Evaluating The Adverse Health Effects of Herbal Medicine on Swiss Albino Mice: A Comprehensive Hepatotoxicity and Nephrotoxicity Analysis

doi:10.21203/rs.3.rs-4813609/v1

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Evaluating The Adverse Health Effects of Herbal Medicine on Swiss Albino Mice: A Comprehensive Hepatotoxicity and Nephrotoxicity Analysis

https://doi.org/10.21203/rs.3.rs-4813609/v1

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Herbal medicine (HM) has been utilized extensively to a wide range of illnesses for thousands of years and has significantly contributed to the health of human beings. The harmful health consequences or degree of toxicity of herbal medication are not demonstrated according to testing, manufacturing, and labeling standards of Food and Drug Administration. The aim of the study was to investigate the hepatotoxicity and nephrotoxicity of herbal medicine - Amlasia: a well‑known Hamdard herbal formulation – on swiss albino mice. For fourteen days straight, the Amlasia solution was administered orally at two separate dosages (0.5 ml per kg and 2.5 ml per kg body weight). Paracetamol was employed for a single lethal dosage of hepato-renal toxicity and allopathic medications Maxpro were utilized as standard. Enzyme levels such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), triglyceride, total protein (TP), bilirubin, albumin (ALB), blood urea nitrogen (BUN), creatinine, and urea were measured in order to evaluate liver and kidney impairment. Besides, oxidative stress markers (reduced glutathione, malondialdehyde, catalase activity) were assessed and finally, cellular toxicity level was evaluated through histopathology analysis. The outcomes for the parameters measuring liver and kidney function showed that, enzyme levels did not significantly change after Amlasia treatment. Moreover, the oxidative stress markers also indicate the non-significant difference as compared with saline control group. Finally, the histopathological analysis of the liver and kidney samples revealed normal cellular levels according to microscopic evaluation. The investigated herbal medicine did not cause hepatotoxic or nephrotoxic disorders in the experimented animals, according to biochemical parameter, oxidative stress marker and histological examination. It was concluded that, the investigated herbal medicine was therefore found to be pharmacologically safe.

Herbal medicine

Hepatotoxicity

Nephrotoxicity

Biochemical Parameter

Liver

Kidney

Herbal medicines were the primary curing medication of human ailments approximately a century ago. Globally, herbal treatments are becoming more and more popular. About 75–80% of the world's population still relies mostly on herbal medicine for primary healthcare, particularly in developing nations [1]. The Secretariat of the Convention on Biological Diversity estimates that the global market for herbal products was worth $60 billion in 2000 [2]. With a consistent growth rate of between 3% and 12% annually, the global market for herbal treatments was valued at US over $83 billion in 2008 (see: http://www.nutraceuticalsworld.com )). The information that is currently available indicates that there is an extensive market for traditional Chinese medicine and that Chinese herbs are shipped to more than 175 nations and territories, including the US, Japan, South Korea, India, Germany, and the Netherlands [3]. Approximately 500 different kinds of medicinal plants can be found in Bangladesh; more than 250 of these species are now used to make traditional herbal remedies. Furthermore, about 80% of Bangladesh's rural residents rely on these traditional herbal treatments as their main source of healthcare [4]. Amlasia is a special formulation made of valuable natural active ingredient amlaki juice, pipul and honey which is very effective in treating indigestion, indigestion, flatulence, heartburn, acidity, anemia, heart disease and jaundice [5]. According to estimates, 25% of contemporary medications—including aspirin, artemisinin, ephedrine, and paclitaxel—are derived from herbs that were originally used traditionally [6]. Nonetheless, the majority of herbal products' safety and effectiveness are not well supported by scientific research [7]. The safety of herbal medications has drawn the attention of regulatory bodies due to reports of severe side effects, such as allergic responses, hepatotoxicity, and renal failure [8]. Hepatotoxicity is the term used to describe liver damage or malfunction brought on by an excess of medications or xenobiotics [9]. The substances known as hepatotoxins or hepatotoxicants are those that harm the liver.

Hepatotoxicants are exogenous substances that have clinical significance. They can include industrial toxins, natural substances like microcystins, overdoses of specific medications, herbal therapies, and nutritional supplements [10], [11]. Certain medications have the potential to harm an organ when used in excess or occasionally even when started within recommended dosage ranges. A drug's withdrawal from the market is most frequently due to liver damage, which has been linked to over 900 different medications. 50% of acute liver failures and 5% of hospital admissions are attributable to drug-induced liver damage. Liver transplantation or death occur in almost 75% of cases of idiosyncratic medication responses [12]. The detrimental effect of chemicals on renal function is known as nephrotoxicity [13]. These materials can include molds and fungus, medications of addiction like cocaine, metals like mercury, arsenic, and lead, antibiotics like aminoglycosides, and cancer therapies like cisplatin. It has many risk factors that vary depending on the treatment regimen being considered as well as underlying patient characteristics. It is critical to raise public awareness about herbal medicine throughout the world due to the potentially dangerous consequences associated with using supplements that have not been approved by the Food and Drug Administration (FDA). Herbal therapy uses natural chemicals with varied degrees of adverse effects and relatively complex active components. Because some people believe herbal treatments are harmless, doctors and patients may ignore the fact that some of these herbal medicines are known to induce nephrotoxicity. Other plant alkaloids, such as aristolochic acids, are also nephrotoxic components found in plants. Furthermore, it is known that anthraquinones, flavonoids, and glycosides found in herbs can be harmful to the kidneys [14]. Nephrotoxicity linked to herbal medicine can appear in the kidneys as urothelial carcinoma, CKD, nephrolithiasis, rhabdomyolysis, acute kidney damage, and Fanconi syndrome. The inherent toxicity of herbs, improper handling or storage, adulteration, heavy metal contamination, improper dosage, and interactions with other treatments are some of the factors that cause herbal medicines to be nephrotoxic. It is unknown how frequently nephrotoxic herbal medicines cause kidney damage.

