# Toxicological Evaluation and Protective Effects of Ethanolic Leaf Extract ofCassia spectabilis DC on Liver and Kidney Function of Plasmodium berghei-Infected Mice

Authors: Wiwied Ekasari; Anisah Mahardiani; Nindya T. Putri; Tutik S. Wahyuni; Heny Arwati
Journal: Veterinary Medicine International
 (2022)

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## Abstract

Currently, the presence of antimalarial drug resistance has become a major obstacle in the treatment of malaria. To overcome the problem, a series of studies are needed to find new antimalarial drugs from plants. Previously, 90% ethanolic extract ofCassia spectabilis DC (EECS) leaves have been reported to have antimalarial activity in vitro against Plasmodium falciparum and in vivo against Plasmodium berghei ANKA. The research is conducted to find out the toxicity and protective effects of EECS on the liver and kidneys of mice infected with P. berghei ANKA. The acute and subacute toxicity tests were carried out on healthy mice that were given EECS at a dose of 150 mg/kg BW. An antimalarial activity test was carried out at doses of 150 and 200 mg/kg BW in P. berghei-infected mice. Regarding hepatomegaly, further plasma levels of hepatic enzyme were analyzed, as well as histopathological observation of the liver to determine the effect of the extract on liver. The kidney was observed histopathologically as well. The acute toxicity test of EECS showed that there was no mouse died at the highest dose, indicating safe for the mice. The subacute toxicity based on the histology data showed no significant difference in the liver and kidney of mice between the tested group and the healthy group. The histological and enzymatic effect of EECS in mice infected with P. berghei showed the histological and enzymatic effect that improved liver function and the histopathological effect on kidneys with the highest activity at a dose of 200 mg/kg BW compared with the negative control. The results showed the EECS was not toxic in mice and repaired the liver and kidney functions of P. berghei ANKA-infected mice, indicating a good candidate for antimalarial drug development.


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## Body

## 1. Introduction

Until this day, the problem ofPlasmodium 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].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 compound of C. spectabilis DC have been tested in vitro for their antimalarial activities. The 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 in relation to overcoming malarial drug resistance has been continued by examining the safety and effects of improvingC. 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 subacute toxicity tests [17–20]. The liver has an important role in regulating physiological processes. This 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 injected by malarial parasite-infected femaleAnopheles mosquito bite. During blood feeding, infected female Anopheles injects the sporozoite stage of parasite [25, 26]. After about an hour traveling in 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. The 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 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 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. The 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]. The cases of AKI in the complication of malaria are known to contribute to a high mortality rate, which is about 75% of cases. The 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].The toxicity of 90% ethanolic extract ofC. 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

TheC. 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). The specimen was deposited as the herbarium in the Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Universitas Airlangga. The leaves were rinsed thoroughly with tap water to remove extraneous contaminants, dried at 45°C, and ground into powder with a grinder. The 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. The extraction solvent was separated, filtered through filter paper, and evaporated under reduced pressure by rotary evaporation. The 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. The animals were housed under standard conditions and fed with a stock diet and water ad libitum. The 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. The animals were divided into three groups, each group containing five mice. The 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]. The animals were observed for 28 days for any sign of toxicity. At the end of 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 ofPlasmodium 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

This experiment was a suppressive test based on Peters [44]. This suppressive test was done prior to the evaluation of the EECS on the plasma level of SGOT and SGPT. The animals were infected with P. berghei ANKA and divided into five groups of A, B, C, D, and E. The 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. The histopathological observation was performed to find out the toxicological effect of EECS on the degenerative and necrotic cells of the tissues. The damages were graded based on Gibson-Corley et al. [45].


### 2.8. Statistical Analysis

The 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 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.


