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Print version ISSN 0102-695X
Rev. bras. farmacogn. vol.21 no.6 Curitiba Nov./Dec. 2011 Epub Aug 12, 2011
Fariba H. AfsharI,II; Abbas DelazarI,II Omar JannehIII; Hossein NazemiyehIV; Ardalan PasdaranIV; Lutfun NaharV; Satyajit D. Sarker*,VI
IDrug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
IIBiotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
IIIDepartment of Biomolecular and Sport Science, Coventry University, England, UK
IVSchool of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
VDrug Discovery and Design Research Division, Department of Pharmacy, School of Applied Sciences, University of Wolverhampton, West Midland, UK
VIDepartment of Pharmacy, School of Applied Sciences, University of Wolverhampton, West Midland, UK
Artemisia species (Asteraceae), widespread throughout the world, are a group of important medicinal plants. The extracts of two medicinal plants of this genus, Artemisia scoparia Waldst. & Kit. and A. spicigera C. Koch, were evaluated for potential antimalarial, free-radical-scavenging and insecticidal properties, using the heme biocrystallisation and inhibition assay, the DPPH assay and the contact toxicity bioassay using the pest Tribolium castaneum, respectively. The methanol extracts of both species showed strong free-radical-scavenging activity and the RC50 values were 0.0317 and 0.0458 mg/mL, respectively, for A. scoparia and A. spicigera. The dichloromethane extracts of both species displayed a moderate level of potential antimalarial activity providing IC50 at 0.778 and 0.999 mg/mL for A. scoparia and A. spicigera, respectively. Both species of Artemisia showed insecticidal properties. However, A. spicigera was more effective than A. scoparia.
Keywords: Artemisia scoparia, Artemisia spicigera, Asteraceae, DPPH, antimalaria, insecticide.
The genus Artemisia L., one of the largest and most widely distributed genera of the family Asteraceae, comprises about 200-400 species of herbs and shrubs, the most notable being A. annua for producing the antimalarial drug artemisinin (Lee et al., 2000; Kim et al., 2002; GRIN Database, 2010). Artemisia species are distributed in the northern hemisphere, especially, along the Mediterranean coast of Europe, Southwest of Asia and Africa (Trease & Evans, 2002; Salido et al., 2004; Ferchichi et al., 2006). This genus, with its common Persian name "Dermane" and the common English name "Wormwood", includes approximately 34 species that are found wild all over Iran (Mozaffarian, 1996; Ghasemi et al., 2007). Several coumarins, flavonoids, phenylpropanoids, sterols and terpenoids (specially sesquiterpenes and monoterpenes), and their glycosides were isolated from this genus, and shown to have antimalarial, antiviral, antitumor, antipyretic, antihaemorrhagic, anticoagulant, antiinflammatory, antioxidant, antihepatitis, antiulcerogenic, antispasmodic and anticomplementary activities (Kim et al., 2002; Lee et al., 2003).
Artemisia scoparia Waldst. & Kit. (common name: "Redstem wormwood") and A. spicigera C. Koch (common name: "Sluggish wormwood") are two Iranian species, the former being a faintly scented slender branched biennial herb, and the latter an odorous plant (Mozaffarian, 1996). While A. scoparia possesses anticholesterolemic, antipyretic, antiseptic, antibacterial, cholagogue, diuretic, purgative and vasodilator properties (Safaei-Ghomi et al., 2005; Mirjalili et al., 2007; Singh et al., 2009), A. spicigera has antiseptic and stomachic properties (Guvenalp et al., 1998). The essential oils of A. scoparia show strong insecticidal activity against stored-product insects (Negahban et al., 2006), and that of A. spicigera has significant antioxidant property (Can-Baser & Buchbauer, 2009). As part of our on-going phytochemical and bioactivity studies on medicinal plants from Iran (Razvi et al., 2008; Modaressi et al., 2009; Nazemiyeh et al., 2009; Asnaashari et al., 2010; Delazar et al., 2006, 2007, 2010a-c), we now report on the antimalarial, free-radical-scavenging and insecticidal activities of different extracts of A. scoparia and A. spicigera.
Material and Methods
The aerial parts of Artemisia scoparia Waldst. & Kit. and A. spicigera C. Koch, Asteraceae, were collected from a place near the Aras river and Jolfa at E: 45° 17', N: 38° 39' (altitude of 700-750) in Eastern Azerbaijan province (Iran) during November 2009. The identity of the plants was confirmed by anatomical examination in comparison with the herbarium specimens (voucher nos. TUM-ADE 702 and TUM-ADE 705, respectively) retained in the School of Pharmacy, Tabriz University of Medical Science, Iran.