The number of reported adverse events and probable toxicity of herbal medications has increased along with their use. These undesirable side effects can be caused by: (i) pharmacodynamics side effects, which are typically detectable and often predictable; (ii) pharmacovigilance or pharmacodynamics side effect detection; (iii) hypersensitivity, allergic, and idiosyncratic reactions; (iv) mid- and long-term toxic effects, such as liver, renal, cardiac, and neurotoxicity; and (v) genotoxicity and teratogenicity (detectable by in vitro and in vivo toxicological studies or by pharmacovigilance). The lack of comprehensive testing for pharmacology and toxicology in many herbal items on the market means that pharmacovigilance is critical in identifying adverse responses. Unexpected toxicity of herbal products is another concern that persists because of quality problems, such as using low-quality herbal material, misidentifying or incorrectly processing herbs, and supplying contaminated or adulterated goods or herbs [15]. Updating the regulations to mandate GMP standards for manufacturing can help to mitigate these quality difficulties to some extent. Still, low-quality items are likely to be an issue because medicinal plants and products are imported from a variety of nations with varying production standards and regulatory compliance. The regulatory authorities are now concerned about the safety of herbal medications due to reports of serious side effects, such as hepatotoxicity, renal failure, and allergic responses [8], [16]. The World Health Organization created guidelines for the monitoring of herbal safety within the current pharmacovigilance framework after realizing the growing significance of the use of herbal medicines globally.

The purpose of this study was to identify potential harmful health consequences, such as hepatotoxicity and nephrotoxicity, of herbal medicines used to treat acidity in a particular region of Bangladesh. The workflow of this study were presented in Fig. 1.

2.1 Animals and Housing Requirements

For this investigation, adult Swiss albino mice in good health, weighing 20–30 g, of either sex, were employed. We obtained adult mice of both sexes that were produced in the Central Animal House of the Department of Genetic Engineering and Biotechnology at the University of Rajshahi. During the acclimatization process, standard environmental parameters were maintained, including temperature (26 ± 2°), relative humidity (45–55%), and a 12-hour light and dark cycle. The animals were given unlimited access to clean water and regular pelletized feed while living in hygienic settings. To reduce any nonspecific stress, the animals were acclimated to laboratory conditions for one week before the experiment.

2.2 Ethical Permission

The experimental protocol, which has the registration number (#0012, March 15, 2023) and the certified memo number (336(16)/320/IAMEBBC/IBSC), was approved by the Institutional Animal, Medical Ethics, Biosafety and Biosecurity Committee (IAMEBBC) of the Institute of Biological Sciences, Rajshahi University, Rajshahi-6205, Bangladesh, for experimentation on animals, humans, microbes, and living natural sources.

2.3 Grouping and Experimental Protocol

Twelve different groups, each with six mice, were used for this experiment. The grouping comes after this arrangement.

Group 1 was administered oral 10 ml per kg of normal saline for 14-days of treatment duration.
Group 2 was administered oral 10 ml per kg of normal saline for 12-days of treatment duration before the induction of hepatotoxicity with a single oral paracetamol dose of 500 mg per kg on the 13th day.
Group 3 was administered oral 10 ml per kg of normal saline for 12-days of treatment duration before the induction of nephrotoxicity with a single oral paracetamol dose of 500 mg per kg on the 13th day.
Group 4 was administered oral as the treatment groups and treated with Amlasia (Hamdard medicine) at 0.5 ml per kg body weight for 14 days for liver functions investigations.
Group 5 was administered oral as the treatment groups and treated with Amlasia (Hamdard medicine) at 2.5 ml per kg body weight for 14 days for liver functions investigations.
Group 6 was administered oral as the treatment groups and treated with Amlasia (Hamdard medicine) at 0.5 ml per kg body weight for 14 days for kidney functions investigations.
Group 7 was administered oral as the treatment groups and treated with Amlasia (Hamdard medicine) at 2.5 ml per kg body weight for 14 days for kidney functions investigations.
Group 8 was administered oral as the treatment groups and treated with Maxpro at 0.7 mg per kg body weight for 14 days for liver and kidney functions investigations.

2.4 Measurement of Body Weight and Organ Weights

Mice's body weights were measured both before and after the experimentation for each group. The animals were sacrificed by cervical dislocation, and their kidneys and livers were removed, cleaned with phosphate buffer, and patted dry with tissue paper. An electronic balance was used to weigh the liver and kidney.

2.5 Liver Index

At the time of sacrifice, the mice's body and liver weights were measured. The liver weight was divided by the body weight and the resulting percentage was then calculated to find the liver weight to body weight (LW/BW) ratio.

2.6 Kidney Index

At the time of sacrifice, the mice's body and kidney weights were measured. The kidney weight was divided by the body weight and the resulting percentage was then calculated to find the kidney weight to body weight (KW/BW) ratio.

2.7 Collection of Specimen

To get blood samples from the animals, cardiac punctures were done. To make sure the animals were comfortable, they were anesthetized before the sacrifice. Blood samples were taken directly into plain sample vials for blood chemistry analysis after their bodies were delicately opened using a dissecting equipment to reveal their beating hearts. Centrifugation was applied to the obtained blood samples for 15 minutes at 3800 rpm. The goal of this procedure was to extract the serum—which contains a number of components. The resultant sera, or supernatants, were cautiously poured into a different plain sample vial. Following that, these sera were frozen and kept for additional examination. Liver and kidney organ specimens from each of the twelve animal groups were collected in order to evaluate the in vivo antioxidant activity. These materials were collected using universal sample bottles and stored in an ice-cooled (-80° C) environment to preserve their integrity until the antioxidant assays could be performed. Additionally, some liver and kidney tissue were kept for histological examination in a 10% formalin solution.

