Searching for antimalarial agent from Indonesian Combretum indicum and Magnolia figo

The archipelagic country of Indonesia has been an endemic area of malaria in which the Indigenous people of Indonesia have used medicinal plants to fight against plasmodial parasites. The study focused on two medicinal plants of Indonesia, namely Combretum indicum and Magnolia figo. Phytochemical, spectroscopic, and bioactivity assay protocols were performed. The experiments resulted in the major components detected were terpenoids and phenolic constituents. The bioassay indicated significant antimalarial potency of the crude methanol extract of leaves of Combretum indicum and Magnolia figo. Keywords—Combretum indicum; Magnolia figo; Malaria; Plasmodium falciparum


I. INTRODUCTION
Indonesia has been one of the malarial endemic countries in South East Asia with an estimated number of cases of 1.2 million in 2017 [1]. More than 25 Anopheles species infested Indonesia were malarial vector. Indonesia reported a significant improvement in malaria control [2]. More than 50% districts in Indonesia were free from malaria in which left 72% population inhabited malarial free-region. Nevertheless, malarial endemicity is projected to remain high in Eastern Indonesia (Fig. 1).
Malaria involves infective parasites Plasmodium falciparum, P. vivax, P. malariae, P. ovale in which P. falciparum is responsible for the highest mortality [3]- [6]. New malarial parasite Plasmodium knowlesi were firstly reported from South East Asia [3]- [6]. In 2016, 216 million malarial cases was reported globally which 14.6 million cases was in South East Asia [6]. Antimalarial resistant Plasmodium has made malaria control and eradication to become more complicated. Falciparum malaria superbug occured in Cambodia poses a serious threat [7], [8].
The majority of antimalarial agents have been derived from natural products obtained from medicinal plants. The first antimalarial, quinine, was obtained from the bark of cinchona tree, a native tree from Southern America [9], [10]. Bioprospecting study on Indonesian medicinal plants used by the indigenous people of Indonesia in malarial fever therapy have revealed significant results (Fig. 2). This exploration was successfully isolated samaradine Y from Quassia indica with IC50 value of 0.014 M [11]. This compound was more active than a standard malarial drug, chloroquine with IC50 value of 0.29 M [12]. Location of previous bioprospecting study to reveal antimalarial constituents from endemic medicinal plants [12] In this study, two medicinal plants of Indonesia (Combretum indicum and Magnolia figo, Fig. 3) were evaluated for their phytochemical constituents and pharmacological activity against malarial parasite. Combretum indicum is a shrub and well distributed across the archipelago of Indonesia where the species pronounced as "wudani" by people in Sumatra, bidani by Sundanesse, ceguk by Javanesse, rabet dani by Maduresse, tigao by Buginese, and kunyi rhabet by the Indigenous people of small Sunda Islands [14]. The seeds were traditionally prepared as an anthelmintic agent. Magnolia figo was one of famous flower producing plant in Indonesia which is locally named as cempaka. The leaves are traditionally used in malarial fever therapy, whereas the flowers are commonly used as analgesic to treat headache [15].

B. Phytochemical study
Crude methanol extract was developed in a Thin Layer Chromatography (TLC) plate using dichloromethane as developing solvent. Vanillin-H2SO4 staining agent was deployed to visualize the chemotype which red color indicated for phenols present, grey for sugar, purple for terpenoids. Alkaloid present was detected using Dragendorff's reagent with red-orange color.

C. 1 H-NMR study
A portion of sample (5 mg) was completely dried through high vacuum apparatus under silica gel chamber. Dried sample was dissolved in CD3OD and 1 H-NMR was recorded in Jeol NMR 400 MHz.

D. Anti-plasmodium bioassay
Sample (1 mg) was dissolved in DMSO (100 uL) and a serial dilution was performed to gain 1000, 100, 10, 1 ug/mL Parasite used in the study was Plasmodium falciparum 3D7 strain at ring stadium with parasitemia level of ± 1%. Crude extract sample (2 uL) at concentration series was loaded into 96 well plate. Parasite (198 uL) was added in which the last concentration as 100, 10, 1, 0.1 and 0.01 ug/mL. The plate was then allocated into a chamber with mixed gass (5% O2, 5% CO2 and 90% N2) prior incubation at 37℃. The culture was then collected, stripped and stained with 10% giemsa reagent. Percentage inhibition was calculated based on formula below: Note: Xu=% growth of sample, Xk=%growth of negative control. IC50 was obtained as concentration which inhibit 50% growth of the parasite.

