IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA 4.0 International License Available online at IJTID Website: https://e-journal.unair.ac.id/IJTID/ Vol. 11 No. 1 January–April 2023 Original Article Moringa oleifera Leaf Ethanol Extract Inhibits Toxoplasma gondii Tachyzoites Replication Laura Wihanto1* , Gladdy Lysias Waworuntu1, Cecilia Putri Tedyanto1 , Heni Puspitasari2 1Department of Microbiology and Parasitology, Faculty of Medicine, Universitas Katolik Widya Mandala Surabaya, Surabaya, Indonesia 2Toxoplasma Study Group, Institute of Tropical Disease, Universitas Airlangga, Surabaya, Indonesia Received: January 18th, 2023; Revised: March 17th, 2023; Accepted: March 17th, 2023 ABSTRACT The various infection routes of Toxoplasma gondii that are close to daily life strongly support the incidence of toxoplasmosis. The emergence of drug-resistant Toxoplasma gondii strains raises future concerns. Moringa leaf ethanol extract has been shown to have several anti-pathogen activities, which could have an anti-Toxoplasma effect. This research was conducted to analyze the anti-Toxoplasma effect of moringa leaf ethanol extract against tachyzoites replication in Toxoplasma gondii and the correlation between extract doses with the number of tachyzoites. Mice were divided into five groups. The negative control group (Group I) received CMC-Na solution. The positive control group (Group II) received spiramycin 100 mg/kg BW. The treatment groups received moringa leaf ethanol extract 250 mg/kg BW (group III), 500 mg/kg BW (group IV), and 1000 mg/kg BW (group V), respectively. Mice were injected with 1 x 105 tachyzoites/0.1 mL/mice intraperitoneally on the first day. Moringa leaf ethanol extract and spiramycin were given orally once daily for three days. The number of tachyzoites in the intraperitoneal fluid was calculated on the fifth day. The results have shown that there were significantly lower differences (P < 0.05) in group IV (P = 0.021) and group V (P = 0.022) compared to group I. There was also a significant negative correlation between the extract doses and the number of tachyzoites (P = 0.000; r = -0.781). Moringa oleifera leaf ethanol extract has an anti-Toxoplasma effect by inhibiting the tachyzoite replication at 500 mg/kg BW and 1000 mg/kg BW. Keywords: Moringa oleifera; tachyzoites; Toxoplasma gondii Highlights: This research provides the first study that proved the effectiveness of Moringa oleifera leaf ethanol extract in inhibiting Toxoplasma gondii tachyzoites replication. How to Cite: Wihanto, L., Waworuntu, G. L., Tedyanto, C. P., Puspitasari, H. Moringa oleifera Leaf Ethanol Extract Inhibits Toxoplasma gondii Tachyzoites Replication. Indonesian Journal of Tropical and Infectious Disease. 11(1). 35–43. Apr. 2023. DOI: 10.20473/ijtid.v11i1.42672 * Corresponding Author: laura@ukwms.ac.id https://e-journal.unair.ac.id/IJTID/ https://orcid.org/0000-0001-7746-7678 https://orcid.org/0000-0003-3440-9886 https://orcid.org/0000-0002-0060-8820 36 IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA 4.0 International License Laura Wihanto, et al. Moringa oleifera Leaf Ethanol Extract INTRODUCTION Toxoplasma gondii is an obligate apicomplexan intracellular protozoan that causes toxoplasmosis in warm-blooded animals, including humans. It has been reported that approximately 30-50% of the world’s human population is infected by this parasite.1 The prevalence of toxoplasmosis seropositivity in humans has also reached 32,6% among 9 of 47 primary health centers in Makassar, Indonesia.2 The high incidence of toxoplasmosis makes this disease a global health problem that needs attention because it can cause severe clinical manifestations in immunocompromised patients and permanent fetal disability.3 The various infection routes of T. gondii that are close to daily life could strongly support the incidence of toxoplasmosis. This zoonotic infection can occur in several ways: accidentally ingesting cat faeces that contain oocysts; eating undercooked meat that contains tissue cysts; transplacental transmission from an infected mother to a fetus; and