INTRODUCTION:

Indonesia is a tropical country with the second-largest biodiversity in the world. The area of tropical forests in Indonesia is around 143 million hectares, which is a habitat for 80% of the world's medicinal plants¹. In Indonesia, there are about 30,000 plant species, of which 7,000 are thought to have medicinal properties². According to The World Health Organization (WHO), about 80% of people from developing countries use traditional medicine to treat a disease³. The 2018 Basic Health Research reported that as many as 49% of Indonesia's population used herbal plants as traditional medicine⁴.

The use of traditional medicine has increased after the COVID-19 pandemic because it is widely effective in increasing endurance⁵.

One of the plants that can increase the immune system is Moringa (Moringa oleifera Lam). Its leaves have positive compounds, including vitamins, phenolic acids, flavonoids, isothiocyanates, tannins, and saponins, which act as potent antioxidants⁶. Based on the results of research conducted by Dillasamola et al.⁷, Moringa leaf extract serves as an immunostimulant in mice (dose of 10, 30, and 100mg/kg BW) because it can increase macrophage activity. It is reported that administration of Moringa leaf extract increases the number of CD4+ T cells and the relative number of CD8+ T cells⁸. In addition, several studies have established that the ethanol extract of Moringa leaves can be used as an antimicrobial⁴, anticancer⁹, analgesic¹⁰, antidiabetic¹¹, antioxidant¹², antihypertensive¹³, and antithyroid agent¹⁴.

Many people have used Moringa leaves as a traditional medicine consumed with boiled water and Moringa leaves soaked in water¹⁵. Even though it comes from nature, the safety of using Moringa leaves cannot be guaranteed because all compounds entering the body will have distinct influences¹⁶. There are several reports of unwanted effects from using Moringa as a therapy, including the occurrence of anaphylaxis¹⁷, acute pancreatitis¹⁸, and hyperpigmentation¹⁹.

Due to the high cost of modern medicines and their side effects, herbal medicine from plants has become a mainstay of medical therapy²⁰. Plant-based drugs are readily available, less expensive, safe, and efficient and rarely have any side effects²¹. According to the Indonesian Ministry of Health²², traditional medicines are safe, efficacious, and of good quality. Tests carried out to determine the safety of conventional drugs include toxicity tests, one of which is the subacute toxicity test. The subacute toxicity test aims to determine whether there is a toxic effect after repeated exposure to the test preparation for a certain period²³. One of the parameters to determine the presence of harmful effects is observing the histopathology of organs in the kidney²⁴. Kidneys are organs that have various vital roles in the body²⁵. Their primary role is excretory function through different regulatory mechanisms like prostaglandin synthesis, which helps maintain the glomerular filtration rate (GFR)26 and maintain the homeostatic balance²⁷.

Research results from Osman et al.²⁸ showed that the acute toxicity test of the ethanol extract of Moringa leaves in mice had an LD50 value of 6,616.67mg/kg BW. Researchers tested the safety of Moringa leaf ethanol extract (Moringa oleifera Lam.) as immunostimulant doses for rats, in volumes of 7, 21, and 70mg/kg BW, by increasing the highest dose to 140 mg/kg BW to examine if there is any toxic effect upon increasing the dose. The parameters observed were kidney histology, which assessed the histology score and determined the ratio of kidney organs as a supporting parameter. The results of this study are expected to provide an overview of the safety of Moringa leaf extract (Moringa oleifera Lam.) on repeated use or routine use. It can also be used as basic information for further research.

MATERIALS AND METHOD:

Materials:

Moringa oleifera Lam. leaves were authenticated by Dr Nurainas at Herbarium Universitas Andalas (ANDA), Department of Biology, Faculty of Math and Natural Sciences, Universitas Andalas, through letter number 533/K-ID/ANDA/XI/2022. The study was conducted by using Aquadest (Andeska laboratory), 70% distilled ethanol (Andalas laboratory), Haematoksilin and Eosin colouring (Pupick Med), NaCl physiological (PT Widatra Bhakti), Na CMC 1%, buffer formalin (Leica), alcohol 80% (Brata Med), alcohol 95% (Brata Med), ethanol absolute (Brata Med), xylol 80% (Merck), xylol 95% (Merck), xylol 100% (Merck), paraffin (Merck), balsam kanada (DPX mountant).

