Parasitol Res (2014) 113:197–209 DOI 10.1007/s00436-013-3644-4
ORIGINAL PAPER
Strong larvicidal potential of Artemisia annua leaf extract against malaria (Anopheles stephensi Liston) and dengue (Aedes aegypti L.) vectors and bioassay-driven isolation of the marker compounds Gaurav Sharma & Himanshi Kapoor & Madhu Chopra & Kaushal Kumar & Veena Agrawal Received: 1 October 2013 / Accepted: 9 October 2013 / Published online: 25 October 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract Malaria and dengue are the two most important vector-borne human diseases caused by mosquito vectors Anopheles stephensi and Aedes aegypti, respectively. Of the various strategies adopted for eliminating these diseases, controlling of vectors through herbs has been reckoned as one of the important measures for preventing their resurgence. Artemisia annua leaf chloroform extract when tried against larvae of A. stephensi and A. aegypti has shown a strong larvicidal activity against both of these vectors, their respective LC50 and LC90 values being 0.84 and 4.91 ppm for A. stephensi and 0.67 and 5.84 ppm for A. aegypti. The crude extract when separated through column chromatography using petroleum ether-ethyl acetate gradient (0–100 %) yielded 76 fractions which were pooled into three different active fractions A, B and C on the basis of same or nearly similar R f values. The aforesaid pooled fractions when assayed against the larvae of A . stephensi too reported a strong larvicidal activity. The respective marker compound purified from the individual fractions A, B and C, were Artemisinin, Arteannuin B and Artemisinic acid, as confirmed and characterized through FT-IR and NMR. This is our first report of strong mortality of A. annua leaf chloroform extract against vectors of two deadly diseases. This technology can be scaled up for commercial exploitation.
G. Sharma : H. Kapoor : V. Agrawal (*) Department of Botany, University of Delhi, Delhi 110007, India e-mail:
[email protected] M. Chopra Dr. B.R. Ambedkar Centre for Biomedical Research, University of Delhi, Delhi 110007, India K. Kumar National Centre for Disease Control, Delhi 110054, India
Introduction Malaria and dengue are two of the most important vectorborne diseases, endemic to the tropical and subtropical regions causing a serious health concern, mainly in the developing countries. Nearly, 216 million cases of malaria were reported in 2010, leading to death in 655,000 cases (WHO 2012a). Besides malaria, dengue has been ranked as another important vector-borne viral disease which has increased to 30-folds over the past five decades. On an average, around 50–100 million cases of it are reported every year (WHO 2012b). Anopheles stephensi Liston, is the major human malarial mosquito vector prevalent in several countries including the Middle East and South Asia (Bian et al. 2013), which harbours and transmits the malarial protozoan parasite Plasmodium falciparum (Corby-Harris et al. 2010). In addition to malaria vector, Aedes aegypti L. is another leading mosquito vector for RNA viruses which causes dengue and yellow fever (Arensburger et al. 2011). As these vectors transmit two crucial diseases, so they are of immense medical importance. Of the various strategies adopted to curb malaria and dengue, vector control is an effective measure of checking the intensified spread of these diseases. The non-availability of proper vaccines against dengue virus makes vector control the only potential preventive approach to be followed to manage the spread of disease. In case of malaria, cure through drugs is prevalent over vector control, but as mosquitoes are developing resistance to these drugs, again switching over to vector control is beneficial (Townson et al. 2005). Vector control generally deals with eliminating the probable carriers of diseases maybe mammals, birds or insects like mosquitoes. Though for mosquito vector control, various synthetic chemicals and repellents such as DDT, malathion, organochlorine, organophosphate, deltamethrin and synthetic
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pyrethroid insecticides are used (Senthilkumar et al. 2009). But, due to their toxic and hazardous nature, such chemicals can cause a threat to the ecosystem and non-target organisms, including humans and also, mosquitoes develop resistance to these drugs very soon. Plant-based insecticides and larvicidal compounds are proving as potent alternatives for vector control due to their biodegradable nature, pest specificity and non-toxic effects on other organisms (Bagavan et al. 2008). Several such compounds and extracts have been evaluated for their bioefficacy against vectors of various diseases. In recent times, a number of reports have appeared regarding the larvicidal activities of herbal-based drugs/ extracts against various mosquito vectors like malarial, filarial, encephalitis, dengue, etc. (Babu and Murugan 1998; Murugan and Jeyabalan 1999; Choochote et al. 2004; Das et al. 2007; Pandey et al. 2007; Bagavan et al. 2008; Chapagain et al. 2008; Senthilkumar et al. 2009; Hallert et al. 2012; Sharma et al. 2012; Warikoo and Kumar 2013; Cheah et al. 2013). However, there is no report on the strong larvicidal activity of Artemisia annua extract on malaria and dengue vectors, though it has been extensively used against malarial parasites (Plasmodium falciparum , Plasmodium vivax). Realizing the current scenario of deadly mosquito vectors where the available drugs are becoming ineffective, it is the exigency to explore the newer plant-based potential drugs that are not only cost-effective but eco friendly to the flora and fauna vis a vis synthetic drugs. The current investigation has been carried out to explore the bioefficacy of A . annua leaf extract against two vectors A. stephensi and A. aegypti. Besides, isolation of the bioactive compounds has also been done from the active fractions, effective against the carriers of two serious human diseases, malaria and dengue. To the best of our knowledge, this is a first report of the most effective larvicidal activity of A. annua extract against A. stephensi and A . aegypti.