III. RESULTS AND DISCUSSION
As part of antimalarial project, this paper considered reporting two species, namely Combretum indicum and Magnolia figo. Among two species in this study, the Indonesian medicinal plant inventory indicated only leaves of Combretum indicum to constitute whereas the sample in this study revealed no alkaloid was detected. The Dragendorff remains versatile alkaloid detecting reagent with limit detection as less as 0.1 ppm. Therefore, the antimalarial activity was suggested from non-alkaloid content in which TLC-based analysis using vanillin reagent figured the crude methanol extract of the leaves of Combretum indicum producing a distinct purple color at less polar and orange at polar retention regions. These are a clear indication that the leaves constitute terpenoid and phenolic components. The High-Pressure Liquid Chromatography (HPLC) profile of the crude extract (Fig. 4) showed at least major components with UV chromophore containing compounds. A sensitive malarial parasite, Plasmodium falciparum 3D7 was grown under crude methanol extract in which 100% inhibition was obtained at 100 µg/mL concentration producing a significant IC50 value of 4.12 µg/mL. Further extensive isolation work is necessary to reveal the major and minor constituents of the crude methanol extract.
Although Combretum indicum is the accepted name, its synonym Quisqualis indica was found in several publications revealed it constituents including the isolation of a volatile alkaloid quinoline-4-carbonitril from flower. The alkaloid was reported to possess a significant anti-inflammatory and antioxidant activities [16]. Anticancer active components was previously recorded from the leaves, 25-O-acetyl-23,24dihydro-cucurbitacin F [17]. The effectiveness of Combretum indicum in controlling malarial vector was previously reported with no anti-plasmodial activity found [18].
Dragendorff treatment showed no orange color, suggesting no nitrogen containing compounds was present in the crude methanol extract. Based on phytochemical profiling using vanillin reagents, the crude methanol extract of Magnolia figo clearly indicated the presence of terpenoids based on deep purple color. Glycoside was a minor chemotype components as 19 no strong grey spot was produced under the same protocol. Reddish color on the test showed the presence of phenolic type compounds. The HPLC chromatogram (Fig. 5) showed major peaks existed in retention time below 50% acetonitrile in water indicated the high presentation of phenolic constituents. Proton NMR spectrum (Fig. 6) analysis on the crude methanol extract indicated peaks at 7 ppm for aromatic protons, sugar components were represented by peaks at around 3-5 ppm and terpenoid constituents were represented by peaks at around 0.5-2 ppm. Anti-plasmodial bioassay revealed the crude methanol extract of Magnolia figo has a significant activity with IC50 value of 13.42 µg/mL. Previous research on this species were commonly reported under Michelia figo. The leaves from Taiwan was previously reported to constitute a sesquiterpene lactone (11,13dehydrolanuginolide), alkaloid ((-)-nuciferine, (-)-anonaine, and N-methylcorydaldine), steroids (β-sitostenone, stigmasta-4,22-dien-3-one), benzenoids (p-hydroxybenzaldehyde, phydroxybenzoic acid, methylparaben, vanillin), chlorophylls (pheophytin a, pheophorbide a, pheophytin b, pheophorbide b, aristophyll-C, 132-hydroxy-(132-S)-pheophytin a) [19]. However, information regarding their antimalarial were limited. Bisbenzylisoquinoline alkaloid magnolin (Fig. 7) from leaves of Thai M. figo were previously reported to possess antimalarial activities against both sensitive Plasmodium falciparum FCR3 and resistant Plasmodium falciparum K1 strains with IC50 value of 0.16 and 1.51 µM, respectively [20].  [20] Compared to the previously studied samples, the Indonesian origin samples in this study did not show alkaloid components, which suggest other chemotype migh contribute to the claims.

IV. CONCLUSIONS
The study revealed the antimalarial potency of the crude methanol extract of Combretum indicum and Magnolia figo with significant activity against Plasmodium falciparum 3D7. Further research is necessary to reveal the chemotypic constituents which responsible for the claims. Nevertheless, these validated the traditional claims of antimalarial medicinal plants used by the indigenous people of Indonesia.