Air-dried and ground aerial parts of A. scoparia and A. spicigera (120 g each) were individually Soxhlet-extracted successively, with n-hexane, dichloromethane (DCM) and methanol (MeOH) (1.1 L each). All these extracts were separately concentrated using a rotary evaporator at a maximum temperature of 45 °C.
Heme biocrystallisation and inhibition assay for potential antimalarial activity
The potential antimalarial activity of plant extracts was evaluated by the method described by Fitch et al. (1999) with some modifications (Tripathi et al., 2004). Briefly, varying concentrations (0-2 mg/mL in 10% DMSO) of the extracts were incubated with 300 µM of hematin (freshly dissolved in 0.1 M NaOH), 10 mM oleic acid and 10 µM HCl. The reaction volume was adjusted to 1000 µL using 500 mM sodium acetate buffer, pH 5. Chloroquine diphosphate was used as a positive control. The samples were incubated overnight at 37 ºC with regular shaking. After incubation, samples were centrifuged (14,000 x g, 10 min, at 21 °C) and the hemozoin pellet repeatedly washed with sonication (30 min, at 21 °C; FS100 bath sonicator; Decon Ultrasonics Ltd.) in 2.5% (w/v) SDS in phosphate buffered saline followed by a final wash in 0.1 M sodium bicarbonate, pH 9.0, until the supernatant was clear (usually 3-5 washes). After the final wash, the supernatant was removed and the pellets were re-suspended in 1 mL of 0.1 M NaOH before determining the hemozoin content by measuring the absorbance at 400 nm (Beckmann DU640 spectrophotometer) using a 1 cm quartz cuvette. The results were recorded as % inhibition (I%) of heme polymerization/crystallization compared to positive control (chloroquine) using the following formula: I% = [(ABAA)/AB] x 100, where AB: absorbance of blank; AA: absorbance of test samples.
The hydrogen atoms or electrons donation ability of the extracts was determined spectrophotometrically by the bleaching of purple coloured methanolic solution of DPPH (2,2-diphenyl-1-picrylhydrazyl) as a reagent (Takao et al., 1994). 2,2-Diphenyl-1-picrylhydrazyl, molecular formula C18H12N5O6, was obtained from Sigma-Aldrich (Germany). DPPH (4 mg) was dissolved in MeOH (50 mL) to obtain a concentration of 80 μg/mL. The MeOH extracts of plants were dissolved in MeOH and the n-hexane and DCM extracts were in chloroform to obtain a concentration of 1 mg/mL. Dilutions were made to obtain concentrations of 0.25, 0.125, 0.0625, 0.03125, 0.015625, 0.0078125, 0.00390625, 0.001953125, 0.000976563, 0.000488281 mg/mL. Diluted solutions were mixed with DPPH (1 mL) and allowed to stand for 30 min for any reaction to occur. The UV absorbance of samples and blank (without sample) was recorded at 517 nm. The experiment was performed in duplicate and the average absorption was noted for each concentration. The same procedure was followed for the positive control, quercetin. Percent inhibition of the free radical DPPH (I %) was calculated in the following way: I% = [(ABAA)/AB] x 100, where AB: absorbance of blank; AA: absorbance of test samples. Concentration providing 50% inhibition (RC50) was calculated from the graph plotting inhibition percentage against test sample concentrations (Takao et al., 1994; Delazar et al., 2006; Kumarasamy et al., 2007).
Insecticidal activity assay
Adults of Tribolium castaneum (red flour beetle) were supplied from a laboratory culture, which were reared on a mixture of whole wheat flour and maize flour at the ratio of 1:1 in glass containers containing 0.5 kg of the mixture. All species were reared at 27±2 °C, 12% moisture content in continuous darkness for about three weeks without exposing to insecticides. Adults used in the experiments were 1-3 weeks old and of mixed sex.
The insecticidal activity of the n-hexane, DCM and MeOH extracts of A. scoparia and A. spicigera was evaluated by the method described previously (Loschiavo et al., 1963) with minor modification (Freedman et al., 1982). The extracts were prepared at concentrations of 20, 40 and 80 mg/mL in respective extraction solvents. The filter paper (9 cm of diameter) received 1 mL of these extracts, and was placed on a Petri dish (9 cm of diameter). The control was treated with pure solvents. Solvent was allowed to evaporate. Ten non-sexed adults of Tribolium castaneum (Tenebrionidae) were placed in each Petri dish, maintained under 27±0.5 °C, 12% moisture content and 12 h photo phase. The experimental design was completely randomized, with three replicates. Insect mortality was evaluated after 4, 8, 24, 48 h of exposure to impregnated filter paper. Beetle responses to treated discs versus control discs were converted to "percentage of mortality".