## 2.1. Plant Material and Preparation of EECS

TheC. 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). The specimen was deposited as the herbarium in the Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Universitas Airlangga. The leaves were rinsed thoroughly with tap water to remove extraneous contaminants, dried at 45°C, and ground into powder with a grinder. The 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. The extraction solvent was separated, filtered through filter paper, and evaporated under reduced pressure by rotary evaporation. The 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. The animals were housed under standard conditions and fed with a stock diet and water ad libitum. The 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. The animals were divided into three groups, each group containing five mice. The 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]. The animals were observed for 28 days for any sign of toxicity. At the end of 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 ofPlasmodium 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

This experiment was a suppressive test based on Peters [44]. This suppressive test was done prior to the evaluation of the EECS on the plasma level of SGOT and SGPT. The animals were infected with P. berghei ANKA and divided into five groups of A, B, C, D, and E. The 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. The histopathological observation was performed to find out the toxicological effect of EECS on the degenerative and necrotic cells of the tissues. The damages were graded based on Gibson-Corley et al. [45].


## 2.8. Statistical Analysis

The 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 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.


## 3. Results

### 3.1. Acute Toxicity Test

No mice died were observed within 24 hours postoral administration of the extract. The behavioral observation of the mice mediated no toxicity sign effects such as paw licking, hair erection, reduction in feeding activity, and increasing respiratory rate [46, 47].Based on the number of animals’ death from the three doses of extract (see Table1), yielding r values were 0, 0, and 0, assuming there was no animal died at the highest dose (5,000 mg/kg BW).Table 1
Number of animals’ deaths in the acute toxicity test of EECS on BALB/c mice in 24 hours.
Doses (mg/kg BW)Number of deaths (r)1,25002,50005,0000


### 3.2. Subacute Toxicity Test

The gross examination of the liver showed hepatomegaly, but there was no enlargement of the kidneys. The size and cell morphology of liver and kidney in both the treated and control groups were similar among the groups (see Figure1). The results of histopathological changes in the liver and kidney tissues are summarized in Table 2. Statistically, there was no significant difference (p>0.05) and histopathological changes were observed in the liver and kidney tissues of the test groups when compared to the control groups.Figure 1
Histological section of the liver (a–d) and kidney (e–h) of mice (section stained with H&E, ×400). Green arrow: degenerative cell, yellow arrow: necrotic cell. (a and e) Single dose. (b and f) Five times of dose. (c and g) Ten times of dose. (d and h) Normal control groups.
(a)(b)(c)(d)(e)(f)(g)(h)Table 2
Lesion scores of liver and kidney of BALB/c mice in subacute oral toxicity of EECS.
OrganMean score of lesionsGroupsAsymptotic significance (p<0.05)EECS1 × 150 mg/kgEECS5 × 150 mg/kgEECS10 × 150 mg/kgNormal controlLiverNecrosis2.34 ± 0.10a2.29 ± 0.30a2.03 ± 0.92a2.37 ± 0.34a0.838Degeneration2.20 ± 0.16a2.43 ± 0.21a,b2.77 ± 0.60b,c2.26 ± 0.25a0.029∗Mean score2.27 ± 0.132.36 ± 0.252.40 ± 0.762.31 ± 0.290.433KidneyNecrosis1.94 ± 0.32a1.09 ± 0.60b1.54 ± 0.63a,b1.60 ± 0.48a,b0.060Degeneration1.14 ± 0.49a1.74 ± 0.19b1.93 ± 0.71a,b1.86 ± 0.46b,c0.065Mean score1.16 ± 0.401.41 ± 0.391.73 ± 0.671.73 ± 0.470.062Data are expressed as mean ± standard deviation (n = 7). The superscript (∗) denotes that the values were significantly different from control (p<0.05; Kruskal–Wallis test for global comparison of organ lesions among groups). The superscripts (a, b, c) indicate that the values in the same row with different superscript letters were significantly different (p<0.05; Mann–Whitney test). EECS: 90% ethanolic leaf extract of Cassia spectabilis DC.