2.8 Assessment of Liver and Kidney Functions

The measurement of certain enzymes that the liver produces is known as hepatic enzyme activity. In addition to identifying liver diseases and tracking the efficacy of therapies, measuring the activity of liver enzymes can yield important information regarding liver health. Alanine amino transaminase (ALT), aspartate amino transaminase (AST), alkaline phosphatase (ALP), triglyceride (TG), Total protein (TP) and liver total Bilirubin were determined using the standard diagnostic kits as per the manufacturer’s protocol. To analyze the functions of kidney the level of Creatinine, blood urea nitrogen (BUN), serum albumin and urea were also determined using the standard diagnostic kits as per the manufacturer’s protocol.

2.9 Assessment of Hepatic and Renal Oxidative Stress

Colorimetric techniques were used in accordance with conventional protocols to detect reduced glutathione (GSH), malondialdehyde (MDA) levels, and catalase activity in the liver and kidney homogenates of all groups.

2.9.1 Reduced Glutathione (GSH) Contents

Glutathione, or GSH, is an essential antioxidant molecule that can be found in all human cells. Using the Ellman method, spectrophotometric analysis was used to measure reduced glutathione (GSH) in the liver and kidneys [17]. After thawing, tissue fragments were homogenized using glass-teflon homogenizers in 1 mL of homogenizing solution (250 mM sucrose, 20 mM Tris-HCl, 1 mM dithiothreitol, pH 7.4) on ice. The homogenates were centrifuged for two hours at 15,000 rpm and 4°C. As a chromogen, 5, 5-Dithiobis 2-nitrobenzoic acid was utilized, and its absorbance was measured at 412 nm after it had been reduced.

2.9.2 Standard Curve for Reduced GSH Contents

The reduced glutathione was dissolved in distilled water to yield a µmol/L stock solution, which was then used to make the reduced GSH standard. To create the working solution and obtain the reduced GSH standard curve, the stock solution was mixed with distilled water to yield 83.6, 41.8, 20.9, 10.45, 5.22, 4.86, 2.43, 1.22, and 0.61 µmol/L.

2.9.3 Determination of Lipidperoxidation

According to Ohkawa et al.'s method [18], 1 mL of 0.67% thiobarbituric acid was used to determine the levels of malondialdehyde (MDA) in the liver and kidneys. Tris-HCl (50 mM, pH 7.4) was used to homogenize liver and kidney tissue. The homogenate was centrifuged for 10 minutes at 12,000 g. After the supernatant was removed, 50 µl of homogenate, 500 µl of 0.67% TBA (0.57 thiobarbituric acid added to 100 mL of distilled water and heated to 50–60°C to aid in dissolving), and 500 µl of 20% trichloroacetic acid were combined. For twenty minutes, the mixes were incubated in a bath of boiling water. The reaction mixture was centrifuged at 4000g for 10 minutes after cooling to room temperature, and the absorbance of the supernatant was measured at 532 nm.

2.9.4 Standard Curve for Malondialdehyde (MDA)

To generate the MDA standard, 50 µL of 1,1,3,3 tetraethoxypropane (TEP) was dissolved in 25 mL of 40% ethanol, yielding an 8360 µmol/L stock solution. To generate the working solution, 100 µL of the stock solution was combined with 20 mL of 40% ethanol. This resulted in an MDA standard of 41.8 µmol/L. The MDA standard was then further diluted with distilled water to yield MDA standard curve values of 20.9, 10.45, 5.22, 4.86, 2.43, 1.22, and 0.61 µmol/L.

2.9.5 Catalase (CAT) Activities

The enzyme known as catalase (CAT) is present in almost all living things. The modified Aebi et al. method [19] was used to measure the CAT activity in the liver and kidneys spectrophotometrically. Following tissue homogenization in 50 mM phosphate solutions on ice, 0.1 mM H₂O₂ was added. The homogenates underwent a 10-minutes centrifugation at 2000 rpm and 4°C. Spectrophotometric measurements were used to determine the rate of H₂O₂ breakdown at 240 nm. A unit of catalase activity was defined as the quantity of enzyme required to break down one µmol of H₂O₂ for every milligram of soluble protein in a minute at 30˚C.

2.9.6 Calibration Curve for Catalase (CAT) Activities

The calibration curve for catalase enzyme was obtained from Adnan Mukhtar et. al; 2022.

2.10 Histopathological Investigations

Understanding the cellular and tissue-level alterations linked to different diseases and disorders is made possible by studying histopathology. After being washed with normal saline, the liver and kidney samples were stored in 10% v/v neutral buffered formalin solution. A section of kidney and liver tissue was removed, dried in ethanol at varying percentages (80%, 90%, 100%, and 70%), and then cleaned with xylene. After that, the paraffin-embedded fixed tissues were cut into 5 µm slices with a rotating microtome and stained with hematoxylin and eosin (H&E) dye. The liver and kidney sections were subjected to histological evaluation using a light microscope equipped with a camera. Based on pathologic lesions in the corresponding sections stained using the H&E method, the degree of drug-induced liver and kidney damage was assessed. The primary histological abnormalities taken into consideration for the liver were cytoplasmic vacuolation, cellular infiltration, necrosis, pyknotic nuclei, and sinusoidal dilatation. On the other hand, cytoplasmic vacuolation, necrosis, membrane thickness, hemorrhage, and cellular infiltration were the main histological anomalies in the kidney. The degree of histopathological damage was evaluated for each group of the liver and kidney based on severity.