Preparation of Extraction:

A total of 5 kg of fresh Moringa leaf samples were sorted and cleaned from impurities and then air-dried to obtain dry samples, that could be crushed easily. Furthermore, dry leaves are crushed using a grinder without causing damage or losing the required chemical content until a fine powder of moringa leaves is obtained²⁹. The powder was macerated using 70% ethanol solvent (1:10) for 24 hours (stirring occasionally during the first 6 hours, then let stand for 18 hours). Maceration was carried out three times using half the solvent from the first maceration for the second and third rounds of maceration. All the macerate is collected to evaporate the solvent using a rotary evaporator until a thick extract is obtained.

Characterisation of Moringa oleifera Lam Extract:

Organoleptic:

The extracts obtained were tested organoleptically using observations of the five senses, which described the extract's smell, shape, taste, and colour²⁹.

Yield:

The yield is calculated by weighing the Moringa leaf crude drug (simplified) to obtain mass (A), followed by weighing the obtained extract to obtain mass (B)²⁹. The yield calculation can be calculated using the below formula:

Yield = (B/A) x 100 %

Determination of moisture content:

Approximately 10g of extract is weighed in a pre-weighed container. It is dried at 105̊ C for 5 hours and weighed again. Subsequently, it is again dried at 105℃ for 5 hours and reweighed. The process of drying and weighing is continued 1 hour apart until the difference between two consecutive weighings is less than 0.25%²⁹.

(w1-w0) - (w2-w0)

Moisture content = ----------------------------- x 100%

(w1-w0)

Determination of total ash content:

Almost 2 to 3g of the extract is weighed carefully and is put in a crucible that has been incinerated gently incandescent until the charcoal is exhausted, cool, and weighed. If the charcoal cannot be removed, hot water is added, stirred, and filtered using filter paper and the rest of the filter is put back in the same crucible. The filtrate is placed into the crucible, steamed, and ignited until a constant weight is obtained at 800±25ºC. The ash content of the material dried in the air is calculated with the following equation²⁹.

(w2-w0)

Total ash content = ------------------------------- x 100%

(w1-w0)

Determination of acid-insoluble ash content:

The derived ash is boiled with 25mL of dilute hydrochloric acid LP for 5 minutes to determine the total ash content. The acid-insoluble part is collected, filtered through ash-free filter paper, washed with hot water, and incandesced in a crucible until the weight remains at 800±25̊C. The acid-insoluble ash content of the air-dried material is calculated with the following equation²⁹.

(w2-w0)

Acid insoluble ash content = ---------------------- x 100%

(w1-w0)

Phytochemical screening of Moringa oleifera Lam. extract:

Flavonoid examination:

1mL of extract is put in a test tube and then a few drops of concentrated HCl and a little Magnesium powder are added to it. The orange-red to purple-red tint indicates that the extract contains flavonoids¹¹.

Phenolic examination:

1mL of extract is put in a test tube and then FeCl₃ 1% is added to it. The formation of a blue-black colour characterises positive results¹¹.

Saponin examination:

1mL of test extract is put in a tube and shaken vertically for 10 seconds. If a stable foam is formed, it is positive for saponins¹¹.

Steroid and triterpenoid screening:

The extract is dissolved in 3mL of chloroform and then 2mL of anhydrous acetic acid and 2mL of concentrated sulfuric acid are added through the tube wall. If a brownish or violet ring is formed at the restriction of two solvents, it indicates the presence of triterpenoids, while if a bluish-green colour is formed, it indicates the presence of steroids¹¹.

Alkaloid screening:

A few mg of extract is collected and a few drops of Wagner's reagent is added to it. Positive results are characterised by the formation of a brown precipitate¹¹.

Test preparations:

Dose planning:

The ethanol extract of Moringa oleifera Lam. will be administered to male white rats at doses of 7, 21, and 140mg/kg BW. The dose is given based on the dose used in the immunostimulant activity test of Moringa oleifera Lam. leaf ethanol extract, which uses doses of 10, 30, and 100mg/kg BW in male white mice⁷. There is a difference in the highest dose used in order to observe the activity when the dose was increased to 140 mg/kg BW and whether it caused any toxic effects on the liver. The test formulations will be administered orally daily for 7 days, 14 days, and 21 days.

Preparation of 1% Na CMC suspension:

500 mg of Na CMC is weighed and sprinkled on 10mL of hot water in a heated mortar. It is left untouched for 15 minutes and then grounded until homogeneous. Next, aquadest is added until the volume is 50mL.

