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A possible anti-inflammatory mechanism of ethyl acetate extracts of Teucrium stocksianum Bioss
© Mukarram Shah. 2015
Received: 15 June 2015
Accepted: 26 August 2015
Published: 29 August 2015
Teucrium stocksianum (T. stocksianum) is one of the important members of the genus Teucrium which contains numerous biologically active compounds. Traditionally, it is used for the treatment of fever, pain, as expectorant and blood purifier. Researchers are trying to discover plants origin, novel and safe remedies for the management of various ailments. The present study was aimed to determine the possible anti-inflammatory mechanism of ethyl acetate extract of T. stocksianum.
Preliminary, the ethanolic extract and sub-fractions were screened for anti-inflammatory potential at doses of 100, 200 and 300 mg/kg (i.p) body weight, using carrageenan induced paw edema test in mice. In-order to determine the possible mechanism of anti-inflammatory effect, the ethyl acetate fraction was ascertained with different phlogistic agents like histamine, bradykinin, prostaglandins E2 and arachedonic acid via paw edema test in mice.
The ethanolic extract and sub-fractions of T. stocksianum displayed marked to moderate anti-inflammatory activity in a carrageenan induced paw edema test in mice. Among the sub-fractions, ethyl acetate fraction (EAF) demonstrated excellent (66 %) anti-inflammatory action at the highest tested dose (300 mg/kg) that reached to the maximum value at 3rd hour after carrageenan injection and remained significant (***P < 0.001) till 5th hour of test sample administration. EAF revealed moderate effect against the paw edema induced by histamine (31.048 %) while non-significant results (18.148 %) were observed against the edema induced by bradykinin. The extract demonstrated significant (66.23-73.076 %) anti-inflammatory potential against the edematogenic effect of prostaglandin E2. Moreover, the extract also significantly inhibited (51.33 %) the paw edema induced by arachedonic acid.
Our results suggest that the EAF has dual action and produced the anti-inflammatory effect by blocking both pathways of arachedonic acid metabolites (cyclooxygenase and lipoxygenase). Thus validating the traditional use of T. stocksianum and could provide a source of novel, effective and safe drug for the treatment of inflammation.
In the plant kingdom nature has bestowed the remedies of ailments. According to WHO, approximately 70-80 % population of developing countries acquire their primary pharmaceutical care from medicinal plants . Medicinal plants have diversified pharmacological potentials due to the presence of various classis of phytochemicals .
Lamiaceae is a famous cosmopolitan family comprising of about 200 genera and more than 4000 species disseminated all over the world particularly in the Mediterranean and mountainous region . Phytochemicals evaluation of the family Lamiaceae revealed the presence of different classes of secondary metabolites. The most common compounds among the species are terpenoids, iridoids, phenolics compounds and flavonoids [4, 5]. T. stocksianum Boiss is a key member of the Lamiaceae family. It is used in folk medicine for the treatment of fever, sore throat, as expectorant, diabetes, foot burning sensation, body coolant and blood purifier [6, 7]. In our previous work we extracted the essential oils from T. stocksianum which showed excellent analgesic activity . The methanolic extract, sub-fractions and crud saponins of T. stocksianum exhibited marked antioxidant, analgesic, cytotoxic, phytotoxic and insecticidal activities [9–11]. It has been documented that the ethyl acetate fraction of the crude methanolic extract of this plant has shown significant antidiabetic effect in Alloxan induced diabetic rabbits, which endorse the folkloric use of the plant . Islam et al, explored the antiulcerogenic and cytoprotective effects of the alcoholic extract of T. stocksianum . Radhakrishnan et al  determined the anti-inflammatory effect of the ethanolic crude extract of the aerial part of T. stocksianum. But the detail mechanism of anti-inflammatory potential is not been reported elsewhere. In order to determine the possible anti-inflammatory mechanism, crude ethanolic extract and its subsequent fractions were initially screened for anti-inflammatory activity. Finally, the most potent fraction i.e. ethyl acetate was selected for the determination of possible anti-inflammatory mechanism using various inflammatory phlogistic agents in mice.
T. stocksianum was collected in the month of May 2012 from District Swat in the province of Khyber Pakhtunkhwa (KPK), Pakistan and was identified by Professor Dr. Nasrullah, Department of Botany, University of Malakand, Pakistan. Voucher specimen was deposited in the Herbarium of the same Department having reference number H.UOM.BG.199. Plant was washed with tape water, shad dried and pulverized to coarse powder. About 2 kg of the plant material was extracted with ethanol (80 %), yielded 7.5 % (150 g) crud extract. The ethanolic crud extract were suspended in distilled water and fractionation was carried out using successive solvent-solvent extraction method. This resulted in 26 g (1.30 %), 38 g (1.90 %), 47 g (2.35 %) and 23 g (1.15 %) of n-hexane, chloroform, ethyl acetate and aqueous fraction respectively.
