acetate fraction of the leaves of Bidens pilosa B. pilosa plants were collected from a suburb of Yaounde (Eleveur) in September 2011 and authenticated at

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the National Herbarium of Cameroon by comparison to the voucher specimen (no. 65112/HNC) deposited in 2005. The ethyl acetate fraction was prepared by macerating 1,000 g of air-dried leaves for 2 days in 5 l of methylene chloride/methanol (1:1). After filtration, the collected extract was concentrated using a HEIDOLPH W2000 rotary evaporator, giving about 60 g of greenish dough. This extract was exhausted in 500 ml of ethyl acetate, and after concentration using a rotary evaporator, it gave 12.7 g of ethyl acetate fraction of B. pilosa (The´ophile et al. 2006). A stock solution concentrated at 20 mg/ml was prepared daily by dilution with 1 % DMSO. Animals Mus musculus mice of both sexes aged between 80 and 90 days and weighing 20–30 g, and Wistar rats weighing 100–140 g, aged between 50 and 60 days, were obtained from the university’s animal holding facility. Animals were housed in standard environmental conditions under a 12:12 h light/dark natural cycle. All animals had free access to water and standard rat chow. In all experiments, each group was made up of five animals. Prior authorization for the use of laboratory animals was obtained from the Cameroon National Ethics Committee (ref. no. FWIRB00001954). Drugs and chemicals Indomethacin (Sigma), tramadol, acetyl salicylic acid, promethazin, cyproheptadin, carrageenan (Sigma), dextran (Sigma), histamine (Sigma), serotonin (Sigma) and capsaicin were used. Pharmacological experiments All reference drugs and the extract were administered by the oral route. Analgesic activity Acetic acid-induced writhing assay Mice were divided into five groups of five animals each and selected as described by Collier et al. (1968). The three first groups received different doses of the ethyl acetate fraction of B. pilosa (50, 100 and 200 mg/kg), the fourth group received the standard drug, acetylsalicylic acid (20 mg/kg), and the last group received vehicle (1 % DMSO). Writhing was induced by administering 10 ml/kg of acetic acid solution (0.7 %) by the intraperitoneal route

Analgesic and antiinflammatory activities

30 min after treatment. After acetic acid injection, the mice were observed in a transparent box, and the number of writhes was counted for a period of 15 min. The percentage inhibition of writhing was calculated using the following ratio: (control mean–treated mean)/control mean 9 100) (Koster et al. 1959).

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Antiinflammatory activity Evaluation of antiinflammatory properties of B. pilosa extract was carried out by phlogistic substances (carrageenan, dextran, histamine and serotonin) in rats. Carrageenan-induced paw edema assay

Hot-plate assay Five groups of mice were given EABp at doses of 50, 100 or 200 mg/kg, DMSO 1 % (control) and the last received the standard drug tramadol (20 mg/kg). Thirty minutes after injection, mice were placed into a 10-cm-wide glass cylinder, thermostatically set at 55 ± 0.5 °C in a precision water bath (Galeotti et al. 1997). Latency to lick a hind paw or jump out of the apparatus was recorded with a stopwatch for the control and drug-treated groups. Capsaicin-induced neurogenic pain The capsaicin-induced pain test was performed in mice that had been individually exposed to the previously described observation chamber for 5 min. Using a minimum of restraint, 20 ll of 32 mg/ml capsaicin in 0.9 % saline was injected subcutaneously into the dorsal hind paw of mice pretreated (30 min) with EABp (50, 100 and 200 mg/kg b.w.) and vehicle (DMSO 1 %) or the standard drug tramadol (20 mg/kg). The mouse was then put back into the chamber, and the time the animal spent licking the injected paw or leg was recorded for the first 5 min after capsaicin injection (Sakurada et al. 1992). Formalin-induced pain This test was carried out as described by Hunskaar and Hole (1987). Animals were injected subcutaneously with 20 ll of formalin into the dorsal hind paw. EABp (50, 100 and 200 mg/kg), vehicle (DMSO 1 %) and acetyl salicylic acid (20 mg/kg) were administered 30 min before formalin injection. The time the mice spent licking or biting the injected paw or leg was recorded. On the basis of the response pattern described by Dongmo et al. (2005), two distinct periods of intensive licking activity were identified. The first period (early phase) was recorded 0–5 min after the injection of formalin, and the second period (late phase) was recorded 20–30 min after the injection. The percentage inhibition of licking was calculated by the formula (C-T)/C 9 100; where C represents the vehicle-treated control group value for each phase and T represents the treated group value for each phase (Hunskaar and Hole 1987).

