Antiplasmodial potential of Eucalyptus obliqua leaf methanolic extract against Plasmodium vivax : An in vitro study

: Malaria is an intraerythrocytic parasitic disease caused by the genus Plasmodium of which Plasmodium vivax and Plasmodium falciparum are the major species. The high cost and associated side e ﬀ ects of antimalarial drugs triggered research about medicinal plants to develop alter - native and low - cost drugs with lesser side e ﬀ ects. Therefore, this study was designed to investigate the antiplasmodial activity of the Eucalyptus obliqua L ’ Hér. leaf extract against P. vivax and its phytochemicals in in vitro . The methanolic extract of E. obliqua was prepared and di ﬀ erent concentra - tions of the crude extract and phytochemicals were used against P. vivax . The methanolic extract of E. obliqua showed profound antiplasmodial activity ( LD 50 0.084mg/mL; 80.04% ) at 0.1mg/mL concentration after 24h. Alkaloids, ﬂ avonoids, saponins, and tannins were found in the E. obliqua methanolic extract. Only alkaloids at the concentration ( 0.1mg/mL ) exhib - ited 60.93% inhibition of P. vivax. The methanolic extract of E. obliqua exhibits antiplasmodial activity in vitro . However, invivo e ﬃ cacy is an important aspect in the testing of medicinal plants against parasitic infections and should be evaluated in future.


Introduction
Malaria is a parasite-borne intraerythrocytic infection that affects more than half of the world population [1,2].It is caused by hemoparasites of the genus Plasmodium and is transmitted by the female anopheles' mosquito [3].It is estimated that 60% of the total population of Pakistan resides in areas with high malaria incidence [4].In Pakistan, Plasmodium vivax is responsible for 64% of malaria incidence [5].The resistance of malarial parasites to many available antimalarial drugs is a major monetary constraint in combating malaria [6].The current commercially available antimalarial drugs are associated with various side effects.Moreover, the expenses associated with the conventional approaches of managing malaria are reasonably high particularly for individuals living in low-income countries like Pakistan.Therefore, new drugs are required to avert the complications posed by drug-resistant Plasmodium strains [7].
For centuries, medicinal plants have been used to treat human diseases worldwide like stomach pain, diabetes, hyperacidity, gonorrhea, dysentery, cystitis, urethritis, laryngitis, leucorrhoea, inflammation, bronchitis, tuberculosis, wounds, and many others.More than 80% of the population of both developing and developed countries rely on traditionally synthesized herbal drugs [8].The need and utilization of medicinal plants in China, South Africa, and India have dramatically increased in the last few years.A lot of studies have been carried out on the sustainable use and conservation of medicinal plants in these countries [9].Some studies have been published in Pakistan on the antiplasmodial potential of medicinal plants.
Medicinal plants offer viable alternatives with fewer side effects, are reasonably cheaper, and easily available.Pakistan has a distinctive geography and has a wide range of diversity in climatic zones due to which it is rich in the diversity of plants.It is estimated that Pakistan has more than 6,000 species of higher plants among which 12% have medicinal importance.These medicinal plants are also exported to other countries [10].Studies in Pakistan have revealed that eucalyptus leaves collected from the Soon Valley in Khushab, Pakistan, have been traditionally used against malaria infection [11].
The family (Myrtaceae) of eucalyptus plants is a rich source of biologically active compounds, such as steroids, alkaloids, tannins, saponins, terpenes, flavones, flavonoids, polyphenolics, phenolics, triterpenoid, fatty acids, lignins, vitamin C, anthraquinone, glycosides, anthocyanin, coumarins, cardiac glycosides, and volatile oils [12,13].Eucalyptus obliqua L'Hér. is a fast-growing evergreen plant, named after its oblique leaves.E. obliqua is well known for the isolation of antimalarial compounds extracted from its leaves and is also used widely for draining swamps due to its water-absorbent ability [14,15].
The local names of E. obliqua are Sufaida and Laachi in Pakistan [16].E. obliqua has potential antimalarial ability as it is being traditionally used as folk medicine in many parts of the world [17].It is also reported that oils and secondary metabolites of E. obliqua exhibit antimicrobial and antifungal properties.Most of the species of Eucalyptus are also known for their medicinal importance and are used in the treatment of malaria, microbial infections, and dysentery [18,19].There is a dire need to assess medicinal plants for antiplasmodial agents which may be a source of an alternative antiplasmodial drug(s).Therefore, this study aimed to investigate the antiplasmodial activity of E. obliqua leaf extract and phytochemicals against P. vivax in vitro.

Plant collection and methanolic extract preparation
Fresh leaves of E. obliqua were collected from its natural habitat in the southern district of Kohat, Khyber Pakhtunkhwa, Pakistan (coordinates latitude: 33.6060705292903 and longitude: 71.46785718724671).The plant leaves were rinsed with fresh water and were identified by a taxonomist at the Department of Botanical and Environmental Sciences, KUST, Kohat, and a sample voucher number: MEB397 was given and has been deposited.The leaves were shade dried and ground in an electric grinder.The methanolic extract was prepared as described by ref.
[20] with slight modifications.Briefly, 10 × 100 mL of leaf powder was mixed in absolute methanol in a beaker.The suspension was kept in an electric shaker for 48 h at room temperature.
The suspension was filtered through Whatman's filter paper one and the filtrate was processed in a rotary vacuumed evaporator (BUCHI Rotavapor R-200, Switzerland) at 40°C.The concentrated crude extract was obtained and stored at 4°C for further analysis.

