Reversal Effects of N-Acetyl Cysteine on Moringa oleifera Leaves-Induced Sub-Acute Hepatotoxicity in Wistar Albino Rats

Background: M. oleifera is a highly valued medicinal plant used widely from time immemorial to treat various ailments. However, with continued un-stan-dardized use of the plant leaves, studies have reported organ toxicity to the liver, kidney and the heart. As communities continue to use M. oleifera leaves for its medicinal and nutritional values, there is need to find an antidote for its hepatotoxicity. Aim: The study established the reversal effect of N-Acetyl Cysteine (NAC) on M. oleifera aqueous leaf extract-induced hepatotoxicity in Wistar albino rats. Methods: Twenty-four (24) rats received a toxic dose (8.05 g/kg bwt) of M. oleifera leaf extract for 28 days to cause sub-acute hepatotoxicity. They were divided into 4 groups of 6 rats each. Group I received 1 ml normal (control group), Group II received 1000 ng/kg NAC, Group III received 1200 mg/kg NAC and Group IV received 1500 mg/kg NAC. Another group showed reduced inflammation after treatment with NAC for 3 and 7 days in the M. oleifera and paracetamol induced hepatotoxic rats. Conclusion: NAC can reverse M. oleifera leaf aqueous extract-induced sub-acute hepatotoxicity in Wistar Albino rats.


Introduction
Traditional medicines especially using medicinal plants still remain a key component in human life. However, there are growing concerns related to the safety and toxicities of these herbal medicines derived from the medicinal plants. These herbal medicines cause various organ toxicities such as liver damage with a high incidence of mortality and morbidity. Among the commonly used medicinal plants is Moringa oleifera (M. oleifera). It was initially grown in tropical & subtropical countries and more recently, worldwide [1]. It is part of a consortium of medicinal plants that 80% of the world's population depends on as first line therapy for disease alleviation [2]. In many regions of Africa, it is widely consumed as self-medication by patients with diabetes, hypertension, or HIV/AIDS [3]. All the plant parts are traditionally used for different purposes, but the leaves are generally the most used. The rural communities in Uganda, use its leaves to treat 24 common medical conditions, including diabetes mellitus, asthma, bronchitis, mastitis, skin conditions, worm infestations, and HIV/AIDS symptoms among others [4]. M. oleifera's leaves ability to treat most of these conditions has been attributed to the various phytochemical compounds present in the plant parts including its leaves [4]. They are reported to be a rich source of alkaloids and oxalates, and these have been reported to be toxic to the body including the liver [5], thus requiring other medications to reverse these toxic effects. Studies done earlier found out that the LD50 of M. oleifera leaves' aqueous extract was 16.10 g/kg [6]. A sub-acute toxicity profile done on rats whereby 1/2 lethal dose (8.05 g/kg), 1/4 LD50 (4.0 g/kg) and 1/16 LD50 (2.0 g/kg) of M. oleifera leaves aqueous extract was given intragastrically to different groups of rats for 28 days. They found hepatotoxicity, renal toxicity and heart toxicity in rats that received 1/2 lethal dose while the doses below that were not affected. It was concluded that a dose of 8.05 g/kg caused sub-acute organ toxicity [7]. The reversal of these hepatic toxic effects induced by phytochemical free radical can be achieved by use of antioxidants. Several antioxidants have been used with varying degrees of success. However, not surprisingly, N-acetyl cysteine (NAC), which supplies the cysteine necessary for glutathione synthesis, has proven more effective in treating disease-associated to reactive free radicals organ damage [8]. N-Acetyl cysteine belongs to a family of antioxidant compounds that are commonly used to manage a number of drug or disease-related toxicities, including glutathione defi-ciency and acetaminophen toxicity [9]. Although toxic levels of M. oleifera are known to cause organ toxicity, limited studies have examined the possibility of reversal of these effects. Moreover, no study has been done to document the effect of NAC in M. oleifera aqueous leaf extract induced hepatotoxicity. Therefore, this study established the reversal of N-acetyl cysteine on M. oleifera aqueous leaf extract induced-sub-acute hepatotoxicity in Wistar Albino rats.

Study design and setting
It was a laboratory-based experimental study whereby M. oleifera leaves were The laveas were air dried in a shade until constant weight was attained. They were pound using a motor and pestle into course powder for extraction.

