Phytochemical Analysis, Antimalarial Properties, and Acute Toxicity of Aqueous Extracts of Trisamo and Jatu-Phala-Tiga Recipes

Phuwajaroanpong, Arisara; Chaniad, Prapaporn; Plirat, Walaiporn; Konyanee, Atthaphon; Septama, Abdi Wira; Punsawad, Chuchard

doi:https://doi.org/10.1155/2023/6624040

Advances in Pharmacological and Pharmaceutical Sciences

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Abstract Introduction Materials and Methods Results Discussion Conclusions Data Availability Ethical Approval Conflicts of Interest Authors’ Contributions Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2023 | Article ID 6624040 | https://doi.org/10.1155/2023/6624040

Phytochemical Analysis, Antimalarial Properties, and Acute Toxicity of Aqueous Extracts of Trisamo and Jatu-Phala-Tiga Recipes

Arisara Phuwajaroanpong,^1,2Prapaporn Chaniad,^1,2Walaiporn Plirat,^1,2Atthaphon Konyanee,^1,2Abdi Wira Septama,³and Chuchard Punsawad^1,2

Academic Editor: Mohd Esa Norhaizan

Received08 Jun 2023

Revised06 Aug 2023

Accepted04 Sept 2023

Published15 Sept 2023

Abstract

Drug resistance remains a significant problem that threatens antimalarial drug treatment. Hence, the challenge is to find new effective antimalarial drugs. Based on our previous study, aqueous extracts of trisamo (TSM) and jatu-phala-tiga (JPT) had good in vitro antimalarial activities, and these recipes contain multiple beneficial pharmacological effects that could be useful for malaria therapy. Therefore, this study aimed to investigate the antimalarial activity and toxicity of the aqueous extracts of TSM and JPT in mouse models. The aqueous extractions were carried out using the decoction method. Compound identification was conducted using LC-QTOF-MS analysis. The antimalarial activities of TSM and JPT at doses 200, 400, and 600 mg/kg were evaluated against Plasmodium berghei ANKA infection using a four-day suppressive test. The toxic effects of oral administration of the extracts at 2 g/kg dose were determined using an acute toxicity test. The chemical constituents of TSM contained 83 compounds, whereas JPT contained 84 compounds. All doses of the extracts exhibited a significant suppression () of the parasite compared to the negative control in a four-day test. The maximum activities were observed at 600 mg/kg dose with 67.02% suppression for TSM and 79.34% for JPT, followed by 400 mg/kg dose (57.63% for TSM and 64.79% for JPT) and then 200 mg/kg dose (52.35% for TSM and 54.46% for JPT). In addition, there were no significant differences () in the RBC, MCV, and MCH levels of mice receiving JPT extract compared to the uninfected control. The WBC level of mice receiving 400 and 600 mg/kg of TSM, and 200 and 400 mg/kg of JPT, was significantly () lower than the infected control, and the extracts did not significantly prevent the loss of platelets. For the acute toxicity test, there were no signs of toxicity or deaths in mice, and there were no differences in the histology, weight, or enzyme biochemistry of the liver and kidney between the extract and vehicle groups. However, the platelet count in the extract-treated mice was significantly higher than that in the control group. In conclusion, this study suggests that aqueous extracts of TSM and JPT have potent antimalarial activities and could be promising as new candidates for antimalarial drug development.

