Environmental Toxicology and Pharmacology 26 (2008) 92–95 Contents lists available at ScienceDirect Environmental Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/etap Anti-inflammatory activity of four solvent fractions of ethanol extract of Mentha spicata L. investigated on acute and chronic inflammation induced rats P. Arumugam ∗ , N. Gayatri Priya, M. Subathra, A. Ramesh Department of Genetics, Dr. A.L.M. Post Graduate Institute of Basic Medical Sciences, University of Madras, Chennai 600113, India a r t i c l e i n f o Article history: Received 11 August 2007 Received in revised form 14 February 2008 Accepted 18 February 2008 Available online 23 February 2008 Keywords: M. spicata Solvent fractions Acute and chronic toxicity Anti-inflammation a b s t r a c t Anti-inflammatory effects of four solvent fractions of ethanol extract of Mentha spicata were evaluated in acute and chronic inflammation induced in Wistar albino rats. Lipid peroxidation (LPO) and some antioxidants produced during chronic inflammation were quantitated. Hexane (320 mg/kg of body weight in 25% DMSO), chloroform (320 mg/kg body weight in 25% DMSO), ethyl acetate (160 mg/kg body weight in 25% DMSO), aqueous (320 mg/kg of body weight in ddH2 O) fractions, two negative control groups (25% DMSO and ddH2 O) and two anti-inflammatory drugs (Diclofenac: 25 mg/kg of body weight; Indomethacin: 10 mg/kg of body weight both in ddH2 O) were administered by oral intubations to the eight groups of rats consisting six animals, each. In acute study, 1% carrageenan was injected subcutaneously in the subplantar region of the right hind paw after 1 h of administration of test doses. The increased paw edema was measured at 0.5, 1, 2, 4, 8, 16 and 24 h intervals. In the chronic study, the oral administration was carried out for seven consecutive days. On eighth day, four sterile cotton pellets (50 mg each) were implanted subcutaneously in the dorsal region of the rats. On the sixteenth day, the rats were sacrificed and the cotton pellets with granulomatous tissue were dissected out and weighed (fresh and dry). Both in chronic and acute inflammation, ethyl acetate (EAF) and aqueous fraction (AF) were effective. EAF is comparable with the positive standards in chronic inflammation. The results indicate that EAF’s anti-inflammatory activity is largely due to its ability to modulate in vivo antioxidants. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Mentha spicata L., also known as spearmint belongs to the Lamiaceae (labiatae) family. The genus Mentha consists 25–30 species; and spearmint is the most common among them (Bhattacharjee, 1998). The plant is a glabrous perennial with creeping rhizomes and can grow up to 1 ft height. In India, it is commonly called as pudina and is widely used in culinary preparations to add flavor and aroma (Choudhury et al., 2006). Mint oil is of economic importance and is widely used in pharmaceutical, cosmetic, food, confectionary and beverage industries (Kanatt et al., 2007). Spearmint oil contains monoterpenoids like carvone, limonene, menthone, menthol, pulegone, dihydrocarveol and s-carvone. Some of them were found to possess high antioxidant activity than ␣-tocopherol (Elmasta et al., 2006). The plant is also known for its ability to enhance memory (Adsersen et al., 2006). The boiled leaves extract is being used to relieve hiccup, flatulence, giddiness and as remedy for inflammation, bronchitis, and to control vomiting during pregnancy (Kumar and Chattopadhyay, 2007). Apart from being ∗ Corresponding author. Tel.: +91 44 2450 2430x5212; fax: +91 44 2450 2344. E-mail address: arumugamgenetics@yahoo.co.in (P. Arumugam). 