Preparation of suspension of Moringa leaf ethanol extract (Moringa oleifera Lam.):

1g of extract is suspended in 50mL of 1% Na CMC. Then, the test suspension is diluted according to the concentrations of each designed dose. The volume of the test formulation injection should be adjusted based on the body weight of the test animals.

Administration of Test Preparations:

The animals were divided into four groups as follows:

Group I: Only 1% Na CMC was administered

Group II: Administered extract with a dose of 7mg/kg BW

Group III: Administered extract with a dose of 21mg/kg BW

Group IV: Administered extract with a dose of 140 mg/kg BW

Each group consisted of 9 rats, and the treatment was given for 7, 14, and 21 days respectively. On the 8th, 15th and 22nd days, the animals were sacrificed by dissection, and then their kidney organs were taken, and the organ ratio was calculated. After that, histopathological preparations were made using the paraffin method and stained with haematoxylin and eosin to be observed under a microscope.

Kidney Histology Analysis:

a. The rats were sacrificed and their kidney organs were removed. They were then rinsed with physiological NaCl solution. The organs were transferred into the buffered formalin solution.

b. The kidney organ was cut cross-sectionally into several pieces that represented the whole part of the kidney. Each organ tissue was dehydrated with 80%, 95%, and absolute alcohol solutions for 1 hour.

c. The object was transferred into the absolute alcohol solution: xylol (1:1), xylol 1 and xylol 2, each for 1 hour for clarification.

d. The object was kept in the infiltration solution in an incubator at 56-60° C. Xylol: paraffin, paraffin I, paraffin II and paraffin III (each for half an hour).

e. Embedding (planting) - The object was put in a metal mould or paper box filled with liquid paraffin, heated in an incubator, and then allowed to cool and solidify.

f. Section (slices) - A paraffin block was placed in the holder and then thinly sliced with a microtome knife (5-6 µm).

g. Afiniting (adhesion) - The slide was rubbed with Mayer's albumin and was subjected to some incisions, dripped with water and stretched over the hot plate until the incision expanded.

h. Staining with Erlich's haematoxylin and eosin was carried out in the following way: Xylol I, xylol II (5 minutes each), absolute alcohol I, absolute II, 80%, 95%, and 100% (3 minutes each), Erlich's haematoxylin for 10 minutes and then washed with running water.

i. Furthermore, the preparations were dipped in eosin for 5 minutes and in absolute alcohol III and absolute IV (each for 2 minutes), xylol III and xylol IV for 1 minute, and washed with running water.

j. Mounting (closure) – To save the tissue from drying out, it is dripped with an entellan (adhesive) in the form of Canadian balsam, covered with a cover glass, and then dried.

k. The preparation is labelled to the right of the object and then examined under a microscope after which, a microscopic photo is taken.

Examination of Kidney Histology Preparations:

Examining kidney histology preparations was done microscopically with a magnification of 400. Histological assessment is a semi-quantitative of microscopic changes in the kidney organ, which was carried out by scoring (value) based on the changes that occur. Scoring can be given in Table 1.³⁰

Data Analysis:

Data analysis on kidney histology was carried out descriptively, and the ratio of kidney organs was analysed using a statistical test of two-way ANOVA analysis of variance between dose and duration of administration. Duncan Multiple Range Test (DMRT) yielded significant results. Significance was taken at p<0.05.

Table 1. Kidney histology scoring.

Score	Damage Rate	Description
0	Normal	Normal histology
1	Minimal	<10%, tubular epithelial degeneration without necrosis
2	Mild	<25% tubular epithelial degeneration, necrosis, and accompanying abnormalities
3	Moderate	<50% tubular epithelial degeneration, necrosis, and accompanying abnormalities
4	Severe	<75% tubular epithelial degeneration, necrosis, and accompanying abnormalities
5	Very severe	>75% tubular epithelial degeneration, necrosis, and accompanying abnormalities

Ethical Clearance:

Ethical approval for this study was obtained from the Faculty of Medicine Ethics Committee, Universitas Andalas, through letter number 65/UN.16.2/KEP-FK/2023 dated 14 February 2023.

RESULT AND DISCUSSION:

The process of making the extract was done by maceration, particularly because it can be carried out on many samples, the process is simple and easy, does not require special equipment, and does not go through a heating process to minimise chemical changes of specific compounds or degradation of metabolites so that they are suitable for thermolabile compounds³¹. Moringa leaf simplicia was macerated using 70% ethanol solvent³². This solvent is used because the sample is dry and has a relatively small water content; the 30% water content helps break down the cell walls so that the penetration of ethanol into cells is faster and optimal³³.

