The use of microfluorometric method for activity-guided isolation of antiplasmodial compound from plant extracts
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- Shuaibu, M.N., Wuyep, P.A., Yanagi, T. et al. Parasitol Res (2008) 102: 1119. doi:10.1007/s00436-008-0879-6
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In vitro antiplasmodial activity of methanolic extracts of 16 medicinal plants was evaluated by fluorometric assay using PicoGreen. The IC50s, as determined by parasite DNA concentration, ranged from <11 to >200 and <13 to >200 μg/ml for Plasmodium falciparum 3D7 and K1, respectively; and the most active extracts were those from Anogeissus leiocarpus and Terminalia avicennoides (<11–≥14 μg/ml). Aqueous, butanolic, ethyl acetate, and methanolic fractions of these two extracts revealed butanolic fraction to have a relatively better activity (IC50, 10–12 μg/ml). Activity-guided chromatographic separation of the butanolic fraction on Sephadex LH-20 followed by nuclear magnetic resonance and correlation high-performance liquid chromatography revealed the presence of known hydrolysable tannins and some related compounds—castalagin, ellagic acid, flavogallonic acid, punicalagin, terchebulin, and two other fractions. The IC50s of all these compounds ranged between 8–21 μg/ml (8–40 μM) against both the strains. Toxicity assay with mouse fibroblasts showed all the extracts and isolated compounds to have IC50 ≥ 1500 μg/ml, except for Momordica balsamina with <1500 μg/l. All the extracts and isolated compounds did not affect the integrity of human erythrocyte membrane at the observed IC50s. However, adverse effects manifest in a concentration-dependent fashion (from IC50 ≥ 500 μg/ml).
Malaria, caused by protozoan parasites of the genus Plasmodium, affects up to 500 million individuals and kills over one million people every year, particularly children in sub-Saharan Africa (WHO 2007). Despite over a hundred years of drug development, malaria remains one of the most devastating infectious diseases in the world. The development of drug resistance to the potent and affordable drugs like chloroquine, lack of concerted vector control strategy, and absence of licensed vaccine, has left many developing countries without sustainable options for malaria control; and chemotherapy remains the only prompt and effective option (Sirima and Gansane 2007). Therefore, there is a pressing need for search of newer antimalarial compounds and targets.
Plant extracts are still widely used in the treatment of malaria and other ailments, in Africa and other continents. In Africa, up to 80% of the population uses traditional medicine for primary health care. In Ghana, Mali, Nigeria, and Zambia, the first line of treatment for 60% of children with high fever resulting from malaria is the use of herbal medicines at home (WHO 2003). Verification of the efficacy of folk medicines has led to the isolation of hitherto excellent antimalarials, quinine and artemisinin (and their derivatives); from Chinchona spp and Artemisia annua, respectively (Bruce-Chwatt 1988; Fleming 1999; Woerdenbag et al. 1990). This has made natural products presumed to have a rich source of antimalarial compounds or their pharmacophores.
In vitro activities of different plant extracts against chloroquine-susceptible (3D7) and -resistant (K1) strains of Plasmodium falciparum and toxicity on mammalian cell
Plant species and name
Mean IC50 in μg/ml against P. falciparum strain
Cissus populnea (sp)
Materials and methods
Plants and extracts preparations
The 16 plants were collected in Bauchi state, northern Nigeria. They were all identified by traditional healers and we collected them. The botanical names of the plants were identified and confirmed by the herbarium, Department of Biological Sciences, Ahmadu Bello University, Zaria, Nigeria. The plant material (leaves, roots, or stem) were air-dried and powdered, and 100 g of each plant part were extracted with 200–300 ml of methanol at room temperature for about 72 h. The MeOH extracts were each filtered and the filtrate evaporated to dryness. These crude extracts were used for the in vitro antiplasmodial activity screening.
