The Effectiveness of Local Plants from Lom and Sawang Ethnics as Antimalarial Medicine

Native people or ethnic societies that live in endemic malaria islands such as in Bangka Island and Belitung Island have used many medicinal plants to cure malaria. Leaves of kesembung (Scaevola taccada (Gaertn Roxb), roots of kebentak (Wikstroemia androsaemofolia Decne), and roots of medang mencena (Dapniphyllum laurinum (Benth) are the examples. This research was aimed to investigate the present of some biochemical compound and evaluate the antimalarial activity of ethanol extract of the plants against Plasmodium falciparum 3D7 in vitro. The IC50 level was determined through visual observation under microscope over 5000 of giemsastained erythrocytes then analyzed by probit analysis. Results showed that kebentak root ethanol extract was effective to inhibit P. falciparum 3D7 with level 0.485 μg/ mL. Furthermore, the IC50 level of kesembung leaves and medang root were 44.352 μg/mL and 1486.678 μg/mL respectively. Phytochemical test result showed that kebentak leaf ethanol crude extract contained triterpenoid, kesembung root contained phenol and tannins; moreover, medang root contained alkaloid, saponin, and triterpenoid.


INTRODUCTION
Indonesia as one of developing countries embraces the local wisdom in people daily activities such as the utilization of medicinal plant to cure disease.In Indonesia, medicinal use of natural materials has been done by the ancestors and it has been passed down from one generation to the next generation.Every society in every region has been using a variety of useful plants in their environment (Ifandi et al., 2016).Sawang ethnic is native society that inhabits Belitung Island, while Lom ethnic inhabits Bangka Island.The study about plant utilization in medical treatment by local people in both islands has been delivered by Tim penulis Ristoja (2013aRistoja ( , 2013b)).
Bangka and Belitung islands are the endemic area of malaria.The number of people suffering for malaria increase twofold in the last two decades due to resistance of new Plasmodium falciparum strain to chloroquine and its derivatives (Trape et al., 2002).The efforts in order to explore new potential antimalarial plants are needed to overcome the resistance of malarial parasites (Bankole et al., 2016).Historical records show that mostly antimalarial drugs are derived from medicinal plants, namely Artemisin derived from Artemisia annua.This plant has been used by society in China.Previous study about extract of belilik fruit and root (Brucea javanica Merr.) origin from Bangka showed optimal inhibition to Plasmodium falciparum growth with IC 50 < 0.01 µg/mL (Helmi & Susanti, 2013b).
Kesembung lives in beach ecosystem, where it will not disrupted by the waves and will get optimum sun-light.This plant is herbaceous with widened upper, shiny green yellowish colour, curvy edge, wax-coated surface, spherical to elliptical shape, and rounded tip leaves.Size of these leaves is around 16.5-30 x 7.5-9.5 cm (Kontz et al., 1996).According to Meijin (2009), Scaevola genus contains several active compounds such as coumarins, terpenoids, iridoids, alkaloids, and flavonoids.Plants in this genus have shown biological activities such as antiviral, antitumor, and antibacterial.Taxonomically, this plant is material used were sulfuric acid, alcohol, acetic acid glacial, diethyl eter, Dragendorff (mix Bi(NO 3 ) 2 . 5H 2 O in nictric acid and Ki solution), ethanol, FeCl 3 1%, H 2 SO 4, HCL, kloroform, and ammonia.The analysis were including alkaloid test by Robinson method (1995).The other active compounds that detected were saponin, triterpenoid, steroid, flavonoid, and phenol through Harborne method (1996).
Firstly, master solution was made in DMSO and diluted into series of concentration with medium RPMI 1640 enriched with 10% plasma, 25 mM HEPES, and 25 mM NaHCO 3 until the concentration in culture wells were 100.10, 1. 0.1, and 0.01 µg/ml.Cells were cultured with 5% of hematocrit and 1% of parasitemia.Cultures were harvested after 48 hours of incubation then smeared into object glass with 20% of giemsa staining.Furthermore, IC 50 level was determined after the percentage of parasitemia and probit analysis result were obtained (Fidock et al., 2004, Ekasari et al., 2009).
Phytochemical compounds are substances that can protect plant from diseases.Naturally, plants produce these compounds to protect their self from damage, but human explore and use them in diseases prevention (Builder et al., 2014).Several secondary metabolites have antiplasmodic activity (Saxena et al., 2010).Qualitative test through colorimetry showed that kesembung leaf contain phenol, tannins, and triterpenoids.In addition, medang mencana root contained alkaloids, saponins, and triterpenoids, while kebentak root contained only triterpenoid (Table 2).Therefore the three plants have different kind of active compound contents, but they all have triterpenoids.According to Harborne (1996), triterpenoids are consisted of long hydrocarbon chain C 30 that cause non-polarity.This compound has cyclic structure that mostly consisted of alcohol, aldehyde, or carboxylic acid.Normally, it has no co-   (-).Not detected Numbers of active compounds have been isolated; their antiplasmodic activity has been proven through in vitro and in vivo.For example, quinine from Cinchona ledgeriana is one of antimalarial drug that has been used until these days (Bell, 2005).According to Bero et al., (2009), most compounds that show positive result in phytochemical test are antimalarial substances.Commonly, those compounds are alkaloids, terpens, quassinoids, flavonoids, limonoids, calcons, peptides, xanthones, quinones, coumarines, and the other antimalarial substances (Kaur et al., 2009).Fidock et al. (2004) stated that six targets of antimalarial agents are cytosol (folic metabolism, glycolysis, protein synthesis, gluthatione metabolism in cytosol, and signal transduction), parasites membrane (phospolipids metabolism and membrane transport), food vacuole (hem polymerization, haemoglobin hydrolysis, free radical releasing), mitochondria (electron transports), apicoplast (protein synthesis, DNA synthesis, transcription, type II fatty acid biosynthesis, isoprenoid synthesis, and protein famesilation),and extracellular (erythrocyte invasion).