Maceration was carried out repeatedly until a clear macerate was obtained, and then the macerate was evaporated with a rotary evaporator until a thick extract was obtained. The solvent evaporation process from the extract aims to obtain a more concentrated extract to increase the compound concentration and facilitate storage.

The organoleptic test obtained is a viscous green-brown extract with a characteristic odour and bitter taste. The bitter taste in the extract is due to several compounds, such as flavonoids and terpenoids³⁴.

From 650 grams of simplicia powder, 116.1 grams of viscous extract was produced, resulting in an extract yield of 17.86%. The yield produced met the standards set in the Indonesian Herbal Pharmacopoeia, which is not less than 9.2%³².

The resultant moisture content of the extract obtained was 9.15%. The water content obtained met the requirements. According to the Indonesian Herbal Pharmacopoeia, drying losses and moisture content of Simplicia and Moringa leaf extract are no more than 10%³². Water content that exceeds this range can cause the growth of microbes, which can reduce the stability of the extract³⁵.

The results were 5.01% and 0.468% in testing the total and acid-insoluble ash content, respectively. This value meets the requirements according to the standards set by the Indonesian Herbal Pharmacopoeia, where the total ash content of the concentrated extract of Moringa leaves is not more than 9%, and the acid-insoluble ash content is not more than 0.9%³². The ash content obtained reflects the toxic minerals contained in the extract³⁵.

The results obtained in testing the total ash content and acid-insoluble ash content were 5.01% and 0.468%, respectively. This value meets the requirements according to the standards set by the Indonesian Herbal Pharmacopoeia, where the total ash content of the concentrated extract of Moringa leaves is not more than 9% and the acid-insoluble ash content is not more than 0.9%³². The ash content obtained reflects the total minerals contained in the extract which are toxic³⁶.

Plant extracts and their phytoconstituents have been shown to have biological activity³⁷. The phytochemical screening test qualitatively showed that the ethanol extract of Moringa leaves contained secondary metabolites of alkaloids, flavonoids, phenolics, terpenoids and steroids and did not contain saponins. According to research by Swathi³⁸, Moringa plants contain alkaloids, phenolics, saponins, terpenoids, steroids, and flavonoids. Differences in the content of chemical compounds obtained can be influenced by several factors, such as demographic conditions, nutrition, and the environment in which a plant grows³⁹.

Administration of test preparations:

As many as 36 white male rats were divided into four large groups. Group I rats were the control group given 1% Na CMC suspension, group II rats were given the extract at a dose of 7mg/kg BW, group III was given the extract at a dose of 21mg/kg BW, and group IV rats were given the extract at a dose of 140mg/kg BW. Each group consisted of 9 rats, which were further divided into three subgroups comprising 3 rats: group A, which was a group of rats which were treated for seven days and then sacrificed on the eighth day; group B was given treatment for 14 days and then sacrificed on the 15^th day, and group C was treated for 21 days and sacrificed on the 22^nd day. Test materials were prepared and administered daily by oral route once a day.

The subacute toxicity test of the ethanol extract of Moringa leaves can be determined by observing the histology of the kidney. Histology has been used as a research base for non-clinical safety testing, identifying changes related to the diagnosis and evaluating the response to therapy⁴⁰. Testing of kidney histology was chosen because the kidneys are susceptible to the toxic effects of drugs and chemicals. The kidneys receive about 25% of the blood from the cardiac output, so they often encounter large amounts of chemicals easily⁴¹.

Administration of ethanol extract of Moringa leaves to the ratio of kidneys in male white rats:

Based on the results of calculations on the ratio of kidneys using IBM SPSS, it was found that the duration of administration had a significant effect on the ratio of kidney organs (p<0.05). However, the dose had no significant effect on the ratio of kidney organs (p<0.05). Likewise, with the effect of the interaction between dose and duration of administration (p<0.05), the DMRT result showed no significance between the control group and the groups administered with a dose of 7 mg/kg BW, 21mg/kg BW, and 140mg/kg BW, respectively. Thereafter, there is a difference between the group of test animals that were given the dose for 7 days and those that were given the dose for 14 days and 21 days.

