Sarfo-Antwi F, Adam A. R. L, Larbie C, Emikpe B. O, Suurbaar J. Anti-Inflammatory and Antioxidant Potential of Ageratum conyzoides Ethanol Extract Against Carbon Tetrachloride-Induced Renal Toxicity in Rats. Biomed Pharmacol J 2025;18(1).

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Manuscript received on :14-09-2024
Manuscript accepted on :18-12-2024
Published online on: 27-01-2025

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Reviewed by: Dr. Hina Imran
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Frederick Sarfo-Antwi ^1*, Abdul-Rashid Lansah Adam ¹, Christopher Larbie ², Benjamin Obukowho Emikpe ³ and Jennifer Suurbaar ¹

¹Department of Biochemistry and Molecular Medicine, School of Medicine, University for Development Studies, Tamale, Ghana.

²Department of Biochemistry and Biotechnology, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.

³Department of Veterinary Pathology, School of Veterinary Medicine, Kwame Nkrumah University of Science and Technology, Kumasi, Ghana.

Corresponding Author E-mail: fsarfoantwi@uds.edu.gh

DOI : https://dx.doi.org/10.13005/bpj/3135

Abstract

Background: Kidney diseases remain a significant global health challenge, prompting researchers to explore the therapeutic potential of medicinal plants for treating various ailments, including renal disorders. This study aimed to evaluate the anti-inflammatory and antioxidant effects of the ethanolic extract of Ageratum conyzoides leaves (ESE) on kidney damage induced by carbon tetrachloride (CCl₄) in a rat model. Methods: Haematological and biochemical indicators were used to evaluate the nephroprotective efficacy. Rat kidney homogenates were used to assess antioxidant and anti-inflammatory biomarkers. The histological data was used to evaluate the amount of kidney injury. Results: Pro-inflammatory cytokine expression increases brought on by CCl₄ insult were dramatically and dose-dependently inhibited by ESE treatment. A significant reduction in lipid peroxidation of kidney tissue brought the concentration of defensive antioxidant enzymes to near-normal levels. The beneficial activities of ESE on CCl₄-induced hepatotoxicity damage were validated by histopathological analysis of the kidney; the maximum percentage of liver protection was observed when ESE was induced at a dosage of 500 mg/kg b.wt. Conclusion: According to the findings, ESE protects the kidneys against CCl₄-induced toxicity through anti-inflammatory and anti-oxidative effects. This response possibly may be due to the presence of phytochemicals.

Keywords

Biochemical; Cytokines; Homogenates; Kidney; Peroxidation

Introduction

The kidney is the main metabolic organ responsible for removing waste products, undesirable substances, and potentially dangerous substances from the human system¹.

Its high blood circulation and intricate cellular transport networks cause these compounds to accumulate inside nephron epithelial cells, making them highly vulnerable to the harmful effects of drugs and environmental pollutants².It is necessary for animal tissues to constantly cope and deal with highly reactive oxygen species (ROS), which are generated during various metabolic physiological processes. Examples of these radicals include hydrogen peroxide, hydroxyl radicals, superoxide anion, and others. It seems that life requires some free radical generation, however, the overproduction of free radicals could lead to oxidative stress. According to a study³, oxidative pathways associated with the cytochrome P450 enzyme system, which are involved in the hepatic metabolism of pharmaceuticals, are responsible for almost half of all cases of acute liver failure in Ghana. In several sectors, carbon tetrachloride (CCl₄) is a common chemical solvent. Hira and ⁴ reported that kidney damage brought on by CCl₄ is similar to damage brought on by other common causes of renal deterioration, including alcohol, chemicals, viruses, and autoimmune illnesses. Kidney disease is a very prevalent and burdensome condition, and plant-based treatments have shown to be promising. It has been discovered that many plants and their extracts possess antioxidant properties, which can help mitigate kidney damage by preventing processes like inflammation, fibrosis, and oxidative stress from occurring⁴.