### 3.3. Effect of EECS on SGOT and SGPT

The effect of EECS on biochemical parameters showed that the EECS intake induced a significant increase in plasma levels of SGOT at the dose of 150 mg/kg BW and was out of the normal range. The normal range of SGOT was 59–247 U/L and SGPT in mouse was 28–132 U/L [48], while the plasma level of SGPT was still in the normal range. At a dose of 200 mg/kg BW, the plasma levels of either SGOT or SGPT were in the normal range. There was a very sharp increase in the plasma levels of SGOT and SGPT in the negative control group, indicating liver damage (see Table 3).Table 3
Differences in plasma level of SGOT and SGPT ofP. berghei ANKA-infected mice treated with EECS compared with the control groups.
GroupsParasitemia (%)Suppression (%)SGOT (U/L)SGPT (U/L)D0D4Negative control1.24 ± 0.6914.42 ± 7.86—658.75 ± 99.52167.75 ± 32.37EECS 150 mg/kg2.13 ± 1.198.77 ± 4.4251.85384.75 ± 73.9875.75 ± 35.52EECS 200 mg/kg0.98 ± 0.649.42 ± 3.2758.93216.00 ± 48.8042.00 ± 4.50Chloroquine2.30 ± 1.440.14 ± 0.28100.00175.50 ± 45.5941.25 ± 5.60Healthy control———162.50 ± 59.1897.25 ± 44.70Data are expressed as mean ± standard deviation (n = 4). No significant difference between the groups (p>0.05; one-way ANOVA followed by Tukey’s multiple comparison test). D0: day before the treatment; D4: fourth day after treatment; SGOT: plasma glutamic oxaloacetic transaminase; SGPT: plasma glutamic pyruvic transaminase; EECS: 90% ethanolic leaf extract of Cassia spectabilis DC.The observations on the morphology of liver and kidneys of mice showed the enlargement of the liver (hepatomegaly) and showed differences in color, size, and texture in the liver. The morphology of kidneys showed no enlargement and was similar either in color, size, or texture among groups. Due to these reasons, the biochemistry examination of the kidney was not performed. Physically, there was an improvement in the liver of mice treated with 150 mg/kg BW of extract when compared to the control, although it was not as good as in mice treated with 200 mg/kg BW of extract. In addition, the appearance of the liver treated with 200 mg/kg BW extract was almost as same as the condition of experimental mouse liver treated with chloroquine and the healthy control group, where brownish-red and smooth texture of surface has appeared.The results of histopathological changes in the liver and kidney tissues are summarized in Table4. There were no significant histopathological changes observed in the liver and kidney tissues of the mice when compared to the control. The histopathological changes in liver and kidney are shown in Figures 2 and 3.Table 4
Lesion scores of liver and kidney ofP. berghei ANKA-infected mice treated with EECS.
OrganMean scores of lesionsGroupsNegative controlEECS150 mg/kgEECS200 mg/kgChloroquineHealthy controlLiverNecrosis1.30 ± 0.26a0.75 ± 0.34a,b0.60 ± 0.16b0.65 ± 0.10b0.50 ± 0.12bDegeneration2.45 ± 0.38a1.45 ± 0.19b1.10 ± 0.35b1.20 ± 0.28b,c0.65 ± 0.10cMean score1.87 ± 0.321.10 ± 0.260.85 ± 0.250.92 ± 0.190.57 ± 0.11KidneyNecrosis1.75 ± 0.50a1.55 ± 0.41a,b1.65 ± 0.19a1.40 ± 0.28b1.20 ± 0.36cDegeneration1.30 ± 0.74a,b1.20 ± 0.52a,b1.20 ± 0.16a,b1.10 ± 0.26a1.45 ± 0.68bMean score1.52 ± 0.621.37 ± 0.461.42 ± 0.171.25 ± 0.271.32 ± 0.52Data are expressed as mean ± standard deviation (n = 7). No significant difference between the groups (p>0.05; Kruskal–Wallis test). The superscripts (a, b, c) indicate that the values in the same row with different superscript letters were significantly different (p<0.05; Mann–Whitney test). EECS: 90% ethanolic leaf extract of Cassia spectabilis DC.Figure 2
Pathological lesions around the central vein of experimental mouse liver (section stained with H&E, ×400). (a) Normal hepatocyte cells around the central vein. (b) Degenerative nuclear hepatocyte. (c) Inflammatory cell infiltration. (d) Necrotic hepatocyte.
(a)(b)(c)(d)Figure 3
Histopathological appearances of kidney (section stained with H&E, ×400). Green arrow: degenerative cell, yellow arrow: necrotic cell, blue arrow: normal cell. (a) Negative control group. (b) 150 mg/kg BW of extract. (c) 200 mg/kg BW of extract. (d) Chloroquine. (e) Healthy control group.
(a)(b)(c)(d)(e)


