Effect of Ethanolic Extract of Annona muricata Leaves on Inflammation, Seizure and Liver Enzymes Following Isoniazid Toxicity in Male Wistar Rats.Effect of Ethanolic Extract of Annona muricata Leaves on Inflammation, Seizure and Liver Enzymes Following Isoniazid Toxicity in Male Wistar Rats.

July 3, 2024

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Table of Contents

Greener Journal of Medical Sciences

Vol. 14(2), pp. 94-100, 2024

ISSN: 2276-7797

https://gjournals.org/GJMS

Article’s title & authors

**Effect of Ethanolic Extract of Annona muricata Leaves on Inflammation, Seizure and Liver Enzymes Following Isoniazid Toxicity in Male Wistar Rats.**

Emmanuel Nonso Ezeokafor¹, Roy Chinwuba Uchefuna¹, Ikechukwu Emmanuel Ebuka¹, Ogbuokiri Doris Kasarachi², Precious Ebisintei³, Enendu Angela Chika¹, Aralu Obed Chinwendu⁴, Okoye Kosisochukwu Peter¹, Chidinma Esther Okafor¹

^1. Department of Human Physiology, Nnamdi Azikiwe University, Nnewi Campus.

^2. Department of Anatomy, Nnamdi Azikiwe University, Nnewi campus.

^3. Department of Biological Sciences, University of Africa Toru-Orua.

^4. Department of Physiology, Faculty of Basic Medical Science, Abia state Uturu.

ARTICLE INFO	ABSTRACT
Article No.: 061824087 Type: Research Full Text: PDF, PHP. HTML, EPUB, MP3	Isoniazid is an antibacterial that works by inhibiting bacterial growth. It is most commonly used for the treatment of tuberculosis. The inflammation process is a complex defence mechanism of the body which occurs as a response to trauma, heat, chemicals, bacteria and/or virus. Annona muricata is a medicinal plant widely used in traditional medicine for its antibacterial, antioxidant and anti-inflammatory properties. This study investigated the effects of Annona muricata, a medicinal plant, on inflammation and toxicity following Isoniazid administration. Forty four male wistar rats were used in the study. Two hundred and fifty grams of dried Annona muricata leaf was macerated in 1000 mls of 95% absolute ethanol for 48-hours and was administered orally at a dose of 100 mg/kg, 500 mg/kg and 1000 mg/kg. Inflammation was induced by injecting 0.5 ml of egg white in the hind paws and was observed at an interval of thirty minutes. 150 mg/kg of Isoniazid was administered intraperitoneally thirty minutes after extract administration and then observed for two hours for convulsion and death. Data was analysed using ANOVA followed by post hoc Fisher’s LSD and values were considered significant at p<0.05. SEM. The findings of the study revealed a significant decrease in AST levels, liver weight, seizure and itching levels. It also demonstrated a significant decrease in paw sizes at 60-120 minutes. However, further experiments should be carried out to confirm the findings of this study and the mechanism behind the decrease in seizure and itching levels.
Accepted: 20/06/2024 Published: 03/07/2024
Corresponding Author Dr Emmanuel Nonso Ezeokafor* E-mail: e.ezeokafor@ unizik.edu.ng Tel: +234 803 098 7212
Keywords: Isoniazid, Annona muricata, Wistar Rats

INTRODUCTION

Inflammation is a complex process. It occurs as a response to trauma, heat, chemicals, bacteria or other phenomena and is mediated by a variety of electrically charged signal molecules produced locally by mast cells, nerve endings and platelets (Hards et al.,1994). Characteristics include; heat, redness, swelling, pain and loss of function. Inflammation is a part of the defence mechanism of the body however, it has the potential to become harmful and uncontrolled at times like in rheumatoid arthritis and many other autoimmune inflammatory conditions (Chen et al., 2017).

A convulsion occurs when nerve cell activity in the brain is disrupted, causing muscles to involuntarily contract and spasm, resulting in sudden, violent, and irregular movements of the body (Moawad, 2022). It may be associated with any number of medical conditions, including epilepsy, a head injury, severe fever, an inflammatory brain infection, exposure to toxins, and certain medications. Diagnosis by a doctor such as a neurologist or infectious disease specialist is required to determine the reason it has occurred and to ascertain the appropriate treatment plan (Stafstrom and Carmant, 2015).

