Histomorphological and Biochemical Effects of Ethanolic Extract of Monodora myristica Seed (African Nutmeg) on Some Liver Function Parameters Using Albino Wistar Rats

Aim: This study was carried out to investigate the effects of Monodora myristica on the physiological status of the liver using albino Wistar rats as a model. Study Design: This research was conducted at the Department of Human Physiology, Faculty of Basic Medical Sciences, University of Port Harcourt, Nigeria; between May and October, 2014. Methodology: Thirty six (36) albino Wistar rats weighing between 180 – 220 g were used in the study. They were grouped into 3 groups of 12 rats each (2 test groups and a control group). The animals were fed with standard feed and clean water. In addition, those of test groups 2 and 3 also received 400 mg/kg and 200 mg/kg of ethanolic extract of M. myristica seed respectively on daily Original Research Article basis for twenty eight days (four weeks). After each week of administration, three rats from each group were sacrificed, blood samples collected by cardiac puncture for biochemical analysis of some liver function parameters (alkaline phosphatase (ALP), alanine transaminase (ALT), aspartate aminotransferase (AST), serum total protein and serum albumin). The liver of the animals were also examined for histological study. Results: The results from the biochemical assay were subjected to statistical analysis and it showed a significant ( p <0.05) increase in ALT values, non significant increase in AST and ALP values, and a non significant effect on total serum protein and serum albumin. The histological study of the liver showed hepatocytes and periportal inflammations as well as cytoplasmic swelling, which are indications of alteration on the normal physiological status of the liver. Conclusion: Findings from this study showed that the extract produced an adverse alteration in the normal histomorphology and biochemical make-up of the liver. Therefore, the plant should be used with caution, especially when used for medicinal purposes.


INTRODUCTION
The use of medicinal plants as therapy for health conditions is an age long practice [1]. This usage has gained prominence worldwide over the last three decades and has been estimated that at present over two third of the developing countries' population relies on plant preparation as medicines to take care of their health needs [2]. This surge in the use of medicinal plants as therapy is believed to be due to their accessibility and affordability and the perceived failure of synthetic drugs in the treatment of some chronic diseases like hypertension, diabetes, arthrosclerosis etc. [2]. According to Akerele [3], about 65-80% of the world's population in developing countries, due to poverty and lack of access to modern medicine, depend essentially on plants and their formulations for their primary health care.
Medicinal plants and their formulations have continued to attract attention as a result of the strong speculations and belief that they are safe and very efficacious [4,5]. This strong speculation and assumption has led to the indiscriminate use of medicinal plants and their formulations especially in developing countries, like Nigeria [1].
M. myristica is a species of plant which belong to the family of Annonaceae [6]. M. myristica forms a large branching tree with a gray-barked trunk and reach 35 m high in nature. It has large leaves (35 cm long and 18 cm wide) at the end of its branches. The leaves are purple at first but turn a smooth deep green on the upper side with paler green underneath. They are prominently veined and the petiole is purplish [7]. This is widely distributed from Africa to Asia, Central and South America and Australia [8,9]. It is native to Central, East and West Africa [10]. M. myristica grows very well in the ever green forest of West Africa and in Nigeria are most prominent in the Southern part [11,12]. Its common names include; Calabash nutmeg, African nutmeg, False nutmeg, Jamaican nutmeg, while its local names include Ehuru or Ehiri (Igbo), Ariwo (Yoruba) [8], Erhe (Urhobo), Ehinawosin (Ikale), Uyengben (Edo) [10]. M. myristica seed is oblongoid in shape and pale brown in colour with a thin seed coat and hard kernel. Phytochemically, M. myristica seeds have been reported to contain secondary metabolites like saponins, tannins, flavonoids, glycosides, alkaloids and steroids [12][13][14]. They also contain minerals like potassium, sodium, magnesium, phosphorus and iron [12]. They have also been reported to contain amino acids like phenylalanine, tyrosine, arginine, glutamic acid, asparagines, vitamin C and E, and sugars [15,16]. M myristica has been used as herbal plant and spices since ancient times. It is used in the treatment of hemorrhoids, stomach ache, fiber pain, constipation [17,8]. And also to control passive uterine hemorrhage after childbirth [18,19,8]. It has also been associated with antisickling effectiveness [16]. As spices, the seeds are grinded and used in cooking pepper soup and stew [12,20]. The seeds are also used as an aromatic and stimulating addition to medicines and snuff. In Central African Republic, the seeds are used as condiment and drug in the treatment of headache and hypertension [21]. The direct action of these plant extracts is on the liver, which is central to drug metabolism [22].
The liver is the largest internal and very vital organ in the body, constituting about 2.5% of an adult's body weight. The liver plays an important role in maintaining blood glucose levels. It also regulates the circulating blood lipids by the amount of very low density lipoproteins (VLDLs) it secretes. Liver takes up numerous toxic compounds and drugs which may include medicinal plant formulations from the portal circulation [23]. Many drugs and metabolites are hydrophobic, the liver converts them into hydrophilic compounds and in the process some may adversely affect the liver. Such drugs or metabolites are said to be hepatotoxic, and their effects on the liver are determined by measuring the plasma concentration of some biochemical compounds and enzymes called the liver markers, produced by the liver. These liver markers include total serum protein, albumin, triglycerides, total cholesterol, high and low density lipoproteins, and liver enzymes like alkaline phosphatase, aspartate transaminase and alanine transaminase, etc.
Presently, some research studies have showed that some of these used medicinal plants adversely affect some vital organs in the body while exhibiting their therapeutic potentials [24]. Consequently, it has become imperative to ascertain the effects of plants used as herbs on the physiological status of vital organs. Hence, the objective of this study to investigate the effects of the ethanolic extract of M. myristica seed (African Nutmeg) on the physiological status of the liver using albino Wistar rats.