Ageratum conyzoides (AC), is a persistent weed that is widespread in tropical and subtropical areas⁵. Despite its reputation as a weed, the nutritional and medicinal potential of A. conyzoides remains largely unexplored⁶. Analgesic, hemostatic, insecticidal, and antimicrobial properties have all been demonstrated for this Asteraceae family herbal plant⁷. In this study, we investigated the anti-inflammatory and antioxidant activities of Ageratum conyzoidesethanoic crude extract against carbon tetrachloride using a rat model. 

Materials and Methods

Plant Collection and authentication

The Northern Region of Ghana’s Nyankpala Reserve Forest was the source of the hand-picked A. conyzoides leaves. The leaves were verified by a and a voucher specimen (UDS/ HM57/ 2023 /L011) was deposited at the herbarium. They were properly cleaned with water three times, dry at room temperature, and ground to powdered form.

Preparation of extracts

One hundred grams of powdered sample of A. conyzoides leaves were macerated using 1000 ml of 99.99 % hydroethanolic . For 24 hours at 25°C, it was shaken continuously with a shaker (Gallenkamp, England). Centrifugation was employed to process the supernatant for 20 minutes at 1106 x g at ambient temperature. Following centrifugation, the supernatants were combined and concentrated using a rotary evaporator.

Preliminary Phytochemical analysis

The phytochemical components of A. conzyoides leaves powder and ethanolic crude extracts () were identified by applying conventional techniques, as stated by Donkor and colleagues⁸. The phytochemicals that were investigated included triterpenes, alkaloids, flavonoids, saponins, polyphenols, and tannins.

Evaluation

Forty-five (45) male rats, weighing between 120 and 150 g, were split up into 9 groups and treated for 7 days (n = 5). Table 1 shows the groups and treatments. The rats, which were acquired from the Noguchi Memorial Institute for Medical Research’s Animal Experimentation Unit, were housed at room temperature (25 ±1°C) and humidity (50–60%) for five days before the experiment. They also had a 12-hour light/day cycle maintained. Agricare, Kumasi, Ghana, provided them with a standard laboratory rat diet, and they had unrestricted access to clean . The study was approved by the University for Development Studies Institutional Review Board (UDSIRB) with an ethically approved number of UDSIRB/188/23.

The animals were given 50 mg/kg intraperitoneal pentobarbitone to induce anesthesia on the eighth day. Through cardiac puncture, blood was obtained into EDTA (ethylenediaminetetraacetic acid)-2K tubes for hematological examination. For biochemical analyses, portions of the blood were obtained into gel-separator tubes. Afterward, the rats were euthanized, and the kidneys were removed for examination by histopathologists. Antioxidant enzymes, oxidative stress indicators, and pro-inflammatory cytokines were all measured in kidney homogenates. The animals’ carcasses were interred in a specific location following institutional ethical standards in the area.

Table 1: Animal Groups and Treatments

Change in body weight of rats

The expression below was used to determine the body weights of rats after treatment:

Effect of extracts on rat kidney

The absolute organ weight (AOW) of the rat’s kidney was calculated and used to determine the Relative Organ Weight (ROW) as shown below: 

Hematological and Biochemical analyses

The animals’ hematological profiles were measured using an automated Sysmex XS-1000ianalyzer. Serum was extracted by centrifuging blood samples in activated gel tubes for 15 minutes at 1500x g after they had been allowed to clot. A completely automated Flexor E chemistry analyzer (VitalScientific, Japan) was used to carry out the biochemical analysis. Urea and creatinine were the twobiochemical markers that were examined. The following approach was used to calculate thepercentage of nephroprotection utilizing the primary kidney functional indicators, such as relativekidney weight, urea, and creatinine. 

Antioxidant and oxidative stress biomarkers

After homogenizing the liver (1.0 g) in 10 mL phosphate buffer (0.1 M, pH 7.4) and centrifuging at 10,000 rpm for 20 min at 4°C, the post-mitochondrial fraction was extracted (PMF). The supernatant was collected and utilized for antioxidant assays such as reduced glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), and myeloperoxidase (MPO) using methods as stated by Sarfo-Antwi and colleagues⁶.