other possibilities, such as receiving a blood transfusion or an organ transplant from an infected donor.4 Recent studies in clinical cases of toxoplasmosis have shown that drug resistance in T. gondii is ongoing. The emergence of drug-resistant T. gondii strains raises future concerns, not only in terms of treatment failure but also of increasing clinical severity in immunocompromised patients.5 Using natural ingredients such as plants or fruits as herbal medicine can be an alternative. This alternative is also considered less toxic than synthetic drugs and is better in terms of economy, practicality, and accessibility. Research on natural resources in Indonesia should always be carried out due to the vast and abundant biodiversity in Indonesia, which has excellent potential to bring benefits to the health sector. Moringa (Indonesian: kelor) or Moringa oleifera is a plant often found in Indonesia. Parts of the plants that can be utilized are roots, stems, fruits, flowers, seeds, and leaves. In vitro study of M. oleifera seeds has been shown to inhibit the replication of tachyzoites.6 The leaves are part of the plant that is often consumed by Indonesian people and have been shown to have anti- inflammatory, antifungal, and antibacterial effects.7-9 It also acts as a larvicidal.10 A phytochemical analysis of M. oleifera leaf ethanol extract revealed alkaloids, phenolics, flavonoids, tannins, saponins, and terpenes as their bioactive compounds.8,11-13 It has rutin as its major flavonoid, gallic acid as its major phenolic acid, and lutein as its major carotenoid. Several alkaloid compounds were also detected, such as pyrazoline alkaloids, piperidine alkaloids, and quinoline alkaloids.14,15 Quinoline alkaloids are one of the typical deoxyribonucleic acid (DNA) intercalating alkaloids that have cytotoxic and antiparasitic effects from their intercalating actions between the nucleotide pairs of the parasite.16 M. oleifera leaf ethanol extract could have an anti-Toxoplasma effect through its DNA- damaging compounds. Therefore, this research was conducted to prove the potential of M. oleifera leaf ethanol extract as the new anti-Toxoplasma drug against tachyzoites replication in T. gondii. MATERIALS AND METHODS Experimental Materials and Tools M. oleifera leaves were purchased from and identified by the Technical Implementation Unit of the Herbal Laboratory, Materia Medica Batu, East Java, Indonesia (reference number 074/656/102.20-A/2022). The RH strains of T. gondii tachyzoites were obtained from the Institute of Tropical Disease, Airlangga University, Surabaya, Indonesia. Deutschland-Denken-Yoken (DDY) mice (male, 20-30 grams, 2-3 months old) were purchased from a certified local experimental animal breeder. Other materials used in this research were spiramycin (500 mg, 37 IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA 4.0 International License Indonesian Journal of Tropical and Infectious Disease, Vol. 11 No. 1 January–April 2023: 35–43 Spirasin®, SANBE, Bandung, Indonesia) and sodium chloride 0.9% (Otsu®, PT Otsuka Indonesia, East Java, Indonesia). Tools used in this research were Neubauer counting chamber (0.1 mm depth, Assistent®, Germany), cover glass (22 x 22 mm, OneLab®, Indonesia), light microscope (Nikon®, Nikon Corporation, Japan), disposable syringe 3 cc (Terumo®, Terumo Company, Tokyo, Japan), sterilized falcon tubes (NEST®, NEST Biotech, China), micropipette (DLAB®, DLAB Scientific Co., Ltd., Beijing, China), and hand tally counter (MARAS®, Togoshi Seiki, Taiwan). Plant Extraction M. oleifera leaves were washed, dried at 40oC temperature using an oven, and ground into powder. The powder was macerated with 96% ethanol for 24 hours while being stirred occasionally. The first maceration results were filtered, and the residue was re- macerated with the same stage until the second maceration results were obtained. Both maceration results were mixed and evaporated using a rotary evaporator at 40oC until they became a dense mass.17 Phytochemical Analysis Table 1. Chemical Reaction Tests for Some Bioactive Compounds from