1. Introduction

Malaria remains one of the most serious illnesses in tropical and subtropical regions. It is a vector-borne disease caused by obligate intracellular Plasmodium parasites and is transmitted through the bite of infected female Anopheles mosquitoes [1]. Five species of Plasmodium, P. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi, can cause diseases in humans [2]. Malarial paroxysm is a distinct clinical feature of the disease caused by the rupture of infected red blood cells (RBCs) [2]. Common symptoms include fever, chills, and headaches, whereas the major complications of severe malaria include cerebral malaria, pulmonary edema, acute renal failure, severe anemia, bleeding, liver injury, and death [2]. According to the World Malaria Report of 2022, the estimated number of deaths globally was 619,000, and 247 million cases were reported [1]. The African region of the World Health Organization (WHO) has the highest malaria burden, accounting for approximately 95% of the global cases [1]. However, the case incidence has dropped since 2000 before increasing in 2020 due to a service interruption during the COVID-19 pandemic. Although effective treatment is a key factor in combating this disease, drug resistance poses a significant risk to the control and elimination of malaria [3]. After the emergence of chloroquine resistance in the 1960s, the WHO recommended artemisinin-based combination therapy (ACT) as the first-line treatment for uncomplicated malaria [4], which included artemether-lumefantrine (AL), artesunate-amodiaquine (AS-AQ), artesunate-mefloquine (AS-MQ), artesunate-sulfadoxine-pyrimethamine (AS-SP), dihydroartemisinin-piperaquine (DHA-PPQ), and artesunate-pyronaridine (AS-PY) combinations [3, 4]. The various treatments were determined based on the areas of resistance. Currently, the emergence of partial artemisinin resistance is of great concern and has been observed in countries in the WHO African region and the Greater Mekong Subregion (GMS) [1]. A treatment failure rate of >10% for AL in Burkina Faso and Uganda and for DHA-PPQ in Burkina Faso has been observed [1]. In GMS, mutations associated with SP resistance have been observed; hence, the failure of AS-SP could be of concern [1]. In addition, a high prevalence of mutations associated with partial artemisinin resistance was found in Myanmar and Thailand, and high rates of DHA-PPQ plus primaquine treatment failure were found in Sisaket Province, Thailand, which led the province to change its first-line drug to AS-PY in 2020 [1]. Novel strategies are needed to eradicate malarial parasites to overcome the emergence of drug-resistant parasites. Utilizing traditional medicine is one of several interesting ideas, especially polyherbal or herbal recipes, owing to their positive effects as a result of synergistic interactions [5]. Our previous study on the in vitro antiplasmodial properties of aqueous and ethanolic extracts of ten herbal traditional recipes reported that aqueous extracts of trisamo (TSM) and jatu-phala-tiga (JPT) exhibited good antimalarial activities [6]. Both are traditional herbal recipes that have been used for several centuries in Thailand [7]. The TSM is composed of three Terminalia species: Terminalia bellirica (Gaertn.) Roxb., Terminalia chebula (Roxb. ex DC.), and Terminalia arjuna (Roxb. ex DC.). Trisamo means “three” (tri-) fruits of Terminalia species (-samo), and these plants belong to the family Combretaceae [7]. The common names of T. bellirica, T. chebula, and T. arjuna are beleric myrobalans, chebulic myrobalans, and arjuns, respectively [7]. TSM is indicated for promoting good general health and relieving abdominal bloating and is also used as an antipyretic, expectorant, and rejuvenator [8]. Several biological benefits of the ingredients in the TSM recipe have been reported which include antipyretic, antibacterial, antioxidant, anti-inflammatory, antihyperglycemic, anticlastogenic, immunomodulatory, analgesic, radioprotective, gastrointestinal motility-promotion, cardioprotective, antiaging cytoprotective, anticancer, antidiabetic, wound-healing, and antinociceptive properties [8–11]. The meaning of JPT corresponds to the benefits of four fruits: Jatu means “four,” phala means “fruits,” and tiga means “benefits” or “usefulness.” The four fruit ingredients include Phyllanthus emblica Linn. (P. emblica), T. bellirica, T. chebula, and T. arjuna. Indian gooseberry or P. emblica belongs to the family Euphorbiaceae and is commonly used in Ayurvedic systems for its many beneficial characteristics such as antidiabetic, antimicrobial, anti-inflammatory, and antiaging properties [7, 12]. JPT is well known for its antioxidant activity, and it is used as an antipyretic, laxative, stomachic, colon cleanser, detoxifying agent, health promotion agent, and rejuvenator in Thai traditional medicine [13, 14]. Scientific evidence has revealed that JPT has antimutagenic, cardioprotective, radioprotective, hepatoprotective, anti-inflammatory, and antiobesity properties [15, 16]. In addition, the TSM and JPT recipes consist of numerous secondary metabolites such as flavones, alkaloids, phenols, tannins, coumarin, terpenoids, glycosides, and saponins [17–19]. Several classes of phytoconstituents from natural products are responsible for their antimalarial activity [20]. Alkaloids, including terpenoidal, quinolone, and isoquinoline alkaloids, were identified with promising antimalarial activity [21]. The antimalarial action of plant flavonoids is believed to act by inhibiting fatty acid biosynthesis and the influx of L-glutamine-myoinositol in the infected red blood cells [22]. Terpene and coumarin derivatives have been reported to have potent antimalarial activities [23, 24]. Regarding the abovementioned, the phytoconstituents deposited in the TSM and JPT recipes may provide great potential for antimalarial activities. Thus, this study aimed to investigate the antimalarial activity and toxicity of TSM and JPT in a mouse model.