## 3.1. Acute Toxicity Test

No mice died were observed within 24 hours postoral administration of the extract. The behavioral observation of the mice mediated no toxicity sign effects such as paw licking, hair erection, reduction in feeding activity, and increasing respiratory rate [46, 47].Based on the number of animals’ death from the three doses of extract (see Table1), yielding r values were 0, 0, and 0, assuming there was no animal died at the highest dose (5,000 mg/kg BW).Table 1
Number of animals’ deaths in the acute toxicity test of EECS on BALB/c mice in 24 hours.
Doses (mg/kg BW)Number of deaths (r)1,25002,50005,0000


## 3.2. Subacute Toxicity Test

The gross examination of the liver showed hepatomegaly, but there was no enlargement of the kidneys. The size and cell morphology of liver and kidney in both the treated and control groups were similar among the groups (see Figure1). The results of histopathological changes in the liver and kidney tissues are summarized in Table 2. Statistically, there was no significant difference (p>0.05) and histopathological changes were observed in the liver and kidney tissues of the test groups when compared to the control groups.Figure 1
Histological section of the liver (a–d) and kidney (e–h) of mice (section stained with H&E, ×400). Green arrow: degenerative cell, yellow arrow: necrotic cell. (a and e) Single dose. (b and f) Five times of dose. (c and g) Ten times of dose. (d and h) Normal control groups.
(a)(b)(c)(d)(e)(f)(g)(h)Table 2
Lesion scores of liver and kidney of BALB/c mice in subacute oral toxicity of EECS.
OrganMean score of lesionsGroupsAsymptotic significance (p<0.05)EECS1 × 150 mg/kgEECS5 × 150 mg/kgEECS10 × 150 mg/kgNormal controlLiverNecrosis2.34 ± 0.10a2.29 ± 0.30a2.03 ± 0.92a2.37 ± 0.34a0.838Degeneration2.20 ± 0.16a2.43 ± 0.21a,b2.77 ± 0.60b,c2.26 ± 0.25a0.029∗Mean score2.27 ± 0.132.36 ± 0.252.40 ± 0.762.31 ± 0.290.433KidneyNecrosis1.94 ± 0.32a1.09 ± 0.60b1.54 ± 0.63a,b1.60 ± 0.48a,b0.060Degeneration1.14 ± 0.49a1.74 ± 0.19b1.93 ± 0.71a,b1.86 ± 0.46b,c0.065Mean score1.16 ± 0.401.41 ± 0.391.73 ± 0.671.73 ± 0.470.062Data are expressed as mean ± standard deviation (n = 7). The superscript (∗) denotes that the values were significantly different from control (p<0.05; Kruskal–Wallis test for global comparison of organ lesions among groups). The superscripts (a, b, c) indicate that the values in the same row with different superscript letters were significantly different (p<0.05; Mann–Whitney test). EECS: 90% ethanolic leaf extract of Cassia spectabilis DC.