evaporate to dryness in vacuum. The yield of solvent free gummy extract relative to dry starting material was recorded. Different dilutions of the extracts were made (50, 25, 12.5, 6.25, 3.125 and 1.5625 ppm) through serial dilution with autoclaved sterile water mixed with triton (10 μL/L of water).
Materials and methods
Isolation of larvicidal compounds from crude extract
Plant material and preparation of crude extract
Preparation of extract
Seeds of A . annua were obtained from National Bureau of Plant Genetic Resources (NBPGR), Delhi, India and were subsequently sown in the seed beds of Botanical Garden, Department of Botany, University of Delhi. The leaves from the field-grown plants were collected for preparation of the extract, once they reached maturity. The leaves were dried in the oven at 36 °C for 48 h and the dry material was ground into a fine powder using grinder. Thereafter, the materials were filtered through muslin cloth and were extracted twice with the respective solvents (20 mL/g of the dry weight of the material) overnight with continuous stirring over a magnetic stirrer. The extract concentrate was further allowed to
Mature leaves (400 g) of micropropagated A . annua plants were collected from the field, washed thoroughly under running water and were dried under shade. The dried leaves were subsequently crushed to powdered form in an electric grinder (Inalsa Technologies, Delhi). The dried powder (50 g) was fed into a soxhlet apparatus for extraction with hexane for 48 h. After 48 h, the filtered extract obtained from soxhlet was concentrated in a rotary vacuum evaporator. The solvent free extract (4 g) was partitioned three times between n-hexane (12 ml/g) and 20 % aqueous acetonitrile (MeCN) (4 ml/g) presaturated with each other. The combined 20 % aqueous MeCN was back washed using 10 % of its volume with
Solvents used for extraction Four different solvents, viz. methanol, ethanol, chloroform and acetone (Merck, Germany) were selected for the preparation of crude extracts from the in vivo leaves of A. annua plants (Fig. 1). Mosquito larvicidal assay of crude extract Bioassays were conducted following the standard World Health Organization larval susceptibility test method (WHO 2005). Bioassays were carried out in four replicates (25 larvae per replicate) with positive control and control run simultaneously at 28±20 °C, RH 70–80 % and 14:10 h light/dark conditions in the laboratory. Larvae used in these test were from the National Centre for Disease Control, Delhi (Fig. 1). Data on larva mortality were recorded for 24 h posttreatment and expressed as percent mortality. Mortality in the treatment was corrected with control mortality using Abbot's formula (Abbot 1925). The lethal concentration for 50 % mortality (LC50), lethal concentration for 90 % mortality (LC90) and 95 % confidence limits were calculated by using probit analysis (Finney 1979). Fourier transform spectrometry analysis Fourier transform infrared spectrometry (FT-IR) of the crude extract yielding maximum bioefficacy was obtained with Fourier transform (FT-IR) spectrometry instrument (spectrum RXI), resolution 4.00 cm−1, detector: LiTaO3.
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Fig. 1 Diagrammatic representation of the bioassay conducted with leaf extract in different solvents against A. stephensi and A. aegypti mosquito vectors
presaturated hexane. Removal of H2O from the MeCN phase was accomplished using solid NaCl (7 g/100 ml 20 % aqueous MeCN). Evaporation of the MeCN in vacuo provided an oily yellowish-brown residue (1 g). This MeCN phase extract was used for isolation of larvicidal compounds.