Isoniazid is a drug of choice for the treatment of pulmonary and extrapulmonary tuberculosis, and is used for the chemoprophylaxis of tuberculosis. Typically it is used in combination with other antituberculosis agents to prevent or delay the appearance of resistance. It is one of the few drugs that is bactericidal for all populations of the tuberculosis organism. Isoniazid is a prodrug. It acts by inhibiting the formation of the mycobacterial cell wall. Isoniazid must be activated by KatG, a bacterial catalase-peroxidase enzyme in Mycobacterium tuberculosis (Suarez et al., 2009).

A plant is said to be medicinal when its biologically active compounds have shown considerable pharmacological activities such as antioxidant, antimicrobial, anti inflammatory, anticancer, antiviral, anti-allergic and vasodilatory properties (Rustaiyan et al., 2011). An example is Annona Muricata. The plant derived herbal compounds have a long history of clinical use, better patient tolerance and acceptance. Their high ligand binding affinity to the target introduces the prospect of their use in chemo preventive applications; in addition they are freely available natural compounds that can be safely used to prevent various ailments. Plants became the basis of the traditional medicine system throughout the world for thousands of years and continue to provide mankind with new remedies.

MATERIALS AND METHOD

Location of the study: This study was carried out in the animal house, department of Human Physiology, Faculty of Basic Medical Sciences, College of Health Sciences, Nnewi campus, Nnamdi Azikiwe University.

Materials: 44 male wistar rats, laboratory coat and gloves, rice dusts, centrifuge (search tech instruments, british standard) model 80-2, thermostat oven dhg-90 23a, pec medical, USA, rotary evaporator (digital) tt-52 techmel and techmel usa, thermostatic water bath, beakers and measuring cylinders, 2ml hypodermic syringes, plain bottles, electronic weighing balance, m-methlar model m311l, China, watt-man number1 filter paper, refrigerator (haier thermocool), cages, oral cannula, rat feeds (vital pelleted finisher), egg white, AISY Absorbent Cotton Wool(Hydrophilic), 95% Absolute Ethanol AR JHD: (Guangdang Guanghua Chemical factory,China), Chloroform AR JHD: (Guangdang Guanghua Chemical factory, China).

Plant Sample Collection and Identification: The leaves of soursop were harvested from a local farm in Okofia, Nnewi LGA of Anambra state. It was sent to the Botany Department Awka where it was identified by a botanist and the soursop leaves were registered as NAUH-04B. Shade dried leaves were pulverised into fine powder using an electric blender and stored in an air-tight container for further use.

Preparation of Ethanolic Extract of Annona Muricata: The Annona muricata leaves were harvested from a local farm in Okofia, Nnewi Local Government Area, Anambra State. The Annona muricata leaves were dried at an ambient temperature. The dried Annona muricata leaves were milled into a coarse form using a local grinder. Two hundred and fifty-(250) grams of the Annona muricata leaf was macerated in 1000mls of 95% absolute ethanol (JHD Chemicals, Guangdong, China) for 48-hours. It was then filtered using a clean porcelain cloth and further filtered using Filter paper (Whatman Qualitative Filter paper, No. 1, Sigma Aldrich; WHA1001042, USA). The filtrate obtained was concentrated using a rotary evaporator (Digital TT-52; Techmel&Techmel, USA), which was further dried using a laboratory oven (DGH-9023A, PEC MEDICAL, USA) at 45ºC into a gel-like form. The extract was preserved in a refrigerator (Haier Thermocool) for further usage. The extract procedure was done according to the method described by Attar and Abu-Zeid (2013).

Preparation of Isoniazid Solution and Induction of Convulsion

The effect of ethanolic Annona muricata leaves extract on Isoniazid -induced convulsion was assessed as described by (Corda et al., 1982). The animals were divided into four groups and were given (100, 500 and 1000 mg/kg) of ethanolic Annona muricata leaves extracted orally respectively. Thirty minutes later, the rats in all groups were given Isoniazid (150 mg/kg) intra peritoneally and were observed for two hours for convulsion and death.

Induction of Inflammation Using Egg White

Inflammation was induced by injection of 0.2ml of egg white in hind paw 30 minutes before administration of ethanolic extract of Annona muricata leaves; the size of individual hind paw was determined using vernier calliper, which was observed at 30 minutes interval for a period of 2 hours. The hind paw of the rats in group A, B, C, D and E were measured using a vernier calliper and recorded before the egg white was injected into the hind paw. This was done according to the method described by (Barung et al., 2021).