2.11 Statistical Analysis of Data

The statistical package for social sciences (SPSS) version 23 (SPSS Inc., IL, USA) was utilized to evaluate all the data that had been collected. The results were presented using figures and tables. The presentation of continuous variables was given as mean ± standard error. The analysis of variance test was used to see if the mean values for two or more continuous variables representing various different groups showed any form of statistically significant association. For statistical comparisons, one-way analysis of variance (ANOVA) was employed, followed by a least significance difference (LSD) post hoc test. When a p value was less than 0.05, it was considered statistically significant.

3.1 Measurement of body weights and organ weights

To examine the effects of Herbal medicine on the weights of body and organs were measured. The body weight was taken before and after treatment. No significant difference in body weight was observed between the saline control and paracetamol-treated group in hepatotoxicity analysis group for 0 day and 14th day (Fig. 2 a & Table S1). There was no significant body weight difference were observed in nephrotoxicity analysis group for 0 day and 14th day (Fig. 2 b & Table S2). The weight of liver for paracetamol treated group was significantly (p > 0.05) increased when compared with saline control group. There was no significant liver weight difference between saline control group and herbal medicine (Amlasia with the dose of 0.5 ml per kg & 2.5 ml per kg) treated group (Fig. 3 a & Table S1). No significant difference in kidney weight was observed in all the treatment and experimental group (Fig. 3 b & Table S2).

3.2 Liver and kidney index

The liver index was significantly (p > 0.05) increased in paracetamol treated group as compared with saline control group. Amlasia herbal medicine at the dose of 0.5 ml per kg group indicates the significant (p > 0.05) increased liver index when compared with saline control group. Besides there was no significant difference observed in the rest of the treatment group when compared with saline control group (Fig. 4 a & Table S1).

No significant kidney index was observed between the paracetamol and saline control group. Moreover, there was no significant kidney index difference present in the Amlasia at the dose of 2.5 ml per kg treated group but Amlasia at the dose of 0.5 ml per kg (p > 0.05) indicates increased level when compared with saline control group (Fig. 4 b & Table S2).

3.3 Evaluation of liver and kidney functions

The serum biochemical parameters for the control group and several experimental groups are shown in Table S3 & S4. Biochemical parameters revealed that PCM (500 mg per kg BW) administered orally once caused hepatotoxicity and nephrotoxicity after 48 hours.

3.3.1 Alanine Transaminase (ALT)

Paracetamol treated group mice indicates that the liver function marker ALT were significantly (p > 0.05) higher as compared with the saline control group. At the dose of 0.5ml per kg and 2.5ml per kg BW of Amlasia was significantly reduced (p > 0.05) compared with the saline control group and also able to cause significant reduction (p < 0.05) of the serum ALT liver enzymes when compared with the paracetamol treated group. The allopathic drugs Maxpro induces the reduced enzyme level (p > 0.05) when compared to saline control group and paracetamol treated group (Fig. 5 a & Table S3).

3.3.2 Aspartate Transaminase (AST)

Mice treated with paracetamol (500mg per kg) alone developed significant hepatocellular damage as evidenced from increase in serum level of AST, when compared with saline control group. At the dose of 0.5ml per kg and 2.5ml per kg BW of Amlasia was significantly reduced (p > 0.05) compared with the saline control group and also able to cause significant reduction (p < 0.05) of the serum AST liver enzymes when compared with the paracetamol treated group. The allopathic drugs Maxpro induces the reduced enzyme level (p > 0.05) when compared to saline control group and paracetamol treated group (Fig. 5 b & Table S3).

3.3.3 Alkaline Phosphatase (ALP)

The hepatic injury induced by paracetamol resulted in a significant increase in serum ALP values in comparison to saline control group (P < 0.05). Amlasia at the dose of 0.5 ml per kg and 2.5 ml per kg BW exhibited the lower ALP enzyme levels when compared with both saline control and paracetamol treated group. Moreover, showed slightly lower ALP enzyme levels when compared with allopathic group Maxpro (Fig. 5 c & Table S3).

3.3.4 Total Bilirubin

Paracetamol overdose (500mg per kg) caused statistically significant increase (p < 0.05) in the liver enzyme bilirubin compared with the saline control group. For the 0.5 ml per kg and 2.5 ml per kg dose of Amlasia, the level of total bilirubin slightly high when compared with saline control group but reduced when compared with paracetamol treated group. Moreover, both of the dose of Amlasia exhibited reduced bilirubin level as compared with Maxpro (Fig. 5 d & Table S3).

3.3.5 Triglyceride

It was observed that paracetamol overdose caused statistically significant increase (p < 0.05) in the liver enzyme triglyceride compared with the saline control group. The level of triglyceride for the 0.5 ml per kg and 2.5 ml per kg dose of Amlasia decreases when compared with saline control group but reduced significantly (p < 0.05) when compared with paracetamol treated group. In another case, the both dose of Amlasia 0.5 ml per kg and 2.5 ml per kg exhibited similar level of triglyceride enzyme when compared with Maxpro (Fig. 5 e & Table S3).

3.3.6 Total Protein

Furthermore, the injury induced by paracetamol resulted in a significant increase in serum total protein values in comparison to saline control group (P < 0.05). But in 2.5 ml per kg dose of Amlasia, the level of total protein decreased significantly (P < 0.05) when compared with the both saline control and paracetamol treated group. Amlasia at the dose of 0.5 ml per kg exhibited similar level of total protein and at the 2.5ml per kg exhibited reduced level of total protein when compared with Maxpro (Fig. 5 f & Table S3).