Inflammatory cytokine assay

Using homogenized liver samples, measurement of anti-inflammatory proteins of specific cytokines, namely Tumour Necrosis Factor-alpha (TNF-α), Transforming Growth Factor-Beta1 (TGF-β1), Nuclear Factor Kappa B (NFk-β), and Cyclo-oxygenase 2 (COX-2), were tested using appropriate ELISA kits. TNF-α, NFkβ, and COX-2 ELISA kits were obtained from RayBio – tech, Inc. (Norcross, GA, USA) and TGF-β1 kits were from Aviscera Bioscience, Inc. (Santa Clara, CA, USA). 

Histopathological studies

The kidney sections were embedded in paraffin, fixed in 10% buffered formalin, and then cut into 4 m sections from each block. For histological analysis, the paraffin-embedded slices were stained with hematoxylin and eosin. Light microscopy (Olympus Manual System Microscope BX43) was used to conduct blinded examinations of each sample from each group while being seen at 100x and 400x magnification.

Statistical Analysis

Multiple comparison tests using Tukey were performed after a one-way ANOVA with GraphPad Prism. Statistical significance was established at the 5% level (p < 0.05), and the results were displayed as mean ± SEM.

Results

Preliminary Phytochemical screening

Table 2 below shows the phytochemicals present in the extract and raw powder. Alkaloids and tannins were the most abundant phytochemicals present in the extract.

Table 2: Phytochemical composition of raw powder and ESE

Key: Present (+); Absent (-). 

Body weight after Treatment

Rats treated with CCl₄-treated only, observed a substantive reduction in weight as shown in Table 3.

Table 3: Variation in Weight of Rats

Mean ± SEM (n=5) is used to express values. The significance level is “a” at p<0.05-0.001 relative to the normal group and “b” at p<0.05-0.001 when relative to the CCl₄-treated group alone.

Relative Weight of Organs Following Therapy

Figure 1 illustrates that in comparison to the normal group, the relative organ weight of animals treated with CCl₄ only increased significantly (p 0.05).

Figure 1: Treatment’s Impact on Relative Organ Weight. The results are presented as mean ± SEM (n=5). Statistical significance “a” between 0.05 and 0.001 in comparison to Normal; “b” between 0.05 and 0.001 in comparison to the CCl4-treated group only. Click here to view Figure

The impact of therapy on hematological parameters

The hematological indices were measured to assess the effect of the CCl₄, silymarin, and ESE on the rats. In general, therapy had no significant effect on the rats’ hematological indicators, as indicated in Table 4.

Table 4: Treatment’s Impact on Haematological Markers

The data are shown as mean±SEM (n=5). Significant differences between the Normal and CCl₄-treated groups were statistically indicated by “a” at p<0.05-0.001 and “b” at p<0.05-0.001 respectively.

The impact of remedies on serum urea and creatinine

Figure 2 shows that rats administered CCl₄ alone had substantially higher creatinine and urea levels than the normal group.

Figure 2:  Effects of treatments on serum creatinine and urea. The findings (n=5) are shown as mean ± SEM. Statistical significance: “a” compares to Normal at p<0.05-0.001, and “b” compares to the group that received only CCl4 at p<0.05-0.001. Click here to view Figure

Impact of therapy on percentage protection

Figure 3 depicts the kidney-protective impact of Silymarin and ESE at 100, 250, and 500 mg/kg b.wt. against CCl₄induction. ESE at 500 mg/kg b.wt provided the most effective therapy against CCl4 at 96%, surpassing standard silymarin at 93%.

Figure 3: shows the percentage of protection provided by silymarin and all doses of ESE against carbon tetrachloride. The individual bar represents the average percentage protection of the kidney biomarkers. Click here to view Figure

Treatments’ effects on antioxidant indicators

Table 4 presents the effects of therapy on oxidative stressors and renal antioxidants. When comparing the CCl₄-treated group to the Normal group, there was a notable rise in the oxidative stress biomarker (MDA) and a substantial decrease in the antioxidant assay (CAT, SOD, and GSH) following CCl₄ administration. Comparing the CCl₄ group to the co-treated groups with ESE at all dosages, however, revealed substantial (p<0.05) differences in antioxidant and oxidative stress biomarker levels.