In the present study Swiss albino mice of either sex (25-30 g) were used. Animals were procured from the Pharmacology section of National Institute of Health, Islamabad, Pakistan. Animals were maintained in suitable cages under controlled laboratory conditions of 23-25 °C with 12 h light/dark cycle and had a free access to food and water during acclimatization period. The experimental procedures were approved by the ethical committee of the Department of Pharmacy University of Malakand, KPK, Pakistan.
Chemicals/drugs and solubility
In this study we used Carrageenan, Histamine, Chlorpheniramine maleate, Bradykinin acetate, Arachedonic acid, Caffeic acid and Prostaglandin E2 which were purchased from Sigma Chemicals Company, USA. While Acetylsalicylic acid used was of Reckit & Colman, Pakistan (Aspirin).
Stock solution of Bradykinin was prepared with 70 % ethanol and was further diluted with 0.1 % ethanol. Arachedonic acid was dissolved in carbonate buffer (Na2CO3, 0.2 M, pH 8.5). Caffeic acid solution was prepared in 10 % dimethylsulphoxide (DMSO). The ethanolic extract and subsequent fractions of T. stocksianum were prepared as 100, 200 and 300 mg in 10 ml of 10 % DMSO. Rests of the chemicals were dissolved in 0.9 % normal saline solution.
Anti-inflammatory activity of Teucrium stocksianum in mice
Mechanism of anti-inflammatory activity of Teucrium stocksianum in mice
The experimental animals of either sex were randomly divided in to various groups. The animals received intraperitoneally injection of 10 % DMSO or 0.9 % saline or chlorpheniramine maleate 25 mg/kg (antihistaminic) or 100 mg/kg caffeic acid (lipoxygenase inhibitor) or aspirin 100 mg/kg or 300 mg/kg ethyl acetate fraction of T. stocksianum. After 30 min of the above intraperitoneal administration, paw inflammation was induced in the right hind paw of mice by sub planter injection of 0.1 ml of histamine (1 mg/ml) or arachedonic acid (0.5 % w/v) or bradykinin (20 μg/ml) or prostaglandin E2 (0.01 μg/ml). Paw volume of each mouse was immediately measured before and after the sub planter administration of different irritants (inflammatory agents) at 1, 2, 3 and 4 h.
Statistical analysis and calculations
All the results obtained were articulated as mean ± SEM of 06 animals. One-way analysis of variance (ANOVA) followed by post hoc Dunnett’s test multiple comparison test was applied for the comparison among various groups and Student’s t test to determine the significance of differences between two means. Differences with P ≤ 0.05 and lower between groups were considered significant.
Nearly all the edemogens produced the peek inflammatory response at the 3rd h on the hind paw of control animal, followed by decline in swelling at 4th h. Moreover, the inflammatory response evoked by Histamine, Bradykinin, Prostaglandin E2, Arachedonic acid and Carrageenan, were not significant in the presence of vehicles, 10 % DMSO or normal saline in mice, consequently the observations were pooled (n = 30).
Results and Discussion
Concentration dependent anti-inflammatory effect of ethyl acetate fraction of T.stocksianum (TSEAF) in carrageenan induced paw edema test
DMSO 10 %
0.220 ± 0.109
0.233 ± 0.08
0.235 ± 0.08
0.236 ± 0.03
0.236 ± 0.02
0.193 ± 0.076
0.173 ± 0.061
0.158* ± 0.05
0.163* ± 0.09
0.181 ± 0.104
0.160* ± 0.051
0.13*** ± 0.051
0.116*** ± 0.05
0.130*** ± 0.04
0.135*** ± 0.07
0.160* ± 0.106
0.098*** ± 0.05
0.080*** ± 0.02
0.096*** ± 0.04
0.098*** ± 0.04
0.130*** ± 0.06
0.101*** ± 0.08
0.070*** ± 0.02
0.081*** ± 0.05
0.096*** ± 0.07
Carrageenan induced inflammation is a well-established non-specific protocol for the determination of anti-inflammatory action of medicinal agents. Inflammation induced by carrageenan is believed to be biphasic. The first phase is mediated by the release of serotonin and histamine while the second phase is mediated by leukotrienes and prostaglandins produced by tissue macrophages and bradykinin which peek at 3 h . To ascertain the possible mechanism of inhibition, anti-inflammatory activity of EAF (300 mg/kg) was evaluated against various phlogistic agents including histamine, bradykinin, prostaglandin and arachedonic acid.