Fractionation, purification, and isolation
Air-dried powdered stem bark (1.2 kg each) of Terminalia avicennoides and of Anogeissus leiocarpus were macerated in methanol at room temperature for 48–72 h. The methanolic crude extract yield (162.13 g and 114.1 g) for T. avicennoides and A. leiocarpus was suspended in water and successively partitioned with butanol and ethyl acetate. The insoluble precipitate formed during the solvent partitioning was dissolved in DMSO. Five grams of the butanolic fraction was chromatographed on the Sephadex LH-20 column with water containing increasing proportions of methanol (0→100%, 20% stepwise elution) to give six fractions. Thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis indicated that fraction 1 (0.74 g, 15%) contained sugars and triterpene glycosides, fraction 2 (0.63 g, 13%) was further separated by silica gel column chromatography to give two compounds. Nuclear magnetic resonance (NMR) spectral analysis revealed that one was ellagic acid rhamnoside and the other was triterpene glycoside; however, further structural elucidations were not completed. Fraction 3 (0.31 g, 6%) was a mixture of tannins and phenol carboxylic acids. Fraction 4 (0.40 g, 8%) was a mixture of flavogallonic acid bislactone (co-TLC, HPLC) and punicalagin (HPLC), and fraction 5 (1.30 g, 26%) contained ellagic acid (TLC, HPLC) and tannins detected as a broad hump of the base line on HPLC analysis. The major constituent of fraction 6 (0.69 g, 13.8%) was found to be terchebulin by NMR spectral comparison (Lin et al. 1990; Tanaka et al. 1986a, b). Unknown tannins and phenol carboxylic acid was also detected in fraction 6, while the butanolic fraction of A. leiocarpus revealed castalagin as the major compound along with ellagic and flavogallonic acids (Tanaka et al. 1996).
1H and 13C NMR, 1H–1H COSY, NOESY, HSQC, and HMBC spectra were recorded with a Unity plus 500 spectrometer (Varian, Palo Alto, CA, USA) operating at 500 MHz for 1H- and 125 MHz for 13C-NMR spectroscopy. The 1H and 13C NMR spectra were also measured using a JEOL JMN-AL400 (JEOL, Japan) operating at 400 MHz for 1H- and 100 MHz for 13C-NMR. FABMS were recorded on a JMS DX-303 spectrometer (JEOL), and m-nitrobenzyl alcohol or glycerol was used as the matrix.
Chromatography and HPLC analysis
Column chromatography was conducted on Sephadex LH-20 (Pharmacia Fine Chemical). TLC was performed on 0.2 mm thick precoated Kieselgel 60 F254 plates (Merck) with benzene/ethyl formate/HCO2H (1:7:1, v/v) with spots detected by UV illumination, sprayed with 2% ethanolic FeCl3, or 10% H2SO4 reagent. Analytical HPLC was performed on a Cosmosil 5C18-AR II column (Nacalai Tesque; 250 × 4.6 mm i.d.), with gradient elution at 10–30% (30 min) and 30–75% (15 min) of CH3CN in 50 mM H3PO4 (flow rate, 0.8 ml/min; detection, JASCO photodiode array detector MD-910).
Screening for antiplasmodial activities
The methanolic extracts were dissolved in DMSO as a stock solution of 5 or 10 mg/ml and diluted to concentrations ranging from 1.0–500 μg/ml. Different concentrations were assessed for antiplasmodial activities in 96-well plates in 48-h time course. Crude extracts, partially purified and isolated compounds were all dissolved in DMSO and diluted in complete RPMI 1640. Various concentrations of the diluted extracts and compounds were aliquoted into different wells of 96-well microplates and 5% hematocrit culture of 1.5–2% parasitemia was added to a final volume of 200 μl and incubated for 48 h. Known antimalarials, artemisinin and CQ were also dissolved in their respective solvents and their activities were assessed. Background effect of each plant extract and compound were measured in the absence of the parasites. This was conducted by measuring the fluorescence of each extract or compound in the absence of the parasites and the fluorescence value was subtracted from the value obtained in the presence of the parasite and drug. All assays were carried out in replicate.
Plasmodium culture medium
Complete RPMI 1640 medium (Sigma, St. Louis, MO), containing l-glutamine and 25 mM HEPES, supplemented with10 mg/ml gentamicin, 50 mg/liter hypoxanthine, 0.225% NaHCO3 and 0.5% Albumax II (GIBCO), and adjusted to a pH of 7.3 to 7.4 (Fidock et al. 2004). Cultures were maintained in type O+ human red blood cell suspensions obtained from healthy local donors at Nagasaki University Hospital, Japan and 5% hematocrit was prepared in a complete RPMI 1640 (Sigma, St. Louis, MO).
Two strains of Plasmodium falciparum (3D7 and K1) were used and were provided by Dr. Mitamura of International Medical Centre, Tokyo, Japan. 3D7 is chloroquine (CQ)-sensitive and K1 is CQ-resistant. The two strains were maintained in vitro by a modification of the method of Trager and Jensen, 1976. The parasite density was maintained below 2–2.5% parasitemia under an atmosphere of a gas mixture containing 5% CO2, 5% O2, and 90% N2 at 37°C.