3.3.7 Creatinine

Acute paracetamol overdose caused statistically significant increase (p < 0.05) in the total bilirubin levels compared with the saline control group. In another case, the level of creatinine for the 0.5 ml per kg and 2.5 ml per kg dose of Amlasia similar when compared with saline control group but reduced significantly (p < 0.05) when compared with paracetamol treated group. The both dose of Amlasia 0.5 ml per kg and 2.5 ml per kg exhibited similar level of creatinine enzyme when compared with Maxpro (Fig. 6 a & Table S4).

3.3.8 Blood Urea Nitrogen (BUN)

It was observed that paracetamol overdose caused statistically significant increase (p < 0.05) in the kidney enzyme BUN compared with the saline control group. The different dose of Amlasia at the dose of 0.5 ml per kg and 2.5ml per kg BW was significantly reduced (p > 0.05) compared with the saline control group and also cause significant reduction (p < 0.05) of the serum BUN enzymes when compared with the paracetamol treated group. Moreover, the both dose of Amlasia 0.5 ml per kg and 2.5 ml per kg exhibited similar level of BUN enzyme when compared with Maxpro (Fig. 6 b & Table S4).

3.3.9 Urea

Acute paracetamol overdose caused statistically significant increase (p < 0.05) in the urea levels compared with the saline control group. It was observed that the different dose of Amlasia at the dose of 0.5 ml per kg and 2.5ml per kg BW was significantly reduced (p > 0.05) compared with the saline control group and also cause significant reduction (p < 0.05) of the urea level when compared with the paracetamol treated group. The both dose of Amlasia 0.5 ml per kg and 2.5 ml per kg exhibited similar level of urea when compared with Maxpro (Fig. 6 c & Table S4).

3.3.10 Albumin (ALB)

It was observed that paracetamol overdose caused statistically significant increase (p < 0.05) in the ALB level compared with the saline control group. At the 0.5 ml per kg dose of Amlasia, it significantly increased (p < 0.05) the ALB level when compared with the saline control group. But at the dose of 2.5 ml per kg exhibited increased ALB level (not significant) when compared with the saline control group. Amlasia at the dose of 0.5 ml per kg exhibited reduced level of ALB and at the 2.5ml per kg exhibited similar level of ALB when compared with Maxpro (Fig. 6 d & Table S4).

3.4 Evaluation of hepatic antioxidant activity and oxidative stress

3.4.1 Reduced Glutathione (GSH) Content

Acute acetaminophen overdose produced a statistically significant reduction in the reduced glutathione (GSH) levels of the acetaminophen-treated group (p < 0.05) compared with the saline control group. In another case, at the dose of 0.5ml per kg of Amlasia the results showed no difference compared with saline control group. But in 2.5 ml per kg dose of Amlasia, the level of the reduced glutathione increased significantly (P < 0.05) when compared with the both saline control and acetaminophen treated group. The both dose of Amlasia 0.5 ml per kg and 2.5 ml per kg exhibited reduced level of reduced glutathione when compared with Maxpro (Fig. 7 a & Table S5). Figure S1 indicates calibration curve of reduced glutathione (GSH) contents.

3.4.2 Malondialdehyde (MDA) Content

In this study, it was observed that acute paracetamol overdose produced a statistically significant increase in the levels of malondialdehyde (MDA) antioxidant enzyme of the acetaminophen-treated group (p < 0.05) compared with the saline control group. At the 0.5 ml per kg dose of Amlasia, it increased the MDA level when compared with the saline control group but significantly increased (p < 0.05) when compared with acetaminophen treated group. Besides, at 2.5 ml per kg dose of Amlasia, the level of MDA increased significantly (P < 0.05) when compared with the saline control group and lowered (not significant) compared with acetaminophen treated group. In comparison with the Maxpro, the 0.05 ml per kg dose of Amlasia showed no difference but slightly higher value (not significant) indicated by the 2.5 ml per kg dose of Amlasia group (Fig. 7 b & Table S5). Figure S2 indicates calibration curve of malondialdehyde (MDA) contents.

3.4.3 Catalase activity

It was observed that paracetamol overdose caused statistically significant decrease (p < 0.05) in the catalase activity level compared with the saline control group. In another case, at the dose of 0.5ml per kg and 2.5 ml per kg of Amlasia the results showed no difference compared with saline control group. No significant difference was found Amlasia group when compared with allopathic standard Maxpro group (Fig. 7 c & Table S5). Figure S3 Calibration Curve of catalase activity (Adnan Mukhtar et. al; 2022).

3.5 Evaluation of renal antioxidant activity and oxidative stress

3.5.1 Reduced Glutathione (GSH) Content

Furthermore, the injury induced by paracetamol resulted in a significantly decrease in GSH level in comparison to saline control group (P < 0.05). At the dose of 0.5 ml per kg and 2.5ml per kg BW of Amlasia exhibited no difference in the level of GSH compared with the saline control group and statistically significant (P < 0.05) increased GSH level exhibited by 0.5 ml per kg dose of Amlasia but not significant increased GSH level exhibited by 2.5 ml per kg dose of Amlasia compared with paracetamol treated group. In comparison with the Maxpro, the both dose of Amlasia 0.5 ml per kg and 2.5 ml per kg exhibited the increased (not significant) level of GSH contents (Fig. 8 a & Table S6).

3.5.2 Malondialdehyde (MDA) Content

Also, acute paracetamol overdose produced a statistically significant increase in the malondialdehyde (MDA) levels of the paracetamol treated group (p < 0.05) compared with the saline control group. At the dose of 0.5 ml per kg and 2.5ml per kg BW of Amlasia exhibited increased level (not significant) of MDA contents compared with the saline control group and also exhibited no differences when compared with paracetamol treated group. The both dose of Amlasia 0.5 ml per kg and 2.5 ml per kg exhibited similar level of MDA when compared with Maxpro (Fig. 8 b & Table S6).

