Journal of Ethnopharmacology 140 (2012) 230–233 Contents lists available at SciVerse ScienceDirect Journal of Ethnopharmacology jo urnal homepage: www.elsevier.com/locate/jethpharm Antioxidant, anti-inflammatory and anti-nociceptive effects of Ammannia baccifera L. (Lythracceae), a folklore medicinal plant a b a,∗ Nataraj Loganayaki , Perumal Siddhuraju , Sellamuthu Manian a Department of Botany, School of Life Sciences, Bharathiar University, Coimbatore 641046, TN, India b Department of Environmental Sciences, School of Life Sciences, Bharathiar University, Coimbatore 641046, TN, India a r t i c l e i n f o a b s t r a c t Article history: Ethnopharmacological relevance: Ammannia baccifera L. has been reported as folklore remedy for the Received 23 June 2011 treatment of inflammation and tumor in the state of Rajasthan, India. Received in revised form 13 October 2011 Aim of the study: The present study was designed to investigate the antioxidant, anti-inflammatory and Accepted 2 January 2012 anti-nociceptive effects of the methanol extract from the aerial parts of Ammannia baccifera under in vitro Available online 20 January 2012 and in vivo models. • Materials and methods: The in vitro antioxidant activity of the extract was measured using DPPH , super- Keywords: oxide, hydroxyl and nitric oxide radicals. The anti-inflammatory activity was evaluated using carrageenan Ammannia baccifera Anti-inflammatory induced paw edema. Analgesic activity of the methanol extract was estimated against acetic acid-induced Antioxidant writhing and hot plate methods. Paw edema Results: The IC50 value for free radical scavenging activity of this extract was significantly superior over Hot plate the positive standards butylated hydroxyl anisole (BHA) and rutin. The extract exhibited significant anti- inflammatory and analgesic activities at the dose of 100 and 200 mg/kg p.o. The analgesic effect of the higher dose of the extract (200 mg/kg) was comparable with the standard drugs aspirin and morphine. Conclusion: The present study scientifically validated the traditional use of this plant against inflamma- tion. © 2012 Elsevier Ireland Ltd. All rights reserved. 1. Introduction rhamnetin and its 3-rhamnosylglucoside were found to be present in Ammannia multiflora (Balraj and Nagarajan, 1981). In the present Ammannia baccifera L. belonging to the family Lythraceae is study, we investigated the analgesic and anti-inflammatory prop- distributed commonly through out India, often as a weed in rice erties of the methanol extract of Ammannia baccifera using several fields. In Ayurveda, the leaves are considered to have laxadive, experimental models with mice and rats for the purpose of validat- stomachic, hepatopathy and aphrodisiac properties and are use- ing its ethnomedical use. Natural active principles, endowed with ful for treating strangury. In Unani they are used as an appetizer anti-inflammatory activity, frequently exhibit antioxidant power; and for the treatment of rheumatic pains and fevers. In Siddha the moreover many plant extracts interacting with several biosynthetic whole plant is used in the treatment of glandular swellings, leuko- pathways of inflammation mediators in general possess antioxi- rrhea, snake-bite poisoning abscesses, ulcers and polyuria. Leaves dant property (Speroni et al., 2006). For this reason, the extracts contain lawsone as the major chemical constituent. The herb is were also assessed for antioxidant capacity, using different in vitro reported to possess anti-typhoid, anti-tubercular and anti-tumor chemical methods. properties (Parrotta, 2001; Khare, 2007). The different extracts of the plant are reported to have antimicrobial activity against human and plant pathogenic fungi (Ray et al., 2004). Four flavonol and three 2. Materials and methods flavone glycosides quercetin 3-␤-d-glucoside, rutin, luteolin 7-␤- d ␤ d -glucoside, isorhamnetin 3-rutinoside, apigenin 7- - -glucoside, 2.1. Plant material vitexin and kaempferol 3-rhamnoglucoside were isolated and char- acterized from Ammannia coccinea (Graham et al., 1980). Free Aerial part of Ammannia baccifera were collected from local areas of Coimbatore district, Tamil Nadu, India, authenticated and deposited in the Botany Herbarium, Bharathiar University ∗ (BUH006148). The plant material was shade dried, powdered and Corresponding author. Tel.: +91 9442170766; fax: +91 422 2422387. E-mail address: manian [email protected] (S. Manian). used for methanol extraction. 0378-8741/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2012.01.001 N. Loganayaki et al. / Journal of Ethnopharmacology 140 (2012) 230–233 231 2.2. Preparation of extract Group 1: received the vehicle saline (10 ml/kg p.o.). Group 2: treated with standard drug. The plant material was successively extracted with petroleum Group 3: treated with methanol extract of Ammannia baccifera ether (dewaxing) and methanol using soxhlet apparatus. The sol- (100 mg/kg p.o). vent was removed and used for further experiment. Group 4: treated with methanol extract of Ammannia baccifera (200 mg/kg p.o). 