## 3.3. Effect of EECS on SGOT and SGPT

The effect of EECS on biochemical parameters showed that the EECS intake induced a significant increase in plasma levels of SGOT at the dose of 150 mg/kg BW and was out of the normal range. The normal range of SGOT was 59–247 U/L and SGPT in mouse was 28–132 U/L [48], while the plasma level of SGPT was still in the normal range. At a dose of 200 mg/kg BW, the plasma levels of either SGOT or SGPT were in the normal range. There was a very sharp increase in the plasma levels of SGOT and SGPT in the negative control group, indicating liver damage (see Table 3).Table 3
Differences in plasma level of SGOT and SGPT ofP. berghei ANKA-infected mice treated with EECS compared with the control groups.
GroupsParasitemia (%)Suppression (%)SGOT (U/L)SGPT (U/L)D0D4Negative control1.24 ± 0.6914.42 ± 7.86—658.75 ± 99.52167.75 ± 32.37EECS 150 mg/kg2.13 ± 1.198.77 ± 4.4251.85384.75 ± 73.9875.75 ± 35.52EECS 200 mg/kg0.98 ± 0.649.42 ± 3.2758.93216.00 ± 48.8042.00 ± 4.50Chloroquine2.30 ± 1.440.14 ± 0.28100.00175.50 ± 45.5941.25 ± 5.60Healthy control———162.50 ± 59.1897.25 ± 44.70Data are expressed as mean ± standard deviation (n = 4). No significant difference between the groups (p>0.05; one-way ANOVA followed by Tukey’s multiple comparison test). D0: day before the treatment; D4: fourth day after treatment; SGOT: plasma glutamic oxaloacetic transaminase; SGPT: plasma glutamic pyruvic transaminase; EECS: 90% ethanolic leaf extract of Cassia spectabilis DC.The observations on the morphology of liver and kidneys of mice showed the enlargement of the liver (hepatomegaly) and showed differences in color, size, and texture in the liver. The morphology of kidneys showed no enlargement and was similar either in color, size, or texture among groups. Due to these reasons, the biochemistry examination of the kidney was not performed. Physically, there was an improvement in the liver of mice treated with 150 mg/kg BW of extract when compared to the control, although it was not as good as in mice treated with 200 mg/kg BW of extract. In addition, the appearance of the liver treated with 200 mg/kg BW extract was almost as same as the condition of experimental mouse liver treated with chloroquine and the healthy control group, where brownish-red and smooth texture of surface has appeared.The results of histopathological changes in the liver and kidney tissues are summarized in Table4. There were no significant histopathological changes observed in the liver and kidney tissues of the mice when compared to the control. The histopathological changes in liver and kidney are shown in Figures 2 and 3.Table 4
Lesion scores of liver and kidney ofP. berghei ANKA-infected mice treated with EECS.
OrganMean scores of lesionsGroupsNegative controlEECS150 mg/kgEECS200 mg/kgChloroquineHealthy controlLiverNecrosis1.30 ± 0.26a0.75 ± 0.34a,b0.60 ± 0.16b0.65 ± 0.10b0.50 ± 0.12bDegeneration2.45 ± 0.38a1.45 ± 0.19b1.10 ± 0.35b1.20 ± 0.28b,c0.65 ± 0.10cMean score1.87 ± 0.321.10 ± 0.260.85 ± 0.250.92 ± 0.190.57 ± 0.11KidneyNecrosis1.75 ± 0.50a1.55 ± 0.41a,b1.65 ± 0.19a1.40 ± 0.28b1.20 ± 0.36cDegeneration1.30 ± 0.74a,b1.20 ± 0.52a,b1.20 ± 0.16a,b1.10 ± 0.26a1.45 ± 0.68bMean score1.52 ± 0.621.37 ± 0.461.42 ± 0.171.25 ± 0.271.32 ± 0.52Data are expressed as mean ± standard deviation (n = 7). No significant difference between the groups (p>0.05; Kruskal–Wallis test). The superscripts (a, b, c) indicate that the values in the same row with different superscript letters were significantly different (p<0.05; Mann–Whitney test). EECS: 90% ethanolic leaf extract of Cassia spectabilis DC.Figure 2
Pathological lesions around the central vein of experimental mouse liver (section stained with H&E, ×400). (a) Normal hepatocyte cells around the central vein. (b) Degenerative nuclear hepatocyte. (c) Inflammatory cell infiltration. (d) Necrotic hepatocyte.
(a)(b)(c)(d)Figure 3
Histopathological appearances of kidney (section stained with H&E, ×400). Green arrow: degenerative cell, yellow arrow: necrotic cell, blue arrow: normal cell. (a) Negative control group. (b) 150 mg/kg BW of extract. (c) 200 mg/kg BW of extract. (d) Chloroquine. (e) Healthy control group.
(a)(b)(c)(d)(e)