M. Oleifera Leaf Ethanol Extract7,17 Constituent Method Alkaloids Mayer test Flavonoids Ammonium test Phenolics Ferric chloride test Steroids Lieberman-Burchad test Saponins Froth test Tannins Ferric chloride test The phytochemical analysis was performed to determine secondary metabolites (alkaloids, flavonoids, phenols, steroids, saponins, and tannins) present in the M. oleifera leaf ethanol extract using the color and precipitate reaction methods (Table 1). Thin-Layer Chromatography Analysis Figure 1. Retention factor (Rf) Values Calculation Formula in Thin-Layer Chromatography Analysis. The thin-layer chromatography (TLC) analysis was performed to isolate the specific compounds present in the M. oleifera leaf ethanol extract. The extract was applied on silica gel 60 F254 plates as the stationary phase. The mobile phases were (chloroform: methanol: water) (50:65:10) for alkaloids and steroids, (n-butanol: acetic acid: water) (4:1:5) for flavonoids, phenols, and tannins. After leaving the developed plates to dry, they were observed under Ultra-Violet (UV) light at both 254 nm and 366 nm, then sprayed with iodine reagents to detect the bands.18 The movement of the separated compounds was expressed by retention factor (Rf) values, which were calculated by the formula (Figure 1). Animals All the mice have been declared healthy by the veterinarian. The mice were acclimated for one week under laboratory conditions in wire-covered cages with paddy husk as bedding at a temperature of 24±4oC, relative humidity of 44-56%, and 12 hours of light and dark cycle. Four mice per cage were given free access to distilled water and standard mouse food. 38 IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA 4.0 International License Laura Wihanto, et al. Moringa oleifera Leaf Ethanol Extract Parasite Culture The tachyzoite culture was performed in vivo on male DDY mice. They were maintained by routine intraperitoneal passage every 72 hours. The number of tachyzoites was determined by counting them in a counting chamber, then diluted to sodium chloride 0.9% solution before being inoculated into the experimental mice.19 The toxoplasmosis induction used in this research was 1 x 105 tachyzoites/0.1 mL/mice. Toxoplasmosis Drug Reference This research used spiramycin as the toxoplasmosis drug reference.20 It was administered orally at a dose of 100 mg/kg BW. The tablets were crushed into powder and dissolved with sodium carboxymethyl cellulose (CMC-Na) solution until they became a homogenous suspension. Experimental Design and Protocol Mice were divided into five T. gondii- infected groups (n = four mice for each group). The infected group were divided as follows: negative control group (Group I) received CMC-Na solution 0.5 mL/mice orally as a placebo, positive control group (Group II) received spiramycin 100 mg/kg W, group III received M. oleifera leaf ethanol extract 250 mg/kg BW, group IV received M. oleifera leaf ethanol extract 500 mg/kg BW, and group V received M. oleifera leaf ethanol extract 1000 mg/kg BW. Mice in the infected group were injected with 1 x 105 tachyzoites/0.1 mL/mice intraperitoneally on the first day. M. oleifera leaf ethanol extract and spiramycin were diluted into 0.5 mL of CMC-Na solution and given orally once daily for three days from the second to the fourth day. On the fifth day, all mice were sacrificed with cervical dislocation, and the intraperitoneal fluid was collected to count the tachyzoites. Intraperitoneal Fluid Collection The outer skin of the peritoneum was cut using scissors and tissue forceps, then gently pulled back to expose the inner skin lining the peritoneal cavity. The peritoneal cavity was washed with 3 mL of normal saline. The abdomen was shaken slowly to dislodge the tachyzoites into the saline solution. Aspiration of the intraperitoneal fluid was carried out using a syringe.21 Count of Parasites The number of parasites was carried out by blind-direct examination using a counting chamber at 400x magnification of a light microscope.19 Blind-direct examination means the counter does not know from which group the sample was taken. The mean of tachyzoites was expressed in a multiplication factor of 104. Statistical Analysis The research results were analyzed using Statistical Product and Service Solutions software (IBM Corp., Armonk, NY) version 25. The significant differences were statistically determined using the Kruskal- Wallis test, followed by the Mann-Whitney test. Values at P < 0.05 are considered significant. The Pearson correlation coefficient (r) was used to determine the correlation between extract doses and the number of tachyzoites. 