2. Materials and Methods

2.1. Management and Preparation of TSM and JPT Recipes

T. bellirica, T. chebula, T. arjuna, and P. emblica were bought at a Thai pharmacy store in the southern Thai province of Nakhon Si Thammarat’s Muang District. The morphological identification of plants was confirmed by a botanist, and the deposited specimens SMD074002003 (T. bellirica), SMD070006007 (T. chebula), SMD070006002 (T. arjuna), and SMD209003007 (P. emblica) were at Walailak University in Thailand’s School of Medicine’s Department of Medical Sciences. The fruits were dried for 3 days in an oven (Memmert Model SFE 600, Schwabach, Germany) after being washed with tap water. Each fruit was ground using a herb grinder (Taizhou Jincheng Pharmaceutical Machinery Co., Ltd., Model: SF, Jiangsu, China). The TSM and JPT recipes were prepared according to Thai herbal pharmacopeia [7, 25]. The TSM recipe was prepared by mixing T. bellirica, T. chebula, and T. arjuna, in a 1 : 1 : 1 ratio, and the JPT recipe was prepared by mixing T. bellirica, T. chebula, T. arjuna, and P. emblica in a 1 : 1 : 1 : 1 ratio.

2.2. Aqueous Extraction Method

Aqueous extractions of TSM and JPT were performed using the decoction method [26, 27]. For each recipe, 60 g of plant material suspended in 600 mL of water was extracted by boiling for 30 min. Then, filtration through filter paper (Whatman, Buckinghamshire, England) was used to separate the liquid from the marc. Subsequently, the marc was re-extracted twice by boiling in 600 mL of water for 30 min. The rotary evaporator (Rotavapor, Buchi, China) was used to concentrate the combined filtrate at 45 rpm and 45°C. Then, the extract was dried at −89°C in a freeze-drying apparatus (Martin Christ, Germany). The crude extract was weighed, and the yield was determined as follows:

2.3. Compound Identification Using Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry (LC-QTOF-MS) Analysis

Chromatographic separations were accomplished according to our previous study [6]. The temperature in the column was fixed at 25°C. The mobile phase A was made up of 0.1% formic acid in water, and mobile phase B was made up of acetonitrile. A flow rate of the mobile phase was 0.20 mL/min, and the injection volume was 2 μL. The MS conditions involved an electrospray ionization (ESI) probe in negative mode with a scanning range of 100−1,200 m/z. Agilent MassHunter Workstation Software V8 was used to process the data. Compound identification was based on the similarity score, which was achieved by matching the retention times and mass data of an unknown compound to the reference spectra in a METLIN mass spectra library (Agilent Technologies). A similarity score of >90% was employed to identify the compounds deposited in the extracts.

2.4. Animals and Management

Male ICR mice that were 6–8 weeks old were obtained from Nomura Siam International Co., Ltd., in Bangkok, Thailand. All mice were acclimatized for 1 week under strictly hygienic laboratory conditions. Housing settings included cycling light and dark for 12 hours each cycle, with a regulated room temperature of 23 ± 2°C and humidity levels of 50−70%. Mice were given unlimited access to food pellets and clean drinking water.