This test is based on the thickness variation of the back paw of the animals after the application of carrageenan (Winter et al. 1962). The amount of edema was measured with a digital caliper as described by Favacho et al. (2011). Thus, the different experimental groups (5 rats/group) were treated topically with EABp (50, 100 and 200 mg/kg b.w.), indomethacin (10 mg/kg positive control) or DMSO 1 % (negative control). To induce edema, 0.1 ml subplantar injection of 1 % carrageenan was applied to the posterior right paw of each rat, 1 h after treatment of each group. The paw thickness (mm) of rats was measured before the application of the proinflammatory substance and hourly for up to 6 h after application of the edematogenic stimulus with a digital caliper (Stainless Hardened). Percentages of inhibition were obtained for each group using the following ratio: [(Tt–To)control-(Tt–To)treated]/(Tt–To)control 9 100, where Tt is the average thickness for each group and To the average thickness obtained for each group before any treatment. Dextran-induced rat hind paw edema assay The animals were treated as during the carrageenaninduced paw edema assay where dextran (0.1 ml, 1 % w/v in normal saline) was used in the place of carrageenan (Mandal et al. 2000). Histamine-induced rat hind paw edema assay The right feet of the rat were treated by subplantar injection of 0.1 ml of 1 % w/v of freshly prepared histamine in normal saline (Mandal et al. 2000). The positive control received the standard drug promethazin (1 mg/kg). Serotonin-induced rat hind paw edema assay In the serotonin model, edemas of the right hind paw of the rat were induced by subplantar injection of 0.1 ml of 1 % w/v freshly prepared serotonin in normal saline. The groups and the treatment procedure of the animals were the same as for the carrageenan-induced rat hind paw edema model, and the paw volume was observed (Mandal et al. 2000). The positive control group received the standard drug cyproheptadin (2 mg/kg).

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High-performance liquid chromatography (HPLC) fingerprint of the ethyl acetate fraction from Bidens pilosa The high-resolution mass spectra were obtained with an LTQOrbitrap Spectrometer (Thermo Fisher, USA) equipped with HES I-II source. The spectrometer was attached with an Agilent (Santa clara, USA) 1200 HPLC system consisting of an LC pump, PDA detector (k = 205 nm), auto sampler and column oven. The analysis was performed using a Nucleodur Gravity column (1.8 lm particle size) from Macherey–Nagel (Du¨ren, Germany) with a H2O (?0.1 % HCOOH ? 10 mM NH4Ac) (A)/acetonitrile (?0.1 % HCOOH) (B) gradient (flow rate 300 ll/min). Samples were analyzed by using a gradient program as follows: 90 % A isocratic for 2 min, linear gradient to 100 % B over 13 min, after 100 % B isocratic for 5 min, the system returned to its initial condition (90 % A) within 0.5 min and was equilibrated for 4.5 min. After obtaining a chromatogram, the chemical structures were drawn using the Chemdraw software.

Fig. 1 Effects of AEBp on acetic acid-induced abdominal contractions. Each column represents the mean of five animals ± SEM. ***p \ 0.001, significantly different from the negative control group. CN negative control, B.p Bidens pilosa, ASP aspirin

Statistical analysis The Sigma Stat software version 2.03 was used for statistical analysis. The analysis of variance test (one-way) was used to calculate the statistically significant differences between groups and was followed by Student’s t test. Results obtaining a p \ 0.05 were considered statistically significant (Asongalem et al. 2004).