Table 5: Effect of Treatment on Antioxidant Makers

The results are shown as mean ± SEM (n=5).  “a” represents the statistical significance when compared to the Normal group, and “b” represents the statistical significance when compared to the CCl₄-treated group only.

Treatment impact on inflammatory markers

Inflammatory markers were substantially higher in the group treated with only CCl₄ than in the normal group (p<0.05-0.001).

Figure 4: depicts the impact of therapy on inflammatory biomarkers. Data are presented as mean ± SEM (n=5). “a” indicates a significant distinction from the normal group (P < 0.05 – 0.001), while “b” indicates a significant variance from the CCl4-treated group.  Click here to view Figure

Impact of Therapy on Liver Histology

The kidneys of the normal group recorded no undesired morphological changes; nevertheless, the group receiving CCl₄only had significant diffuse tubular ectasia with coagulation necrosis. Animals who received 500 mg/kg bwt of ESE recovered almost normal architecture in place of these abnormalities as shown in Figure 4.

Figure 5: depicts the impact of therapy on kidney histology. (a) – (d) show no visible damage; (e) indicate diffuse tubular ectasia with epithelial coagulation necrosis; (f) – (h) show mild coagulation necrosis; (j) display no observable lesion.  Click here to view Figure

Discussion

Numerous articles have shown that a variety of mutagens and carcinogens can create peroxide radicals which can initiate the formation of cancer and other health disorders^9-12.

The current study sought to assess any potential safeguards provided by ESE against CCl₄-induced oxidative stress, which causes damage to the kidneys in rats. Phytochemical analysis of ESE and raw powder revealed the existence of alkaloids, phenols, flavonoids, triterpenes, tannins, as well as saponins. CCl₄ is a widely used, trusted, and well-known chemical that can harm the liver and kidney tissue^13-15. As expected, the CCl₄-treated group had a significant reduction in body weight at termination because it interfered with the animal’s normal metabolism of fat, protein, and carbohydrates. Additionally, a decrease in appetite directly affects body weight reduction. However, co-treatment with ESE at all dosages resulted in a significant increase in body weight. Figure 1 illustrates how co-treatment with extracts returned kidney weight to a nearly normal range, despite the possibility that the negative effects of CCl₄ caused the rats’ kidneys to enlarge and allow materials like fatty acids and glycerol to enter the kidney cells. According to past research, CCl₄ treatment increased several organs, including the kidney and liver^16-18.

The current investigation found that serum creatinine and urea levels were considerably higher in rats who received CCl₄ induction as shown in Figure 2. Lipid peroxidation caused a loss in membrane integrity and the accumulation of indicators like creatinine and urea in damaged kidneys brought on by CCl₄ intoxication. However, pre-treatment with silymarin and ESE at all doses resulted in a significant reduction in these markers.

This was confirmed by the histological observation, which revealed widespread tubular ectasia in the CCl₄ alone group together with coagulation necrosis of epithelium and kidney interstitial inflammation as shown in Figure 5. As seen in Figure 3, these results indicate that ESE and silymarin can protect nephrocytes from CCl₄-induced injury. The maximum protection effect of the kidney was achieved with pretreatment ESE at 500 mg/kg b.wt. Erythropoietin synthesis by the kidney is disrupted by CCl₄, which also alters erythrocyte viability and morphology¹⁹.In this study, the reduction of hemoglobin synthesis and the reduction of erythrocyte life may have contributed to the anemia associated with CCl₄ as seen in Table 4. Similar findings postulated that CCl₄ direct detrimental effects on the cell membrane were the reason for the rats’ shortened life span^20-23.

