In Vitro and In Vivo Antimalarial Evaluations of Myrtle Extract, a Plant Traditionally Used for Treatment of Parasitic Disorders

Based on the collected ethnobotanical data from the Traditional Medicine and Materia Medica Research Center (TMRC), Iran, Myrtus communis L. (myrtle) was selected for the assessment of in vitro and in vivo antimalarial and cytotoxic activities. Methanolic extract of myrtle was prepared from the aerial parts and assessed for antiplasmodial activity, using the parasite lactate dehydrogenase (pLDH) assay against chloroquine-resistant (K1) and chloroquine-sensitive (3D7) strains of Plasmodium falciparum. The 4-day suppressive test was employed to determine the parasitemia suppression of the myrtle extract against P. berghei   in vivo. The IC50 values of myrtle extract were 35.44 µg/ml against K1 and 0.87 µg/ml against 3D7. Myrtle extract showed a significant suppression of parasitaemia (84.8 ± 1.1% at 10 mg/kg/day) in mice infected with P. berghei after 4 days of treatment. Cytotoxic activity was carried out against mammalian cell lines using methyl thiazol tetrazolium (MTT) assay. No cytotoxic effect on mammalian cell lines up to 100 µg/mL was shown. The results support the traditional use of myrtle in malaria. Phytochemical investigation and understanding the mechanism of action would be in our upcoming project.


Introduction
Malaria is a classic example of a disease that affects the productivity of individuals, families, and the whole society [1]. The number of deaths due to malaria was estimated to be 655000 in 2010 [2]. Malaria was highly endemic in the Caspian areas in the north and Persian Gulf littoral and plain areas in the south parts of Iran. It had been widely prevalent for a long time in the country [3]. At present the southern area of Iran (Figure 1), accommodating numerous emigrants including Afghanis and Pakistanis, is considered a high risk region in the area. According to the Ministry of Health of Iran the total number of malaria cases in Iran had been estimated to be 2900 cases in 2010 [4].
A dramatic recrudescence of malaria is ongoing due to the increasing resistance of mosquito vectors to insecticides and resistance of parasites, mainly Plasmodium falciparum, to available modern drugs [5]. Since malaria chemotherapy is complicated by drug-resistant strains of Plasmodium, new antimalarial agents are needed [6]. Traditional treatments may well prove to be the source of new antimalarial agents in view of the success with the two important chemotherapeutic agents, quinine and artemisinin, both of which are derived from plants [1]. Iran has an honorable past in traditional medicine. One of the most significant ancient heritages is sophisticated experience of people who have tried over millennia to find useful plants for health improvement, with each generation adding its own experience to this tradition [7].
High risk region This study is based on the ethnomedicinal data of the indigenous people of Kohgiluyeh-va-Boyer Ahmad province of Iran (unpublished data), where aqueous extract of myrtle is known as antiparasitic agent. Myrtle has been previously reported for various ailments like gastric ulcer, diarrhea, dysentery, vomiting, rheumatism, hemorrhage, deep sinuses, leucorrhoea, and cosmetic purposes [8]. In this study the in vitro and in vivo antiplasmodial activities and in vitro cytotoxic effect of the plant were assessed. The methanolic extract was selected for this experiment avoiding possible elimination of active compounds.

Collection and Extraction
. Myrtus communis L. was collected in its natural habitats. Botanical identification was performed by voucher specimen (TMRC 1169) deposited at the herbarium of the Traditional Medicine and Materia Medica Research Center (TMRC). The aerial parts of the plant were allowed to dry in shadow until desiccated. Plant sample was crushed into powder using a hammer mill and stored at room temperature in appropriate container.
10 g of powdered dried aerial parts was macerated in methanol with constant shaking at the room temperature for 24 hours. The filtrate was evaporated to dryness and used for further assessments.