Fig. 2 Effect of AEBp on reaction time when animals were placed on the hotplate. Each column represents the mean of five animals ± SEM. ***p \ 0.001, significantly different from the negative control group. CN negative control, B.p Bidens pilosa, TRA tramadol

Results Analgesic effects of the ethyl acetate fraction of B. pilosa The extract at doses of 50, 100 and 200 mg/kg b.w. showed a significant (p \ 0.01) antinociceptive effect in all four different models for nociception. Acetic acid-induced writhing The EABp showed a significant (p \ 0.001) inhibition of acetic acid-induced writhes in mice at all of doses compared to controls. However, the effect of AEBp was greater than that observed for the non-steroidal antiinflammatory drug acetylsalicylic acid (20 mg/kg) (Fig. 1). Hot plate test EABp compared with controls delayed the reaction time on the hot plate. Thus, mean reaction time ± SEM was 4.80 ± 0.33 (s) in the control group, and it increased

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Fig. 3 Effect of AEBp on reaction time when animals were injected by capsaicin. Each column represents the mean of five animals ± SEM. ***p \ 0.001, significantly different from the negative control group. CN negative control, B.p Bidens pilosa, TRA tramadol

significantly (p \ 0.001) in a dose-dependent manner to 12.40 ± 0.36 (s), 18.40 ± 0.22 (s) and 20.80 ± 0.33 (s) in groups treated with 50, 100 or 200 mg/kg EABp

DMSO 1 % dimethyl sulfoxyde 1 %; B. pilosa Bidens pilosa; Indo indomethacin The value in brackets represents the inhibition percentage

(55.68) (59.07) (64.51)

(51.42)

1.56 ± 0.17** 0.94 ± 0.10**

(48.13) (46.77)

0.44 ± 0.04** 10 Indo

(11.93)

0.66 ± 0.08**

1.71 ± 0.19**

(12.15)

1.11 ± 0.09**

(25)

200

3.10 ± 0.46 1.88 ± 0.13 100

0.93 ± 0.13

(9.37) (6.07) (12.09)

3.48 ± 0.30

3.19 ± 0.14 2.01 ± 0.06

2.12 ± 0.05

1.09 ± 0.09

1h 0.5 h

Paw thickness differences (mm)

1.22 ± 0.15 0

50

Table 1 shows that the administration of carrageenan (0.1 %) produced visible edema in the paw of rats that was

DMSO 1 %

Carrageenan-induced rat hind paw edema

B. pilosa

Antiinflammatory effects of the ethyl acetate fraction of B. pilosa

Dose (mg/kg)

The nociceptive response of control mice lasted 42.80 ± 0.75 (s) after the first phase (neurogenic pain) and 12.60 ± 0.50 after the second phase (inflammatory pain). Treatment of mice with EABp (50, 100 or 200 mg/kg) significantly reduced (p \ 0.01) the time spent licking the paw following either phase of the formalin-induced nociception. Acetyl salicylic acid (20 mg/kg body weight) used as a positive control caused a significant reduction (p \ 0.001) by 42.86 % only in the second phase (Fig. 4).

Treatment

Formalin-induced pain

Table 1 Effects of EABp on carrageenan-induced rat hind paw edema

The time spent licking the paw following capsaicin injection was significantly (p \ 0.001) and dose dependently reduced by the administration of EABp (50, 100 or 200 mg/kg) from control values of 59.40 ± 1, 21 (s) to 54.00 ± 0.55 (s), 49.80 ± 0.58 (s) and 45.40 ± 0.51 (s), respectively. Tramadol (20 mg/kg), the positive control for the test, significantly reduced (p \ 0.001) the licking time of mice by 41.20 ± 0.37 (s) (Fig. 3).