2.5. Testing for Antimalarial Activity

The antimalarial activity was evaluated using Peters’ 4-day suppressive test [28]. The rodent malaria parasite, P. berghei ANKA strain, was provided by Thomas F. McCutchan and obtained from BEI Resources, NIAID, NIH. Inoculation was initiated by intraperitoneal (IP) injection of 0.2 mL of infected blood into the donor mice. Once the parasitemia level reached 20–30%, blood was collected to infect recipient mice. To evaluate the in vivo antimalarial activity against early infection, 40 mice received IP injection of 0.2 mL of 1 × 10⁷P. berghei infected cells, whereas five mice were injected with 0.2 mL of normal saline solution as uninfected controls. Forty infected mice were randomly divided into eight groups (five mice per group), which included negative (received phosphate-buffered saline (PBS)) and positive (received 25 mg/kg chloroquine) control groups, and six experimental groups (received TSM or JPT). Aqueous extracts of TSM and JPT were administered at doses 200, 400, and 600 mg/kg. Oral administration was started at 3 h and then at 24, 48, and 72 h post-infection. On day 4, all mice were anesthetized by inhalation of 2% isoflurane in oxygen and euthanized immediately after blood collection by cardiac puncture. Thin blood smears were made from blood samples to determine the percentage of parasitemia. In addition, blood samples were used to determine changes in hematological parameters, including red blood cell (RBC) count, hemoglobin (HGB), hematocrit (HCT), mean cell volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), platelet (PLT) count, and white blood cell (WBC) count. Hematological analysis was carried out with an automatic AU480 chemistry analyzer (Beckman Coulter, USA). Parasitemia was monitored by Giemsa-stained thin blood smears, which were then viewed under a light microscope (Olympus CX31, Model CX31RBSFA, Tokyo, Japan) with oil immersion (100× magnification). The following formula was used to obtain the parasitemia and suppression percentages:

2.6. Acute Toxicity Test

This test was performed in accordance with the Organization for Economic Cooperation and Development (OECD) guideline No. 425 [29] according to a previously described method [6, 26, 30]. Three groups of five mice each were formed from a total of fifteen mice. PBS was used as the vehicle control for Group I. TSM and JPT were administered at a dose of 2 g/kg to Groups II and III, respectively. All mice were weighed before receiving the extract or PBS. To assess the toxicity of the extracts, 2 g/kg of the extract was administered after the mice had fasted for 3 h. The mice were observed immediately after feeding and then carefully observed for 30 min. Behavioral changes, signs of toxicity, and mortality were observed twice daily for 14 days. On day 14, all mice were weighed, anesthetized with 2% isoflurane, and euthanized through cardiac puncture. Blood samples were collected from the heart for hematological and biochemical analyses. The liver and kidneys were removed and weighed to determine the relative organ weights. Relative organ weight was calculated using the following formula; thereafter, the organs were used for histopathological analysis.

2.7. Hematological, Biochemical, and Histopathological Assessment in Acute Toxicity Test

Blood was collected into two types of tubes including EDTA and serum clot activator tubes. Blood in EDTA tubes was used for the hematological analysis (RBC, HGB, HCT, MCV, MCHC, MCH, PLT, and WBC), whereas serum were used for evaluation of renal and hepatic functions, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), and creatinine. Blood samples were analyzed using an AU480 chemistry analyzer (Beckman Coulter, USA). For histopathological assessment, the liver and kidneys were fixed in 10% formalin, and hematoxylin and eosin staining was performed as previously described [31–33].

2.8. Statistical Analysis

SPSS for Microsoft Windows (version 17.0; IBM, Armonk, NY, USA) was used to conduct the statistical analyses. The mean ± standard error of the mean (SEM) is used to express all data. The data were examined for normality of distribution before being subjected to a one-way analysis of variance (ANOVA), with a significance level of .

3. Results

3.1. Percentage Yield of TSM and JPT Recipes

The percentage yield of the aqueous extracts of JPT (40.12) was slightly higher than that of TSM (39.62). The TSM appeared as a dark brown solid, and JPT was a light brown crumbly solid.

3.2. Compound Composition of TSM and JPT Detected by LC-QTOF-MS Analysis

The compounds in the TSM and JPT extracts were tentatively identified using LC-QTOF-MS analysis. Tables 1 and 2 show the compounds found in the aqueous extracts of TSM and JPT, respectively. The chemical constituents of TSM contained 83 compounds, whereas JPT contained 84 compounds. Figures 1 and 2 show the peak chromatograms of TSM and JPT, respectively.

3.3. Effects of the Extracts on Percentage Parasite Suppression

Percentage parasitemia and effects of crude extracts on the percentage suppression of P. berghei infection are shown in Table 3. The standard drug administered at a concentration of 25 mg/kg eliminated 100% of the blood-stage parasites. Administration of TSM and JPT extracts exhibited significant () dose-dependent percentage parasite suppression compared to the negative control, with mean suppression percentage ranges 52.35–67.02% for TSM and 54.46–79.34% for JPT. In addition, percentage suppression at all doses of TSM was significantly lower than that in the chloroquine group (), whereas the percentage suppression at 600 mg/kg JPT showed no significant difference () compared to chloroquine administration.

