Anti-inflammatory effect of Acmella uliginosa on acute inflammation activities in mice

Norsafinah M.T. , Nurfarah Diyanah A.K.A. , Munirah M.M. , Nur Mahirah Liyana D. Wan Ahamad Azran W.S.b, Hui Ming O.c, Prof Mohd Roslan S.c
a a a a a

Foundation in Science and Technology Royal College of Medicine Perak, Kolej MARA Kuala Nerang, 06300 Kuala Nerang, Kedah Darul Aman b Department of Biology of UniKL Kolej MARA Kuala Nerang, 06300 Kuala Nerang, Kedah Darul Aman c Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

___________________________________________________________________________ Abstract: Ethnopharmacological relevance:Acmella uliginosa (Sw.) Cass. is a medicinal herbaceous plant that is commonly used by the Malay community in Malaysia to relieve pain often associated with mouth ulcers, toothache, sore throat, and stomach ache. Aim:The study was carried out to investigate the anti-inflammatory effect of the methanolic extract of Acmella uliginosa (Sw.) Cass. flowers (MEAU) using carrageenan-induced paw edema test in mice. Materials & methods:Chemical (methanol, aspirin, absolute ethanol, carrageenan). The extract was given via oral administration at doses of 3, 10, 30 and 100mg/kg. A total of 36 mice were used and the mice were divided into 6 groups with 6 mice in each group. Results:It was demonstrated that intraperitoneal administration of MEAU at a dose of 3, 10, 30 and 100mg/kg produced significant dose-dependent inhibition of paw edema induced by carrageenan. Conclusion:The results from the present study appear to support the folkloric belief in the medicinal properties of Acmella uliginosa (Sw.) class which concluded that MEAU possessed anti-inflammatory effect against inflammation. Abstrak: Perkaitan ethnopharmacological :Acmellauliginosa (Sw.) Cass. adalah sejenis tanaman herba ubat yang umum digunakan oleh masyarakat Melayu di Malaysia untuk menghilangkan rasa sakit di mana sering dikaitkan dengan radang mulut, sakit gigi, sakit tekak, dan sakit perut. Objektif:Kajian ini telah dilakukan untuk menyiasat anti-inflamasi dari ekstrak methanol Acmella uliginosa (MEAU) dengan menggunakan karaginan-ujian edema yang diinduksi pada tapak kaki tikus. Bahan-bahan dan kaedah:Bahan kimia (methanol, aspirin, etanol mutlak, karaginan). Ekstrak MEAU telah diberikan melalui kaedah oral pada dos 3, 10, 30, dan 100mg/kg. Sebanyak 36 ekor tikus telah digunakan dan tikus-tikus tersebut telah dibahagi kepada 6 kumpulan dengan 6 ekor tikus dalam setiap kumpulan.

Keputusan:Ini telah menunjukkan bahawa pemberian intraperitoneal MEAU pada dos 3, 10, 30 dan 100mg/kg menghasilkan kebergantungan dos yang menghalang edema pada tapak kaki tikus dengan signifikan oleh karagenan. Konklusi:Hasil dari kajian ini telah terbukti menyokong kepercayaan masyarakat bahawa Acmella uliginosa (Sw.) terdapat ciri-ciri perubatan yang menyimpulkan MEAU mempunyai kesan anti-inflamasi terhadap keradangan.

Introduction: Acmella uliginosa (Sw.) Cass. Is aperennial herbaceous plant belonging to the daisy family, Asteraceae, which is indigenous and widely distributed in the tropic sand sub-tropics especially in the WestIndies, Venezuela, Brazil, Africa, Indonesia and Malaysia (Pandey et al., 2007). In Peninsular Malaysia, it is popularly known as Subang Nenek or Butang Baju Siti Fatimah . It grows in a bun- dance as a naturalized weed on open hill sides and the rocky shores of rivers. When consumed, its flowers and leaves have a pungent taste that accompanied by tingling and numbness. In Malaysia, A. uliginosa has been generally used as a traditional herbal medicine for its analgesic and antispasmodic properties. The use of the flowers of A. uliginosa in particular,are more common than other parts of the plant, and are widely used as a remedy for the relief of pain especially in mouth ulcers, toothache, sore throat and stomach ache. The flowers and/or the leaves are crushed and its paste is topically applied to the affected areas caused by insectbites to all eviate itch, redness and swelling. Despite the widespread use of this plant in the Malay s folk medicine, yet there is no scientific investigation reported to support its biological properties. Therefore, in the light of its reported analgesic activity, the present study was carried out to evaluate the antinociceptive property of the methanolic extractof A. uliginosa flowers using both chemical and thermal-induced nociception models in mice.