Study Design
This research was conducted at the Department of Human Physiology, Faculty of Basic Medical Sciences, University of Port Harcourt, Choba, Nigeria; between May and October, 2014.

Preparation of Ethanolic Extracts of Monodora myristica Seed
The ethanolic extract of M. myristica seeds was prepared according to Akinwunmi and Oyedapo [8]. The seeds of M. myristica were dehulled (the coat removed), and rid of bad seeds and dirt. Thereafter, the seeds were milled to fine powder using manual engine grinder (Model Corene, A.5 lander YCIA S.A). The milled sample of the plant were soaked in 5 L of 80% ethanol for 48 hours, thereafter filtered with Whatman No. 1 filter paper to separate the filtrate from the residue. The filtrate was concentrated under reduced pressure in a vacuum at 45°C using a rotary evaporator (Searl Instruments Ltd. England) into the ethanolic extract used in the study.

Experimental Design
Thirty six (36) male albino Wistar rats weighing (180 -220 g) were randomized into three (3) groups of 12 rats each. Group I served as the Control, and received water and normal feed only. Group II received 400 mg/kg of the extract in addition to water and feed. Group III received 200 mg/kg in addition to water and feed. The doses of the extract (400 mg/kg and 200 mg/kg) were chosen based on a previous work that determined the LD 50 of M. myristica seed to be >5000 mg/kg [26]. The extract was daily administered via oral route between 8 am to 10 am throughout the period of administration.
Three rats from each group were sacrificed after every seven days, that is, on day 8, day 15, day 22 and day 29 days. After each sacrifices, blood was collected by cardiac puncture into lithium heparin tubes for biochemical analysis, while the liver of each rat was collected via abdominothoracic dissection into plain tubes containing buffered formalin for histological study.

Biochemical Parameters
The collected blood samples were centrifuged at 5000 rpm for 10 minutes to obtain clear serum for the biochemical analysis. The serum supernatant was then carefully aspirated with needled syringe and stored in a plain sample tubes for biochemical analysis. The biochemical analysis for serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), serum alkaline phosphatase (ALP), total serum protein and serum albumin were done using Mindray Auto-analyzer machine (Model: BS -800M) in the laboratory of the Department of Chemical Pathology at the University of Port Harcourt Teaching Hospital following standard laboratory procedures.

Histopathological Examination
Histopathological examination was done following the method outlined previously [27]. Harvested organ (liver) from each animal were cut and fixed in buffered neutral formalin (10%). The tissues were dehydrated in ascending grades of ethanol (70%, 80%, 90%, 95% and 100%), cleared in 2 changes of Xylene, impregnated with 2 changes of molten paraffin wax, and finally embedded in wax. Tissue sections of 4-5 µm in thickness were cut with a microtome and stained with hematoxylin and eosin.