Data was analyzed by one way ANOVA using SPSS-12 for Windows. Differences in means were estimated by means of repetitive measures followed by Bonferroni and Dunnet's post hoc test and expressed as statistical mean±standard deviation. Differences between means were regarded significant at p<0.05.
Results and Discussion
Malaria-infected erythrocytes are characterized by a high rate of production of ferriprotoporphyrin IX (heme) as a result of the ingestion and digestion of host cell haemoglobin (Famin et al., 1999). The parasite utilizes hemoglobin as its primary food source during intraerythrocytic development and proliferation (Francis et al., 1997). Heme is released as a toxic material of this process (Tripathi et al., 2001). It affects cellular metabolism by inhibiting enzymes, peroxidizing membranes and producing oxidative free radicals (Rathore et al., 2006). An important mechanism for the detoxification of heme is the formation of dark micro crystals of hemozoin in the food vacuoles of malaria parasites, and commonly referred to as malaria pigment (Kalkanidis et al., 2002; Egan & Ncokazi, 2005; Rathore et al., 2006). Earlier this process used to be known as "heme polymerization" (Tripathi et al., 2001). It has been revealed that the structure of hemozoin (and its synthetic equivalent, β-hematin) is a cyclic dimer of ferriprotoporphyrin IX (heme). Several studies have shown that chloroquine and most of other antimalarial compounds inhibit β-hematin formation under different conditions (Egan & Ncokazi, 2005). The heme biocrystallization and inhibition assay is based on the above facts (Fitch et al., 1999; Tripathi et al., 2004).
In this study the extracts of A. scoparia and A. spicigera were assessed for potential antimalarial activity using the heme biocrystallization and inhibition assay. The n-hexane and the MeOH extracts of both plants did not show any significant inhibition of heme biocrystallization properties. Unexpectedly, the extracts promoted heme bioscyrstallization, suggesting the lipid-like properties of this extract. However, the DCM extracts of A. scoparia and A. spicigera, in comparison with the blank, showed significant potential antimalarial effects (Figure 1). At lower concentrations (0.1-0.4 mg/mL), the observed absorbances of the DCM extracts were higher than the blank which might be a consequence of other fatty acids present in extracts causing synergistic effect with oleic acid in the test. At higher concentrations (>0.4 mg/mL), the DCM extracts inhibited heme biocrystallization. At higher concentrations (>0.4 mg/mL), the DCM extracts illustrated potent antimalarial effects. The IC50 values of the DCM extracts were 0.778 (A. scoparia) and 0.999 (A. spicigera) mg/mL and that of the positive control chloroquine was 0.043 mg/mL.
The free-radical-scavenging activity of the extracts of the aerial parts of A. scoparia and A. spicigera were evaluated in vitro by the DPPH assay. In this assay, the extracts were able to reduce the stable radical DPPH· to the yellow colored diphenylpicrylhydrazine. The method is based on the reduction of methanolic DPPH solution in the presence of a hydrogen-donating scavenger through the formation of the non-radical form DPPD-H (Kumarasamy et al., 2007). All test extracts were capable of scavenging DPPH free radicals and exhibited a concentration-dependent activity pattern. The order free-radical-scavenging potency of the extracts were MeOH>DCM>n-hexane (Table 1). The extracts of A. scoparia were more potent than those of A. spicigera. Quercetin, a known natural free-radical scavenger, was used as a positive control. The DPPH radical-scavenging activities of the MeOH extracts were quite remarkable and comparable to that of quercetin [RC50 values 0.0317±0.5 mg/mL (A. scoparia), 0.0458±0.5 mg/mL (A. spicigera) and 0.0039±0.5 mg/mL (quercetin). Polar extracts (MeOH) exhibited stronger activity than non-polar extracts. The MeOH extracts might contain phenolics compounds, e.g. flavonoids, coumarins or phenylpropanoids, which might have contributed toward the significant free-radical-scavenging activity of these extracts.
Both Artemisia species showed insecticidal effects (Table 2). However, effect of A. spicigera was greater than that of A. scoparia. Among the extracts of A. spicigera, the DCM extract displayed the most potent activity. Previous studies reported the insecticidal effect of terpenoids and phenylpropanoids present in different species of Artemisia (Abdelgaleil et al., 2008; Liu et al., 2010). The possible presence of such compounds in the DCM extract of A. spicigera might be responsible for the insecticidal activity, as these compounds are better extracted by DCM.
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Received 3 Aug 2010
Accepted 30 Dec 2010
* Correspondence: Satyajit D. Sarker, Department of Pharmacy, School of Applied Sciences, University of Wolverhampton, MM Building, Molineux Street, Wolverhampton WV1 1SB, West Midland, UK. E-mail: firstname.lastname@example.org. Tel. +44 (0) 19 0232 2578