2h

Capsaicin-induced hind paw licking

Values are mean ± SEM (n = 5) for control and mean ± SEM (% of inhibition) (n = 5) for each treatment, * p \ 0.05; ** p \ 0.01 experimental groups compared with the control group

0.28 ± 0.05**

(52.87)

(91.95) (84.61) (73.14) (72.76)

0.44 ± 0.06**

(40.90) (43.14)

0.94 ± 0.25** 1.28 ± 0.03**

(58.51)

1.64 ± 0.33*

(23.56) (13.63)

1.69 ± 0.26* 1.99 ± 0.15**

(8.86) (10)

1.95 ± 0.19**

2.66 ± 0.20 2.47 ± 0.18 3.19 ± 0.10 4.23 ± 0.28*

2.72 ± 0.19

(21.83) (3.49) (7.71) (8,51)

2.76 ± 0.12

2.82 ± 0.25 3.48 ± 0.09

3.23 ± 0.09 4.30 ± 0.12

3h

(p \ 0.001). Tramadol (20 mg/kg), the positive control of the test, significantly increased (p \ 0.001) the latency time to jump to 18.00 ± 0.28 (s) (Fig. 2).

4.68 ± 0.33

5h 4h

Fig. 4 Effect of AEBp on reaction time when animals were injected by formalin. Each column represents the mean of five animals ± SEM. ***p \ 0.001; **p \ 0.01, significantly different from the negative control group. CN negative control, B.p Bidens pilosa, ASP aspirin

3.50 ± 0.59

109

6h

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Fig. 5 Effect of EABp on dextran-induced rat hind paw edema. Each column represents the mean of five animals ± SEM. *p \ 0.05; **p \ 0.01, significantly different from the negative control group. CN negative control, B.p Bidens pilosa, Indo indomethacin

measurable 30 min after induction with a maximal peak after 3 h. EABp treatment (200 mg/kg) 30 min prior to the application of the edematogenic stimulus (carrageenan) antagonized the formation of edema during the 6-h experiment and reached a maximum peak of edema after 3 h, when the inhibition was 58.51 % (p \ 0.01). The group treated with topical indomethacin at 10 mg/kg had a 91.95 % inhibition of the maximal peak of edema (p \ 0.01) at the 6th hour. Dextran-induced rat hind paw edema There was a dose-dependent reduction in dextran-induced rat paw edema at 50, 100 and 200 mg/kg b.w. of EABp. The doses of 200 mg/kg b.w. significantly (p \ 0.05) decreased in the dextran-induced paw edema with the maximal inhibition percentage of 39.26 % at ‘ h as shown in Fig. 5. Indomethacin (10 mg/kg b.w.) showed a maximum inhibition of 59.11 % at the 2nd hour.

Fig. 6 Effect of EABp on histamine-induced rat hind paw edema. Each column represents the mean of five animals ± SEM. *p \ 0.05, **p \ 0.01, significantly different from the negative control group. CN negative control, B.p Bidens pilosa, Pro promethazin

Histamine-induced rat hind paw edema EABp at the doses of 100 and 200 mg/kg significantly (p \ 0.05) reduced the edema formation of the rat paw 1 h after histamine injection. The effects were dose-dependent at the doses tested (50, 100 and 200 mg/kg b.w,) with an inhibitory percentage of 10.86, 21.39 and 32.18 %, respectively (Fig. 6). Standard drug promethazin (1 mg/kg) significantly decreased (p \ 0.01) the paw edema with a maximum inhibition of 41.48 % 1 h after injection. Serotonin-induced rat hind paw edema EABp at the dose of 100 and 200 mg/kg b.w. significantly inhibited (p \ 0.05) serotonin-induced rat paw edema with an inhibition percentage of 56.11 and 44.60 %, respectively. Standard drug cyproheptadin (2 mg/kg b.w.) significantly (p \ 0.01) decreased the paw edema with a maximum inhibition of 56.11 % (Fig. 7).