3.4. Effects of the Extracts on Hematological Changes in 4-Day Suppressive Test

The results of the hematological changes are shown in Figure 3. The indices of uninfected mice were used to represent hematologic reference values at the normal levels. Hematological alterations between normal and infected controls showed significant differences in RBC, MCV, and MCH levels (), while hemoglobin, hematocrit, and MCHC levels were not significantly different among all groups. Mice administered a standard drug demonstrated significantly higher RBC levels (), but MCV and MCH levels were significantly lower () than those in the infected controls. TSM administered at a dose of 400 mg/kg revealed a significant decrease in RBC (), but JPT administration did not show a significant decrease in RBC compared with uninfected mice and those administered chloroquine. The differences between MCV and MCH levels showed that mice receiving JPT at all doses exhibited significantly () lower levels than the infected control, and no difference was observed when compared to the chloroquine group. The MCV and MCH levels in the TSM group were significantly () higher than those in the uninfected and chloroquine groups. Administration of 400 mg/kg TSM resulted in a significant increase in MCV, compared to 400 and 600 mg/kg of JPT, and all doses of TSM resulted in a significant difference in the MCH level compared to 400 mg/kg of JPT. The platelet counts of infected mice and mice that received the extracts were significantly () lower than those of the normal control. Compared to the infected control, only the positive control group showed a significant increase in platelet counts (). In addition, chloroquine also produced a significant () decrease in the WBC count compared to that in infected mice. The extracts did not prevent a significant () loss of WBC compared with chloroquine. TSM (400 and 600 mg/kg) and JPT (200 and 400 mg/kg) were significantly decreased when compared to the infected control.

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Figure 3

Effects of TSM and JPT on hematological parameters in the 4-day suppressive test: (a) RBC count, (b) hemoglobin levels, (c) hematocrit levels, (d) MCV levels, (e) MCH levels, (f) MCHC levels, (g) WBC count, and (h) platelet count. Data are presented as the mean ± SEM (n = 5 per group). There were statistically significant differences at , ^acompared with uninfected mice, ^bcompared with negative control group receiving PBS, ^ccompared with the positive control group receiving CQ, ^dcompared with TSM 200 mg/kg, ^ecompared with TSM 400 mg/kg, ^fcompared with TSM 600 mg/kg, ^gcompared with JPT 200 mg/kg, ^hcompared with JPT 400 mg/kg, and ⁱcompared with JPT 600 mg/kg.

3.5. Clinical Observations, Analysis of Bodyweight, and Organs’ Weights in Acute Toxicity Test

Oral administration of TSM or JPT at a dose of 2 g/kg in mice did not produce any significant changes in clinical signs compared to the vehicle control. There were general physical and behavioral appearances such as bright eyes, erect ears, normal body posture, and grooming. Signs of toxicity, such as diarrhea, tremors, convulsions, ataxia, or unusual behaviors, were not observed throughout 14 days. No mortality occurred during the experiment; therefore, the mean lethal dose (LD₅₀) of the aqueous extract of TSM and JPT administered via the oral route was higher than 2 g/kg. When compared to the vehicle control, the actual body weight, percentage of body weight change, and relative organ weights of the liver and kidney of mice administered that the extracts showed no significant difference () (see in Table 4).

3.6. Effects of the Extracts on Hematological and Biochemical Changes in Acute Toxicity Test

Hematological results revealed that the platelet counts of the TSM and JPT groups were significantly higher than that of the vehicle control group (see Figure 4). Biochemical parameters of the liver and kidney function tests are presented in Table 5. No significant differences in BUN, CREA, AST, ALT, or ALP levels were observed among the groups.

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3.7. Effects of TSM and JPT on Histopathology in Acute Toxicity Test

Figure 5 shows the histopathological examination of the liver and kidneys in the acute toxicity test at a dose of 2 g/kg. Figures 5(a) and 5(b) show the normal structure of the liver and kidney histology, respectively, which were obtained from mice in the control group. In comparison, liver and kidney sections showed no differences between the control and mice treated with TSM and JPT. Liver sections (Figures 5(a), 5(c), and 5(e)) revealed normal hepatocytes without hepatic congestion, inflammatory cell infiltration, or sinusoidal dilatation. Kidney sections (Figures 5(b), 5(d), and 5(f)) showed unchanged glomeruli and renal tubules without vascular congestion.