Anti-inflammatory effect of ethyl acetate extract of T.stocksianum, in bradykinin and prostaglandin induced paw edema test
0.251 ± 0.09
0.220 ± 0.0093
0.198 ± 0.0105
0.210 ± 0.01125
0.225n.s ± 0.012
0.201n.s ± 0.054
0.175n.s ± 0.08
0.201n.s ± 0.0109
0.231 ± 0.06
0.240 ± 0.057
0.251 ± 0.040
0.260 ± 0.0052
0.078*** ± 0.065
0.075*** ± 0.043
0.733*** ± 0.04
0.070*** ± 0.036
Anti-inflammatory effect of ethyl acetate extract (EAF) of T.stocksianum, in histamine induced paw edema test
0.270 ± 0.036
0.248 ± 0.06
0.231 ± 0.065
0.230 ± 0.081
0.0767*** ± 0.055
0.105*** ± 0.062
0.0783*** ± 0.075
0.183 ± 0.088
0.221 ± 0.08
0.171** ± 0.08
0.185* ± 0.088
0.218 ± 0.060
Anti-inflammatory effect of ethyl acetate extract of T.stocksianum, in arachedonic acid induced paw edema test
0.5 % w/v
0.2050 ± 0.061
0.2300 ± 0.077
0.2516 ± 0.030
0.2500 ± 0.025
0.1666* ± 0.102
0.1150** ± 0.071
0.0816** ± 0.060
0.0783** ± 0.030
0.1816 ± 0.074
0.1416* ± 0.074
0.1233** ± 0.076
0.1216** ± 0.030
0.1983 ± 0.047
0.1983 ± 0.087
0.215 ± 0.020
0.235 ± 0.042
In conclusion, this research study revealed that the ethyl acetate fraction of T. stocksianum possesses significant anti-inflammatory activity via dual inhibition of both cyclooxygenase and lipoxygenase pathways of arachedonic acid metabolites. Moreover, highly significant effect was observed via prostaglandin inhibition. These results support the folkloric use of the plant in the treatment of various inflammatory conditions.
We are grateful to Pakistan Council of Science and Industrial Research (PCSIR), Peshawar, KPK, Pakistan for giving us access to Pharmacology laboratory and technical assistance on Plethysmometer (used for anti-inflammatory activity).
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- Shirwaikar A, Verma R, Lobo R, Shirwaikar A. Phytotherapy-safety aspects. Nat Prod Radiance. 2009;8(1):55–63.Google Scholar
- Shah S, Shah SMM, Ahmad Z, Yaseen M, Shah R, Sadiq A, et al. Phytochemicals, in vitro antioxidant, total phenolic contents and phytotoxic activity of Cornus macrophylla Wall bark collected from the North-West of Pakistan. Pakistan J Pharm Sci. 2015;28(1):23–8.Google Scholar
- Ali S, Nasir Y. 1991. Flora of Pakistan 1990:191-192.Google Scholar
- Richardson P. The chemistry of the Labiatae: an introduction and overview. In: Advances in Labiate Science. United Kingdom: Royal Botanic Gardens, Kew; 1992. p. 291–7.Google Scholar
- Zgórka G, Głowniak K. Variation of free phenolic acids in medicinal plants belonging to the Lamiaceae family. J Pharm Biomed Anal. 2001;26(1):79–87.View ArticlePubMedGoogle Scholar
- Ibrar M, Hussain F. Ethnobotanical studies of plants of Charkotli hills, Batkhela district, Malakand, Pakistan. Front Biol China. 2009;4(4):539–48.View ArticleGoogle Scholar
- Wazir SM, Saima S, Dasti AA, Subhan S. Ethanobotnical importance of salt range species of district karak, Pakistan. Pakistan J Plant Sci. 2007;13:29–31.Google Scholar
- Shah SMM, Ullah F, Shah SMH, Zahoor M, Sadiq A. Analysis of chemical constituents and antinociceptive potential of essential oil of Teucrium Stocksianum bioss collected from the North West of Pakistan. BMC Complement Altern Med. 2012;12(1):244.PubMedPubMed CentralGoogle Scholar
- Shah SM, Sadiq A, Ullah F. Antioxidant, total phenolic contents and antinociceptive potential of Teucrium stocksianum methanolic extract in different animal models. BMC Complement Altern Med. 2014;14(1):181.View ArticlePubMedPubMed CentralGoogle Scholar