## 4. Discussion

Currently, the optimal use of available drugs and the development of a new approach to antimalarial chemotherapy are still needed. With serious consideration for safety issues, researches on phytochemistry, pharmacology, and biochemistry in medicinal plants traditionally used as antimalarial are urgently needed [49]. The plants that show good antimalarial activities that have been recorded until today come from 1,277 plants from 160 plant families [50, 51].The acute toxicity test of the EECS showed that the EECS is not toxic to the BALB/c mice up to 5,000 mg/kg BW of dose, since there was no mouse died due to this highest dose of EECS. Following this test, the subacute toxicity test showed that there was no abnormality in mouse mobile activities and in water and food consumption.In addition, the subacute toxicity test was based on the liver and kidney histopathology results (see Figure1). The data obtained based on microscopic observations were analyzed statistically in the form using the Kruskal–Wallis statistical test to determine the differences in all population groups. Based on the results of the Kruskal–Wallis test, which evaluated liver necrosis damage, it was known that there were no significant differences between the groups. This could be seen from the value of p>0.05. Then, the Mann–Whitney statistical test was also performed to determine the location of differences between the groups. Based on the results of the Mann–Whitney test on the level of liver necrosis damage, it was known that there were no significant differences between the values of p>0.05. Likewise in liver degeneration damage, it could be seen that the four treatment groups showed no significant differences because there were no significant differences (not toxic to the liver).The suppressive test of the EECS in this current research was performed to find out the effect of EECS on the function of the liver based on the plasma level of SGOT and SGPT. The doses of EECS used in this test were based on the previous suppressive test [9]. Furthermore, the plasma level of SGOT and SGPT in EECS-treated infected mice was maintained in the normal range compared with the controls and based on the reference of normal BALB/c mice [52], especially at the dose of 200 mg/kg BW. The statistical analysis showed no significant difference (p<0.05) in plasma level of SGOT and SGPT when compared with the healthy control group. These results indicated that the EECS was effective in reducing plasma levels of SGOT and SGPT to the normal levels. The excessive hepatic enzymes were released by the injured hepatocytes due to malarial infection [53]. In this study, the necrosis and degraded cells in the liver were repaired by the administration of EECS followed by the reduction in plasma level of SGOT and SGPT. These results indicated a very good liver functional improvement due to the EECS administration.Apart from blood analysis, the histopathological analysis provides supportive evidence for biochemical and hematological assessment [41]. Histopathological examination is the standard for evaluating treatment for pathological changes in tissues and organs [54]. Histopathological assessment of the effect of EECS on the liver samples treated in this study showed both necrosis and degeneration of hepatocytes and kidney cells (see Table 4). It is likely that these changes may reflect beneficial cellular adaptations of the extract to host tissues.Based on the results, all doses showed significant differences in kidney lesion scores when compared to the controls (see Table4). There was a significant difference between chloroquine and the healthy group in necrotic and degenerative cells of kidney. This significant difference shows that chloroquine provides a therapeutic effect on kidney repair in P. berghei-infected mice.In the absence of significant differences between the treated group and chloroquine (see Table4), it can be concluded that the therapeutic effect is not too different from being used as an alternative to antimalaria, which can reduce kidney damage. Furthermore, a dose of 200 mg/kg BW showed better liver repair activity than a dose of 150 mg/kg BW because there was no significant lesion score in the dose of 150 mg/kg BW compared with the negative control.The EECS has been proved to be not toxic in mice and was able to repair the liver and kidney functions in mice infected withP. berghei ANKA. The ability of EECS in heme detoxification caused the parasite clearance [11] followed by further repairing of tissue in the main organ affected by the malarial infection such as liver and kidney as well as the plasma level of hepatic enzyme.


## 5. Conclusion

The EECS was not toxic in mice and repaired the liver and kidney functions ofP. berghei ANKA-infected mice similarly to chloroquine, indicating a good candidate for antimalarial drug development.



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*Source: 6770828-2022-02-15.xml*