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4. Discussion

As antimalarial drug resistance has been a major problem in malaria control, effective vaccines are unavailable. Therefore, new treatments are urgently needed. Our previous report showed that aqueous extracts from TSM and JPT have potent antiplasmodial activity against P. falciparum [6]. As a result, the current study sought to assess the antimalarial properties and acute toxicity of aqueous extracts of TSM and JPT in mouse models.

For in vivo antimalarial testing, the antimalarial activities of TSM and JPT were investigated at 200, 400, and 600 mg/kg using a 4-day suppressive test. The highest average percentage parasite suppression of 600 mg/kg JPT was 79.34%, and the extracts at all doses significantly suppressed parasite growth compared with the infected control. However, only JPT at 600 mg/kg showed no significant difference () compared to chloroquine, which may imply that the effect of JPT at 600 mg/kg is similar to that of chloroquine. The biological properties of plant extracts are known to be mediated by phytocomponents [34]. Based on the LC-MS analysis, our findings are consistent with those of previous studies. Chebulinic acid, gallic acid, ellagic acid, quinic acid, and luteolin were found in the fruits of T. bellirica, T. chebula, and T. arjuna extracts [8, 35, 36]. In addition, the phytocomponent of P. emblica was reported to have tannins such as chebulic acid, gallic acid, and punicalagin; flavonoids such as luteolin and quercetin derivatives; polyphenolics such as ellagic acid; and phenolics such as chebulinic acid [12, 37–39]. Ellagic acid has been reported to possess antioxidant, anti-inflammatory, antimutagenic, antiproliferative, and antimalarial properties [40, 41]. Chebulinic acid has been reported to have numerous biological activities, including antidiabetic, antifibrotic, anti-inflammatory, antitumor, antiatherogenic, antioxidant, antiulcer, hepatoprotective, and antiviral properties [42]. Quinic acid has an important antibacterial effect [43]. Accordingly, the compound described above, or the other compounds present in TSM and JPT might exert antimalarial activities through individual or synergistic effects. In addition, the antimalarial activity of the extracts in this study showed that the percentage parasite suppression of JPT was higher than that of TSM. The reason for this could be the extra ingredient from P. emblica in the JPT recipes. P. emblica is important in traditional medicinal systems, and its various pharmacological benefits have been reported, including antimicrobial, antioxidant, anti-inflammatory, antipyretic, antitusive, antiatherogenic, anticancer, antidiabetic, antiaging, cardioprotective, gastroprotective, nephroprotective, neuroprotective, chemopreventive, analgesic, and immunomodulatory properties [44]. Furthermore, JPT and its components have been reported to exhibit strong antioxidant activity [45]. Based on previous evidence, we suggest that P. emblica improves the antimalarial property of the extract.

Furthermore, this study investigated the effects of the extracts on hematological parameters during malaria infection because hematological abnormalities are considered a characteristic of malaria, especially RBCs [46]. The differences in RBC, MCV, and MCH between the uninfected and infected groups were significant, and chloroquine improved these blood parameters to normal ranges compared to the uninfected control. The reduction of RBCs in infected mice may be caused by the destruction or sequestration of RBCs, or reduction of RBC production in the bone marrow [9]. The significant increases in MCV and MCH were indicative of malaria-induced macrocytic anemia [47, 48]. The RBC count in the 400 mg/kg TSM group was significantly lower than that in the uninfected control. The MCV and MCH of all TSM extract doses differed significantly from those of the uninfected control. However, the RBC count, MCV, and MCH of mice that received JPT showed no significant differences compared to the uninfected control. This finding implies that only the JPT extract can prevent malaria-induced macrocytic anemia. The ability to improve RBC and related parameters may be due to the inhibitory effects of the drug and its extracts on parasite growth. WBC and related parameters play an important role in infectious diseases [46]. WBC responds to infectious agents, and thrombocytopenia is a common feature of Plasmodium infection, which is associated with several mechanisms, such as endothelial damage and isolated platelet consumption [46]. The platelet count of mice in the infected control group was significantly reduced, but the WBC count was increased, when compared with the uninfected control group. The extracts from TSM and JPT showed a significant and dramatic decrease in platelet count compared to the normal control and chloroquine. This could mean that extracts at doses 200, 400, and 600 mg/kg did not maintain platelet homeostasis during malaria infection. In terms of WBC parameters, TSM reduced the WBC count in a dose-dependent manner. This finding may indicate that a decrease in the WBC count is associated with a reduction in the percentage of parasites. Interestingly, 600 mg/kg JPT resulted in the highest WBC count and the highest percentage parasite suppression among the extracts. This result implies that the antimalarial activity of JPT at this dose may be attributed to the activation of immune cells, which is consistent with a previous study [12]. P. emblica, an ingredient in the JPT recipe, has been reported to exhibit the ability to enhance immunity [12]. Consequently, JPT extract not only exhibited stronger antimalarial activity but also exhibited greater maintenance of RBC parameters than the crude extract from TSM.