A sorbitol synchronized culture, 1.5–2% parasitemia and hematocrit 5% were used to test serial dilutions of plant extracts in 96-well round bottom culture plates. Parasites were allowed to grow in the presence and absence of extracts, fractions or compounds for a 48-hour incubation period in a final volume of 200 μl, after which a 150 μL aliquot of culture was transferred to a new 96-well flat bottom plate. Fifty microliters of the fluorochrome mixture, which consists of PicoGreen® (Molecular Probes, Inc., Eugene, OR) dissolved in TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 7.5) and 2% Triton X-100 diluted with distilled, DNAse-free water (as described by the manufacturer) was then added to liberate and label the parasitic DNA. The plates were then incubated for 30–60 minutes in the dark. The fluorescence signal, measured at 485/20 nm excitation and 528/20 nm emission (Yolanda et al. 2004) using Perkin Elmer ARVO™ MX-1420.
The counting of parasites and estimation of parasitemia was performed by making thin smear and Giemsa staining.
Toxicity toward newborn mouse heart-derived cells (NBMH) was assessed with cells plated in 48-well plates at 105 cells/well. Stock cell cultures were maintained in 25-cm3 flasks and sub-cultured to the appropriate split ratio by mild trypsinization once in 7–10 days. The cells were allowed to settle and start confluence formation for 24–48 h. Microscopically, the fibroblasts had a normal appearance and showed normal cell growth rates. The concentration of a compound which provoked a >75% reduction in cell viability compared to the control cells after 48 h incubation was considered as MIC for the cells. After adherence, the medium was removed and replaced by media containing the different concentrations of the extracts or compounds. The plates were incubated with different concentrations (10–2,000 μg/ml) of the extracts or compounds for 24–96 h at 37°C in a humidified 5% CO2 incubator. Control cells were incubated with culture medium alone and with solvent, DMSO at a final concentration of less than 1%. The wells were assessed microscopically for cell growth and MIC was determined. A week after incubation with the different extracts, the medium was replaced with a fresh one and cell growth was monitored. Selectivity indices (SI) were calculated by dividing the MIC values of the NBMH versus the IC50 values for the parasites.
On normal erythrocytes
Effect on human erythrocyte membrane was conducted as described by Ziegler et al. 2004. Non-parasitized erythrocytes were incubated in 96-well microtitre plates in the medium containing different concentrations of the extracts, fractions, and isolated compounds. Control wells contained erythrocytes in the medium alone or in the medium containing less than equal 1% of DMSO. After 48 h of incubation, 20 μL samples were spread on microscope slides, allowed to dry, fixed with methanol, and stained with Giemsa for light microscopy.
Data were analyzed using Microsoft Excel 2003. Relative Fluorescence Unit (RFU) was calculated by subtracting the control (fluorescence from the 5% hematocrit) and background effects (fluorescence from the individual plant extracts or fractions). RFU was converted to percentage (%) by taking the negative control that was incubated in the absence of extract, fraction, or any of the compounds as 100% parasitemia (fluorescence unit in the presence of extract / fluorescence unit of control in the absence of extract × 100). A concentration–response curve (% parasitemia versus log concentration) was plotted for each of the extracts and 50% inhibitory concentration (IC50) was calculated there from, and is defined as the drug concentration that results in 50% relative fluorescence decrease as compared to control in the absence of any compound.
Of the 16 extracts tested, seven exhibited weak or no activity (IC50 > 50 μg/ml), six were moderately active (IC50 ≥ 15–50 μg/ml) and three (IC50 < 15 μg/ml) relatively exhibited a better activity as observed with reference to the IC50 obtained for the CQ-resistant K1 strain.
In vitro activities of different solvent fractions against chloroquine-susceptible (3D7) and -resistant (K1) strains of Plasmodium falciparum and toxicity on mammalian cell
Mean IC50 in μg/ml against P. falciparum strain
In vitro activities of different isolated tannins against chloroquine-susceptible (3D7) and -resistant (K1) strains of Plasmodium falciparum and toxicity on mammalian cell
Mean IC50 in μg/ml(µM) against P. falciparum strain
These plants are usually used in different forms, mostly drunk as a decoction for treating many symptomatic ailments. We tried to check the possible toxicity of the different extracts in a concentration-dependent fashion using fibroblast obtained from new born mouse heart (NBMH). Most of the extracts did not show serious toxic effect up to concentration of 1,000 μg/ml; neither did the fractions nor the identified compounds, except for Momordica balsamina which was less than 1000 μg/ml (Tables 1, 2, 3). The viability of the cells was monitored using phase contrast microscopy and concentration-dependent changes were observed. The concentration of the extract or compound which provoked a >75% reduction in cell viability compared to the control cells after 48 h incubation was considered as MIC for the cells (NBMH). These changes were normally observed microscopically within the range of 1,500 μg/ml.