Phytochemical analysis
The extract of E. obliqua was subjected to analysis and extraction of alkaloids, flavonoids, and saponins as described by ref. [21], and tannins as described by ref. [22].
2.3 P. vivax culture and maintenance P. vivax strains (confirmed in this study) were used to maintain the culture at the Molecular Parasitology and Virology Laboratory of KUST, Khyber Pakhtunkhwa, Pakistan.A blood medium mixture of 200 µL with 2% hematocrit, consisting of McCoy's 5A medium (Life Technologies, VIC, Australia) supplemented with 20% human serum, was used for culturing.The parasites were cultured in a candle jar at 37.0°C.Incubation was deemed successful and stopped when ≥40% of the ringstage parasites had reached the mature schizont stage (≥4 distinct nuclei per parasite).Gentamicin sulfate (5 µL) was also added to the culture.Thick blood films were prepared on glass slides and stained with 5% Giemsa solution for 30 min and examined microscopically.Daily, infected erythrocytes were inoculated into a fresh complete medium to propagate the culture [23].

In vitro antiplasmodial activity
The in vitro antiplasmodial activity of the methanolic extract and phytochemicals was carried out as previously described by ref. [24].The antiplasmodial activity was performed in 96 microplates.Chloroquine and proguanil were used as positive controls while microplates with parasitized culture and without drug/plant extract served as the negative control.Five concentrations (0.02, 0.04, 0.06, 0.08, and 0.1 mg/mL) of plant extract, phytochemicals, and drugs were used [25,26].The microplates were shaken gently and kept in a candle jar to increase the concentration of CO 2 .The plates were placed in an incubator at 37°C for 24 h.After 24 h the supernatant was removed from each microplate and red blood cells were picked with a micropipette to prepare thin smears using the Giemsa stain and was observed under a microscope (Figure 1) [27].

Statistical analysis
Maturation percentage and inhibition percentage were evaluated using the following formula [24].The mean inhibition and standard deviation were calculated using Statistix 9 software.LD 50 was calculated by using an online tool AAT Bioquest (https://www.aatbio.com/tools/ld50-calculator).
The phytochemical analysis revealed the presence of alkaloids, flavonoids, saponins, and tannins in E. obliqua (Table 2).Of this, only alkaloids at the concentration (0.1 mg/mL) exhibited about 60.93% growth inhibition of P. vivax which was comparable to the control chloroquine (Table 3).Over the last hundreds of years, there has been an observed increase in research focusing on the use of medicinal plants as natural remedies for infectious diseases.A variety of novel antimalarial compounds have been discovered as a result of the extensive research on medicinal plants [28].This study explored the antiplasmodial potential of E. obliqua methanolic extract and phytochemicals against P. vivax in vitro.
The E. obliqua methanolic extract profoundly inhibited (80.04%)P. vivax at the highest tested concentration.The extract impacted the parasite in a dose-dependent manner.This study is quite in agreement with the study of Sabiu and Ashafa [17] who reported the antiplasmodial potential of E. obliqua against the Plasmodium species.Previous studies have confirmed the antimalarial potential of eucalyptus plants as they inhibited more than 50% of the malarial parasite [29].Three extracts (aqueous, n-butanol, and ethyl acetate) of E. globulus remarkably reduced the growth of protozoan parasites.However, the impact was dosage and extract type-dependent [30].Scientists have analyzed and evaluated the effect of various kinds of solvents, to extract bioactive compounds from plants [31].Methanol is the most preferred solvent for plant extraction possibly due to its polar nature that ensures the release of several bioactive compounds from plants [32,33].It has been scientifically proven that highly polar solvents should be used to extract bioactive compounds with a high level of accuracy [31].The effects of the active compounds derived from plants depend mainly upon the solvent used for herbal formulation [33].
Eucalyptus oil was found effective against the asexual stages of Plasmodium falciparum in vitro [34].Furthermore, eucalyptus plants are globally used as a traditional medicine against malarial infection [35,36].Therefore, it could be considered a potential antimalarial agent and is recommended for further in-depth analysis.
The phytochemical analysis revealed the presence of alkaloids, flavonoids, saponins, and tannins.The alkaloids extracted from E. obliqua was found to be responsible for the antiplasmodial activity and it inhibited parasite growth in vitro.The same classes of compounds have also been identified in the ethanolic leaf extracts of Eucalyptus citriodora and methanolic leaf extracts of Eucalyptus camaldulensis [37,38].Alkaloids and flavonoids of different plants have proven antiplasmodial activities.Alkaloids are actively involved in stopping the process of protein synthesis in the Plasmodium species [39].Similarly, flavonoids have also profound efficacy against P. falciparum [40].The presence of these classes of compounds validates the antiplasmodial activity of E. obliqua in this study.However, further studies analyzing the isolated pure compounds of the mentioned classes are highly recommended to better understand the underlying antiplasmodial activity of E. obliqua and related plant species.

Conclusion
This study concludes that the methanolic extract of E. obliqua exhibits antiplasmodial activity in vitro.However, in vivo efficacy is an important aspect in the testing of medicinal plants against parasitic infections and should be evaluated in future.Moreover, additional studies are also invited to elucidate and isolate antiplasmodial compound(s) from E. obliqua leaf extracts.
developed schizonts for the experimental group No. of developed schizonts for control 100 Inhibition percentage 100 Maturation percentage

Figure 1 :
Figure 1: Healthy and affected schizonts/cytotoxic activity under the microscope.

Table 1 :
The in vitro antiplasmodial activity of Eucalyptus obliqua methanolic crude extract

Table 2 :
The phytochemical constituents of Eucalyptus obliqua methanolic crude extract

Table 3 :
In vitro antiplasmodial activity of phytochemicals against Plasmodium vivax