Although natural products have been used to treat several diseases since ancient times, the negative effects of plant products must be considered. According to the results of the toxicity study, there were no deaths or physical or behavioral changes for 14 days, thereby indicating that the LD₅₀ of TSM and JPT was greater than 2 g/kg. TSM and JPT were classified as relatively low acute toxicity hazards in Category 5 according to the international system of chemical classification [49]. The effects of the extracts on changes in body weight and organ weight are sensitive indicators for general health status and organ damage in animals [50, 51]. We found no significant changes in body weight or organ weight when compared with the vehicle control at the endpoint. In addition, this study focused on the negative effects of the extracts on the hematological markers, biochemical enzymes, and pathology of the liver and kidney. For all blood parameters, only the platelet count was significantly increased in mice treated with TSM and JPT compared to the vehicle control. This result implies that the extract at a dose of 2 g/kg may have an effect on platelet enhancement activity, suggesting that this effect may be beneficial for improving thrombocythemia in blood diseases such as malaria. To ensure the safety of the extracts, the biochemical enzymes and pathology of the liver and kidney were assessed. The liver and kidneys play a dominant role in drug metabolism and elimination after ingestion. The liver is well known as the primary organ for parasite development in the pre-erythrocytic stages, resulting in stiffness of the infected liver cells [52]. Acute kidney injury is a well-known significant organ dysfunction caused by malaria infection, and hematological abnormalities are regarded as a key feature of malaria infection [46, 53]. There were no significant changes in biochemical enzymes and histology of the liver and kidneys. This finding implies that the extracts were not associated with nephrotoxicity or hepatotoxicity caused by herbal medicine. Regarding the results of the toxicity test, aqueous extracts of TSM and JPT showed clear evidence that the extracts were considered safe in mice when administered at 2 g/kg, and these extracts may provide great choices for antimalarial drug candidates because they are safe for major cells that can be damaged by the Plasmodium parasite, such as hepatocytes and RBCs.

5. Conclusions

This study demonstrates that aqueous extracts of TSM and JPT exert potent antimalarial activities against P. berghei and are considered safe for oral administration. Therefore, TSM and JPT should be considered as an alternative treatment for malaria. Further experiments should be conducted to test the antimalarial activity in nonhuman primates and in clinical trials.

Data Availability

The data associated with this study are included within the published article. Additional files are available from corresponding authors upon request.

Ethical Approval

The animal study protocol was approved by the Institutional Review Board (or Ethics Committee) of Walailak University, National Research Council of Thailand (NRCT) (protocol number: WU-ACUC-65049).

Conflicts of Interest

The authors declare that there are no conflicts of interest.

Authors’ Contributions

A.P., P.C., and C.P. designed the research studies. A.P., P.C., W.P., A.K., and C.P. carried out the experiments. A.P., P.C., and C.P. analyzed data. A.P., P.C., and C.P. reviewed statistical analysis. A.P., P.C., and C.P. drafted the original manuscript. P.C., A.W.S, and C.P. reviewed and edited the manuscript. All authors read and approved the final version of the manuscript.

Acknowledgments

This work was supported by the Walailak University Ph.D. Scholarships for High-Potential Candidates to Enroll in Doctoral Programs (contract no. HP004/2021) and the Walailak University Graduate Research Fund (contract No. CGS-RF-2022/06).

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