Antiplasmodial activity of chalcone derivatives compound through phagocytosis of kupffer cells in experimental malaria hosts

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INTRODUCTION
Malaria is a global infectious disease caused by the Plasmodium and transmitted by the Anopheles mosquito. Malaria still posed a considerable burden on global health, especially in Africa and Asiatic countries. In Indonesia, there was a decreasing trend of malaria morbidity in 2009-2016 from 1.8/1,000 at risk population in 2009 to 0.84/1,000 at risk population in 2016. However, some provinces in Indonesia still have a considerable burden from malaria. For example, Papua is one of the provinces with the highest Annual Parasite Incidence (API) with 45.85 per 1,000 inhabitants. 1 The main problem of malaria is not only because of its mortality and impact on national productivity but also due to the drug resistance found in P.falciparum and P. Vivax. Furthermore, vaccine development is also hampered by the complex life cycle of P. Falciparum. 2, 3 This phenomenon has ignited extensive research to find an effective alternative of current antimalarials. The ideal antimalarial drugs should fulfill several criteria, including low toxicity and high efficacy in combating the major species of Plasmodium.
Fortunately, there have been several promising compounds that had been evaluated for their antimalarial properties. Chalcones (1,3-diaryl-2-propen-1-ones) are secondary metabolites of flavonoids found in several plant species and have antimalarial activity. 4,5 Antimalarial activity of chalcones was investigated after a report on the research results stating potent antimalarial activity in vitro and in vivo from a compound, namely Licochalcone A, an isolated compound from the root of Chinese licorice. 6,7 Suwito et al. had designed several chalcones derivatives as an inhibitor in ferredoxin (Fd) interaction with ferredoxin-NADP + reductase (NFR), which is a crucial redox system in P. Falciparum's survival. 8 The result indicated that one of the synthesized compounds (E)1-(4 aminophenyl)-3-(2,3-dimethoxy phenyl) prop-2-en-1-one had a remarkable inhibitory activity against Plasmodium through molecular docking. They also found that the amino group of the aminomethoxy derivative of chalcones played an essential role in inhibition through electrostatic interactions and can form a more stable complex with NFR compared to Fd. Likewise, an in vitro study found that (E)1-(4 aminophenyl)-3-(2,3-dimethoxy

Antiplasmodial activity of chalcone derivatives compound through phagocytosis of kupffer cells in experimental malaria hosts
Lilik Wijayanti 1 , Paramasari Dirgahayu 2 , Yulia Sari 3 , Danus Hermawan 4 , Ida Nurwati 5 Introduction: Malaria is still one of the major health problems, specifically due to drug resistance in Plasmodium, which encourages extensive research to find effective alternatives. One of the new antimalarial compounds is chalcone-derivative compound (E)-1-(4-aminophenyl)-3-(2,3-dimethoxy phenyl)prop-2-en-1-one. However, its potency is still needed to be evaluated. Therefore, this study aimed to determine the efficacy and identify the pharmacological mechanism of this chalcone-derivative. phenyl)prop-2-en-1-one had good antiplasmodial activity, selectivity index, and plasmodial growthinhibiting effect. 9 Finally, the in vivo study also confirmed the efficacy of this substance, in which it was shown that the effective dose (ED50) for this substance was at 17.36 mg/kgBW/day. 10 Overall, the initial evidence showed that (E)1-(4 aminophenyl)-3-(2,3-dimethoxy phenyl)prop-2-en-1-one has a potent antimalarial ability.
Other studies had also revealed that (E)1-(4 aminophenyl)-3-(2,3-dimethoxy phenyl)prop-2en-1-one can alter the formation of hemozoin and stomatocytes. 11 However, this compound's effect on host-immunity is not yet investigated, especially immunity mediated by Kupffer cells. On the other hand, it has been known that plasmodium infection can induce host immunity and one of the immune responses involved is mediated by Kupffer cells in the liver. 12 Therefore, this study was aimed to evaluate the effect of (E)1-(4 aminophenyl)-3-(2,3dimethoxy phenyl)prop-2-en-1-one toward Kupffer cells phagocytosis in the presence of plasmodium infection.