Statistical Analysis
The data were statistically analyzed using SPSS VERSION 20.0, and was analyzed for statistical significance by one way Analysis of Variance (ANOVA) followed by Dunnett's post-test for comparison with control group. P values less than 0.05 were considered to indicate statistical significance.

RESULTS
The result of serum ALT concentrations of rats administered 400 mg/kg and 200 mg/kg doses of the ethanolic extract of M. myristica is presented in Table 1. The result showed a dose dependent significant p<0.05 increase in the test groups in comparison with the control group.
The result of serum AST concentrations of rats administered 400 mg/kg and 200 mg/kg doses of the extract is presented in Table 2 above. The result showed a non significant p>0.05 increase in the test groups in comparison with the control group. The effects of 400 mg/kg and 200 mg/kg doses of the extract on serum ALP of rats is presented in Table 3. The result showed a non significant increase in the test groups in comparison with the control group.
The results of serum total protein and serum albumin of rats administered 400 mg/kg and 200 mg/kg doses of the extract are presented in Tables 4 and 5 respectively. The results showed non significant effects.

DISCUSSION
This study is aimed at investigating the effects of ethanolic extract of Monodora myristica on the physiological status of the liver using albino Wistar rats as models. Monodora myristica is a species of plant which belong to the family of Annonaceae [6]. This plant has been associated with different medicinal usage. It is used in the treatment of hemorrhoids stomach ache, fiber pain, and constipation [18,9]; control passive uterine hemorrhage after childbirth [19,20] and has also been associated with antisickling effectiveness [17]. The seeds are also used as an aromatic and stimulating addition to medicines and snuff [7,28]. In Central African Republic, the seeds are used as condiment and drug in the treatment of headache and hypertension [22].    Following some present studies showing that some of the commonly used medicinal plants negatively alters the physiological status of other vital organs of the body in the process of discharging their desired medicinal effects [1] [25], hence this study. The physiological status (functionality or health state) of the liver is mainly determine by measuring the plasma levels of some enzymes called the liver enzymes which include alkaline phosphatase (ALP), aspartate transaminase (AST) and alanine transaminase (AST), etc [6,29] and other biochemicals produced by the liver which include total serum protein, albumin, bilirubin, triglycerides, total cholesterol, high and low density lipoproteins, etc. Normally ALT and AST are mainly present and in high concentrations within the liver cells (hepatocytes), damage or destruction of the hepatocytes leads to the release of these enzymes into circulation thereby increasing their plasma levels [30]. Thus, increase in the plasma or serum level of any of these enzymes is an indication of hepatocytes damage. ALP is an enzyme in the cell lining of the biliary ducts of the liver. Its plasma level increases when liver bile duct obstruction is present or there is intrahepatic cholestasis or infiltrative diseases [31].
The result from the biochemical analysis of this study showed that the 400 mg/kg and 200 mg/kg dose of the extract significantly (p<0.05) increased the serum ALT, and in a dose dependent manner (Table 1). This increase is an indication of liver (hepatocytes) damage which may have resulted to the release of ALT from the damaged hepatocytes into blood circulation, as has been reported by previous works [6,29]. This result was supported by the histological study of the harvested liver which shows hepatocytes and periportal inflammations of varying degree as well as cytoplasmic swelling. These negative effects may have occurred as a result of metabolism of the extract by the liver which serves as the primary organ of biotransformation [32] and also agree with the theory of target organ toxicity [33].
Though, both administered doses cause general increase in the serum AST and ALP, but the increase was not significant as shown in Tables  2 and 3. However, this may indicate delayed effect since histological examination of the liver indicated the presence of hepatocytes inflammation and degeneration; periportal inflammation and hepatocytes fatty accumulations (fatty changes).
From the results, it was observed that the ethanolic extract of M. myristica do not have any effect on the total serum protein and albumin as shown in Tables 4 and 5.
The histological study of the harvested liver shows that the ethanolic extract of M. myristica negatively alters the physiological status of the liver in a dose and time dependent manner, as various degrees of hepatocytes and periportal inflammation and degeneration was observed.

CONCLUSION
This study reveals that Monodora myristica seed (at its medicinal doses) shows negative effects on the anatomical physiology of the liver. Therefore, we advised that caution should be taken in employing their medicinal effects. However, these findings only serve as a template for further studies to ascertain the biochemical components of the plant responsible for this impairment of the liver and their mechanism of actions.

CONSENT
It is not applicable.