2.3. Estimation of total phenolics, tannins and flavonoids 2.5.4.1. Acetic acid-induced writhing. The peripheral analgesic drug, aspirin was used as positive control (25 mg/kg, p.o.). Acetic The total phenolic content and tannins of the extract was deter- acid-induced writhing was determined using the method of mined and calculated as g gallic acid equivalents (GAE) from the Dongmo et al. (2005). calibration curve (Makkar, 1999). The total flavonoid content of sample extract was determined following a colorimetric method 2.5.4.2. Hot-plate test in mice. The central analgesic drug morphine and values were expressed as g rutin equivalent (RE)/100 g of (5 mg/kg p.o.), was used as positive control. Latency to exhibit extract (Zhishen et al., 1999). antinociceptive responses was determined by MacDonald et al. (1946). Latency was recorded 15, 30, 45, 60, 90 and 120 min after 2.4. In vitro antioxidant activity administration of the test substances. • The free radical scavenging assays include DPPH (Blois, 1958), 2.6. Statistical analysis Superoxide (Beauchamp and Fridovich, 1971), Hydroxyl (Klein et al., 1991) and Nitric oxide (Sreejayan and Rao, 1997) scavenging The results were expressed as mean ± S. E. M. Statistical anal- activities were measured using standard procedures. ysis was carried out by analysis of variance (ANOVA) followed by Dunnett’s test. All calculations were performed using: SPSS, version 11.0. 2.5. In vivo studies 3. Results 2.5.1. Animals Male Swiss albino mice (25–30 g) and Wistar albino rats The quantitative estimations of total phenolics, tannins and (150–175 g) were used for the study. All animal experiments were flavonoids from methanol extract of Ammannia baccifera revealed conducted with the permission from Institutional Ethical Commit- that the extract has 95.7 ± 1.6, 6.2 ± 4.2 and 43.3 ± 0.1 g/100 g tee (IAEC No. KMCRET/Ph.D/01/2010-11). extract of total phenolics, tannins and flavonoids, respectively. The antioxidant activity of methanol extract of Ammannia bac- • 2.5.2. Acute toxicity cifera was evaluated using DPPH , super oxide, hydroxyl and • Acute oral toxicity studies were performed according to OECD nitric oxide radicals. The IC50 value of DPPH scavenging activ- 421 (Organization for Economic Co-operation and Development). ity of methanol extract of Ammannia baccifera was 8.3 ␮g/mL. This value was lower than those of positive standards BHA (9.7 ␮g/mL) and rutin (17.4 ␮g/mL). Like that, super oxide and 2.5.3. Carrageenan induced paw edema hydroxyl radical scavenging activities also methanol extract of For the experiment, the male Wistar rats (150–175 g) were Ammannia baccifera (25.9 ± 2.1 and 54.6 ± 1.3 ␮g/mL, respectively) divided into four groups (n = 6). The first group received distilled exhibited higher radical scavenging activity over positive stan- water (10 ml/kg p.o.), while the second group was treated with ± ± ␮ dards BHA (79.6 1.3 and 75.7 3.7 g/mL, respectively) and rutin standard drug indomethacin (10 mg/kg p.o.). The third and the (62.3 ± 4.7 and 66.3 ± 2.9 ␮g/mL, respectively). However in nitric fourth groups were administered with the methanol extract of oxide scavenging activity, methanol extract of Ammannia bac- Ammannia baccifera (100 and 200 mg/kg p.o. respectively). Acute ± ␮ cifera showed higher radical scavenging activity (98.6 5.7 g/mL) inflammation was induced and paw volume was measured imme- over BHA (113.9 ± 8.1 ␮g/mL) and comparable activity with rutin diately at 1 h, 2 h, 3 h, 4 h, and 5 h after the carrageenan injection, (94.7 ± 6.3 ␮g/mL). by using digital Lethylsmometer (Winter and Poster, 1957). The extract did not alter the general behavior and failed to pro- duce any mortality even at the highest dose (2000 mg/kg, p.o.) 2.5.4. Analgesic activity studied after 3 days and found to be safe. For the experiment, Swiss albino mice (20–25 g) were divided The inhibitory effects of Ammannia baccifera and endomethacin into four groups (n = 6). on carrageenon induced paw edema are reported in Table 1. For Table 1 Anti-inflammatory effects of methanol extract from Ammannia baccifera on carrageenan induced paw edema in rats. Dose Edema rate Treatment (mg/kg b.w.) 1 h 2 h 3 h 4 h 5 h Vehicle 26.8 ± 7.7 35.4 ± 10.2 49.9 ± 9.2 66.1 ± 13.6 81.7 ± 10.9 * ** * * Indomethacin 25 23.2 ± 6.4 27.2 ± 9.6 27.7 ± 7.4 29.7 ± 9.9 30.3 ± 12.8 (13.4%) (23.2%) (44.4%) (55.1%) (62.9%) * Extract 100 24.4 ± 5.1 29.9 ± 4.7 37.3 ± 16.7 44.9 ± 19.2 44.8 ± 15.8 (8.9%) (15.5%) (25.2%) (32.0%) (43.2%) * ** * * 200 23.9 ± 3.4 26.6 ± 5.7 28.9 ± 8.5 30.1 ± 7.4 33.2 ± 9.9 (10.8%) (24.8%) (42.0%) (54.4%) (59.3%) ± The data represent the mean SEM (n = 6).