2. Methadology 2.1. Plant collection and identification Flowers of A. uligionosa were freshly collected from the Kampung Janda Baik, Pahang, Malaysia in February 2009. The plant material was identified and authenticated taxonomically by Dr. Shamsul Khamis, Biodiversity Unit, Institude of Bioscience, University Putra Malaysia. A voucher specimen of the collected sample was deposited in the departmental herbarium, as voucher, SK1780/09. 2.2. Preparation of extract The fresh flowers (1 kg) were washed with distilled water to remove the dirt and soaked overnight in methanol (99.8%). Then, the material was sonicated and filtered. The filtrate was then concentrated by using large scale rotary evaporator and lyophilization to yield approximately 2% of a greenish black colored methanolic extract of Acmella uliginosa (MEAU). The extract then was stored at -20 C prior to experiments. 2.3. Drugs and chemicals Methanol, aspirin (100 mg/kg), Tween 20, absolute ethanol (100%), acetylsalicylic acid (ASA) and lambda ( )-carrageenan was purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Acetylsalicylic acid (ASA) and lambda ( )-carrageenan were dissolved in saline solution (0.9% NaCl). The methanolic extract of A. uligionosa flowers (MEAU) was dissolved in vehicle (absolute ethanol: Tween 20 in saline; 5:5; v/v) to the desired concentration just before use. MEAU in vehicle or vehicle alone (control) was administered orally with an orogastric cannula, 30 min prior to acute inflammatory testing. The vehicle used alone had no effects on the inflammatory responses in mice.

2.4. Animals Experiments were conducted using adult male ICR mice (25 35 g) purchased from the Institute for Medical Research, Kuala Lumpur. They were housed at 222 C under a 12-h light/ 12-h dark cycle and with access to food and water ad libitum. The animals were acclimatized and habituated to the laboratory for at least a week before testing and were used only once throughout the experiments. Experiments reported in this study were performed in accordance with the current guidelines for the care of laboratory animals and the ethical guidelines for investigations of experimental pain in conscious animals ( Zimmerman, 1983). 2.5. Carrageenan-induced mice paw edema Inflammation of the paw in mice was induced as described previously with slight modification (Fereira SH., 1979) 19 Mice (n=6 per group) received i.p. injection of 1 (3, 10, 30 and 100 mg/kg) and aspirin (100 mg/kg, as positive control) or a similar volume of vehicle

(10 ml/kg) 30 min prior to subplantar injection of 0.02 ml of -carrageenan (dissolved in 0.9% NaCl) into the left hind paw of each mouse. Paw volume was measured with a plethysmometer (Ugo Basile, Italy) immediately prior to the injection of carrageenan and thereafter at an interval of 1 h for a period of 5 h. Edema inhibitory activity was calculated according to the following formula ( Olajide OA, 2003): [(Ct - C0)control (Ct - C0)treated] Percentage inhibition = [(Ct - C0)control] 100

where, Ct=mean paw volume for each group at time t, and C0=mean paw volume for each group before carrageenan injection.

2.6. Statistical analysis The data obtained was statistically analyzed using two-way ANOVA, followed by Dunnett's multiple comparison tests. The results were expressed as the mean S.E.M. to show variation in groups. Differences are considered significant when P 0.05.

3) Results and discussion:

The present study was carried out to assess the anti- inflammatory effect of methanolic extract of Acmella uliginosa (Sw.) Cass. Flowers (MEAU) in acute exudative models of inflammation in mice. The obtained results from the present study showed that MEAU possessed an anti-inflammatory activity in acute inflammatory models. In the present study it was demonstrated that the oral administration of MEAU at the doses of 3, 10, 30, and 100 mg/kg significantly produced dosedependant inhibition of paw oedema induced by carrageenan in mice over a period of 5h with percentage of inhibition at 3h post carrageenan injection of 0%, 25%, 91.67% and 100% respectively (Table 1). While aspirin (100 mg/kg) showed an inhibition of 91.67%.

The carrageenan-induced paw edema formation is a classical model of acute inflammation and it is believed to involve a biphasic event. In general, the early phase (1-2h) is mediated by the release of histamine and serotonin (Crunkhorn P, 1971). While the second phase, (3-5h) is the result of release of kinins and mainly prostaglandins (Van Arman CG, 1965) . In general, the development of oedema induced by carragenan is correlated with the early exudative stage of inflammation, one of the important processes of inflammation (Ozaki Y., 1990 ). Based on the present results, it can be suggested that the inhibitory effect of MEAU on carragenan- induced paw oedema in mice may be due to the suppression of the release of mediators responsible for inflammation including histamine, serotonin, bradykinin, and prostaglandin.

Table 1
Effect of methanolic extract of Acmella uliginosa (Sw.) Cass. Flowers (MEAU) on the carrageenan induced mice paw edema. Each value is the mean of paw volume S.E.M in ml. (n=6)
Paw volume, ml (% of Inhibition) Treatments Dose (mg/kg) Time interval (h) 0 Control (p.o.) MEAU (p.o.) 3 10 30 100 Aspirin (p.o.) 100 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1 0.067 0.009 0.065 0.008 0.053 0.005 0.043 0.010 0.015 0.002*** 0.033 0.011* 2 0.090 0.011 0.090 0.011 0.077 0.010 0.023 0.008*** 0.003 0.002*** 0.010 0.004*** 3 0.125 0.014 0.128 0.010 0.090 0.018* 0.017 0.007 *** 0.002 0.002*** 0.008 0.003*** 4 0.148 0.012 0.120 0.015 0.067 0.017*** 0.003 0.002*** 0.000 0.000*** 0.007 0.004*** 5 0.178 0.012 0.088 0.005*** 0.052 0.018*** 0.000 0.000*** 0.000 0.000*** 0.002 0.002***

*p< 0.05 and ***p< 0.001 when compared with control (ANOVA followed by Dunnett s test)

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