3.5.3 Catalase activity

Furthermore, the injury induced by paracetamol resulted in a significantly decrease in catalase activity level in comparison to saline control group (P < 0.05). The effect of 0.5 ml per kg and 2.5 ml per kg dose of Amlasia, the results showed no difference compared with saline control group. No significant difference was found in Amlasia group when compared with allopathic standard Maxpro group (Fig. 8 c & Table S6).

3.6 Histopathological observations

Histological section of group I (saline control group) showed normal arrangement of hepatocytes with conspicuous nucleus with a central vein (CV) from which chords of hepatocytes were radiating (Fig. 9 a). The portal triad (PT) was seen consisting of hepatic artery, portal vein and bile duct. The region surrounding the central vein in the liver is known as the centrizonal or centrilobular area.

Treatment with paracetamol resulted in significant liver damage characterized by extensive hemorrhagic necrosis, as depicted in Fig. 9 b. The necrotic tissue was particularly prominent in the centrizonal region and was accompanied by the infiltration of inflammatory cells. In addition to exhibiting fatty changes, the hepatocytes displayed hydropic alterations, and the sinusoids demonstrated signs of congestion.

The photomicrograph of the liver from the animals for Amlasia (0.5 ml per kg and 2.5 ml per kg) revealed a preserved architecture of hepatic cells, characterized by clear cytoplasm. The central veins appeared slightly dilated, but within normal limits. Kupffer cells, responsible for immune function within the liver, displayed a normal appearance, and all cells exhibited large nuclei, indicating a healthy state (Fig. 9 c & d).

Histopathological changes in the kidneys were assessed and examined. In the saline control group, no histopathological changes were seen in the kidney regions (Fig. 10 a) they were all in normal appearance.

On the contrary, severe vacuolization and tubular necrosis were seen in proximal and distal tubule cells in the paracetamol administrated group (Fig. 10 b).

The groups treated with Amlasia at doses of 0.5 ml per kg and 2.5 ml per kg showed no histopathological alterations in the kidney regions. The kidney tissues appeared normal in their appearance, indicating the absence of any noticeable changes (Fig. 10 c & d).

Finally, the allopathic standard Maxpro group indicates the normal arrangement of hepatocytes. The hepatocytes displayed distinct nuclei, and from the central vein (CV), cords of hepatocytes radiated outward, forming the characteristic architecture of the liver tissue (Fig. 9 e). Besides, the allopathic standard Maxpro group for the kidney tissue revealed no observable histopathological alterations in the kidney regions. The kidney tissues exhibited a normal appearance, suggesting the absence of any significant changes or abnormalities (Fig. 10 e).

Throughout history, herbal medicine has been utilized for the prevention and treatment of a wide range of disorders. The Indian medicinal systems, such as Ayurveda, Siddha, Unani, and Amchi, among others, employ a vast array of herbs and herbal compositions. The number of reported adverse events and probable toxicity of herbal medications has increased along with their use. The lack of comprehensive testing for pharmacology and toxicology in many herbal items on the market means that pharmacovigilance is critical in identifying adverse responses.

Hepato-renal dysfunction is one of the main, unavoidable variables during drug or xenobiotic disposition among numerous drug therapy problems [20]. Although acetaminophen is typically regarded as a safe medication, hepatotoxicity and nephrotoxicity are the most common side effects [21], which occur at acute overdose [22] and are associated with increased mortality [23]. Numerous investigations have demonstrated a dose- and time-dependent association between acetaminophen injections and biomarkers related to the liver and kidneys [24]. Free radical overproduction has been widely recognized to be linked to liver and kidney damage caused by drugs or chemicals [25], [26]. Parallel to this, 1–2% of patients who overdose on paracetamol develop renal insufficiency. In the majority of instances, paracetamol-induced renal impairment becomes evident after hepatotoxicity [27]. The system of cytochrome p450 and glutathione deposits determines how much paracetamol damages the kidneys. The proximal tubule contains the majority of the cytochrome p450 in the kidney, with very little found in the glomerulus, distal tubes, or collecting duct. On the other hand, the proximal tubule's activities of secretion and absorption result in a higher concentration of hazardous compounds in the tubules than in other areas [28], [29]. Previous studies have documented the mechanism of acetaminophen-induced nephrotoxicity and hepatotoxicity, which indicates that high dosages of paracetamol might lead to renal toxicity and oxidative stress [30]–[34].

The current study's biochemical measures and histological alterations demonstrated paracetamol-induced hepatotoxicity, whereas an increase in serum levels of renal markers suggested renal injury. The toxic effects of paracetamol were observed in mice treated with elevated levels of serum AST, ALT, ALP, bilirubin, creatinine, urea, and uric acid, indicating impaired liver and renal functioning [35].

This study investigated the potential adverse health effects of Amlasia (Hamdard formulation) at the dose of 0.5 ml per kg and 2.5 ml per kg of body weight. The liver enzyme serum ALT is essential for the metabolism of amino acids. Increased ALT levels in the blood are a sign of illness or injury to the liver [36]. In case of Amlasia-treated groups, ALT levels demonstrated significant alterations compared to the control group. At the dose of 0.5 ml per kg and 2.5 ml per kg, there was a significant decrease in ALT levels. These findings suggest a dose-dependent effect of Amlasia on liver function as reflected by ALT levels.

In addition to ALT, serum AST is another liver enzyme that is frequently tested in blood tests to evaluate liver function. While excessive levels of AST in the bloodstream are usually suggestive of liver impairment or injury, the enzyme is also present in other organs, including the heart, muscles, and kidneys. Numerous liver disorders, such as cirrhosis, hepatitis, and liver inflammation, can result in elevated levels of AST. Muscle injury and myocardial infarction (heart attack) are two more conditions that can cause increased AST levels [37].