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Fig. 7 Effect of EABp on serotonin-induced rat hind paw edema. Each column represents the mean of five animals ± SEM. *p \ 0.05, **p \ 0.01, significantly different from the negative control group. CN negative control, B.p Bidens pilosa, Cyp cyproheptadin

High-performance liquid chromatography (HPLC) fingerprint of the ethyl acetate fraction from Bidens pilosa After 25 min of separation, the HPLC fingerprint coupled to the mass spectrometer showed the chromatogram of the ethyl acetate fraction of Bidens pilosa leaves with the peaks showing the relative abundance of different compounds of the fraction (Fig. 8). The study of the chromatogram permitted

Analgesic and antiinflammatory activities

111

RT: 0,0000 - 24,9965 100

NL: 3,55E8 TIC MS bpae

15,1243 17,0575

90

Relative Abundance

80 70 17,4874

60 50 40

17,9662

21,5649

11,1387

30

10,4274 8,7309 1,0058

20

7,1756

6,3883

23,6075

10 0 0

5

10

15

20

Time (min) Fig. 8 HPLC fingerprint chromatography of the ethyl acetate fraction of Bidens pilosa

(a)

(b)

Fig. 9 Chromatogram and mass spectra of quercetin 3, 30 -dimethyl ether 7-0-b-D-glucopyranoside FT-APCI-MS [M ? H]?: m/z 493.1340 (C23H25O12, 493.1346) (a) and chromatogram and mass

spectra of iso-okanin 7-O-b-D-(200 ,400 ,600 -triacetyl)-glycopyranoside, FT-APCI-MS [M ? H] ?: m/z 577.1557 (C27H29O14, 577.1557) (b) 0.0

identifying two known flavonoids, namely quercetin 3,30 dimethyl ether 7-0-b-D-glucopyranoside, retention time 9.83 min, previously isolated and identified by Brandao et al.

(1998) (A), and iso-okanin 7-O-b-D-(200 ,400 ,600 -triacetyl)glycopyranoside, retention time 11.14 min, previously isolated and identified by Wang et al. (1997) (B) (Fig. 9).

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Discussion and conclusion In this study, we evaluated the analgesic and antiinflammatory effects of EABp. For analgesic effects, we used chemical and thermal models of nociception. Acetic acid induces pain by the release of endogenous mediators of pain such as prostaglandins through the activity of the enzyme cyclooxygenase (COX) (Satyanarayana et al. 2004). As such, this model of nociception is one of peripherally mediated pain and therefore should be effectively inhibited by peripheral analgesics through COX inhibition. Our results therefore show that EABp has peripheral analgesic properties similar to acetyl salicylic acid, probably due to inhibition of COX activity and further inhibition of the release of other endogenous pain mediators. However, acetic acid-induced writhing is a non-specific test, responding to analgesics as well as other classes of drugs such as anticonvulsants (Meymandi and Sepehri 2008). Two methods for testing the effects of EABp on central pain were used. The EABp showed a dose-dependent analgesic effect in the hot plate test. This analgesic activity was similar to the effect of tramadol (20 mg/kg). In the capsaicin test, EABp showed a dose-dependent decrease in time spent licking the paw like for tramadol (20 mg/kg). The fact that pre-treatment of mice with EABp inhibited neurogenicinduced pain is an indication of the centrally acting properties of the plant extract. Paola et al. (1997) showed that neurogenic-induced pain may result in stimulation of either Ad or C fibers, or both. Thus, the central analgesic properties of EABp may be due to the inhibition of either Ad or C fibers, or both. Similar results were obtained by Manoj et al. (2012) in the study of the analgesic activity of ethanolic extract of Zizyphus nummularia. Paw formalin injection produces two distinct phases of pain-like behavior: the first phase (0–5 min), followed by a second (20–30 min) after formalin injection. The early phase of intensive pain, which starts immediately after formalin injection, seems to be caused predominantly by activation of C-fibers subsequent to peripheral stimulation (Dongmo et al. 2005). The late phase of moderate pain, which started 20 min after formalin injection and lasted 30 min, appears to be caused by tissue and functional changes in the dorsal horn of the spinal cord (Coderre et al. 1990). In this study, EABp significantly inhibited both phases. Thus, the result obtained from the present study indicates that EABp relieved the pain through both central and peripheral mechanisms. Similar results were obtained by Al Amin et al. (2012) in the study of the analgesic activity of an ethanol extract of Asteracantha longifolia. Carrageenan-induced paw inflammation in rats is considered to be one of the best methods for screening for antiinflammatory properties of a drug (Morris 2003). This model causes an inflammatory response that occurs in three

