In Vivo Antimalarial Test of Artocarpin and in vitro Antimalarial Test of Artonin M Isolated from Artocarpus

The derivative of flavonoid compounds, artocarpin (1) and artonin M (2), were isolated from the root wood of Artocarpus altilis and from the root bark of A. kemando, respectively. Both plants originated from Lampung, Indonesia. The structure of the two compounds has been carefully determined by physical method and spectroscopy techniques of UV, IR, and NMR. The in vivo antimalarial test of artocarpin showed very good Plasmodium activity in female mice, with ED50 value of 34.88 mg/kg body weight (kgBW), whereas the in vitro antimalarial test of artonin M showed very strong activity with IC50 of 0.3 μg/mL (5.967 x 10 -7 M).


Introduction
The antimalarial researches on flavanoid compounds isolated from artocarpus plants have been carried out and reported previously [1][2][3][4]. The compounds isolated from these plants were very interesting to explore as an antimalarial drug. Artonin E, cycloartobiloxanthone, artocarpin, cycloartocarpin isolated from A. altilis have been found to exhibit cytotoxic activity against Plasmodium falciparum. All of these active compounds are prenylated on C3, and the antimalarial activity test previously performed on these compounds were usually in vitro activity test [1][2][3][4].
A. kemando Miq. is one of endemic plants that grows in Indonesia. From this plant, some prenylated flavanoids have been isolated, and these include artomandin, artoindonesianin C, artonol B, and artochamin A [5]. All of these compounds have been found to show cytotoxic activity against KB cell cancer (human oral epidermoid carcinoma); thus, this plant is known as a source of promising anticancer drug [6]. Futhermore, artonin E, artonin O, artobiloxanthone, and cylcoartobiloxanthone have also been found active as anticancer [5][6][7]. However, the antimalarial activity test of the compounds isolated from A. kemando is not available yet.
The previous results of in vitro antimalarial activity test from flavanoid compounds obtained from Artocarpus indicated that many flavanoids were active and considered as antimalaria [1][2][3][4], but no reports have been found for the in vivo antimalarial test. Thus, in this paper, we reported the in vivo antimalarial test of artocarpin isolated from A. altilis and in vitro antimalarial activity of artonin M isolated from A. kemando Miq.

Isolation and Purification of the Compounds
2.6 kg of root wood A. altilis was mashed and was macerated using 16 L of methanol solvent, for 3x24 hours. The maceration results were then filtered and concentrated, obtained 83.46 grams of the extract. The maceration extract was fractionated using Vacuum Liquid Chromatography (VLC), received 2 stages of VLC using Merck 60 Silica Gel adsorbent (35-70 Mesh) and amylose, and eluted with a mixture of ethyl acetate/n-hexane which gradually increased its polarity. Fractionation results produced 4 main fractions (A-D), fraction B 0.9 g, C 1.93 g, and D 13.8 g.
The fractions B and C were VLC and subsequently in CC repeatedly using the same adsorbent and eluent, compound (1), as much as 0.63 grams, and exhibited melting point 185ºC -186.8ºC. TLC with an artocarpine standard using three systems of ethyl acetate / n-hexane eluent 2: 8 (Rf 0.14), acetone / dichloromethane 1: 9 (Rf 0.51), and acetone / n-hexane 3: 7 (Rf 0.37), and one stain was obtained with the same Rf.
2.0 kg fine powder of root bark was macerated with a methanol solvent for 1x24 hours with three repetitions. The results of maceration were evaporated using a rotary evaporator and obtained 149.8 grams of extract. The extract obtained was then fractionated by the VLC method, with adsorbent silica gel and eluent n-hexane and ethyl acetate with variations in polarity increase. The results of the fractionation obtained seven main fractions, A -G. Fraction C received 16.7 grams in VLC further with the same adsorbent and eluent, and after that, the column chromatography was repeated using silica gel adsorbents and variations in n-hexane and ethyl eluents. Acetate and n-hexane and acetone obtained yellow crystals (compound 2) weighing 8.8 mg, with a melting point 248 -251 °C. In TLC compounds (2) with 3 eluent systems, namely n-hexane: acetone: dichloromethane (DCM) (2: 1: 1) (c), acetone: n-hexane (3: 7) (b), EtOAc: n -hexane (2: 8), one stain was obtained.