The findings of our study revealed a notable decrease in AST levels in the experimental group (Amlasia at the dose of 0.5 ml per kg as well as 2.5 ml per kg), suggesting no potential liver injury or impairment when compared to the control group.

Alkaline phosphatase, or serum ALP, is an enzyme that is present in many bodily tissues, although it is most abundant in the kidneys, liver, bones, and intestines. Blood tests frequently measure ALP to evaluate the health of the liver and bones. Increased blood ALP levels may be a sign of bone or liver problems. Liver diseases such as cirrhosis, hepatitis, or blockage of the bile ducts can result in elevated ALP levels. ALP levels can also be raised in bone-related conditions such as Paget's disease or certain bone-related cancers [38].

When comparing the experimental group (Amlasia at the dose of 0.5 ml per kg and 2.5 ml per kg) to the control group, the results showed a substantial drop in ALP levels, indicating no toxicity or abnormalities.

The breakdown of red blood cells produces the yellow-orange pigment known as bilirubin. It is broken down by the liver and released as bile. Blood tests are frequently employed to quantify bilirubin levels in the circulation in order to evaluate the function of the liver and gallbladder. Increased bilirubin levels might be a sign of bile duct issues or liver diseases like cirrhosis or hepatitis [39].

Comparing the bilirubin levels of the experimental group (Amlasia at 2.5 ml per kg and 0.5 ml per kg) with the control group revealed equal levels, ruling out any potential liver damage or decreased bilirubin metabolism. These results imply that there is no probable interference with the regular breakdown and excretion of bilirubin, which might be a sign of normal liver activities or conditions.

Triglycerides are a kind of fat (lipid) that are present in the blood and play a big role in the lipid profile. A blood test is performed to detect triglyceride levels, which are then used to determine an individual's risk for cardiovascular problems. A higher risk of heart disease, stroke, and other cardiovascular issues is linked to elevated triglyceride levels [40].

The analysis of triglyceride levels at different doses of Amlasia-treated groups (0.5 ml per kg and 2.5 ml per kg) demonstrated a similar level compared to the saline control group. These findings suggest a dose-dependent effect of Amlasia on triglyceride metabolism.

Total protein refers to the quantification of all proteins present in a biological sample, such as blood or urine. Its readings can be used to evaluate immune system activity, liver and kidney function, nutritional status, and different illness conditions. Serum total protein levels are measured by blood tests, and abnormal amounts may point to underlying medical issues [42].

Total protein levels at different dosages of Amlasia (0.5 and 2.5 ml per kg) were assessed, and the results showed no discernible variations from the control group, indicating that these treatments had no effect on total protein levels.

Creatinine is a waste product produced by muscles from the breakdown of a compound called creatine. The kidneys remove it from the blood and expel it in the form of urine. In order to evaluate kidney function, serum creatinine levels are often determined using a blood test. Increased amounts of creatinine in the blood may be a sign of renal disease or poor renal function[41].

The analysis of creatinine levels at different doses of Amlasia (0.5 ml per kg and 2.5 ml per kg) demonstrated no significant changes compared to the saline control group, suggesting no potential effects on kidney function.

When the body breaks down proteins, a waste product called blood urea nitrogen (BUN) is created. The kidneys filter BUN, which is then expelled in the urine. The quantity of BUN in the blood may be measured by blood tests, which provides crucial details regarding kidney function and general health. Increased BUN levels can be a sign of dehydration, renal disease, or other illnesses that affect the kidneys (24).

The assessment of BUN levels at two different Amlasia doses (0.5 and 2.5 ml per kg) showed notable differences from the saline control group, suggesting no possible effects on kidney function because the BUN level significantly dropped from the saline control group.

A waste product called urea is created in the liver as a result of the breakdown of proteins. It is filtered by the kidneys and excreted in urine. Blood urea levels can reveal important details about kidney function and general health. Elevated urea levels can be a sign of renal failure, dehydration, or other illnesses affecting the kidneys [42].

The examination of urea levels at varying doses of Amlasia (0.5 ml per kg and 2.5 ml per kg) indicated significant alterations when compared to the saline control group. These findings suggest no potential impact on kidney function, due to the level of urea decreased significantly from saline control group.

In human blood plasma, albumin makes up over 60% of the total protein composition, making it the most prevalent protein. Mostly produced by the liver, it is essential for preserving oncotic pressure, moving different materials (including hormones, fatty acids, and medications), and controlling the body's fluid balance. In many different medical diseases, albumin levels can offer crucial diagnostic and prognostic information. Low albumin levels, or hypoalbuminemia, can be a sign of chronic inflammation, malnourishment, liver illnesses (such cirrhosis or hepatitis), renal diseases, certain malignancies, or conditions that cause protein loss (like nephrotic syndrome or gastrointestinal problems)[43].

The analysis of albumin levels at different doses of Amlasia (0.5 ml per kg and 2.5 ml per kg) revealed no statistically significant changes when compared to the control group. These findings suggest that the administration of Amlasia at various doses did not significantly affect albumin levels in the experimental group.

GSH, or reduced glutathione, is the active form of glutathione that functions as an antioxidant. It is essential for keeping the cellular redox balance stable and shielding cells from oxidative damage. GSH helps the body detoxify toxic substances and is a potent scavenger of free radicals [44]. Numerous pathogenic ailments, such as liver diseases (like cirrhosis and hepatitis), neurological disorders (like Parkinson's and Alzheimer's disease), cardiovascular diseases, and several malignancies have been linked to a drop in GSH levels [45].

When compared to the saline control group, the analysis of reduced GSH content at different doses of Amlasia (0.5 ml per kg and 2.5 ml per kg) did not reveal any significant changes for either the renal or hepatic condition, indicating that these treatments had no discernible effect on reduced GSH levels.