In vivo antiplasmodial activity testing
The in vivo antiplasmodial activity was evaluated using Swiss Mice infected with Plasmodium berghei by the classical 4-day suppressive test. The research protocol had been approved by the Health Research Ethics Committee, Dr. Moewardi General Hospital/ Faculty of Medicine, Universitas Sebelas Maret, Surakarta. P. berghei strain was obtained from the Department of Parasitology, Faculty of Medicine, Universitas Sebelas Maret, Surakarta. The Swiss mice were obtained from the Integrated Research and Testing Laboratory, Universitas Gadjah Mada, Yogyakarta. P. berghei-infected mice erythrocytes were obtained from donor mice and resuspended in RPMI 1640 medium to a volume of 0.2 ml a day before inoculation. Ninety male mice (20-25 g and 6-8 weeks) were inoculated intraperitoneally with 10 7 P. berghei-infected mice erythrocytes. The mice were then divided into nine groups, with ten mice in each group. The first four groups received 10, 20, 40, and 80 mg/kgBW/day of the tested compound. The second four groups were treated with 0.25, 0.5, 1, and 2 mg/kgBW of doxycycline as the positive control. Another group (negative control) was only received aquadest. Each dose of tested compound or doxycycline was given to the mice daily for four consecutive days, starting two hours after inoculation until the third day. A day after the last treatment, a Giemsa-stained thin blood smear from the tail vein was prepared. Parasitemia level was then determined microscopically by counting the number of parasitized erythrocytes out of 200 erythrocytes in the microscope's random fields.

Histological preparations
After treatment, the mice were sacrificed on a determined day by neck dislocation. The liver organs were then taken for histological preparations using the paraffin block method and stained with hematoxylin and eosin (HE). The right lobe of the liver was taken and sliced at the middle of the lobe to get a uniform preparation. Three slices were made from each right lobe of the liver with a thickness of 3-8 µm. The distance between slices with each other was about 25 slices. Three preparations were made from each experimental host and Kupffer cells were counted (400X magnification). Additionally, the number of Kupffer cells that had phagocytosed the infected erythrocytes was also calculated.

Data analysis
The data were compiled and the study groups were compared by using the ANOVA test. Then, a posthoc multiple comparisons test (Tamhane) was also performed to compare the groups against each other. Finally, the number of Kupffer cells were compared using Kruskall Wallis and Man Whitney statistical tests. The p-value ≤ 0.05 was considered significant.

The Relationship between the test substance and the number of Kupffer cells
After analyzing the effect of the chalcone derivative (E)1-(4 aminophenyl)-3-(2,3-dimethoxy phenyl) prop-2-en-1-one toward parasitemia, we assessed the effect of the compound toward Kupffer cells number within the liver. As depicted in Table 2, the number of Kupffer cells was doubled in the mice, which received chalcone derivative at 10 mg/ kgBW, compared to the negative control group (Aquadest). The Kupffer cells were doubled again at 20 mg/kgBW and begin to plateau at the higher dosage. Comparably, doxycycline was also effective in increasing the number of Kupffer cells and it seemed to have higher efficacy than the tested chalcone derivative. The number of Kupffer cells was tripled at dose 0.25 mg/kgBW and increased at a higher dose. The ANOVA showed a significant result, which indicated that there were significant differences between the study groups. However, indepth analysis using a post-hoc test revealed that the chalcone derivative at dose 20 mg/kgBW had a comparable effect to 0.25 mg/kgBW doxycycline. Also, 80 mg/kgBW of chalcone derivative did not significantly differ from 40 mg/kgBW in increasing the number of Kupffer cells. 40 mg/kgBW of chalcone derivative was also had comparable effect with 0.5 and 1 mg/kgBW doxycycline while 2 mg/ kgBW doxycycline proved to be superior to even 80  Africa, and it is estimated that every minute one child dies of malaria. The main problem in the treatment or eradication of malaria to date is the increased resistance of P. falciparum and P. vivax to antimalarial drugs. Meanwhile, the development of malaria vaccines is hampered due to the complexity of P. falciparum's life cycle, and therefore efforts need to be made to develop multistage vaccines. 2 Drug resistance in Plasmodium is continuously reported from various parts of the world. Consequently, the World Health Organization (WHO) has instructed to stop the monotherapy protocol and recommended Artemisin Combination Therapy (ACT) to improve treatment outcomes and reduce mortality. Meanwhile, rigorous research in drug discovery is continuously conducted to find new antimalarial agents as current agents' alternatives. The ideal antimalarial drug should meet several criteria in which one of which is to have mild side effects with low toxicity. Chalcones (1,3-diaryl-2-propen -1-ones) are secondary metabolites of flavonoids that can be found in several types of plants. 13 Chalcones and their derivatives are known to have various interesting biological activities such as antiviral, anti-inflammatory, antimicrobial, antitumor, cytotoxic, analgesic, antifungal, antioxidant, anticancer, and antiplasmodial. 14, 15 Following previous studies, this study found that chalcone derivative effectively reduced parasitemia and increased the number of stomatocytescontaining Kupffer cells. Compared to doxycycline, the tested compound was still inferior and only the highest dosage (80 mg/kgBW) was comparable to 0.5 mg/kgBW doxycycline. Likewise, 40 mg/kgBW of E)1-(4 aminophenyl)-3-(2,3-dimethoxy phenyl) prop-2-en-1-one was also comparable to 0.5 mg/ kgBW doxycycline and 1 mg/kgBW doxycycline while the highest dose of the compound was comparable to 40 mg/kgBW. Nevertheless, we found a novel compound that has a promising prospect as a new alternative antimalarial agent.
Although the mechanism of action of chalcone was not part of this study, it had been proposed that chalcone and its derivatives act as an inhibitor of ferredoxin (Fd) and ferredoxin-NADP+reductase (NFR) interaction. The presence of inhibitors inhibits electron addition to Lyt B resulting in a disruption of the synthesis of isoprenoid and isoprenoid precursors. 8 Additionally, chalcone inhibits hemoglobin digestion by binding to and inhibiting falcipain, an enzyme that plays a role in hemoglobin digestion in Plasmodium's food vacuole. 16, 17 The free heme, which is released as the byproduct of hemoglobin digestion, is usually aggregated into hemozoin by the Plasmodium to  mg/kgBW. The visual depiction of the Kupffer cells in the liver slice is presented in Figure 3.

