SEQUESTRATION OF ERYTHROCYTE INFECTED BY Plasmodium berghei ANKA IN MICE LIVER TREATED WITH ETHANOL EXTRACT OF PEARL GRASS (Hedyotis corymbosa (L.) Lamk)

Background: Malaria is a parasitic infection caused by the Plasmodium genus. Certain Plasmodium species can evade the immune system by sequestering internal organs, including the liver. The ethanolic extract of pearl grass (Hedyotis corymbosa (L.) Lamk) (EEPG) has been reported to have an antimalarial activity in reducing parasitemia and hepatomegaly in Plasmodium berghei ANKA-infected mice. Purpose: To analyze the effect of EEPG administration on the sequestration of P. berghei ANKA-infected erythrocytes in the livers of BALB/c mice. Method: P. berghei ANKA-infected mice were treated with EEPG at doses of 250, 300, and 350 mg/kg BW. The positive and negative control groups received dihydroartemisinin-piperaquine (DHP) 187.2 mg/kgBW and 1% CMCNa, respectively. The treatments were administered for four consecutive days, followed by observation of parasitemia on Giemsa-stained tail blood smears. On day five, mice were sacrificed for liver removal. Sequestrations were observed on HE-stained slides of mouse livers. The differences in sequestration between treatment groups were analyzed using One-way ANOVA and Games-howell post-hoc analysis. The correlation between parasitemia and sequestration was analyzed using the Pearson correlation test. Result: The percentage reduction of the number of infected erythrocyte sequestration in EEPG-treated groups was 81.74%, 77.72%, and 77.70%, respectively, while the positive control group was 91.14%. Parasitemia was correlated with the number of erythrocytes sequestration (p-value < 0.05). Conclusion: EEPG was able to decrease parasitemia along with the decrease in the number of infected erythrocytes sequestration in the liver. These results indicated that EEPG is

Introduction

Malaria is a disease transmitted through the bite of the female Anopheles mosquito and is caused by protozoa of the genusPlasmodium(W.H.O., 2024). In humans, malaria is commonly caused by five species ofPlasmodium, namely (1)Plasmodium falciparumand (2)Plasmodium vivax(Loy et al., 2017), (3)Plasmodium malariae(Plenderleith et al., 2022), (4)Plasmodium ovale(Okafor & Finnigan, 2025), and (5)Plasmodium knowlesi(Bin Said et al., 2022). Species ofP. falciparumaccount for the highest number of malaria-relateddeaths (99%) compared to other species ofPlasmodium. One of the reasons is the ability of this species to evade the immune system by sequestrating and rosetting. Sequestration refers to a phenomenon where infected erythrocytes accumulate in the brain, liver, and lungs(Belachew, 2018);(Viriyavejakul et al., 2014). Theprocess of sequestration inP. falciparuminfection is mediated by the binding ofP. falciparumerythrocytemembrane protein-1 (PfEMP-1) and its receptors on the surface of endothelial cells of microvasculatures in visceral organs, mainly CD36(Fonager et al., 2012)ICAM-1(Cunningham et al., 2017)VCAM-1, E-selectin, and chondroitin sulfate(Mahamar et al., 2017). Inflammatory responses triggered by ruptured infected erythrocytes further support this process by inducing cytokines such as TNF-α, which increase cytoadhesive endothelium surface protein levels like ICAM-1(Gallego-Delgado et al., 2014);(Storm & Craig, 2014). The phenomenon has been reported to occur in various mouse strains infected with various strains ofPlasmodium berghei(Franke-Fayard et al., 2010);(Wardani et al., 2020). Furthermore, Schizont Membrane-Associated Cytoadherence (SMAC) has been reported as a protein that is involved in the sequestration ofP. berghei-infected erythrocytes in mice(Fonager et al., 2012).