Antimalarial test result shows that kesembung has IC 50 44.352µg/mL (Table 3).According to Pouplin et al (2007), an extract could be classified as active parasitemia reducer if it can reduce more than 30% of parasitemia.Therefore, kesembung can be utilized to reduce parasitemia with concentration 10 µg/mL.
Phytochemically showed that kesembung leaf extract contained phenol, tannin, and triterpenoids.Previous researchers reported that those compound having antimalarial activity.Table 3 showed that this extract could inhibit the plasmodium.It was assumed that phenol, tannin and triterpenoid in this extract could inhibit the para-  2011) who explained that phenol increase cellular oxidation in red blood cell and inhibit protein synthesis of parasite when act as antimalarial agent.Builder (2010) taked that these processes will lead to oxidative damage by malaria parasite induction.Because of that, antioxidant activity of phenol increases the effectiveness of antimalarial activity (Builder et al., 2014).
In addition, Syarif (2007) stated that phenol takes role in inhibition of hem polymerization.
Triterpenoid has also been reported as antimalarial agent.A study showed that triterpenoid that isolated from Lansium domesticum had antimalarial activity with IC 50 5.9 µg/mL (Saewan, 2006).Betulinic acid (pentacyclic triterpens) can fuse with erythrocyte membrane then enter into the cell through lipid bilayer, as a result growth and invasion of malaria parasite are inhibited (Ziegler 2004).There is correlation between the increasing of IC level with malaria parasite proliferation; therefore the alterations of erythrocyte membrane and parasite vacuolization are the acts of triterpenoids in antimalarial.Pouplin et al., (2007) stated that triterpenoids inhibit parasites growth by hampering protein synthesis inside parasite cells.Fidock (2004) taked that saponin, flavonoid, and tannin as antioxidant will scavenge free radicals due to damage of parasite invasion.
Medang mencana antimalarial activity test show that it had IC 50 1486.68µg/mL(Table 4), it could be categorized not effective to be used as antimalarial drugs.Although, it was not effective as antimalarial drug but this extract could inhibit the P. falcifarum (table 4).This plant has not optimally studied yet, but it had been proven that it had alkaloids contents such as daphnyphyllin (Hirata, 1975).Alkaloids have been known as natural resources that can be utilized as drug.Quinine is one of alkaloids that has been utilized as antimalarial drug, it is produced by Cinchona succiroba (Kaur et al., 2009).This compound will block the intracellular choline transport that can inhibit parasite growth (Hilou et al., 2006).Besides alkaloid, medang mencena leaf extract contained saponin.Saponin could inhibit parasite growth by inducing the erythrocyte lysis (Widodo & Rahayu, 2010).Perhaps, the increase of concentration and suitable solvent usage in extraction will allow the plant to perform better antimalarial activity.
Kebentak root extract has level of IC 50 0.485 µg/mL (Table 5).Triterpenoid in kebentak root is expected can effectively inhibit P. falcifarum growth.This plant is classified in family Thymelaceae, that is to say that alkaloid is commonly found compound in plants of this family.Phytochemically showed that kebentak root extract contained triterpenoid.Quassinoid is one of triterpens that found abundant in plant of family Simaroubaceae.Several active quassinoid are isolated from Indonesian herbal such as pasakbumin B, pasakbumin C, and eurikumanondari from Eurycoma longifolia Jack.The other quassinoids are bruseajavanin A, dihidrobruseajavanin A, and bruseakantinosid that isolated from Brucea javanica Jack were effective to inhibit the P. falciparum.
Wikstromia has been reported not only contains triterpenoid, but also contains several  Helmi & Susanti (2013a) shows that both extracts and fruit were not effective in P. falcifarum growth inhibition.
The optimum dose of the extract that can inhibit 50% of parasite growth was >100 µg/mL.In spite of this study, it has been revealed that benta root can inhibit parasitemia for more than 30% of P. falciparum parasites with concentration 0.1 µg/mL.A research about antimalarial study of W. indica, plant that share the same genus with kebentak has been conducted.Root of this plant was extracted with BuOH solvent.The solution was fractionized to flavonoids; specifically sikokianin B dan sikokianin C. Sikokianin B and sikokianin C perform excellent inhibition over the growth of P. falciparum with IC 50 yaitu 0.54 µg/mL and 0.56 µg/mL respectively (Nunome et al., 2004).On the other hand, there was no flavonoid detected in kebentak root.

Table 2 .
Phytochemical components of each plant

Table 1 .
Yield of plant extractionType of plant and its part Initial Weight (g) Final Weight (g) Yield (%)

Table 3 .
Percentage of P. falciparum 3D7 growth and the inhibition of Kesembung leaf extract

Table 4 .
Percentage of P. falciparum 3D7 growth and the inhibition of Medang root extract

Table 5 .
Percentage of P. falciparum 3D7 growth and the inhibition of Kebentak root extract

Table 6 .
The IC 50 level of three tested plants Ethanol crude extract of Kebentak root had the highest effectiveness against P. falciparum 3D7 with IC 50 level was 0.485 µg/mL.The IC 50 levels of Kesembung leaf and medang mencena root were 44.352 µg/mL and 1486.678µg/mL respectively.Phytochemical test results show that ethanol crude extract of Kebentak root contains triterpenoid, Kesembung leaf contains phenol, tannins, and triterpenoids, while Medang Mencana root contains alkaloid, saponin, and triterpenoid.