1382-6689/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2008.02.008 a stimulant and carminative, the mint plant is also known for its insecticidal, antimicrobial, antispasmodic and antiplatelet properties (Papachristos and Stamopoulos, 2002; Samarth and Kumar, 2003; Ozgen et al., 2006; Tognolini et al., 2006). Many phenolic, flavonoid and terpenoid compounds have been identified from different extracts of spearmint. The major water-soluble phenolics include: eriocitrin, luteolin, glucoside, apigenin, acacetin, thymusin, thymonin, sideritoflavone and diosmetin (Yamamura et al., 1998). Two new monoterpenoids spicatoside, A and B, were found to possess anti-inflammatory, haemostatic and pain relieving properties (Zheng et al., 2003). In this report we present in vivo anti-inflammatory effects of some solvent extracts of M. spicata assessed against carrageenan and cotton pellet-induced inflammation in rats. 2. Materials and methods 2.1. Animals Healthy, Wistar albino rats of either sex (180–210 g) were purchased from King’s Institute, Chennai and were maintained at the departmental animal house under standard environmental conditions (temperature, 22 ± 2 ◦ C, and 12 h light/dark cycle). The animals had free access to standard pellet diet and water ad libitum; and were maintained in accordance with the Guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India. 93 P. Arumugam et al. / Environmental Toxicology and Pharmacology 26 (2008) 92–95 2.2. Chemicals Malondialdehyde (MDA), folin’s phenol reagent, 5,5′ -dithiobis (2-nitro benzoic acid) (DTNB), 1-chloro 2,4-dinitrobenzene (CDNB) and reduced glutathione were purchased from SISCO Research Laboratories Pvt. Ltd. (Mumbai, India). Carrageenan, Diclofenac and Indomethacin were obtained from Sigma–Aldrich, USA. All other chemicals used in the experiments were of analytical grade. sacrificed using anesthetic ether, and the cotton pellets were dissected out without affecting the surrounding granuloma tissues (Winter and Porter, 1957). Liver tissues were also excised and stored in 0.9% saline at −20 ◦ C for biochemical analysis. The moist pellets were weighed and then dried at 60 ◦ C for 48 h and again weighed. The reduced weights of the cotton pellets observed for the test compounds and anti-inflammatory are compared with that the respective negative and positive controls. This provides a measure to assess the anti-inflammatory effect of the test compounds. 2.3. Extraction of solvent fractions 2.6. Biochemical analysis M. spicata L. was commercially purchased and identified at the Center for Advanced Studies in Botany, University of Madras (voucher number-855). Solvent extraction was carried out as described by Villasenor et al. (2002). Eight hundred grams of shadow dried leaf powder was immersed in 4 L of 95% ethanol and left for 24 h under constant stirring and filtered. This was repeated twice with 2 L of 95% ethanol. Thus, a total of 3.5 L filtrate was collected and concentrated by rota vapor at 40 ◦ C. The yield of ethanol extract (EE) was 42 g (5.25%). The ethanol extract was partitioned between 780 mL of hexane and 130 mL of water (6:1) using separating funnel. This mixture was thoroughly mixed for 15 min and after 6 h the hexane fraction (HF) was collected. The aqueous layer (90 mL) was further fractionated with 540 mL of chloroform (CF) and then with 540 mL of ethyl acetate. All fractions were concentrated in rota vapor. The yield of these fractions was 18, 12 and 6.7 g, respectively and constituted about 43%, 29% and 16% of the ethanol extract. The aqueous fraction (90 mL; AF) was lyophilized (Flexi-Dru ␮p at 50 MT and −85 ◦ C) and weighed 3.5 g (8% of ethanol extract). Ten percent liver tissue homogenate was prepared in Tris–HCl buffer (0.1 M, pH 7.4) for estimation of total protein (Lowry et al., 1951), lipid peroxidation (Ohkawa et al., 1979) and antioxidants—glutathione-s-transferase (Habig et al., 1974), glutathione peroxidase (Rotruck et al., 1973), superoxide dismutase (Marklund and Marklund, 1974), catalase (Sinha, 1972) and reduced glutathione (Moron et al., 1979) fallowing standard protocols. 2.7. Statistical analysis The data was analyzed by one-way ANOVA using SPSS software package (version 12.0) and probability values, P ≤ 0.05 were considered to be significant. Significance of the group mean’s difference was tested using Student–Neuman–Keuls test (SNK test) when ANOVA is significant. 