Pearl grass (Hedyotis corymbosa(L.)Lamk) or also known asOldenlandia corymbosais an herbaceous plant of the Rubiaceae family. Phytochemically analysis has shown that it contains phytochemical proteins, polysaccharides, polyphenols, tannins, flavonoids, saponins, steroids, triterpenes, and glycosides(Das et al., 2019). Traditionally, this plant has been used as an antipyretic, anti-inflammatory, antibacterial, diuretic, anti-stomach ulcer, anti-dysentery, postpartum medicine, and for digestive disorders(Soemardji et al., 2015). The antimalarial properties of pearl grass have demonstrated its ability to inhibit the growth ofP. falciparumin an in vitro culture by methanolic extract of pearl grass(Mishra et al., 2009)as well asP. bergheiin BALB/c mice by Ethanolic Extract of Pearl Grass (EEPG) that has been reported previously by our research group(Putri et al., 2023). However, the effect of pearl grass extract on the sequestration of malaria-infected erythrocytes has not been reported either in human or in mouse malaria. This study aimed to analyze the effect of EEPG on the parasitemia and sequestration of erythrocytes infected withP. bergheiANKA in the livers of BALB/c mice.

Material and Method

Preparation of the Ethanolic Extract of Pearl Grass (EEPG)

The EEPG was prepared by maceration of simplicia in 70% ethanol solvent in the Laboratory of Herbal Materia Medika, Batu City, East Java Province, Indonesia. The doses of EEPG used were 250 mg/kgBW, 300 mg/kgBW, and 350 mg/kgBW in 1% CMCNa(Putri et al., 2023).

Research design

This study was an experimental laboratory research employing a post-test only control group design. The experiment was conducted between March 2022 and May 2022 across three different: Departments Biochemistry, Anatomical Pathology and Medical Parasitology, Faculty of Medicine, Universitas Airlangga. Twenty-five mice were injected intraperitoneally with 200 μL ofP. berghei-infected blood obtained from donor mice.

The mice were then randomly divided into five groups. Group 1 - 3 received 250 mg/kgBW, 300 mg/kgBW, and 350 mg/kgBW of EEPG, respectively. Group 4 served as the positive control group and was treated with 187.2 mg/kgBW of dihydroartemisinin-piperaquine (DHP). Group 5 acted as the negative control group and received 1% CMCNa. All treatments were administered orally once a day for four consecutive days.

On day five, a drop of blood was collected from each mouse’s tail to prepare thin blood smears, which were then stained with Giemsa-stain for parasitemia determination. Subsequently, all mice were sacrificed, and their livers were processed into Hematoxylin Eosin (HE)-stained slides for anatomical pathology examination. The number of infected erythrocyte sequestrations was observed within 5 fields of view per microscopy slide(Wardani et al., 2020). The number of infected erythrocytes sequestration reduction due to EEPG administrations was calculated using the following Formula (1). In which NS: number ofP. berghei-infected erythrocytes sequestration, TG: EEPG treated group and NG: negative control group.

\( \documentclass{article} \usepackage{amsmath} \begin{document} \displaystyle \% \text{ Reduction} = \frac{\text{Mean of NS in NG} - \text{Mean of NS in TG}}{\text{Mean of NS in NG}} \times 100\% \quad \text{....(1)} \end{document} \)

Data analysis

The sequestration data were statistically analyzed using a One-way ANOVA followed by Games-howell post-hoc tests. Correlations between variables were assessed using the Pearson correlation test.

Result

Sequestration ofP. bergheiANKA-infected erythrocytes in mice the liver

Microscopic observation of the HE-stained mice liver tissues revealed a purple color of the nuclei of the hepatic cells, the clear red of healthy erythrocytes, the purple color of infected erythrocytes containing parasites, and the purple color of various leukocytes. An overview of the HE-stained mice livers is presented inFigure 1. Based onFigure 1, sequestrations ofP. bergheiANKA-infected erythrocytes were observed in the sinusoids of the liver. Infected erythrocytes can be distinguished from healthy erythrocytes