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phases: the first phase occurs within the first hour post carrageenan injection and is characterized by the release of histamine and 5-HT. The second phase is mediated by kinins, and finally in a third phase, the mediator is suspected to be prostaglandin (Dongmo et al. 2005). Like indomethacin, EABp (200 mg/kg) was effective against all three phases of carrageenan-induced inflammation. Similar results were obtained by Nguemfo et al. (2009) in the study of antiinflammatory activity of some isolated constituents from the stem bark of Allanblackia monticola. Dextraninduced edema is a well-known experimental model in which the edema is a consequence of liberation of histamine and serotonin from mast cells (Sharma et al. 2010). At 200 mg/kg, the EABp inhibited dextran-induced rat paw edema like indomethacin, a reference drug. Dextran induces fluid and accumulated because of mast cell generation in protein-rich exudate containing a large number of neutrophils (Lo et al. 1982). A dose of 200 mg/kg significantly inhibited (p \ 0.05) histamine and serotonin-induced paw edema in rats. Similar results were obtained by Fotio et al. (2009) in the study of the antiinflammatory property of stem bark aqueous and methanol extracts of Sclerocarya birrea and by Rajavel et al. (2012) in the study of the antiinflammatory effect of a methanolic extract of Oscillatoria annea. These results corroborate those obtained by Masako and Yoshiyuki (2006) in the evaluation of the antiinflammatory and antiallergic activities of leaf aqueous extract of Bidens pilosa, which inhibited dye exudation in rat skin induced by passive cutaneous anaphylaxis and chemical mediators (histamine, serotonin and substance P). However, it is important to highlight the fact that the method used by those authors was less convenient as animals were killed during the experiment. In our study, we also obtained a therapeutic effect at the dose of 50 mg/kg, while a dose of 250 mg/kg was needed by Masako and Yoshiyuki. The qualitative analysis of the fraction by high-performance liquid chromatography (HPLC) coupled to a mass spectrometer identified two bioactive flavonoids of Bidens pilosa, namely quercetin 3,30 -dimethyl ether 7-0-b-Dglucopyranoside, previously isolated and identified (Brandao et al. 1998; Kviecinski et al. 2011), and iso-okanin 7-O-b-D-(200 , 400 , 600 -triacetyl)-glucopyranoside, previously isolated and identified (Wang et al. 1997). These bioactive molecules are known for their antiinflammatory effect (Di Carlo et al. 1999; Toker et al. 2004). Inhibition of cyclooxygenase and lipoxygenase (Robak and Gryglewski 1996) was responsible for the observed antiinflammatory and analgesic effects. It is also reported that quercetin is an effective inhibitor of phospholipase A2, which catalyzes the hydrolysis of phospholipids to release arachidonic acid as the precursor of the inflammatory response (Harborne 1994). Phenolic compounds such as gallic acid could also

Analgesic and antiinflammatory activities

be responsible for the antiinflammatory activity since they inhibit mRNA induction of proinflammatory cytokines (IL1, IL-6), chemokines (CCL-2/MCP-1, CCL-7/MCP-3) cyclo-oxygenase-2 and metalloprotease (Yoon et al. 2013). Therefore, we could draw the conclusion that flavonoids might be one of the main antiinflammatory compounds in the ethyl acetate fraction of Bidens pilosa leaves. These findings may scientifically justify the use of Bidens pilosa for the treatment of pain and inflammation.