39 IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA 4.0 International License Indonesian Journal of Tropical and Infectious Disease, Vol. 11 No. 1 January–April 2023: 35–43 RESULTS AND DISCUSSION Phytochemical Analysis Table 2. Phytochemical Analysis Results of M. oleifera Leaf Ethanol Extract Constituent Result Interpretation Alkaloids Development of cream-yellow precipitate Positive Flavonoids Development of red or pink color Positive Phenols Development of dark green color Positive Steroids Development of blue color Positive Tannins Development of brownish-green color Positive Saponins No formation of stable foam Negative The phytochemical analysis of M. oleifera leaf ethanol extract revealed the absence of saponins and the presence of alkaloids, flavonoids, phenols, steroids, and tannins (Table 2). These results were not aligned with the previous research, which revealed that the M. oleifera leaf ethanol extract contained saponins as its secondary metabolite compound.7,10,12 The absence of saponins in this research could have occurred due to several factors that affected the extraction process. Factors influencing the maceration process results are temperature, solvent types and concentration, duration, and other factors.22 The extraction of M. oleifera leaves using methanol as a solvent with 72 hours of maceration showed positive saponin results on the phytochemical screening.7 Positive saponin results were also obtained in the extraction using ethanol as the solvent with a maceration time of 72 hours.12 Another study using ethanol as a solvent with 48 hours of maceration time showed negative screening results for saponins, which were the same as the results of the phytochemical analysis in this research using the same type of solvent but with 24 hours of maceration time.11 Figure 2. TLC results of alkaloids under UV light (a) 254 nm (b) 366 nm Figure 3. TLC results of flavonoids under UV light (a) 254 nm and (b) 366 nm Figure 4. TLC results of phenols and tannins under UV light (a) 254 nm and (b) 366 nm Figure 5. TLC results of steroids under UV light (a) 254 nm and (b) 366 nm (a) (b) (a) (b ) (a) (b) (a) (b) 40 IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA 4.0 International License Laura Wihanto, et al. Moringa oleifera Leaf Ethanol Extract Table 3. Rf values and colors of peaks of each compound of M. oleifera leaf ethanol extract Constituent Rf values Colors of peaks Alkaloids 0.63 Red 0.73 Blue 0.81 Blue Flavonoids 0.35 Yellow 0.43 Yellow 0.48 Yellow 0.80 Yellow 0.88 Yellow Phenols 0.78 Blackish green Steroids 0.75 Yellowish blue 0.88 Yellowish blue 0.93 Yellowish blue Tannins 0.78 Blackish green The positive bioactive compound results were also confirmed with TLC analysis as shown in Figure 2–5. Alkaloids were detected with Rf values of 0.63, 0.73 and 0.81. Flavonoids were detected with Rf values of 0.35, 0.43, 0.48, 0.80, and 0.88. Phenols were detected with Rf values of 0.78. Steroids were detected with Rf values of 0.75, 0.88, and 0.93. Tannins were detected with Rf values of 0.78 (Table 3). Number of Tachyzoites Figure 6. T. gondii-infected group. Tachyzoites (black arrows) on intraperitoneal fluid from the infected group under a light microscope with 400x magnification. Identification of tachyzoites was carried out based on its distinctive morphology, which is a crescent-like shape, sharp at the anterior and blunt at the posterior, with a length of about 4-8 µm and a width of about 2-3 µm. The color of tachyzoites in the intraperitoneal fluid preparation was clear