- Shah SMM, Sadiq A. Biological activities of crude saponins, methanolic extract and sub fractions of Teucrium stocksianum Bioss collected from North West of Pakistan. Pharmacologyonline. 2014;3:145–52.Google Scholar
- Shah SMM, Sadiq A, Shah SMH, Khan S. Extraction of saponins and toxicological profile of Teucrium stocksianum boiss extracts collected from District Swat. Pakistan Biol Res. 2014;47(1):65.View ArticlePubMedGoogle Scholar
- Rashid M, Bashir S, Mushtaq MN, Khan HU, Malik MNH, Qayyum A, et al. Comparative hypoglycemic activity of different extracts of Teucrium stocksianum in diabetic rabbits. Bangladesh J Pharmacol. 2013;8(2):186–93.Google Scholar
- Islam M, Zakaria M, Radhakrishnan R, Kamil M. Effect of Teucrium stocksianum on gastric ulceration and secretion in rats. Pharm Biol. 2002;40(3):216–20.View ArticleGoogle Scholar
- Radhakrishnan R, Zakaria M, Islam M, Kamil M, Ismail A, Chan K, et al. Analgesic and anti-inflammatory activities of Teucrium stocksianum. Pharma Biol. 2001;39(6):455–9.View ArticleGoogle Scholar
- Muhammad N, Saeed M, Khan H. Antipyretic, analgesic and anti-inflammatory activity of Viola betonicifolia whole plant. BMC Complement Alterna Med. 2012;12(1):59.View ArticleGoogle Scholar
- Adedapo AA, Sofidiya MO, Maphosa V, Moyo B, Masika PJ, Afolayan AJ. Anti-inflammatory and analgesic activities of the aqueous extract of Cussonia paniculata stem Bark. J Nat Prod Res. 2008;2(2):46–53.Google Scholar
- Musa A, Aliyu A, Yaro A, Magaji M, Hassan H, Abdullahi M. Preliminary phytochemical, analgesic and anti-inflammatory studies of the methanol extract of Anisopus mannii (NE Br)(Asclepiadaceae) in rodents. Afric J Pharm Pharmacol. 2009;3(8):374–8.Google Scholar
- Channa S, Dar A, Anjum S, Yaqoob M. Anti-inflammatory activity of Bacopa monniera in rodents. J Ethnopharmacol. 2006;104(1):286–9.View ArticlePubMedGoogle Scholar
- Chawla A, Singh M, Murthy M, Gupta M, Singh H. Anti-inflammatory action of ferulic acid and its esters in carrageenan induced rat paw oedema model. Indian J Exp Biol. 1987;25(3):187–9.PubMedGoogle Scholar
- DiMartino M, Campbell Jr G, Wolff C, Hanna N. The pharmacology of arachidonic acid-induced rat paw edema. Agents Actions. 1987;21(3-4):303–5.View ArticlePubMedGoogle Scholar
- Singh S, Majumdar D. Evaluation of antiinflammatory activity of fatty acids of Ocimum sanctum fixed oil. Indian J Exp Biology. 1997;35(4):380–3.Google Scholar
- Sanchez S, Bartrons R, Rodriguez L, Gonzalez P, Planas ME, Lia A. Protective effect of nifedipine against carrageenan-induced inflammation. Pharmacol. 1998;56(3):131–6.View ArticleGoogle Scholar
- Ali N, Ali SS. Antispasmodic activity of Teucrium stocksianum Boiss. Pakistan J Pharm Sci. 2011;24(2):171–4.Google Scholar
- Kim HP, Son KH, Chang HW, Kang SS. Anti-inflammatory plant flavonoids and cellular action mechanisms. J Pharmacol Sci. 2004;96(3):229–45.View ArticlePubMedGoogle Scholar
- Salminen A, Lehtonen M, Suuronen T, Kaarniranta K, Huuskonen J. Terpenoids: natural inhibitors of NF-κB signaling with anti-inflammatory and anticancer potential. Cell Mol Life Sci. 2008;65(19):2979–99.View ArticlePubMedGoogle Scholar
- Sur P, Chaudhuri T, Vedasiromoni J, Gomes A, Ganguly D. Antiinflammatory and antioxidant property of saponins of tea [Camellia sinensis (L) O. Kuntze] root extract. Phytothe Res. 2001;15(2):174–6.View ArticleGoogle Scholar