Extracts preparation
The extraction process followed the already established extraction procedure on the plant materials [11]. About 300 g of M. oleifera leaf powder were weighed using an electronic weighing scale (Mettler PJ3000, Mettler-Toledo GmbH, Ockerweg, Germany), and then soaked in 1.3 litres of hot water (96˚C) to prevent fungal attacked and allowed to cool while being shaken at 3 hourly intervals for 12 hours. The resulting suspension was filtered using a Whatman No. 1 filter paper in a Buchner funnel. The filtrate was freeze dried at 32 Pa with an original temperature set at −47˚C and then maintained at 0˚C to dry the extract. A freeze dryer (Genesis 12 ES) in the Chemistry department laboratory at Makerere University was used to dry the extract. The extracts obtained were stored in an air tight bottle, wrapped in a silicon paper to prevent moisture and was dissolved in distilled water to make stock solution.
Normal saline was used as the negative control and paracetamol caused hepatotoxicity and was used as a positive control.

Animal handling
Thirty six disease-free Wistar albino rats aged between 6 and 8 weeks of which 24 were randomized into experimental groups. They were bred at the Makerere University College of Veterinary Medicine Animal Resources and Biosecurity Neuroscience & Medicine (CoVAB), animal house, transferred to Department of Medical Physiology animal house at Makerere University College of Health Sciences for the study. The rats were kept at standard laboratory conditions of temperature (25˚C ± 1˚C), relative humidity (45% -55%) having 12 hr light and 12 hr darkness. Standard commercial rat pellets and clean water were provided Ad libitum [10].

Dosing of animals
Twenty-four (24) rats received a toxic dose (8.05 g/kg bwt) of M. oleifera leaf extract for 28 days to cause sub-acute hepatotoxicity. They were divided into 4 groups of 6 rats each. Group I received 1 ml normal saline which acted as a control group, Group II received 1000 ng/kg NAC, Group III received 1200 mg/kg NAC and Group IV received 1500 mg/kg NAC. Another group of 6 rats (Group V) received 0.75 mg/kg Paracetamol to cause hepatotoxicity. For Group V which was a positive control received the prescribed clinical dose of 1200 mg/kg NAC which reverses the hepatotoxicity. All the NAC doses were given once a day intragastric for 7 days.
Reversal effects of NAC on M. oleifera sub-acute hepatotoxicity Groups II to IV were used to test reversal effect of ANC on M. oleifera leaves extract induced-hepatotoxicity and Group V rats were used to confirm the re- NAC was given intragastric once a day for 7 days. Blood was withdrawn from a tail vein and serum harvested on days 1, 3 and 7 for liver enzymes and bilirubin measurements.
On day 7 they were then sacrificed, blood taken from the heart puncture, centrifuged and serum harvested. Serum proteins, bilirubin, AST, ALP, and ALT were measured by the COBA-e-411 Clinical chemistry analyzer instrument (Roche diagnostics, Germany) using methods described by the manufacturer.

Histopathology of the liver
The liver tissues were harvested by soaking cotton wool in concentrated ethyl ether and putting it close to their nostrils which caused them to sleep for good.
The abdominal walls were opened and a piece of liver tissue was removed. They were placed into formalin buffer 10%, processed and sections of 5 mm thickness were prepared by a histopathologist, stained with Hematoxicylin and Eosin and examined under light microscope at ×10 maginification.

Data management & Analysis
Data was entered into a Microsoft Excel spread sheet and exported to Graph-Pad prism 8.0a Software (GraphPad Software Inc., California, USA) for analysis.
Comparisons of means and standard deviations of serum enzymes and serum bilirubin were made between day one against those of day 3 and 7 by the two-way The protocol was approved and numbered SBC-HDREC-575. Ethical practices that govern handling of laboratory animals were adhered to as per international biosafety guidelines and the guidelines for the care and use of laboratory animals (Gordon, 2001).

Results
In this study, the rats that were pre-treated with a toxic dose of 8.05 g/kg of M. oleifera leaf extract and those that received 0.75 mg/kg Paracetamol, showed a significant increase in mean serum liver enzymes and bilirubin levels on day 1. However by day 7 there was reduction in the mean serum liver enzymes and bilirubin      The serum proteins remained in the normal range of 73.30 ± 1.40 to 58.40 ± 4.30 g/L. This means that the liver toxicity developed did not affect the mean total serum proteins.