The kidney-organ ratio was chosen because it is crucial to see the damage produced by chemicals. Organ weight can be a sensitive indicator of the influence of chemical compounds. The problem in interpreting organ weight data is that a compound can affect organ weight and the overall body weight of the animal. Therefore, the relative organ weight ratio is calculated by dividing the weight of each organ by its body weight⁴².

Figure 1. Graph of the effect of dose and duration of administration of ethanol extract of Moringa leaves on the ratio of kidney organs

The administration of ethanol extract from Moringa leaves caused a decrease in the ratio of kidney organs compared to that of the control group. The smallest ratio of kidney organs was found in the 140mg/kg BW dose group on the 21^st day. This is consistent with the results of the renal histology scoring, which increased at a dose of 140mg/kg BW. The obstacle of this parameter is the value of the ratio of kidney organs, which has decreased, while there is cell necrosis on kidney histology examination. Cell necrosis can cause inflammation in the kidney, which can lead to an increase in the value of the ratio of kidney organs. This was due to the rise in the body weight of the test animals, given the ethanol extract of Moringa leaves, compared to the control group. An increase in the body weight of the test animals will cause a decrease in the value of the ratio of the kidney organs.

Administration of ethanol extract of Moringa leaves on kidney histology of male white rats.:

The kidney histology preparations were carried out using the paraffin method, which is the standard method used in making histological preparations. Paraffin is used as an infiltration medium for embedding in the preparation of histological preparations. The paraffin wax can penetrate tissues in liquid form and harden fast when it cools⁴³. The paraffin method produces thin slices of 3 µm, making them easy to observe under a microscope⁴⁴. Previously, the kidney organs were rinsed with 0.9% physiological NaCl so that no blood would stick to the organs, and then the organs were fixed using formalin buffer. The purpose of fixation is to preserve the organ so that it retains the shape of the organ⁴⁵.

After fixation, the kidney organs were cut crosswise and lengthwise, which presented the overall picture of the kidney. The kidney organs were previously observed visually; if there are morphological changes in the kidneys, they must be used as samples. However, in this study, no changes in kidney morphology were found. Haematoxylin and Eosin stains were used to stain sections to make it easier to see under a microscope⁴⁶. Haematoxylin is an alkaline that can colour the cell nucleus blue. Meanwhile, eosin dye is acidic, which can colour the cytoplasm of cells pink. This HE dye combination is often used because it can produce clear microscopic images of cells between the cytoplasm and the cell nucleus⁴⁷.

Renal histology preparations were observed under a light microscope with 400x magnification. Histological assessment is semi-quantitative and is carried out on microscopy changes in the kidney organs using scoring (value) based on the changes that occur. Scoring is determined by looking at the presence or absence of damage to the kidney seen from five randomly selected visual fields. The results of kidney histology scoring are depicted in Table 2.

Based on the results, the highest kidney histological damage score was found at the highest dose, 140 mg/kg BW. Administration of ethanol extract of Moringa leaves for the first 7 days did not significantly affect the value of kidney histological damage, similar to the administration for 14 days. However, histological damage significantly increased when ethanol extract from Moringa leaves was administered for 21 days, especially at 21 and 140mg/kg BW doses.

Figure 2. Comparison of histological picture of the rat kidney with various doses and long given (7 days, 14 days and 21 days) of the test preparation. (G) Glomerulus; (T) Tubules.

According to the results, no experimental animals died before being sacrificed. However, from the observations made on kidney histology, damage was found at doses of 7, 21, and 140 mg/kg BW, which was not found in the control group (-). The control group (-) given 7, 14, and 21 days showed normal kidney microscopic appearance; the tubules and glomeruli were arranged orderly without any signs of inflammation, degeneration, necrosis of the tubular epithelium, or bleeding.

Giving a dose of 7mg/kg BW for 7 days resulted in <10% damage to the tubules and a 21mg/kg BW (score 1). However, at a dose of 140mg/kg BW, there was <25% damage to the tubules (score 2).

The microscopic appearance of Moringa leaf extract for 14 days was not significantly different from that for 7 days. Doses of 7 and 21mg/kg BW resulted in minimal damage to as little as <10% of the tubules (Score 1). However, at a dose of 140mg/kg BW, there was mild damage of <25% to the tubules (Score 2), and then cell degeneration and glomerular dilation were observed when compared to the control group.

