Abstract: Background and Objective: Several species of Conyza bonariensis (C. bonariensis) and related species are used traditionally in the management of pain and inflammatory conditions. The current study was aimed to evaluate the analgesic and anti-inflammatory activities of C. bonariensis in animal models of pain and inflammation. Methodology: For the analgesic effect, acetic acid and formalin-induced models of nociception were employed while the anti-inflammatory effect was assessed in the carrageenan-induced rat paw edema test. The data were analyzed by student’s t-test and one-way ANOVA followed by Tukey-Kramer multiple comparisons. Results: The intraperitoneal (i.p.) administration of the methanolic extract (50-100 mg kg–1), butanol, chloroform and hexane fractions (25-50 mg kg–1 i.p) produced significant inhibition (p<0.01) of the acetic acid-induced writhing in mice and suppressed formalin-induced licking response of animals in the second phase of the test. C. bonariensis (50-200 mg kg–1 i.p.) produced marked anti-inflammatory effect in the carrageenan-induced rat paw edema assay comparable to indomethacin. Among various fractions of the plant, hexane and butonalic acid exhibited significant (p<0.05) anti-inflammatory effects. Conclusion: It is concluded that C. bonariensis possesses analgesic and anti-inflammatory properties and that the bioactive bhytochemicals appeared to be concentrated in the hexane and butonalic fractions.
INTRODUCTION
Conyza bonariensis belongs to family Asteraceae and found in the local range lands of subcontinent and Middle East1. Various parts of C. bonariensis are used in a variety of ailments in the South Asian traditional system of medicine. Leaves are used as laxative, roots used in diarrhea2, cough and flowers are considered as aphrodisiac. Phytochemical studies of C. bonariensis revealed several bioactive constituents including flavonoids, flavonoid glycosides, cyanin glycoside, taraxeryl acetate, β-sitosterol, campesterol, stigmasterol and ergosterol. Conyza filaginoides, a realted specie known as simonillo in Mexico is used in the relief of painful digestive disorders such as dyspepsia, hepatic and biliary colic and has been shown to have analgesic and anti-inflammatory properties3.
The plant has also been investigated pharmacologically for anticonvulsant2, antitumor, hypoglycaemic4 and estrogenic5 activities. The essential oil from C. bonariensis6 and extracts of some related species of the plant such as C. Canadensis7 and C. floribunda8 have shown marked anti-inflammatory effects. The genus Conyza is popular for its medicinal uses as an anti-inflammatory and is rich in bioactive phytochemical constituents9.
The current study was aimed to validate the traditional uses of C. bonariensis in the management of pain and inflammatory conditions.
MATERIALS AND METHODS
Animals: NMRI/Balb-C mice (20-25 g) and wistar rats (200-250 g) of either sex were obtained from the animal house facility Department of Pharmacology, College of Medicine, King Saud University, Riyadh. The animals were housed in plastic cages under standard condition with 12 h light: dark cycle with free access to food and water.
Chemicals: The following chemicals were used in the experiments: Acetic acid, arachidonic acid, carrageenan, diclofenac sodium, indomethacin and were purchased from the Sigma Chemical Co., (St. Louis Mo, USA). Formalin 37% purchased from Fluka Chemie, Switzerland, respectively.
Plant materials: The whole plant of Conyza bonariensis (L.) Cronq. (Asteraceae) was collected from Oghi, Mansehra, Pakistan, in November, 2013 and identified by Mr. Jan Alam (Taxanomist) at the Department of Botany, University of Karachi, Pakistan. A voucher specimen (KUH G. H. No. 68220) has been deposited at the herbarium of the above department.
Extraction and fractionation: The air dried whole plant of Conyza bonariensis (35 kg) was extensively extracted with MeOH (50 L×3) at room temperature. The extract was evaporated to yield the residue (815 g), which was partitioned between n-hexane (110 g), CHCl3 (90 g), EtOAc (95 g), n-BuOH (495 g) and water (25 g). The extracts were stored in freezer at -80°C.
Experimental methods
Acute toxicity tests: Different doses of the plant extract of C. bonariensis were injected intraperitoneally (i.p) to separate groups of six animals. The animals were observed for 1-2 h after administration of the extracts for any acute toxicity symptoms e.g., behavioral symptoms. After 48 h, the highest dose that did not induce any mortality was considered as the maximum non-fatal dose. The number of deaths was counted 48 h after treatment. LD50 value (dose of the extracts producing mortality in the 50% of the experimental animals) was determined wherever applicable by graphical method.
Writhing test: Male adult mice were used in this experiment according to the method of Erol and Demirdamar10, 30 min. After the administration of the extracts, mice were given an i.p. injection of 0.7% v/v acetic acid solution (volume of injection 0.1 mL/10 g). The mice were placed individually into glass beakers and 5 min were allowed to elapse. The number of writhes produced in these animals was counted for 20 min. For scoring purposes, a writhe was indicated by stretching of the abdomen with simultaneous stretching of at least one hind limb. Control animals received normal saline (10 mL kg–1, i.p.) and diclofenac (10 mg kg–1, i.p.) was used as a reference drug.