Experimental Animals

Experimental rats were purchased from the animal house in the department of Veterinary Medicine in the University of Nigeria (UNN) Nsukka. The animals weighed between 160-300g, and were randomly grouped and housed in steel cages and kept at room temperature. The rats had no history of drug consumption, that is; they had not been used for any investigation. The rats were put on standard rat’s pellet (feed) and pure drinking water and allowed to get acclimated for 21 days before the start of the experiment. The study was done in accordance with the guidelines for animal use of the Faculty of Basic Medical Sciences, Nnamdi Azikiwe University.

Experimental Design

Forty four male wistar rats were divided into two different research subgroups; anti-inflammatory subgroup and anti-convulsant subgroup. To check for anti-inflammatory properties of Annona muricata, twenty five male wistar rats were used while to check for the anticonvulsant properties of Annona muricata, twenty male wistar rats were used.

Anti-inflammatory effect of ethanolic extract of annona muricata leaves.

Twenty four male wistar rats were divided into six groups of four, the anti-inflammatory subgroups were designated as groups A, B, C, D, E and F.

· Group A (negative control): (0.2ml) egg white

· Group B (Positive control): Feed + distilled water (10 ml/kg).

· Group C (Low dose): (0.2ml) egg white + extract 100ml.

· Group D (Medium dose):(0.2ml) egg white + extract 200ml.

· Group E (High dose):(0.2ml) egg white + extract 400ml.

· Group F (Very high dose):(0.2ml) egg white+ extract 800 ml.

The hind paw of the rats in group B, C, D and E were measured using a vernier calliper and recorded before the egg white was injected.

Anticonvulsant effect of ethanolic extract of annona muricata leaves.

Twenty male wistar rats were divided into four groups of five, the anticonvulsant subgroup were designated as groups A, B, C, and D.

· Group A (negative control): (150mg) Isoniazid

· Group B (low dose): (150mg) Isoniazid + (extract 100ml for seven days).

· Group C (medium dose): (150mg) Isoniazid + (extract 500ml for seven days).

· Group D (high dose): (150mg) Isoniazid + (extract 1000ml for seven days).

RESULTS

Table 1: effect of ethanolic leaf extract of Annona muricata on liver enzymes (AST, ALT, and ALP) following Isoniazid induced hepatotoxicity

	Aspartate Transaminase (IU/L)	Alanine Transaminase (IU/L)	Alkaline Phosphatase (IU/L)
	MEAN±SEM	MEAN±SEM	MEAN±SEM
Group A (150 mg/kg of Isoniazid)	49.00±1.00	36.33±4.33	78.53±1.88
Group B (150 mg/kg of ISA + 100 mg/kg of EAML)	37.67±0.67^*	29.67±1.33^a	114.33±10.88^*
Group C (150 mg/kg of ISA + 500 mg/kg of EAML)	38.33±2.96^*	35.33±0.88^a	114.23±13.77^*
Group D (150 mg/kg of ISA + 1000 mg/kg of EAML)	37.33±1.67 ^*	38.00±5.29^a	84.76±5.55^a
F-value	9.74	1.06	4.22

Data was analysed using ANOVA followed by post hoc Fisher’s LSD and values were considered significant at p<0.05. SEM: Standard error of mean, significant (^*) and not significant (^a), EAML: ethanolic leaf extract of Annona muricata. ISA: Isoniazid

Table 1 results showed a significant decrease in groups B, C, and D (p=0.002, p=0.003, p=0.002) in AST level when compared to group A. The ALT level showed a non-significant decrease in groups B and C (p=0.216, p=0.845) and group D had a non-significant increase (p=0.746) compared to group A. The ALP result showed a significant increase in groups B and C (p=0.026, p=0.026) while group D had a non-significant increase (p=0.647) compared to group A.