The histological microscopic appearance of the kidneys differed significantly at 21 days, in line with the mean visual field scoring. At a dose of 7 mg/kg BW, there is minimal damage of <10% to the tubules (Score 1). At doses of 21 and 140mg/kg BW, cell degeneration and Bowman's cavities were widened and filled with proteinaceous material. However, at a dose of 21mg/kg BW, it is classified as mild damage with <25% damage to the tubules (Score 2) and a dose of 140mg/kg BW is classified as moderate damage of <50% to the tubules (Score 3).

Damage to the kidneys can be caused by toxic substances that enter the body. The damage can be seen from changes in histological structure, including the occurrence of cell degeneration and cell necrosis. Cell degeneration occurs in the cytoplasm, while cell necrosis occurs in the cell nucleus. Degeneration occurs when a cell loses its standard cell structure due to influences from within or outside the cell. Metabolic disturbances characterise cell degeneration. This causes the accumulation of intracellular and extracellular materials, leading to cell death and is a sign of cell damage due to toxins⁴⁸.

Necrosis is the death of tissue cells due to injury while the individual is still alive. Microscopically, there is a change in the nucleus, namely the loss of the chromatin appearance, the nucleus becomes wrinkled, loses vascularity, the nucleus looks denser, the colour turns dark black (pyknosis), and it is divided into fragments, torn (Karyorrhexis), the nucleus no longer takes on much colour because it is unnoticeably pale (karyolitic). Necrosis is characterised by the destruction of the proximal tubular epithelial cells, where the proximal tubular epithelial cells are sensitive to anoxia and are easily destroyed in case of poisoning due to metabolic remnants excreted by the kidneys. Therefore, changes in kidney histology can be ascertained because of the amount of compounds that enter the body⁴⁹.

Another damage occurs when the Bowman cavity containing protein material is widened. Filtration in the glomerulus induces the entry of small molecules such as water and electrolytes to the Bowman cavity and retains macromolecules such as proteins. Endothelial cells in the glomerulus have a cell surface layer called the glycocalyx, composed of proteoglycans and sialoproteins. Normal glycocalyx will inhibit the escape of protein molecules in the filtration process. As a result, the presence of protein found in Bowman's cavity is caused by damage to the glycocalyx of the glomerular endothelial cells. When kidney damage occurs, the body cannot eliminate excess urine and waste from the body and all the blood electrolytes (such as potassium and magnesium) increase⁵⁰.

In this study, there are still limitations in the form of colour differences that occur in microscopic images. This can happen due to several factors, such as the thickness when cutting using a microtome; the thicker the incision, the darker the resulting colour will be. In addition, it is also influenced by the soaking time and rinsing time when dyeing. The longer the preparation is soaked during staining, the darker the resulting colour will be, and the longer the preparation is rinsed, the paler the colour will be. However, these limitations did not affect the histological results obtained.

CONCLUSION:

The administration of Moringa leaves for 7 days showed kidney histology normal scoring, and 14 days of dosage administration showed a normal–minimal damage score. Meanwhile, the 21-day administration showed a minimal–moderate damage score. Based on the above explanation, it can be concluded that the Moringa leaves’ administration for 7 days, 14 days, and 21 days results in non-significant damage to the kidney organs, which has been proven by the histological picture of the kidney organs.

ACKNOWLEDGEMENT:

The authors thank the Rector Universitas Andalas through the Institute for Research and Service Society. The research was made possible and financially supported under "Penelitian Terapan Unggulan Klaster Riset Publikasi Guru Besar Universitas Andalas" (PTU-KRP1GB-Unand), Batch I, the Year 2023, with Contract Number. T/16/UN.16.19//KO/PTU-KRP1GB-Unand/ 2023 signed on April 4, 2023.

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Received on 04.09.2023 Modified on 24.04.2024

Research J. Pharm. and Tech 2024; 17(11):5531-5539.

DOI: 10.52711/0974-360X.2024.00845

Dosage (mg/kg BW)	Histological damage scores
	7 days					14 days					21 days
	FOV 1	FOV 2	FOV 3	FOV 4	FOV 5	FOV 1	FOV 2	FOV 3	FOV 4	FOV 5	FOV 1	FOV 2	FOV 3	FOV 4	FOV 5
Control	0	0	0	0	0	0	1	0	0	0	0	0	0	1	0
7	1	0	0	1	0	1	0	1	1	0	1	0	0	1	1
21	1	0	1	0	0	1	1	0	0	0	2	1	2	2	2
140	1	1	2	1	2	1	2	2	1	2	2	3	3	2	2
Description	Normal					Normal – Minimal damage					Minimal – Moderate damage