Formalin test: The formalin induced pain test was perform according to method described by Hunskaar and Hole11, mice were injected, 20 μL of 1% formalin in 0.9% saline, subcutaneously into the dorsal hind paw and placed immediately in transparent box for observation. The duration of paw licking was determined between 0-5 min (first phase) and 15-30 min (second phase) after formalin injection. The time in seconds spent in licking and biting responses of the injected paw was noted. Animals were treated (i.p) with different doses of the plant extracts, 30 min prior to administration of formalin. Diclofenac (10 mg kg–1, i.p.) and morphine (5 mg kg–1, i.p.) were used as reference drugs. Naloxone (5 mg kg–1, i.p.) was administered 20 min prior to the extracts or morphine injections. Control animals received the vehicle (0.1 mL/10 g). The paw licking time of the animals was compared to control group and represented as percent inhibition.
Rat paw edema assay (anti-inflammatory activity): The carrageenan induced hind paw edema test was conducted according to the Winter et al.12. Rats divided randomly into different groups of 5-8 animals were injected subcutaneously into the plantar surface of the hind paw with 0.05 mL of freshly prepared 1% carrageenan (prepared in distilled water). Different doses of plant extracts were injected i.p., 30 min before the administration of carrageenan. Indomethacin was used as positive control. The control animals received same volume of the vehicle. Rat paw edema was assessed by volume displacement method (plethysmometer (UgoBasile 7150)) before and after carrageenan injection at 1, 2, 3 and 4 h. Difference in paw volume, determined before and after injection of the phlogistic agent indicated the severity of edema.
The percentage inhibition of the inflammation was determined for each animal by comparison with controls and calculated by the following formula13.
Where "dt" is the difference in paw volume in the drug treated group and "dc" the difference in paw volume in control group. "I" stands for inhibition.
Statistical analysis: The results of the study were expressed as Mean±SEM and statistical significance between control and treated groups evaluated by student’s t-test and one-way ANOVA followed by Tukey-Kramer multiple comparison test. p<0.05 was considered significant.
RESULTS
Acute toxicity test: The intraperitoneal administration of various dose of the plant extracts did not cause any lethality in mice. The maximum non-fatal dose of C. bonariensis was 2 mg kg–1.
Writhing test: The intraperitoneal (i.p) administration of the crude extract C. bonariensis (50 and 100 mg kg–1) caused significant inhibition (p<0.001) of the nociception induced by acetic acid with maximum inhibition 72%. The results were comparable to standard drug diclofenac sodium that produced 62% inhibition at 10 mg kg–1 i.p (Table 1).
Among various fractions of the plant extract, the butanolic fraction was the most potent producing 80% protection at 50 mg kg–1. The chloroform and hexane fractions (50 mg kg–1) caused 75 and 44% inhibition of the acetic acid induced writhing respectively (Table 1).
Formalin test: In the formalin test, the plant extracts of C. bonariensis had no appreciable effect in the first phase but caused dose dependent inhibition of the licking responses in the late phase of formalin test. As shown in Table 2, C. bonariensis extracts protected the animal from painful stimulation of formalin with maximum effect of 61% at 200 mg kg–1. Unlike the effect of chloroform fraction that demonstrated activity in the first phase, the butanolic fraction of C. bonariensis produced significant suppression of the nociceptive responses in the first and second phases of the formalin test with 40 and 63% inhibitions, respectively. Diclofenac (10 mg kg–1 i.p) produced 67% inhibition of the formalin induced nociception in the second phase of the test.
Anti-inflammatory activity: The crude extracts and fractions of C. bonariensis were assessed in the carrageenan induced edema model in rats. The injection carrageenan when injected into subplantar region of the rats paw produced a localized edema that reached to its maximum at the 3rd h after injection. The localized inflammatory response to carrageenan was sustained for 4 h and gradually declined after this time.
As shown in Table 3, C. bonariensis produced marked reduction in carrageenan induced edema (79% at 200 mg kg–1) at the 3rd h of the carrageenan administration. The difference between the paw volume of the control and extracts treated animals was statistically significant (p<0.05, p<0.01) at the 3rd h of observation. The early phase of the edema (1-2 h) was not affected by the plant extracts (data not shown). The standard drugs indomethacin, at 20 mg kg–1 i.p. produced about 66% inhibition of the carrageenan induced edema (Table 3).
The effect of various fractions of the plant extracts was quite variable. Hexane fraction was the most potent producing 47% inhibition of carrageenan edema at 100 mg kg–1 . At the same dose butanolic and chorofrom fraction exhibited 37 and 11% of the anti-inflammatory effect (Table 3).
