Toxicological Evaluation And Protective Effects Of Ethanolic Leaf Extract Of Cassia Spectabilis DC On Liver And Kidney Function Of Plasmodium Berghei-Infected Mice
Oct 30, 2023
1. Introduction
To this day, the problem of Plasmodium parasitic resistance towards existing antimalarial drugs is still a major problem for the eradication of malaria [1–3]. therefore, research on the discovery of new sources of antimalarial drugs, one of which is from medicinal plants, continues to be done [4]. In vivo and in vitro models have long been used in antimalarial testing. In the latter half of the 20th century, Plasmodium berghei or Plasmodium yoelii was used to infect rodents. Meanwhile, P. berghei became the most commonly used species for study at the liver stage, particularly the formation of hypnozoite to investigate malarial recurrence [5–7].
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One of the traditional plants in Indonesia, Cassia spectabilis DC of the Caesalpiniaceae family, has been proved experimentally in vitro against P. falciparum and in treating malaria in vivo against P. berghei [8, 9], indicating that C. spectabilis DC plant is very potential to be further developed as a candidate for antimalarial drugs. Previous works reported that an in vivo test on 90% ethanolic extract of C. spectabilis DC leaf against P. berghei ANKA in BALB/c mice showed that an ED50 value was 131.5 mg/kg BW [9], and it is categorized as very good antimalarial activity [10].
Furthermore, the extract, fractions, subfractions, and isolated compounds of C. spectabilis DC have been tested in vitro for their antimalarial activities. *e active compound of this plant has been successfully identified as a compound that is identical to (–)-7-hydroxycassine, and its in vitro antimalarial activity test showed a very low IC50 of 0.016 μg/mL [11], which is classified as a very strong antimalarial activity [12].
The research on this plant about overcoming malarial drug resistance has been continued by examining the safety and effects of improving C. spectabilis DC leaf extract on the liver and kidney functions in P. berghei ANKA-infected mice. Research studies on the effect of medicinal plants that have antimalarial activity on the liver and kidney functions of mice infected with parasites have been widely carried out [13–16], as well as acute and sub-acute toxicity tests [17–20]. The liver has an important role in regulating physiological processes. is an organ is involved in several vital functions such as metabolism, secretion, and storage. In addition, the liver has an important role in the detoxification and excretion of endogenous and exogenous compounds [21–24].
Malarial infection begins when sporozoites are infected by malarial parasite-infected female Anopheles mosquito bite. During blood feeding, infected female Anopheles injects the sporozoite stage of the parasite [25, 26]. After about an hour of traveling in the human body, sporozoites then enter the liver, attack hepatocytes, and start the asexual cycle of exoerythrocytic schizogony. Inside the liver cells, the parasites multiply asexually, until they reach mature schizont, and finally, a large number of merozoites produce and enter the bloodstream after infecting hepatocyte burst. *e infected hepatocytes causing liver damage due to the rupture of infected hepatocytes and merozoites enter the bloodstream and start the erythrocytic cycle in the red blood cells [27, 28]. Damage that occurs in the liver cells can increase the enzymes that work on liver function, especially transaminase enzyme, and morphological changes in the appearance of the liver [29, 30], such as hepatomegaly. Hepatosplenomegaly is a common feature of malarial infection in humans [31] and mice [32] caused by chronic exposure to malarial parasites. However, no enlargement of the kidney in malaria infection.
The dysfunction of the liver can be detected by hepatocellular transamination of plasma glutamic oxaloacetic transaminase (SGOT) and plasma glutamic pyruvic transaminase (SGPT) released in plasma, or by histological examination of the tissue [33]. The most common damage is the activation of apoptotic cell death or hepatocyte necrosis [34–36]. Clinically significant kidney involvement is associated with the infection by P. falciparum and P. malariae. *e infection of P. falciparum produces acute manifestations, ranging from asymptomatic, up to urinary disorders, and mild electrolyte disturbances for acute renal failure (ARF) or acute kidney injury (AKI) that require dialysis support [27, 37]. *e cases of AKI in the complication of malaria are known to contribute to a high mortality rate, which is about 75% of cases. *e histological study suggests the presence of glomerulonephritis, acute tubular necrosis, and interstitial nephritis as the key hemodynamic factor in malaria-associated AKI [38]. Generally, the degree of kidney dysfunction can be detected by the presence of adequate amounts of protein in the urine and an increase in the plasma urea, creatinine, and plasma electrolyte levels [39].