Malondialdehyde, or MDA, is a marker for lipid peroxidation and oxidative stress in tissues and cells. It is a consequence of the process known as lipid peroxidation, in which MDA is produced when free radicals damage cell membranes. Increased oxidative stress in the body has been connected to a number of illnesses and the aging process; elevated MDA levels may be a sign of this stress [46].

The evaluation of MDA content at different doses of Amlasia (0.5 ml per kg and 2.5 ml per kg) did not reveal any significant changes compared to the saline control group. This suggests that the administration of Amlasia at various doses did not have a notable impact on MDA levels, indicating no significant alterations in lipid peroxidation or oxidative stress.

Catalase is an essential enzyme existing in the cells of nearly all living individuals. It is essential in preventing oxidative damage to cells because it catalyzes the conversion of hydrogen peroxide into oxygen and water. This procedure aids in keeping dangerous reactive oxygen species (ROS) from building up inside of cells [47].

When compared to the control group, the measurement of catalase activity at two different dosages of Amlasia (0.5 and 2.5 ml per kg) showed no discernible variations. This shows that there were no appreciable changes in the antioxidant capacity associated with catalase enzyme function following the ingestion of Amlasia at different dosages, suggesting that catalase activity was not affected.

Findings from histopathology can provide crucial insight into the underlying medical conditions affecting the kidneys and liver. For instance, in liver histology, liver illnesses like cirrhosis, hepatitis, or drug-induced liver damage may be indicated by the presence of necrosis, inflammation, or fibrosis. Abnormalities in the glomeruli, tubules, or interstitium in kidney histology might indicate a number of renal illnesses, such as acute tubular injury, chronic kidney disease, or glomerulonephritis [32].

Histological analysis of liver tissue at two different Amlasia dosages (0.5 and 2.5 ml per kg) did not show any appreciable differences from the control group. The hepatocytes were grouped in a typical manner, the central veins were undamaged, and the cytoplasm was visible. These results suggest that there were no appreciable histological alterations in the liver tissue following the treatment of Amlasia at different dosages.

In comparison to the control group, there were no appreciable differences seen in the histological examination of kidney tissue at different dosages of Amlasia (0.5 ml per kg and 2.5 ml per kg). The glomeruli, tubules, interstitium, and blood arteries in the kidney tissue were all intact, indicating a normal structural appearance. According to these findings, there were no discernible histological changes in the kidney tissue following the administration of Amlasia at various dosages.

The experimental results of the current study indicate that Amlasia at doses of 0.5 ml per kg and 2.5 ml per kg BW contains no hepato-renal toxicity and unfavorable health consequences in comparison to the saline control and paracetamol treated group. According to physical, biochemical from serum, histological, and oxidative stressed markers of liver and kidney tissue indicates the pharmacological safety condition of this herbal medication. In order to confirm the true safety and non-toxic state, these results require further validation using molecular biology analysis and the western blotting technique.

Conflicts of Interest:

There is no conflict of interest disclosed by the authors.

Author Contributions

Md. Murshid Alom contributed to the conceptualization, design, planning, investigation, data analysis, software implementation and manuscript writing. Ariful Islam and Nazmul Islam aided in planning, design, editing and review. Md. Khalekuzzaman and Rashed Zaman: contributed to planning and review. Md. Asadul Islam and Md. Rausan Zamir performed roles in planning, design, editing, reviewing, and handling correspondence related to the manuscript. The paper has been reviewed and approved by all authors in its final published version.

Funding

This research was supported by International Science Programme (ISP), Uppsala University, Sweden, conducted by BAN-05, Bangladesh.

Acknowledgements

The authors are grateful to the Department of Chemistry, University of Rajshahi, Rajshahi 6205, Bangladesh for their support during this research project and for granting them recognition.

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Evaluating The Adverse Health Effects of Herbal Medicine on Swiss Albino Mice: A Comprehensive Hepatotoxicity and Nephrotoxicity Analysis

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Abstract

Figures

1. Introduction

2. Materials & Methods

2.1 Animals and Housing Requirements

2.2 Ethical Permission

2.3 Grouping and Experimental Protocol

2.4 Measurement of Body Weight and Organ Weights

2.5 Liver Index

2.6 Kidney Index

2.7 Collection of Specimen

2.8 Assessment of Liver and Kidney Functions

2.9 Assessment of Hepatic and Renal Oxidative Stress

2.9.1 Reduced Glutathione (GSH) Contents

2.9.2 Standard Curve for Reduced GSH Contents

2.9.3 Determination of Lipidperoxidation

2.9.4 Standard Curve for Malondialdehyde (MDA)

2.9.5 Catalase (CAT) Activities

2.9.6 Calibration Curve for Catalase (CAT) Activities

2.10 Histopathological Investigations

2.11 Statistical Analysis of Data

3. Results

3.1 Measurement of body weights and organ weights

3.2 Liver and kidney index

3.3 Evaluation of liver and kidney functions

3.3.1 Alanine Transaminase (ALT)

3.3.2 Aspartate Transaminase (AST)

3.3.3 Alkaline Phosphatase (ALP)

3.3.4 Total Bilirubin

3.3.5 Triglyceride

3.3.6 Total Protein

3.3.7 Creatinine

3.3.8 Blood Urea Nitrogen (BUN)

3.3.9 Urea

3.3.10 Albumin (ALB)

3.4 Evaluation of hepatic antioxidant activity and oxidative stress

3.4.1 Reduced Glutathione (GSH) Content

3.4.2 Malondialdehyde (MDA) Content

3.4.3 Catalase activity

3.5 Evaluation of renal antioxidant activity and oxidative stress

3.5.1 Reduced Glutathione (GSH) Content

3.5.2 Malondialdehyde (MDA) Content

3.5.3 Catalase activity

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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