References Adjanohoun JE, Aboubakar N, Dramane K et al (1996) Traditional medicine and pharmacopoeia: contribution to ethnobotanical and floristic studies in Cameroon. Organisation of African Unity Scientific, Technical and Research Commission, CNPMS, PortoNovo, p 77 Al Amin Md, Chowdhury IA, Mahbub KMM et al (2012) Antiinflammatory and analgesic activities of Asteracantha longifolia Ness. Ban J Pharm 15(2):171–176 Asongalem EA, Foyet HS, Ekobo S et al (2004) Antiinflammatory, lack of central analgesia and antipyretic properties of Acanthus montanus (Ness) T Anderson. J Ethnopharmacol 95:63–68 Bartolome AP, Villasen˜or IM & Yang WC (2013). Bidens pilosa L. (Asteraceae): Botanical properties, traditional uses, phytochemistry, and pharmacology. Evidence-based complementary and alternative medicine, 340215: 51 Brandao MGL, Nery CGC, Mamao MA et al (1998) Two methoxylated flavones glycosides from Bidens pilosa. Phytochem 48:397–399 Coderre TJ, Vaccarino AL, Melzack R (1990) Central nervous system plasticity in the tonic pain response to subcutaneous formalin injection. Brain Res 535:155–158 Collier HT, Dinnen LC, Johnson CA et al (1968) The abdominal constriction response and it suppression by analgesic drugs in mouse. Br J Pharmacol Chemother 32:295–310 Di Carlo G, Mascolo N, Izzo AA, Capasso F (1999) Flavonoids: old and new aspects of a class of natural therapeutic drugs. Life Sci 65(4):337–353 Dimo T, Azay J, Tan PV et al (2001) Effects of the aqueous and methylene chloride extracts of Bidens pilosa leaf on fructosehypertensive rats. J Ethnopharmacol 76(3):215–221 Dongmo AB, Nguelefack TB, Lacaille-Dubois MA (2005) Antinociceptive and anti-inflammatory activities of Acacia pennata wild (Mimosaceae). J Ethnopharmacol 98:201–206 Favacho HAS, Oliveira BR, Santos KC et al (2011) Anti-inflammatory and antinociceptive activities of Euterpe oleracea Mart., Arecaceae, oil. Rev Bras Pharmacol 21(1):1015–1022 Fotio LB, Dimo T, Nguelefack TB et al (2009) Acute and chronic anti-inflammatory properties of stem bark aqueous and methanol extracts of Sclerocarya birrea (Anacardiaceae). Inflammopharmacol 17:229–237 Galeotti N, Ghelardini C, Papucci L et al (1997) An antisense oligonucleotide on the mouse shaker-like potassium channel kv1.1 gene prevents antinociception induced by morphine and baclofen. J Pharmacol Exp Ther 281:941–949 Harborne JB (1994) The Flavonoids: Advances in Research since 1986. Chapman & Hall, London Honsoul K, Raghu PK, Gou YK (2012) Regulation and implications of inflammatory lymphangiogenesis. Trends Immunol 33(7):350–356