*e toxicity of 90% ethanolic extract of C. spectabilis DC (EECS) leaves in BALB/c mice, followed by the enzyme examinations, may affect histopathology and liver and kidney functions post-EECS administration.
2. Materials and Methods
2.1. Plant Material and Preparation of EECS. *e C. spectabilis
DC leaves were purchased and determined in LIPI (Indonesia Research Centre), Botanical Garden, Purwodadi, East Java, Indonesia (B-160/IPH.06/KS.02/III/ 2019). *e specimen was deposited as the herbarium in the Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Universitas Airlangga. *e leaves were rinsed thoroughly with tap water to remove extraneous contaminants, dried at 45°C, and ground into powder with a grinder. *e extraction was carried out by macerating the powder plant materials (500 g) in a flask containing 2,500 mL of 90% ethanol (at 25–30°C) for 3 × 24 hours. *e extraction solvent was separated, filtered through filter paper, and evaporated under reduced pressure by rotary evaporation. *e ethanolic extract yield of 1000 g dried weight of C. spectabilis DC leaf powder was 10.20% (w/w) and was used in this experiment.
2.2. Animals.
Male BALB/c mice aged 6–8 weeks, weight of 25–30 g, were used in the study. All mice were obtained from Farma Veterinary Center, Directorate General of Livestock and Animal Health Ministry of Agriculture, Surabaya, East Java, Indonesia. *e animals were housed under standard conditions and fed with a stock diet and water ad libitum. *e approval of the study protocol was obtained from the Ethics Committee for Animal Research, Universitas Airlangga, Indonesia, Number 2.KE.181.10.2018.
2.3. Acute Toxicity Test.
EECS was weighted and resuspended with 0.5% sodium carboxymethyl cellulose (Na CMC) to obtain the desired doses. BALB/c mice were fasted for 24 hours before being fed with the extract. *e animals were divided into three groups, each group containing five mice. *e doses for each group were 1,250; 2,500; and 5,000 mg/kg BW, respectively. The general behavior of each mouse was observed continuously for one hour after each dose, intermittently every four hours, and thereafter over a period of 24 hours [40–42].
2.4. Subacute Toxicity Test.
In the subacute toxicity test, mice were grouped into three groups and each group was treated with EECS at once, five times, and ten times of 150 mg/kg BW daily oral dose for 28 days; the normal control group was only given food and drink ad libitum [43]. *e animals were observed for 28 days for any sign of toxicity. At the end of the observational period, all animals were sacrificed under ether anesthesia and vital organs such as livers and kidneys were removed from all animals for gross and histopathological examinations.
2.5. Rodent’s Parasite.
Chloroquine sensitivity of Plasmodium berghei ANKA was obtained from the Institute of Biomolecular Eijkman, Jakarta, Indonesia, and maintained by serial passaging that was used in this study.
2.6. Effect of EECS on the Plasma Level of SGOT and SGPT.
*is experiment was a suppressive test based on Peters [44]. *is suppressive test was done prior to the evaluation of the EECS on the plasma level of SGOT and SGPT. *e animals were infected with P. berghei ANKA and divided into five groups of A, B, C, D, and E. *e animals were treated shortly after infection on day 0 (D0) and continued daily for three days (D1–D3). Group A as a negative control received 0.5% Na CMC orally. Groups B and C as the treated groups were given EECS at a single oral dose of 150 and 200 mg/kg BW, respectively. Group D was a positive control given chloroquine at a single oral dose of 100 mg/kg BW. Moreover, group E as a healthy control was not infected with parasites and was only given food and drink ad libitum. On the fourth day (D4), the blood smears of each mouse were prepared and examined microscopically. Further, plasma was collected prior to the measurement of SGOT and SGPT levels.
2.7. Effect of EECS on Histopathology of Liver and Kidney.
At the end of the observational period, all animals were sacrificed under ether anesthesia prior to the liver and kidney removal. After weighing the organs, the tissues were then fixed in 10% formaldehyde solution and further processed for hematoxylin-eosin (H&E) staining. *e histopathological observation was performed to find out the toxicological effect of EECS on the degenerative and necrotic cells of the tissues. *e damages were graded based on Gibson-Corley et al. [45].
2.8. Statistical Analysis.
*e data were represented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used to compare the normally distributed data among the treatments, followed by a post hoc multiple comparison test when a different significance was obtained. When the data were not distributed normally, the Kruskal–Wallis and the Mann–Whitney U-test were used to assess the differences among treatments.
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