Additionally, the capacity of iron to generate hemoglobin in the mitochondria is inhibited by CCl₄^24-25. Free radical processes are known to be involved in the kidney damage caused by CCl₄²⁵. A significant rise in MDA levels and a significant reduction in antioxidant makers were seen in rats exposed to CCl₄, however, as Table 5 illustrates, rats who received both CCl₄ and ESE concurrently showed a considerable drop in MDA and an increase in antioxidant enzymes. Histological studies have demonstrated that low levels of antioxidant enzymes could cause oxidative stress and lipid peroxidation. In this study, pre-treatment with ESE at all dosages restored normal levels of TNF-α, TGF-β1, NF кβ1, and COX-2, which were significantly elevated owing to CCl₄ toxicity. These outcomes align with a previous investigation on CCl₄ poisoning, whereby molecular discoveries demonstrated increased quantities of TNF-α, TGF-β1, COX-2, IL-17, and IL-23, in addition to overexpression of NF кβ in the hepatic tissues²⁶.

-inflammatory cytokines including TNF-α, TGF-β1, COX-2, IL-17, and IL-23 are produced through the mitogen-activated protein kinase (MAPK) pathway, which is reported to be activated by CCl₄^27-29.Furthermore, CCl₄ participates in the activation of NF-кβ, which regulates the production of several genes linked to inflammation, including adhesion molecules, chemokines, and pro-inflammatory cytokines³⁰.Researchers in this study discovered that lowering the amounts of pro-inflammatory cytokines in kidney homogenates after ESE treatment at all dosages also reduced inflammatory responses. The anti-inflammatory mechanism of ESE therapies is suggested by their reported down-regulation of NF-κβ expression along with associated cytokines and chemokines, hence providing a nephroprotective effect. The protective impact of kidneys through inflammation suppression is indicated by positive regulation of TNF-α production by ESE. Nephrocytes are known to express cyclooxygenase 2 (COX-2), which both attenuates and stimulates cell proliferation in acute kidney damage and only occurs under conditions of prolonged aggressiveness. Furthermore, inflammatory neurotoxicity has been linked to COX-2 activation after lead exposure^31-32.

According to the current study, CCl₄ causes over-production of COX-2 in kidney tissues, which in turn causes excessive inflammation (together with NF-κβ and TNF-α). However, COX-2 expression was downregulated when co-treated with ESE and the standard drug, silymarin. This provides more proof of the extract’s anti-proliferative ability in nephroprotection. The study’s findings demonstrated that ESE had a protective effect at all dosages, with the highest protection occurring at 500 mg/kg b.wt. The biochemical and histological data suggest that ESE is nephroprotective, which is likely due to the phytochemicals present.

Conclusion

Our findings validated the antioxidant characteristics of ESE and showed, for the first time, that these benefits are linked to kidney protection through the suppression of anti-inflammatory cytokines. These findings corroborate previous research that suggested medicinal plants were a good substitute for kidney protection. We concluded that ESE could be essential for its economic use and perhaps further study could look into separating the pure bioactive chemicals that give its medicinal effect.

Acknowledgment

The authors are grateful to the University for Development Studies for providing the laboratory to support this study.

Funding Sources

The author(s) received no financial support for the research, authorship, and/or publication of this article. 

Conflict of Interest

The author(s) do not have any conflict of interest.

Data Availability Statement

This statement does not apply to this article.

Ethical statements

The study was approved by the University for Development Studies Institutional Review Board (UDSIRB) with an ethically approved number of UDSIRB/188/23

Informed Consent Statement

This study did not involve human participants, and therefore, informed consent was not required.

Clinical Trial Registration

This research does not involve any clinical trials

Author Contributions

Conceptualization and methodology: Frederick Sarfo-Antwi, Christopher Larbie, and Abdul-Rashid Lansah Adam. Formal analysis and investigation: Frederick Sarfo-Antwi, Benjamin Obukowho Emikpe, Christopher Larbie and Abdul-Rashid Lansah Adam. Writing of the original draft, review, and editing: Frederick Sarfo-Antwi,  Rashid Lansah Adam and Jennifer Suurbaar. All authors approved the final manuscript.

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