2.2.
In Vitro Antiplasmodial Activity. In vitro screens for compound activity require the ability to culture P. falciparum in human erythrocytes [9]. The chloroquine-resistant (K1) and chloroquine-sensitive (3D7) strains of P. falciparum were continuous subcultured in vitro from cryopreservation and maintained in human red blood cells, diluted to 7% hematocrit with RPMI1640 medium. All cultures were placed in the candle jar at 37 ∘ C under 3% O 2 , 6% CO 2 , and 91% N 2 atmosphere.
In vitro antiplasmodial activity, following the lactate dehydrogenase (LDH) method was assessed [10]. Plasmodium species depend on LDH for the metabolism of carbohydrates. Parasite LDH(pLDH) is used for the conversion of lactate into pyruvate, which is the last step in glycolysis; however, only pLDH can use coenzyme 3-acetyl-pyridine adenine dinucleotide (APAD). At the presence of APAD, the detection of LDH is specific for the parasite enzyme. LDH determination is carried out in the presence of nitro blue tetrazolium (NBT) which is reduced to formazan that is detected at 630 nm.
Starting concentration of chloroquine diphosphate and artemisinin which served as positive controls was 20 mg/mL. Myrtle extract was dissolved in DMSO to produce stock solution of 20 mg/mL. The stock solutions were subsequently diluted with deionized water at twenty different concentrations. Twofold serial dilutions were made in 96-well microtitre plates in duplicate and infected erythrocytes were added to give a 2% hematocrit and 1% parasitaemia. For the infected control, parasitized red blood cells were devoid of myrtle extract, whereas only nonparasitised red blood cells were prepared for noninfected control. Test samples were incubated at 37 ∘ C for 24 hours and subsequently cooled at −20 ∘ C to lyse the red blood cells. The plates were next allowed to reach room temperature. The spectrophotometric assessment of LDH activity was facilitated by adding 20 L nitroblue tetrazolium (NBT) and phenazine ethosulfate mixture to the 100 L MALSTAT reagent. Absorbance was measured with an ELISA plate reader at 630 nm. The percentage inhibition at each concentration was determined and the mean of the IC 50 values of parasite viability was calculated using probit analysis [11].

In Vitro Cytotoxicity Activity.
Preferably, the extract for antiplasmodial investigation should have no cytotoxicity. So the cytotoxicity of myrtle was measured by the colorimetric methyl thiazol tetrazolium (MTT) assay and scored as a percentage of absorbance reduction at 570 nm of treated cultures versus untreated control cultures.
MCF7 (breast adenocarcinoma), HepG2 (hepatocellular carcinoma), WEHI (fibrosarcoma), and MDBK (normal kidney cells) were seeded into 96-well microplates at 10 4 cells per well and allowed to grow for 24 hours. The initial concentration of extract was 100 g/mL in DMSO, which was serially diluted in complete culture medium with two fold dilutions. Different concentrations of the extract were added to each well. Plates were incubated at 37 ∘ C for 72 hours under 5% CO 2 atmosphere. Then the 50 L of MTT-PBS solution in culture medium was added to each well. The plates were further incubated for 4 hours at the same condition. The medium was then removed and replaced with 200 L of DMSO to solubilize the MTT formazan product. The solutions were shaked for 20 min and the absorbance at 570 nm was measured. The IC 50 values were calculated from the drug concentration-response curves. Tamoxifen was used as a positive control with concentrations from 50 to 1.56 g/mL.

In Vivo Antimalarial
Assay. The suppressive activity of the methanol extract of myrtle was assessed using the 4-day suppressive test against Plasmodium berghei infection in mice [12]. All the procedure was accepted by Shahid Beheshti University Ethics committee and in accordance with the principles for laboratory animal use and care in the European community guidelines. Since the advantage of intraperitoneal injection is to be very easy to perform in comparison with other routes of administration, this route was selected for the study. Adult male albino mice, weight 30 ± 3 g, were inoculated with P. berghei, and each mouse received 1 × 10 7 infected erythrocytes by intraperitoneal injection on the first day of the experiment. The mice were randomly divided into experimental test and control consisting of 5 mice per cage. Two control groups were used in this experiment: one was treated with chloroquine at dose of 20 mg/kg as a positive control while the other group was kept untreated given normal saline as placebo. The mice of test group were treated during consecutive day with 10 mg/kg of the sample by intraperitoneal injection for 4 days. On days 5, 8, and 15 of the test, thin blood smears were made from the tail blood of the mice. The blood films were fixed with methanol and stained with Giemsa and then assessed by microscope. Percentage of parasitaemia was counted based on infected erythrocytes calculated per 1000 erythrocytes.

Results
Myrtle extract was prepared from the aerial part of Myrtus communis and was first tested at 20 concentrations on the Plasmodium falciparum K1 and 3D7 strains using pLDH assay. The cytotoxic activity was performed against three cancer cell lines (MCF7, HepG2, and WEHI) and normal cell line (MDBK). The 4-day suppressive test against P. berghei was carried out. The results of the in vitro and in vivo antiplasmodial and cytotoxic activities of the Myrtus communis are shown in Table 1. Myrtle extract showed better antiplasmodial activity against sensitive 3D7 strain with no cytotoxic activity up to 100 g/mL against the selected cell lines.
Percentage of parasitaemia on days 5, 8, and 15 of the experiment in the test group in comparison with placebo group is shown in Figure 2. The average survival days of the test group were 18 ± 0.57 days. All the mice of placebo group died within two weeks of experiment.