Table 2 effect of ethanolic leaf extract of Annona muricata on seizure, itching, and relative brain weight following Isoniazid induced neurotoxicity

	Seizure (Frequency)	Itching (Frequency)	Relative brain weight (g)
	MEAN±SEM	MEAN±SEM	MEAN±SEM
Group A (150 mg/kg of Isoniazid)	45.00±2.88	95.00±2.88	0.70±0.01
Group B (150 mg/kg of ISA + 100 mg/kg of EAML)	26.67±1.67^*	78.00±1.53^*	0.82±0.01^a
Group C (150 mg/kg of ISA + 500 mg/kg of EAML)	14.00±0.57^*	47.33±1.45^*	0.79±0.01^a
Group D (150 mg/kg of ISA + 1000 mg/kg of EAML)	7.00±1.15^*	25.00±2.88^*	0.76±0.03^a
F-value	87.06	184.88	6.59

Table 2 results revealed a significant decrease in groups B, C, and D (p=0.001, p=0.010, p=0.000) in the seizure level when compared to groups A. The result of itching showed a significant decrease in groups B, C, and D (p=0.001, p=0.002, p=0.001) compared to group A. The relative brain weight showed a significant increase in groups B and C (p=0.003, p=0.014), and group D had a non-significant increase (p=0.059) compared to group A.

Table 3 effect of ethanolic leaf extract of Annona muricata on relative liver weight following Isoniazid toxicity

	Relative liver weight (g)
	MEAN±SEM
Group A (150 mg/kg of Isoniazid)	3.98±0.11
Group B (150 mg/kg of ISA + 100 mg/kg of EAML)	4.19±0.09^a
Group C (150 mg/kg of ISA + 500 mg/kg of EAML)	3.76±0.01^a
Group D (150 mg/kg of ISA + 1000 mg/kg of EAML)	3.43±0.15^*
F-value	10.04

Table 3 results showed the relative liver weight had a non-significant increase in group B (p=0.173), group C had a non-significant decrease (p=0.173) and group D indicated a significant decrease (p=0.001) compared to group A.

Table 4 effect of ethanolic leaf extract of Annona muricata on paw-size at 0 and 30 minutes following egg white induced inflammation

	0-minutes (cm)	30-minutes (cm)
	MEAN±SEM	MEAN±SEM
Group A (0.2 ml of Egg white)	2.40±0.17	4.00±0.05
Group B (positive control)	0.98±0.01*	1.08±0.01*
Group C (0.2ml of Egg white + 100 mg/kg of EAML)	2.53±0.06^*	3.93±0.12^a
Group D (0.2ml of Egg white + 200 mg/kg of EAML)	2.30±0.05^*	3.83±0.08^a
Group E (0.2ml of Egg white + 400 mg/kg of EAML)	2.53±0.08^*	3.93±0.12^a
Group F (0.2ml of Egg white + 800 mg/kg of EAML)	2.43±0.07^*	3.76±0.37^a
F-value	0.96	0.26

Table 4 revealed a significant increase in the paw size in groups A, C, D, E, and F (p=0.010, p=0.001, p=0.012, p=0.010, p=0.002) compared to group B at zero minutes. At 30 minutes, a non-significant decrease in the paw-size was revealed in groups C, D, E, and F (p=0.802, p=0.534, p=0.802, p=0.389) compared to group A, while group B had a significant increase compared to group A (p=0.010).

Table 5 effect of ethanolic leaf extract of Annona muricata on paw-size at 60, 90 and 120 minutes following egg white induced inflammation

	60-minutes (cm)	90-minutes (cm)	120-minutes (cm)
	MEAN±SEM	MEAN±SEM	MEAN±SEM
Group A (0.2 ml of Egg white)	3.76±0.07	3.63±0.08	3.60±0.15
Group B (positive control)	1.18±0.01*	1.18±0.01*	1.18±0.01*
Group C (0.2ml of Egg white + 100 mg/kg of EAML)	3.00±0.05^*	2.63±0.27^*	2.70±0.25^*
Group D (0.2ml of Egg white + 200 mg/kg of EAML)	2.90±0.21^*	2.50±0.15^*	2.33±0.18^*
Group E (0.2ml of Egg white + 400 mg/kg of EAML)	2.73±0.17^*	2.20±0.05^*	2.16±0.6^*
Group F (0.2ml of Egg white + 800 mg/kg of EAML)	2.80±0.30^*	2.60±0.30^*	2.36±0.26^*
F-value	5.08	7.38	8.52

Table 5 results showed a significant decrease in the paw size at 60-minutes in groups C, D, E, and F (p=0.015, p=0.008, p=0.003, p=0.004) compared to group A. However, group A showed a significant increase in the paw size compared to group B (p=0.020). At 90-minutes, a significant decrease in the paw size in groups B, C, D, and E (p=0.005, p=0.002, p=0.000, p=0.004) compared to group A. However, group A showed a significant increase in the paw size compared to group B (p=0.004). At 120 minutes, a significant decrease in the paw size in groups B, C, D, and E (p=0.009, p=0.001, p=0.000, p=0.001) compared to group A. However, group A showed a significant increase in the paw size compared to group B (p=0.012).