Antimalarial activity
The in vitro antimalarial assay was performed following the procedures available in the literature and have been used previously in some of our publications [8][9][10]. However, the in vivo antimalarial test was carried out based on the procedure used published methods available in the literatures [11] and have also been used in our previous work [4].

In vivo antimalarial test of artocarpin (1)
The growth of Plasmodium was inhibited upon administering the compound 1, where the higher dosage of compound 1 was used, in addition to the smaller parasitemia average and the higher percentage inhibition average. The dosage that resulted in the highest inhibition of Plasmodium was 100 mg/kgBW, where it resulted in inhibition of 73.66 + 0.12%. In the smallest dosage used, the inhibition of Plasmodium was still observed, although it was very low, with a percentage of 12.60 + https://doi.org /10.37358/Rev. Chim.1949 Rev. Chim., 71 (5) Table 1). The parasitemia level and inhibition percentage of artocarpin is in Figure 7. According to Munoz et al. [12], the in vivo antimalarial activity was categorized as follows: ED50 < 100 mg/kgBW/day is categorized as excellent, ED50 101-250 mg/kgBW/day categorized as good, ED50 251-500 mg/kgBW/day as medium, and ED50 > 500 mg/kgBW/day as inactive. Based on these criteria, compound 1 is categorized as excellent drug; therefore, it will be excellent to be developed as an antimalarial drug in the near future.   (1) In the in vivo antimalarial activity test, some factors that demonstrate an effect on the result of the test exist. The individual factor of mice really affects the growth of P. Berghei, which also gives the varied picture of parasitemia. This condition normally affects the growth of P. berghei in the body of mice. The factor of Plasmodium also affects the number of parasetemia that occur in the mice. The host factor influencing the mice is the body resistance to eliminate Plasmodium from each mouse, while the factor of P. berghei that does not sincronize in the mice and is normally only 10% of P. berghei that was innoculated and can grow [13].
The presence of schizogony erythrocytic cycle on P. berghei inside the visceral organ, making the ring stage and trophozoit, are mostly taken as innoculum. The character of P. berghei also affects the speed of its growth inside the host [14]. Thus, these conditions perhaps is the cause of the parasitemia value being relatively low. The parasitemia negative control was only 2.63 + 0.01 %.
The mechanism of the compound tested as antimalarial was perhaps by inhibition mechanism of the heme polymerization. It has been found that the terpenoid compound will bind to a heme electronic system, and the hydroxyl group will be bound to heme iron [15]. The other mechanism in killing Plasmodium is probably available but which mechanism used is not known exactly.

In vitro antimalarial of Artonin M (2)
The results of in vitro antimalaria of 2 showed that this compound exhibits very high activity against Plasmodium, giving an IC50 value of 0.3 µg/mL (5.976 x 10 -7 M), and this is comparable with the control positive chloroquine with an IC50 value of 3 x 10 -7 M. This value is stronger than the antimalarial drug from the organotin (IV) compound, which has been reported lately [9,10,16] and may be better than other synthetic compounds having antimalarial activity which have been reported by others [17][18][19][20]. The data of activity test can be seen in Figure 8 and Table 2, while the data for the chloroquine are in Figure 9.   Each molecule structure of 1 and 2 exhibits two hydroxyl groups at the meta position on B ring. The hydroxyl group at C-7 on A ring on these two compounds is not on a free state, and they exhibit one uncyclinazed isoprenyl group on the C ring at compound 1 and on furanodihydrobenzosanton at compound 2. The structure of these two compounds is similar for the position of two hydroxyl groups on B ring and the presence of prenyl or geranyl on C ring, thus making these compounds as active as antimalaria.

Conclusions
The result of in vivo antimalarial assay of artocarpin ED50 value obtained was 34.88 mg/kgBW. This value indicated that artocarpin demonstrates very strong antiplasmodial activity. The result of in vitro antimalarial activity of artonin M resulted in strong activity with an IC50 value of 0.3 μg/mL. This IC50 value also is an indication that artonin M has the potential to be developed as an antimalarial drug. The in vivo antimalarial of artocarpin and in vitro antimalarial of artonin M from Artocarpus plant are the first report of antimalarial activity assay. Based on the result reported here, the conclusion exists that A. altilis and A. kemando plants are one of the main sources to obtain the potential compound as antimalarial, which will be very useful for the replacement of some comercial drugs available in the market and caused the antimalarial resistency.