Sixteen medicinal plants used in northern Nigeria were identified; methanolic extracts were prepared and their in vitro biological activities against P. falciparum strains were determined using microfluorometric assay. In Nigeria, the traditional healing system plays an important role in health care delivery and about 70–80% of the population depend on traditional healers for most of their symptomatic ailments, and a good number of plants are available for this purpose (Akah et al. 1998; WHO 2003). The practice of herbal medication is very common among rural dwellers in northeastern Nigeria, where the herbs are sold openly in markets, shops, and through loud speaker advertisement on vehicles (Adamu et al. 2005). Our choice of these plants was influenced by their extensive use as traditional remedies for many symptomatic diseases; where the leaves, stem, or root barks are used as water extracts or decoctions as prescribed by the traditional healers and taken orally.
A number of the plants did not display a desirable in vitro antiplasmodial activity, despite strong claims of their curative effect by the healers and the users as well. A possible explanation could be that the plants act as antipyretics or immune stimulants to relieve the symptoms of the disease, rather than having direct antiparasitic activity (Phillipson et al. 1993). Alternatively, precursors of the active components may be present in the extracts but have to be modified, usually in vivo, before activity is exhibited (Cailean et al. 2004).
Extracts of A. leiocarpus and T. avicennoides, belonging to the family Combretaceae, appeared to display better activity. Although substantial amount of phytochemical research has been carried out on Combretaceae family, there are little or no reports on the effect of their hydrolysable tannins on parasites in vitro. In some reports (Vonthron-Sénécheau et al. 2003), tannins are usually removed because of the general belief that they are non-selective inhibitors of enzymes. However, in this report and like that of Asres et al. (2001a, b), we did not remove the tannins. This is because local consumption of these extracts does not exclude the tannins. We followed the fractions with a better activity and identified hydrolysable tannins to display some antiplasmodial effect. Tannins isolated from Terminalia spp have earlier been reported to possess antimicrobial activity (Taguri et al. 2004), but this is the first report of their antiplasmodial activity from A. leiocarpus and T. avicennoides and is in accord with the reported IC50 of punicalagin, 11.66 μg/ml (Asres et al. 2001b), even though our assay method (Table 3) and punicalagin source are different but from the same Combretaceae family.
These plant materials are generally taken orally as water extracts or sometimes as decoctions for treatment of various diseases and we investigated their toxicity on mammalian cells in vitro. Cytotoxic activity on mouse fibroblasts revealed SI to be within the range of 6–121, 86–181, and 23–180 for methanolic extracts, solvent fraction, and identified compounds, respectively. M. balsamina is the only herbal extract that was obtained from whole plant and showed minimum cytotoxic concentration (MCC) of <1500 μg/ml. This observation may shed some light on why most of these plant materials are used locally as medicines with no toxic effect reported by the users or the healers and is in agreement with other scientifically validated reports that higher concentration of extracts of Terminalia spp are tolerable in vivo, ≥100 mg/kg body weight (Abdullahi et al. 2001; Bizimana et al. 2006; Kamtchouing et al. 2006), and this also corroborates with the common practice of using these plants as chewing sticks (Rotimi et al. 1988; Taiwo et al. 1999). These also support our observations on the effect of extracts or identified compounds on the viability of fibroblasts and integrity of erythrocytes.
Many hydrolysable tannins from different plants were reported to have various biological activities, such as anticancer, antiviral, antibacterial, antiulcer, etc. (Asres et al. 2001b; Govindarajan et al. 2006; Larrosa et al. 2006; Quideau et al. 2004;Yang et al. 2000). Our activity-guided analyses of extracts of A. leiocarpus and T. avicennoides revealed hydrolysable tannins, mainly castalagin, ellagic acid, flavogallonic acid, punicalagin, and terchebulin. P. falciparum K1 is CQ-resistant while 3D7 is CQ-sensitive. However, our results show K1 to have comparable sensitivity in most instances to 3D7, particularly to the isolated tannins. This is not unexpected as CQ and tannins belong to different classes of compounds and this may present ample advantage for CQ-resistant P. falciparum-infected individuals in dealing with the infection. It is therefore likely that these compounds are, or in part, responsible for the observed antiplasmodial activity of A. leiocarpus and T. avicennoides.
This work was supported by Japanese Society for the Promotion of Science (JSPS). MNS is a JSPS fellow (ID#: 05166) and wish to thank, Dr. Haruna Danwanka, Mallam Farouq and Sabo Miri of Abuabakar Tafawa Balewa University, Bauchi, Nigeria; for their cooperation during the crude extract preparations and Dr. Toshihide Mitamura of Research Institute International Medical Center of Japan, Tokyo for the supply of the P. falciparum strains and his useful comment on the work and manuscript.