DISCUSSION

Inflammation is a complex biological response of the body to various pathological stimuli like pathogens and toxic compounds (Chen et al., 2017). Egg-white has been proven to induce inflammation in rat paws by causing edema formation however the references to explain the mechanism behind it remains inadequate. Researchers suspect that the proteins; ovalbumin (54%), ovotransferrin (12%), ovomucoid (11%), ovomucin (3.5%), and lysozyme (3.5%; Abeyrathne, Lee and Ahn, 2013) contained in egg white are the major allergens and serve as the trigger for an inflammatory response (Kim et al., 2015).

Annona muricata is a tropical plant widely characterised for its biological activities (antibacterial, anti-inflammatory, antioxidant, etc). Analysis of egg-white induced inflammation between 0-30 minutes in all groups when compared to the control group showed a significant increase in paw size. This supports the study by Barung, Dumanauw, Duri and Kalonio, 2021 which states the potency of egg-white as an edema inductor. No significant decrease was observed in the groups treated with A. muricata at 0-30 minutes however, a significant decrease was observed in all treated groups at 60-120 minutes. This decrease suggests the efficacy of A. muricata as an anti-inflammatory agent. The effect could be attributed to the flavonoids and terpenoids present in the fruit (Okeke-Nwolisa, Enweani-Nwokelo, Unekwe and Egbuonu 2023).

A seizure occurs due to an uncontrolled, abnormal electrical activity of the brain that may cause changes in the level of consciousness, behaviour, memory, or feelings (Huff and Murr, 2023). A. muricata has been used traditionally as an anticonvulsant and a sedative. Analysis of seizure and itching levels of groups treated with A. muricata after Isoniazid administration showed a significant decrease suggesting the effectiveness of the extract as an anticonvulsant. This agrees with the study by Souza et al., 2018 which indicates a possible interaction between the extract and the GABAergic and monoaminergic systems of the brain possibly as a result of the flavonoids and phenolic acids present.

AST, ALT and ALP are enzymes found in the liver and are markers for hepatocellular damage. AST serves as a more sensitive marker for liver damage because of its abundance when compared to ALT while ALT serves as a more specific marker due to its restricted tissue expression (Jorge, 2013). Analysis of liver enzyme levels of treated groups following Isoniazid induced toxicity showed a significant decrease in AST levels of all groups compared to the untreated group. This suggests the possibility of A. muricata as an ameliorative agent for hepatocellular damage. A significant increase was also observed in ALP levels of groups B and C when compared to group A however, the reason for this increase is not understood.

CONCLUSION

The study demonstrated that ethanolic extract of A. muricata significantly decreased paw size following egg white induced inflammation at 60-120 minutes. The mechanism of this decrease is linked to the flavonoids and terpenoids contained in the extract. A decrease in AST levels, liver weight, seizure and itching levels were also noticed suggesting the possibility of an anticonvulsant activity. The decrease in AST levels suggests that A. muricata could be hepatoprotective which can be attributed to the annonaceous acetogenins it contains. Further experiments should be carried out to confirm the findings of this study and the mechanism behind the decrease in seizure and itching levels.

Acknowledgement

The authors wish thank God for his goodness and appreciate everyone who contributed in one way or the other to ensure this project was a success.

Conflict of Interest

The authors declare no conflict of interests.

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Cite this Article:

Ezeokafor, EN; Uchefuna, RC; Ebuka, IE; Kasarachi, OD; Ebisintei, P; Enendu, AC; Aralu, OC; Okoye, KP; Okafor, CE (2024). Effect of Ethanolic Extract of Annona muricata Leaves on Inflammation, Seizure and Liver Enzymes Following Isoniazid Toxicity in Male Wistar Rats. Greener Journal of Medical Sciences, 14(2): 94-100.

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