113 Hunskaar HS, Hole K (1987) The formaline test in mice: dissociation between inflammatory and non-inflammatory pain. Pain 30:103– 114 Koster R, Anderson M, De Beer EJ (1959) Acetic acid and analgesic screening. Proc Soc Exp Biol Med 18:412–415 Lim KHJ, Yap KB (1999) The prescription pattern of outpatient polyclinic doctors. Sing J Med 40:416–419 Lo TN, Almeida, Beavan MA (1982) Dextran and carrageenan evoke different inflammatory response in rat with respect to composition of infiltrates and effect of indomethacin. J Pharmacol Exp Ther 221:261–267 Longo F, Rakotonirina S, Rakotonirina A et al (2) Evidence of phospholipase C signaling pathway activation by Bidens pilosa leaf aqueous extract on the rat primed estrogenized myometrium. Pharmacol onl 2:12–27 Longo F, Rakotonirina S, Rakotonirina A et al (2008) In vivo and in vitro effects of Bidens pilosa L. (Asteraceae) leaf aqueous and ethanol extracts on primed-estrogenized rat uterine muscle. Afr J Tradit Complement Altern Med 5(1):79–91 Longo F, Wafo TP, Rakotonirina S et al (2011) Ocytocic Effects of Bidens pilosa on primed-estrogenised rat myometrial sarcoplasmic reticulum. Syllabus Rev Sci Ser 2(2):47–56 Mandal CS, Maity TK, Das J et al (2000) Anti-inflammatory evaluation of Ficus racemosa Linn leaf extract. J Ethnopharmacol 72:87–92 Manoj G, Sasmal D, Nagori BP (2012) Analgesic and anti-inflammatory activity of ethanolic extract of Zizyphus nummularia. Res J Med Pl 6(7):521–528 Masako H, Yoshiyuki S (2006) Anti-inflammatory and anti-allergic activities of Bidens pilosa L. var. radiata Scherff. J Health Sci 52(6):711–717 Mbacham W, Evehe M, Bilanda D et al (2005) Synergistic effects in vitro with chloroquine, of methanol extract of Bidens pilosa leaves on resistant Plasmodium falciparum isolate [MIM-BD3144]. J Act Trop 95:S342–S342 Merskey HM (1986) Pain terms. Pain 3:215–221 Meymandi MS, Sepehri G (2008) Gabapentin action and reaction on the antinociceptive effects of morphine on visceral pain in mice. Eur J Anaesthesiol 25:129–134 Morris CJ (2003) Carrageenan-Induced Paw Edema in the rat and mouse. Methods Mol Biol 225:115–121 Nguemfo EL, Dimo T, Dongmo AB et al (2009) Anti-oxydative and anti-inflammatory activities of some isolated constituents from the stem bark of Allanblackia monticola Staner L.C (Guttiferae). Inflammopharmacology 17:37–41 Paola S, Mauro B, Manfredi B et al (1997) Effects of tramadol on immune response and nociceptive thesholds in mice. Pain 72:325–330 Rajavel R, Mallika P, Rajesh V et al (2012) Antinociceptive and antiinflammatory effects of the methanolic extract of Oscillatoria annae. Res J Chem Sci 2(7):53–61 Robak J, Gryglewski RJ (1996) Bioactivity of flavonoı¨ds. Pol J Pharmacol 48(6):555–564 Sakurada T, Katsumata K, Tan-No K et al (1992) The capsaicin test in mice for evaluating tachykinin antagonists in the spinal cord. Neuropharmacology 31:1279–1285 Satyanarayana PSV, Jain NK, Singh S et al (2004) Effect of selective inhibition of cyclooxygenase-2 on lipopolysaccharide induced hyperalgesia. Inflammopharmacology 12:57–68 Sharma US, Sharma UK, Sutar N et al (2010) Anti-inflammatory activity of Cordia dichotoma forst f. seeds extracts. J Pharmac Analys 2(1):1–4 Tan PV, Dimo T, Dongo E (2000) Effects of methanol, cyclohexane and methylene chloride extracts of Bidens pilosa on various gastric ulcer models in rats. J Ethnopharmacol 73(3):415–421

123

114 The´ophile D, Laure NE, Benoıˆt NT et al (2006) Antinociceptive and anti-inflammatory effects of the ethyl acetate stem bark extract of Bridelia scleroneura (Euphorbiaceae). Inflammopharmacology 14:42–47 Toker G, Ku¨peli E, Memisog˘lu M et al (2004) Flavonoids with antinociceptive and anti-inflammatory activities from the leaves of Tilia argentea. J Ethnopharmacol 95(2–3):393–397 Tracy RP (2006) The five cardinal signs of inflammation: colour, dolor, rubor, tumor and penuria (apologies to Aulus Cornelius Celsus, de medica, c. 25). J Gerontol 61:1051–1052

123

A. Fotso et al. Wang J, Yang H, Lin ZW et al (1997) Flavonoids from Bidens pilosa Var. Radiata Phytochem 46:1275–1278 Winter CA, Risley EA, Nuss GW (1962) Carrageenan-induced edemas in hind paw of the rat as an assay for anti-inflammatory drugs. Proc Soc Exp Biol Med 111:544–547 Yoon CH, Chung SJ, Lee SW et al (2013) L’acide gallique, acide polyphe´nolique naturel, induit l’apoptose et inhibe l’expression des ge`nes pro-inflammatoires dans les synoviocytes fibroblastiques de polyarthrite rhumatoı¨de. Revue du Rhumatisme 80(3):271–278 In French