3. Results 2.4. Carrageenan-induced paw edema in rats: acute inflammation The paw edema was induced in rats by injecting 1% carrageenan (a proinflammatory agent; prepared in 0.9% NaCl) subcutaneously in the sub-plantar region of right hind paw, 1 h after administration of the test substances (Brich et al., 1992). The test doses used were based on investigator’s unpublished laboratory data. The test substances are—HF and CF (320 mg/kg of body weight in 25% DMSO), EAF (160 mg/kg of body weight in 25% DMSO), AF (320 mg/kg of body weight in ddH2 O) and two known anti-inflammatory drugs (positive controls): Diclofenac (25 mg/kg of body weight in ddH2 O) and Indomethacin (10 mg/kg of body weight in ddH2 O) were administered by oral intubation. The two control groups received 25% DMSO and DDW. The paw volumes of rats were measured by plethysmometer, before and after injection of 1% carrageenan at different time intervals (0.5, 1, 2, 4, 8, 16, and 24 h). Changes in paw volume, in centimeters (cm), were recorded at above time intervals with reference to the initial volume before administration of the inflammatory agent. 2.5. Cotton pellet-induced granuloma in rats: chronic inflammation Eight groups of six rats of either sex were used. Four solvent fractions (HF, CF and EAF in 25% DMSO and AF in ddH2 O), two anti-inflammatory drugs (positive control: Diclofenac and Indomethacin) and two negative controls (25% DMSO and ddH2 O) were orally administered daily once for seven days to each experimental group of rats. On eighth day, the animals were mildly anesthetized with ether and four sterile cotton pellets (50 mg) were subcutaneously implanted in the dorsal region of rats—two in the axilla and two in the groin regions. On sixteenth day, the rats were 3.1. Anti-inflammatory activity of the solvent fractions The effect of four solvent fractions of ethanol extract of the dried leaves of M. spicata on carrageenan-induced acute inflammation, measured by the increased paw volume (in cm) of the rats at different time periods (0.5, 1, 2, 4, 8, 16 and 24 h) is presented in Table 1. In both the negative control groups (25% DMSO and ddH2 O), the paw volume either remained steady or increased to a negligible extent up to 4 h (DMSO, 0.55 cm and ddH2 O, 0.63 cm) and thereafter declined slowly (at 24 h: DMSO, 0.13 cm and ddH2 O, 0.15 cm). Pretreatment with the antiinflammatory drugs (Diclofenac and Indomethacin) significantly reduced the carrageenan-induced inflammation even at 1/2 h (DCL, ∼39% reduction; IND, ∼53% reduction) and at 16 h, over 95% inflammation was reduced. Indomethacin even at half the dose of Diclofenac is more effective than the later. At 0.5 h, former reduces the inflammation by over 50% of the control value compared to about 39% reduction by the later. Compared with the positive controls, two solvent fractions, hexane and ethyl acetate, reduced the inflammation with less Table 1 Effects of solvent fractions of ethanol extract of Mentha spicata and anti-inflammatory drugs on carrageenan-induced acute inflammation Treatment (mg/kg of body weight) Time intervals (h) 0.5 DMSO ddH2 O 1 2 4 8 16 24 0.58 ± 0.08 0.57 ± 0.05 0.58 ± 0.08 0.57 ± 0.05 0.55 ± 0.05 0.57 ± 0.05 0.58 ± 0.08 0.63 ± 0.12 0.47 ± 0.05 0.48 ± 0.08 0.40 ± 0.06 0.42 ± 0.08 0.13 ± 0.08 0.15 ± 0.08 DCL (25) % 0.35 ± 0.05 −38.6 0.35 ± 0.05 −38.6 0.37 ± 0.05 −35.1 0.30 ± 0.06 −52.4 0.22 ± 0.04 −54.2 0.02 ± 0.04 −95.2 0.0 ± 0.00 −100.0 IND (10) % 0.27 ± 0.05 −52.6 0.27 ± 0.05 −52.6 0.33 ± 0.05 −42.1 0.30 ± 0.08 −52.4 0.20 ± 0.06 −58.3 0.02 ± 0.04 −95.2 0.0 ± 0.00 −100.0 HF (320) % 0.57 ± 0.08 −1.7 0.55 ± 0.05 −5.2 0.55 ± 0.05 0.0 0.47 ± 0.05 −19.0 0.43 ± 0.05 −8.5 0.32 ± 0.04 −20.0 0.12 ± 0.08 −7.7 CF (320) % 0.62 ± 0.08 6.9 0.61 ± 0.08 5.2 0.60 ± 0.06 9.1 0.58 ± 0.04 0.0 0.45 ± 0.05 −4.2 0.38 ± 0.04 −5.0 0.18 ± 0.08 38.5 EAF (160) % 0.53 ± 0.05 −8.6 0.52 ± 0.08 −10.3 0.50 ± 0.06 −91.1 0.40 ± 0.06 −31.0 0.37 ± 0.05 −21.3 0.17 ± 0.05 −57.5 0.02 ± 0.04 −84.6 AF (320) % 0.63 ± 0.05 10.5 0.62 ± 0.08 8.8 0.48 ± 0.04 −15.8 0.42 ± 0.04 −33.3 0.42 ± 0.08 −12.5 0.20 ± 0.06 −52.4 0.07 ± 0.08 −53.3 Values expressed in centimeters are mean ± standard deviation computed over six animals/group. Percentages refer to the change in edema size relative to the negative control groups. 