Histopathology of the liver
The liver histopathological changes stained with of Hematoxylin and Eosin (×10) of all the study animals that received 0.75 mg/kg paracetamol for 28 days were analyzed. The liver tissue had infiltration of lymphocytes around the portal triad ( Figure 5). After treatment with 1200 mg/kg NAC for 7 days, the liver tissue was normal with no lymphocytes infiltration ( Figure 6).  The liver histopathological changes stained with of Hematoxylin and Eosin (×10) of all the study animals that received 8.05 g/kg M. oleifera extract for 28 days were analysed. The liver tissue had infiltration of lymphocytes around the portal triad (Figure 7). After treatment with 1000 mg/kg NAC for 3 days, the liver tissue had mild infiltration of lymphocytes around the portal triad ( Figure   8) and had normal liver tissue with no infiltration of lymphocytes around the portal triad on day 7 (Figure 9).

Discussion
The findings from this study demonstrated that NAC has a capability to reverse the sub-cute hepatotoxicity caused by a toxic dose of M. oleifera extract in Wistar Albino rats. The mean serum liver enzymes (ALT, AST and ALP) and bilirubin levels in the rats treated with NAC following a M. oleifera leaf extract toxic dose, showed a significant (P < 0.05) reduction. The hepatotoxicity that was produced by paracetamol was also reversed. The findings were in agreement with previous studies that focused on the effects of NAC in a variety of biological and pathological processes especially in the management of paracetamol-induced hepatotoxicity [12] [9].

Serum liver enzymes
There was an elevation in liver enzymes (ALT, AST and ALP) in rats given a toxic dose 8.05 mg/kg of M. oleifera leaf extract for 28 days. These results were in agreement with several researchers who have reported perturbations in the activities of ALT, AST, and ALP in liver during M. oleifera leaf aqueous extractinduced hepatotoxicity [13] [14]. The liver is the most vulnerable organ in the body to toxic substances and chemicals and therefore it was affected by toxic levels of M. oleifera leaves aqueous extract with specific elevated transaminase enzymes; Alanine transaminase (ALT), Aspartate transaminase (AST) and Alkaline phosphatase (ALP) [14] [15] [16]. The elevated liver enzymes indicate inflammation or damage to cells in the liver. Inflamed or injured liver cells, leak higher amounts of chemicals, including enzymes such as ALP, AST and ALP, into the bloodstream, resulting into their elevated levels [17].
N-acetyl cysteine may have reversed oxidative stress that was caused by M. oleifera leaf extract, through increased glutathione synthesis, thus providing more substrate for the detoxification [18]. NAC may also have reduced the liver dam-Neuroscience & Medicine age by its ability to promote cell survival through activating extracellular signal-regulated kinase pathway plus directly modifying the activity of several proteins [11].
A rise in alkaline phosphatase indicates damage to the liver cells including the biliary system. Therefore, the increase in serum alkaline phosphate may be considered as a sensitive indicator of cholestasis which is also supported by significant increase in total bilirubin in rats that received M. oleifera aqueous leaf extract [14]. The reduction noted in ALP levels was because of antioxidant properties of NAC.
Serum total protein changes Liver synthesizes plasma protein including albumin, globulin and fibrinogen.
As there were no significant changes in total protein levels in control groups and treatment groups, the synthetic function of the liver was preserved.

Histopathological liver changes
Paracetamol induced-hepatotoxicity histopathological features show infiltration of lymphocytes around the portal triad ( Figure 5). On treatment with NAC for 7 days, the normal liver tissue was observed ( Figure 6). This indicated reversal of the paracetamol induced hepatotoxicity by NAC. In relation to the rats that received M. oleifera aqueous extract for 28 days, the liver tissue features of infiltration shown by of the lymphocytes around the portal triad, confirmed hepatotoxicity in the same way as what happened in the rats that received paracetamol ( Figure 7). This confirms that half LD50 of M. oleifera aqueous extract given orally to rats for 28 days causes hepatotoxicity as was reported by other researchers [7]. The group that received 1500 mg/Kg of NAC for 3 days had normal liver tissue with mild infiltration of lymphocytes around the portal triad ( Figure   8) and had normal liver tissue with no infiltration of lymphocytes around the portal triad (Figure 9). Xenobiotics and intermediates may have disturbed the redox balance and provoked excessive production of ROS in the hepatocytes, which oxidized lipids, proteins, DNA, and other macromolecules causing disruption of cell processes, thus injuring hepatocytes [18].
Due to the fact that M. oleifera leaves are taken by humans and animals due to its nutrition and medicinal values without standardization, there is a great chance of taking in toxic doses of the plant leaves. This study suggests an antidote for M. oleifera leaves over dose.

Conclusion
NAC reverses M. oleifera leaves aqueous extract-induced sub-acute hepatotoxicity in Wistar Albino rats.