| Table 1: | Effect of the methanol extract of C. bonariensis and its fractions on acetic acid-induced writhing in mice |
Values represent Mean±SEM of 10 observations, **p<0.01 and ***p<0.001, compared to control | |
| Table 2: | Effect of the methanol extract of C. bonariensis and its fractions on the formalin-induced licking response in mice |
| Values represent Mean±S.E.M of 12 observations, *p<0.05and ***p<0.001, compared to control | |
| Table 3: | Effect of the methanol extract of C. bonariensis and its various fractions on carrageenan induced paw edema in rats |
Values represent Mean±SEM of edema volume of 15 observations, *p<0.05, **p<0.01 and ***p<0.001, compared to control | |
DISCUSSION
This study was aimed to assess the analgesic and anti-inflammatory activities of C. bonariensis. For the anti-nociceptive effects, the two pain models such as acetic acid induced writhing and formalin induced licking test were used14. Clinically used analgesic drugs such as diclofenac and ibuprofen have been reported to inhibit the acetic acid induced writhing15,16. In the current study, the intraperitoneal administration of the plant extracts of C. bonariensis caused significant inhibition (p<0.01) of the acetic acid induced writhes. Acetic acid increases the prostaglandins levels in the peritoneal fluid17. The inhibitory effects of the plant extracts similar to the effect of diclofenac sodium suggest that it may have occurred via inhibition of prostaglandins action. The analgesic effect was predominantly seen with butanolic and chloroform fractions of the plant extracts indicating that analgesic components are concentrated in these fractions. Similar to the effects of plant extracts, diclofenac sodium, a standard non-steroidal anti-inflammatory drug showed significant activity in this test. Medicinal plants that are used as analgesic in traditional medicine such as, Asparagus pubescens18 and Melastoma malabathricum19, have been shown to decrease abdominal stretching/constriction induced by acetic acid20.
The acetic acid induced pain test is non-selective21.The mechanism of analgesia of the plant extract and its fractions was further investigated in the formalin induced pain assay. Formalin induced pain models useful to understand the mechanism of pain and analgesia22. Formalin induced pain involves two distinct phases, the first phase called the neurogenic phase, where pain is due to direct stimulation of the sensory nerve fiber by formalin while in the second or late the pain is due to release of inflammatory mediators such as prostaglandin and bradykinin11,23,24. The crude extracts and fractions of C. bonariensis inhibited formalin induced pain response in the second phase of formalin test indicating their peripheral action of analgesia. However butanolic fraction additionally exhibited significant analgesic effect in the first phase of the formalin test, which suggests central mechanism of its analgesic effect. This may explain the greater potency of the butonolic fractions in acetic acid and formalin tests. The peripherally acting drugs such as aspirin and diclofenac25, known to attenuate pain by inhibition of cyclooxygenase in arachidonic acid pathways26. The analgesic effect of the plant extract in chemical induced pain models suggests NSAIDs like activity of C. bonariensis plant extracts.
The speculation was further confirmed when C. bonariensis plant extract and it fractions caused significant inhibition of the carrageenan induced paw edema in rats. C. bonariensis and some of its fractions (hexane and butanol) caused significant inhibition of the late phase edema induced by the sub-plantar injection of carrageenan. The carrageenan induced acute inflammation is biphasic, in the early phase (1-2 h after carrageenan injection), edema production is mediated by histamine and serotonin while in the late phase (after 2nd h) inflammatory response is maintained by bradykinin and prostaglandins27. These mediators are well established for their role in inflammation, stimulate the nociceptors and induce pain28. Most of the clinically effective anti-inflammatory drugs are effective in the late phase and has been frequently used to assess the anti-phlogistic effect of the natural products29,30. In the present investigation, C. bonariensis and its fractions exhibited marked anti-inflammatory activity in the late phase of carrageenan induced edema test similar to indomethacin, a standard non-steroidal anti-inflammatory drug (NSAID)31,32. Several natural products and NSAIDS have been reported to inhibit inflammation induced by various phlogistic agents in experimental animal models33-35. NSAIDS such as indomethacin reduces inflammation and arthritic pain by inhibiting prostaglandin synthesis and/or production36. Therefore it is likely that C. bonariensis may contain NSAIDs-like constituents responsible for the observed analgesic and anti-inflammatory properties. Conyza dioscaridis, a related specie has been reported for analgesic and anti-inflammatory activities37. These data substantiate the therapeutic potential of the genus conyza, in pain and inflammatory conditions. These findings suggest that C. bonariensis could prove to be a potential new source of natural drugs with analgesic and anti-inflammatory activities. Further studies are required to investigate the active constituents and possible biochemical mechanisms of the observed analgesic effect of the plant extracts.
CONCLUSION
The results from our current study determine that Conyza bonariensis possesses analgesic and anti-inflammatory properties. The plant extract and its fractions produced marked inhibition of the pain response in animals in the chemical (acetic acid, formalin) induced pain models and carrageenan induced paw edema test. The analgesic and anti-inflammatory effects were similar to that observed with standard NSAIDs suggesting the presence of NSAIDs like constituents in C. bonariensis, while anti-inflammatory activity was concentrated in the hexane and butanol fractions.
SIGNIFICANCE STATEMENTS
This study substantiates the traditional use of C. bonariensis as a remedy of pain and inflammatory conditions. The findings of this study will help natural product researcher to identify the active constituents of this plant as possible novel analgesic and anti-inflammatory drug candidate with good safety and tolerability profile.
ACKNOWLEDGMENTS
The authors acknowledge the support of Prince Abdullah Ben Khalid Celiac Disease Research Chair, Vice Deanship of Research Chairs, King Saud University Riyadh Saudi Arabia.