Discussion
Our previous studies [13,14] and this report lead us to carry out more surveys in Iranian folk medicine and traditional medicine which may help to detect new effective plants. The aim of this survey was the assessment of in vitro and in vivo antimalarial activities and in vitro cytotoxic effect of a plant traditionally used for treatment of parasitic infections.
Based on ethnobotanical data of some provinces of Iran that was carried out at TMRC [13,14] and a study which revealed potential antiplasmodial activity of essential oil of Myrtus communis L. [15], myrtle was selected for this survey.
Myrtus communis L. is an aromatic and medicinal species from the Myrtaceae family. Myrtle is used in folk medicine of Iran for treatment of some diseases such as parasitic disorders and herpes [14,16,17]. Ideally, effective extracts at the blood stage of the malaria parasite should have strong in vitro and in vivo antimalarial activities and should be devoid of cytotoxicity at concentration up to 100 g/mL [18]. Peters test is an appropriate method used to assessment in vivo antiplasmodial activity of plant extracts. Myrtle has strong in vitro antiplasmodial activity (IC 50 = 35.44 and 0.87 g/mL) with no cytotoxicity up to 100 g/mL (Table 1). After four days of treatment the suppressive percentage of parasitaemia with 84.8% was obtained for the methanolic extract of myrtle. This result is interesting when it was compared with the results from other plant extracts reported in the following literatures works.
Chloroform extracts of Artemisia macrivera Linn. and A. maritime Linn. showed antimalarial activity in mice against P. berghei NK65 at dose of 100 mg/kg with average percentage parasitaemia 0.30 ± 0.04 and 0.40 ± 0.05, respectively, on day 5 of the test [21].
Methanolic extracts from 15 medicinal plants in Kenya were screened for their in vivo antimalarial activity in mice against a chloroquine-tolerant P. berghei NK65 at a dose of 500 mg/kg. The best percentage of suppression on day 4 was 59.3% for Toddalia asiatica [22]. Other workers have studied the in vivo antimalarial activity of the methanol extract of Annona senegalensis against P. berghei at the doses of 100, 200, 400, 600, and 800 mg/kg. Chemosuppression of parasitaemia was 57.1%, 59.3%, 76.3%, 89.8%, and 91.1%, respectively [23]. The extracts of Cassia occidentalis, Morinda morindoides, and Phyllanthus niruri were evaluated for their antimalarial activity in vivo against P. berghei ANKA in mice at dose of 200 mg/kg. The most active extract that was from Morinda morindoides reduced parasitaemia by 74% [24]. Extracts obtained from the leaf and stem of Quassia amara and Q. undulate were screened against P. berghei berghei in mice at doses of 100 and 200 mg/kg. The parasite density (%) was between 0.6 and 8.5 on day 5 [25]. For comparison among some plant extracts that were screened for their in vivo antiplasmodial activity using Peters method, myrtle was shown to have dramatic effects at a low dose (10 mg/kg) in day four. Despite the decreasing suppression of parasitaemia on days 8 and 15, still chemosuppression of myrtle extract with 63.1% was observed.
Although several classes of natural products are responsible for the antiplasmodial activity of many plant species used in traditional medicine for the treatment of malaria, the most important and diverse biopotency has been observed in alkaloids, quassinoids, and sesquiterpene lactone. Nonalkaloidal natural compounds from plants with antiplasmodial and antimalarial properties, belonging to the classes of terpenes, limonoids, flavonoides, chromone, xanthone, anthraquinone, and related compounds, were recently reviewed [32]. According to this, flavonoides and steroides from the myrtle might have antiplasmodial activity, and bioassay guided fractionation resulting in isolated active components of Myrtus communis is necessary.

Conclusion
To our knowledge, myrtle extract has not been previously studied for its antiplasmodial activity. Our evaluation of the plant against two strains of Plasmodium falciparum in vitro and P. berghei in vivo proved antimalarial activities with no cytotoxicity up to 100 g/mL. The results suggest that the Iranian ethnic medicinal application of myrtle has a pharmacological basis. Phytochemical investigation and also understanding the mechanism of action would be the next step of this study.