25% DMSO: dimethyl sulfoxide—negative control to HF: hexane fraction, CF: chloroform fraction, EAF: ethyl acetate fraction. ddH2 O: double distilled water—negative control to AF: aqueous fraction, DCL: Diclofenac, IND: Indomethacin. 94 P. Arumugam et al. / Environmental Toxicology and Pharmacology 26 (2008) 92–95 Table 2 Effect of solvent fractions of ethanol extract of Mentha spicata and anti-inflammatory drugs on cotton pellet induced chronic inflammation in rats 3.2. Antioxidant activity of solvent fraction under chronic inflammation Treatment (mg/kg of body weight) Wet weight Dry weight Difference % inhibition DMSO + cotton ddH2 O + cotton Cotton DCL (25) + cotton IND (10) + cotton HF (320) + cotton CF (320) + cotton EF (160) + cotton AF (320) + cotton 945.0 981.3 990.5 332.5 312.2 767.7 835.2 391.2 509.7 212 215.5 232.3 128.2 114.7 189.0 192.7 137.8 155.5 733.0 765.8 758.2 204.3 197.5 578.7 642.5 253.4 354.2 – – – 73.3 74.2 21.1 12.3 65.4 53.7 In vehicle treated groups, lipid peroxidation (LPO) was significantly increased by about 62–75% over the control group (2.39 ± 0.34 nmoles MDA formed/mg protein, P < 0.05; Table 3). Solvent fractions, in particular, ethyl acetate and aqueous fractions effectively decreased the LPO generated by the implanted cotton pellets. All the measured antioxidants were found to be decreased by about 28–43% of corresponding control groups (P < 0.05; Table 3). Among them, GST was most affected (43%) and GPx and GSH least affected (27–31%). The values of the later two enzymes have been increased by two times the control group in ethyl acetate and aqueous fraction treated groups. Thus, these results support the effective chronic anti-inflammatory activity of the two ethanol fractions. ± ± ± ± ± ± ± ± ± 2.1 5.2 1.9 17.7 14.0 5.1 2.5 4.6 3.1 ± ± ± ± ± ± ± ± ± 3.6 2.7 2.7 9.3 10.3 2.5 1.8 2.0 1.9 Cotton: 50 mg; DCL: Diclofenac; IND: Indomethacin; HF: hexane fraction; CF: chloroform fraction; EAF: ethyl acetate fraction; AF: aqueous fraction. Values are in milligrams. Mean ± standard deviation was computed over six animals/group. Percentage inhibition was computed over respective negative control groups. 4. Discussion effectiveness. The reduction by the hexane fraction (0–20%) is smaller compared to reduction observed for ethyl acetate fraction (9–85%). In case of chloroform fraction, the inflammation did not decrease. In fact, the fraction increased inflammation in the range 5–38% of the control values. The aqueous fraction is more effective than the chloroform fraction. In the first 1 h, the aqueous fraction enhances inflammation by about 7–11% of the corresponding negative control values and thereafter, inflammation declines by about 13–53% of the control values. Table 2 shows chronic anti-inflammatory effects of the solvent fractions and the two anti-inflammation drugs, assigned by the granulomatous tissue mass formed around the cotton pellets implanted subcutaneous in the dorsal region of the rats. Changes in the cotton pellets weights (wet weight–dry weight) of the test substances were compared with the negative controls. Both even the anti-inflammatory drugs are equally effective. They have reduced nearly 3/4th the mass of the granulomatous tissue formed around the implanted cotton pellets. Two fractions, ethyl acetate and aqueous fractions, showed significant chronic anti-inflammatory effect. Their effects are in comparable magnitude with the anti-inflammatory drugs. Of the two, ethyl acetate fraction (∼65% reduction) has greater effect than aqueous fraction (∼54% reduction). The hexane and chloroform fractions possessed too small anti-inflammatory activity (less than 20% reduction). Thus, aqueous and ethyl acetate fractions of ethanol extract of M. spicata possess more effective chronic anti-inflammatory activity than acute anti-inflammatory activity. The ethyl acetate fraction is endowed with the compounds that have both chronic and acute inflammatory activity. The anti-inflammatory activity of hexane, chloroform, ethyl acetate and aqueous fractions of ethanol extract of M. spicata was evaluated on carrageenan (acute) and cotton pellet (chronic) induced inflammation in rats. Both ethyl acetate and aqueous fractions were found to be effective in chronic inflammation; whereas, only ethyl acetate fraction is effective in acute inflammation. In acute study, ethyl acetate fraction is effective from 16 h when the inflammation declines by ∼58% and this can go up to 85% at 24 h. Compared with the anti-inflammation drugs, ethyl acetate fraction’s activity is less and delayed. The acute inflammation is produced when water and plasma increases in tissues during arachidonic acid metabolism via cyclo-oxygenase and lipooxygenase enzyme pathways (Moura et al., 2005). The acute inflammation has two phases: the first phase (begins immediately after injection and last one hour) is characterized by the release of histamine and serotonin; and the second phase (begins after one hour and last three hours) is characterized by the bradykinin release via prostaglandins mediator pathways (Garcia-Pastor et al., 1999). The delayed action of ethyl acetate fraction could be due to its ability to inhibit the bradykinin release. On the other hand, the anti-inflammatory drugs were effective during both the phases of acute inflammation. Chronic inflammation is generated when the body fails to respond against inflammatory agents. This leads to fibroblast proliferation and formation of granulomatous tissues (Gepdiremen et al., 2004). Potential chronic anti-inflammatory activity was found in the EAF and AF treated groups (greater than 52%) that were comparable to the anti-inflammatory drugs (∼74%). These fractions may effectively suppress the granulomatous tissues formation as they have highest free radical scavenging activity and contain larger amounts of secondary metabolites like flavonoids and phenolics (Arumugam et al., 2006). Anti-inflammatory compounds inhibit the release of inflammatory mediators (Süleyman et Table 3 Modulation of chronic inflammation induced changes in lipid peroxidation and antioxidants in rat liver tissue by solvent fractions of ethanol extract of Mentha spicata pretreatment Treatment (mg/kg of body weight) LPO Control DMSO + cotton ddH2 O + cotton HF (320) + cotton CF (320) + cotton EAF (160) + cotton AF (320) + cotton 2.39 3.86 4.19 3.03 3.01 2.33 2.40 GST ± ± ± ± ± ± ± 0.34 0.33 0.58 0.60 0.32 0.48 0.33 6.34 3.82 3.60 7.93 7.05 9.15 8.62 GPx ± ± ± ± ± ± ± 0.42 1.12 0.52 0.19 ‘0.60 0.40 0.55 22.22 15.31 15.92 41.50 31.68 42.42 46.13 SOD ± ± ± ± ± ± ± 0.51 0.38 0.29 2.9 3.32 2.89 3.13 13.02 8.42 8.24 11.15 14.69 16.92 15.35 CAT ± ± ± ± ± ± ± 0.71 0.40 0.45 1.19 0.61 1.12 0.45 379.8 242.1 232.2 464.1 408.9 585.2 531 GSH ± ± ± ± ± ± ± 2.54 3.06 2.86 11.6 2.08 4.91 2.38 3.85 2.64 2.80 4.67 5.78 8.26 7.87 ± ± ± ± ± ± ± 0.58 0.47 0.72 0.46 0.49 1.21 0.59 Cotton: 50 mg; Values are mean ± standard deviation; HF: hexane fraction; CF: chloroform fraction; EAF: ethyl acetate fraction; AF: aqueous fraction; LPO: nmoles MDA formed/mg protein; GST: nmoles CNDB conjugated/min/mg protein; GPx: moles reduced glutathione oxidized/min/mg protein; SOD: units/min/mg protein; CAT: ␮moles H2 O2 consumed/min/mg protein; GSH: ␮g/mg protein. P. Arumugam et al. / Environmental Toxicology and Pharmacology 26 (2008) 92–95 al., 2003). Flavonoids were reported to inhibit the cyclooxygenase and lipooxygenase pathways of arachidonate metabolism (Pelzer et al., 1998; Zheng et al., 2003). The inflammatory process was reported to be associated with the generation of reactive oxygen species (ROS) (Jung et al., 2005). In both ethyl acetate and aqueous fraction treated groups, the GSH and GPx levels were increased by two times the control values. Other enzymes also showed higher values relative to the hexane and chloroform fractions treated groups. The results of the present work suggest that the ethanol fractions from the leaves of M. spicata attenuate the chronic inflammation via antioxidxidant activity. In summary, the high polar solvent fractions – ethyl acetate and aqueous fractions have anti-inflammatory activity than the low polar solvent fractions. 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