DISCUSSION
Malaria is a disease caused by plasmodium parasites and is transmitted by the Anopheles mosquito. Malaria can cause anemia, reduce productivity, and cause death, especially in groups with high-risk factors, namely infants, toddlers, and pregnant women. Many deaths occur in children in

ORIGINAL ARTICLE
avoid its toxic effect. Chalcone was also known to inhibit hemozoin synthesis and enhanced its antiplasmodial effect through heme intoxication of the Plasmodium. 18 Also, the degradation of hemin that leaked out into the cytoplasm will inhibit chalcone through the inhibition of glutathione (GSH). 18 Other reports also showed that chalcone attacks the bc1 complex and complex II (succinate ubiquinone reductase) of Plasmodium's mitochondria. 19 This effect will completely disrupt the electron transport chain, which is highly lethal for Plasmodium. Other effects reported include inhibition of Plasmodial development from ring form into a schizont form, inhibiting sorbitolinduced erythrocytes hemolysis infected by parasites, altering the ultrastructure of parasitic mitochondria and inhibiting mitochondrial function. 6, 16 Additionally, our study also showed the immunological effect of chalcone. This study proved that chalcone administration significantly increased the number of Stomatocyte-containing Kupffer cells, which indicated increased phagocytic activity. However, this immunological effect is contrary to previous reports regarding the role of chalcone in host immunity. Chalcone contains two aromatic rings connected by an α, β unsaturated ketone, and reactive keto-ethylonic groups (-CO-CH = CH-) responsible for anti-inflammatory and antimalarial effects. 20, 21 The anti-inflammatory effect of chalcone was reported by Arya et al., who showed that where phenyl-sulfonyl-uranyl-chalcone derivatives inhibited PGE2 production in LPS-induced RAW 264.7 macrophages through selective inhibition of COX-2 activity. Additionally, Singh et al. also reported that 2′,5′-dimethoxy-4-hydroxy chalcone and 3,4-dichloro-2′,5′-diethoxychalcone inhibited NO production in RAW 264.7 macrophages and LPS-induced microglial cells. 22 Therefore, further studies are needed to analyze the difference between our substance with previous studies and the detail of chalcone's immunological effects so the complete pictures of its effect can be unveiled and the treatment strategy can be devised for the clinical trial.

CONCLUSION
In conclusion, the (E)-1-(4-aminophenyl)-3-(2,3-dimethoxy phenyl)prop-2-en-1-one exhibited potent antimalarial activity, possibly via enhancement of the phagocytic activity of Kupffer cells. Further studies are needed to assess the efficacy and the toxicity of this compound in vivo and to evaluate the immunological effect in detail so the complete pharmacological picture of this compound can be obtained.

AUTHOR CONTRIBUTION
Danush Hermawan: processed the ethical clearance, prepared materials in this research, and doing the statistical test; Yulia Sari and Ida Nurwati: performing the research' procedures; Lilik Wijayanti: performing statistical analysis and preparing the manuscript; Paramasari Dirgahayu: finalization of the manuscript.

CONFLICT OF INTEREST
The author declared that there is no conflict of interest regarding all aspects of the study.

ETHICS APPROVAL
This study has been approved by the Health Research Ethics Committee, Dr. Moewardi General Hospital/ Faculty of Medicine, Universitas Sebelas Maret, Surakarta, with letter number 104/II/HREC/2016.