and transparent, accompanied by movement, indicating that the protozoa are still alive and have motility (Figure 6). Table 4. Tachyzoites count results in the intraperitoneal fluid of the T. gondii-infected group (n = four mice per group) Group Infection Mean±SD (x 104) I + 8.91±3.45 II + 2.88±2.14* III + 6.41±1.25 IV + 3.03±1.08* V + 2.28±0.12* Group I: Negative Control Group; Group II: Positive Control Group; Group III: Treatment Group 1; Group IV: Treatment Group 2; Group V: Treatment Group 3; +: Infected; SD: Standard Deviation; *: p < 0.05 compared to Group I Figure 7. Simple scatter plot of tachyzoites count results (y-axis) by the M. oleifera leaf ethanol extract doses (x-axis). The number of tachyzoites tends to increase as the extract doses decrease, with P = 0.000 and r = -0.781 The highest tachyzoites were in group I with a total of 8.91±3.45 x 104. Group II was significantly lower than group I, with a total of 2.88±2.14 x 104. There were two extract treatment groups with a significantly lower number of tachyzoites compared to group I: group IV with a total of 3.03±1.08 x 104 and group V with a total of 2.28±0.12 x 104. The number of tachyzoites in group III, with a total of 6.41±1.25 x 104, was not significantly different compared to group I as shown in Table 4. The toxoplasmosis intraperitoneally induction used in this research was 1 x 105 tachyzoites/0.1 mL/mice. Tachyzoites can invade almost all the host nucleated cells 41 IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA 4.0 International License Indonesian Journal of Tropical and Infectious Disease, Vol. 11 No. 1 January–April 2023: 35–43 and replicate rapidly.3 The cell will eventually suffer damage and rupture due to this state, and the released tachyzoites will continue to look for other cells, perpetuating the cycle. The severity of the clinical symptoms depends on the degree of tachyzoite replication, which means that an individual's immune system is crucial in defining the clinical manifestations.23 Administration of spiramycin at 100 mg/kg BW orally for three days effectively inhibited tachyzoite replication, as proved by the significantly lower difference in the mean number of tachyzoites present in the peritoneal fluid between group II and group I (Table 4). Spiramycin is an antibiotic and an antiparasitic macrolide agent that is considered the drug of choice against T. gondii in pregnancy. The mechanism of action of the drug was to inhibit the synthesis of proteins and the growth of protozoan cells.24 Its effectiveness as a toxoplasmosis drug has also been proven through previous research, which could reduce the number of T. gondii cysts in brain tissues.20 The doses of M. oleifera leaf ethanol extract used in this research were 250 mg/kg BW, 500 mg/kg BW, and 1000 mg/kg BW. The tachyzoite count differences were significantly lower in groups IV and V compared to group I. There was no significant difference in the mean number of tachyzoites between group III and group I (Table 4). The results revealed that M. oleifera leaf ethanol extract at doses of 500 mg/kg BW and 1000 mg/kg BW could inhibit tachyzoite replication, but the dose of 250 mg/kg BW could not. These results occurred because the concentration of chemical properties in M. oleifera leaf ethanol extract increased with increasing doses (Figure 7). Our results are also align with other research on Plasmodium yoelii, in which the higher the dose of M. oleifera leaf ethanol extract, the greater the inhibitory activity against the parasites.25 Quinoline alkaloids (3-methylquinoline) are typical DNA intercalating compounds found in the M. oleifera leaf ethanol extract.15 Their antiparasitic effect occurred through their hydrophobic, aromatic, and planar properties, which allow them to intercalate between the nucleotide pairs of the parasite. These cause mutations, such as deletions or frame-shift mutations, which will disrupt the replication of the parasite.16 If the mutation occurs in an essential protein-coding gene, it causes the death of the parasite.13 This theory also aligns with previous