R f (mobility relative to front) value was calculated applying the following formula: R f ¼ Distance travelled by solute=Distance travelled by solvent
Column chromatography Thin-layer chromatography Thin-layer chromatography (TLC) of the crude extract was carried out to select a suitable solvent system for separation of different compounds present in the crude extract. The glass chamber (Borosil), size 15×5×25 cm was filled with the selected solvent (ethyl acetate–chloroform, 7.5 %), covered and left untouched for 10 min to allow the enclosed air inside the chamber to get fully saturated with solvent vapour. Next, the sample was loaded on the Silica Gel60 F254 TLC plates (Merck; size, 15×2 cm). The spots were allowed to dry. Henceforth, the plate was removed from the chamber and the solvent front was marked immediately with a pencil. The plate was allowed to dry completely, so that the solvent may evaporate. To visualize the different zones/spots of the crude extract resolved on the TLC plate, the plate was exposed to iodine vapours in a tightly closed glass chamber. Finally, the
The solvent system giving the best resolution in TLC was selected as the eluting solvent (mobile phase) while separating the components of the crude extract through column chromatography (CC). The slurry was prepared by mixing 1 g of crude extract with 5 g dry silica gel powder (Merck), 100–200 mesh. A 30-cm-length glass column was taken and a thick layer of cotton was inserted at its bottom with the help of a wire. The column was then fixed to the clamp stand and subsequently filled with the eluent, petroleum ether. The stopcock situated at the bottom was kept open so that the solvent drips continuously at a slow rate. A beaker was kept below the nozzle so as to collect the dripping solvent, which was reused in the entire process of packaging. In the next step, silica powder was mixed with petroleum ether and was poured slowly in the column. This step was repeated several times till a 25-cm-length silica column was packed. The column was
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tapped intermittently to prevent air bubbles from being trapped in it. Subsequently, the powdered slurry was added at the top of the column with the help of a glass rod. Gradually, the slurry settled in the form of a uniform layer above the silica. Adequate care was taken to always maintain a 10-ml layer of solvent above the column so that the silica does not get dry. Once the column was packed with silica, the next step involved separation of different components of the crude extract by running the mobile phase of petroleum ether: ethyl acetate, through the column in the order of increasing polarity (0, 2, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90 and 100 %). The column was run at the flow rate of 0.16 ml/s at room temperature. A total of 76 fractions, of 50 ml each were collected and subsequently concentrated to dryness in a rotary vacuum evaporator. The concentrated fractions were stored in glass vials at room temperature.
fluorescent indicator UV rays (λ =254 nm) and the visible spots were marked with pencil. The respective R f values of different components of each fraction was calculated. The fractions having compounds with similar R f values were mixed together. Finally, three different fractions of the MeCN residue were obtained and labelled as A, B and C. The weight (in milligramme) of each respective fraction was taken and was subsequently tested for larvicidal activity. Nuclear magnetic resonance analysis 1
H nuclear magnetic resonance (NMR) spectra of the three isolated and purified compounds were obtained with NMR instrument (Bruker Spectrospec), DPX-300 MHz using tetramethylsilane (TMS) as internal standard. Bioassay of fractions
Thin-layer chromatography of separated fractions The separated fractions were further analyzed through TLC, following the aforesaid mentioned protocol using hexaneethyl acetate mobile phase in all cases. For visualization of the resolved compounds, the TLC plates were exposed to
Table 1 Larvicidal activity of different extracts of A. annua leaves against late III/early VI instar larvae of A. stephensi
Solvent used for extraction
Concentration of crude extract (ppm)
Mortality (%)
LC50 (ppm) (LCL−UCL)
LC90 (ppm) (LCL−UCL)
χ 2a
Ethanol
50 25 12.5 6.25 3.13 1.56 50 25 12.5 6.25 3.13 1.56 50 25
86 53 36 14 03 02 59 47 14 11 09 04 100 100
19.27 (16.23–22.32)
74.13 (58.23–101.25)
5.25
35.72 (28.18–48.53)
290.27 (173.30–609.83)
6.12
0.84 (0.33 – 1.26)
4.91 (3.73 – 8.58)
1.85**
12.5 6.25 3.13 1.56 50 25 12.5 6.25 3.13 1.56
100 92 87 65 81 73 70 60 55 38
2.09 (0.99–3.28)
168.99 (76.13–728.13)
7.27**
Acetone
Chloroform
LC 50 lethal concentration that kills 50 % of exposed larvae, LC 90 lethal concentration that kills 90 % of exposed larvae, LCL lower confidence limits, UCL upper confidence limits, χ 2 chi-square *P