research, which proved that the moringa seeds extract promotes apoptosis-like death in T. gondii tachyzoites in vitro.6 A simple scatter plot showed a negative correlation between the extract doses and the number of tachyzoites (Figure 7). The decrease in tachyzoite count results, along with increasing doses of M. oleifera leaf ethanol extract, occurred because the concentration of alkaloids in the extract is directly proportional to the dose. The higher concentration of alkaloids causes more intercalated DNA in the parasites, resulting in more disruption in the replication of the tachyzoites. This research proved the effectiveness of M. oleifera leaf ethanol extract in inhibiting tachyzoites replication. Future research needs to conduct more specific studies on the effect of the extract as an anti-Toxoplasma, whether isolating the specific antiparasitic bioactive compound and examining the histopathological variables on T. gondii target organs or other variables of its pathway of antioxidant properties. STRENGTH AND LIMITATION The blind-direct examination method in this research provided minimal occurrence of bias due to the subjective perspective of the researcher. This research also verified and explained the antiparasitic mechanism of M. oleifera leaf ethanol extract through the combination of phytochemical screening, TLC analysis, and the count of parasites. Although we adopt the blind-direct examination for the count of parasites method, it does not provide full objective 42 IJTID, p-ISSN 2085-1103, e-ISSN 2356-0991 Open access under CC-BY-NC-SA 4.0 International License Laura Wihanto, et al. Moringa oleifera Leaf Ethanol Extract results in this research. More objective parameters, such as histopathological or hematological variables, in order to examine the impact of the T. gondii tachyzoites replication, should be carried out in future study to support the results of this research. CONCLUSIONS The M. oleifera leaf ethanol extract has an anti-Toxoplasma effect by inhibiting the tachyzoite replication at 500 mg/kg BW and 1000 mg/kg BW. ACKNOWLEDGEMENT The authors thank the Institution of Research and Community Service of Widya Mandala Catholic University, Surabaya, Indonesia, for providing grants and the Medical Faculty of Widya Mandala Catholic University, Surabaya, Indonesia, for the valuable support throughout this research. ETHICAL CLEARANCE The research protocol was approved by the Health Research Ethics Committee (HREC) of the Medical Faculty of Widya Mandala Catholic University, Surabaya, Indonesia (reference number 0326/WM12/KEPK/DSN/T/2022). This research was carried out following the ethical principles outlined in the Council for International Organizations of Medical Sciences (CIOMS) and World Health Organization (WHO) International Ethical Guidelines for Health-Related Research Involving Humans. FUNDING This research was supported by grants from the Institution of Research and Community Service of Widya Mandala Catholic University, Surabaya, Indonesia (assignment letter number 745/WM01.5/N/2022). CONFLICT OF INTEREST The authors confirm that they have no conflict of interest. AUTHOR CONTRIBUTION Experimental design: LW, GLW, CPT. Extract preparation: GLW, CPT. Materials preparation: LW, CPT. Research implementation: LW, CPT, HP. Research supervision: LW, HP. Data analysis: LW, GLW. Manuscript writing: LW, CPT. Manuscript editing: LW, GLW, CPT, HP. REFERENCES 1. Flegr J, Prandota J, Sovičková M, Israili ZH. Toxoplasmosis - a Global Threat. Correlation of Latent Toxoplasmosis with Specific Disease Burden in a Set of 88 Countries. PLOS ONE. 2014;9(3):e90203. 2. Polanunu NFA, Wahyuni S, & Hamid F. Seroprevalence and associated risk factors of Toxoplasma gondii infection among pregnant mother in Makassar, Indonesia. PLOS ONE. 2021;16(6):e0245572. 3. Wang D, Liu H, Ma X, Ma Y, Li Y, Yang B, et al. Toxoplasma gondii Infection in Immunocompromised Patients: A Systematic Review and Meta-Analysis. Front Microbiol. 2017;8:389. 4. Centers for Disease Control and Prevention (CDC). 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