Effect of Ethanol Fruit Peel Extract of Mangifera Indica on Liver Enzymes of Rats Treated With Methotrexate

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Effect of Ethanol Fruit Peel Extract of Mangifera Indica on Liver Enzymes of Rats Treated With Methotrexate

Medicinal plants have been used for treatment of illness and diseases since the dawn of time. Ethnobotany, the study of medicinal plants refers to plants that possess therapeutics purposes or exert pharmacological effect in the human system. Indigenous culture (Wiart, 2006) (e.g African and Native American) used herbs in their healing ritual while others develop traditional medicinal system in which herbal therapies were used.

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Recently, the World Health Organizations estimated that three quarters of plants provide active ingredients for drugs (WHO, 2005). Examples of some medicinal plants include: Elderberry (Sambucus nigra) berries and leaves have been traditionally used to treat pain, swelling, infections, coughs, and skin conditions and, more recently, flu, common cold, fevers, constipation, and sinus infections (Hasan, 2009). The hematological properties of horsetail (Equisetum arvense) have been utilized to stop bleeding, heal ulcers and wounds, treat tuberculosis and kidney problems. Arnica (Arnica montana) is used as an anti-inflammatory (Ernst, 2011) and for osteoarthritis.

Mangifera indica, commonly known as mango is of a genus of about 30 species in the family of Anacardiaceae of an erect tree of 10-30 metres high distributed throughout tropical and sub tropical regions native to Southern Asia especially eastern India (Gepts, 2009). Some of the ethnomedicinal uses of the extract of root, fruit and leaves of Mangifera indica include anti ulcer, anti inflammatory, anti hemorrhagic, anti cancer, hepatoprotective and also used in the treatment of kidney damage (Gamido, 2004). All these medicinal properties are made possible by the chemical composition of the fruit of the plant (mangifera indica) known as phytochemicals. Many of these phytochemicals are present in mango peel and pulp, these include intriterpene, lupeol which is under basic research for its potential biological effects. An extract of mango branch bark called Vimang, containing numerous polyphenols, has been studied in elderly humans. Mango peel pigments under study include carotenoids, such as the provitamin A compound, beta-carotene, lutein and alpha-carotene and polyphenols, such as quercetin, kaempferol, gallic acid, caffeic acid, catechins and tannins. Mango contains a unique xanthonoid called mangiferin. Phytochemical and nutrient content appears to vary across mango cultivars. Up to 25 different carotenoids have been isolated from mango pulp, the densest of which was beta-carotene, which accounts for the yellow-orange pigmentation of most mango cultivars. Mango leaves also have significant polyphenol content, including xanthonoids, mangiferin and gallic acid. These phytochemical and mineral substances explain its pharmacological properties. The Mangifera indica fruit affects most part of the immune system especially the liver. Liver enzymes are a helpful screening tool, which are an effective modality to detect hepatic dysfunction (Rosen, 2000). Liver disease is often reflected by biochemical abnormalities of 1 of 2 different hepatic systems or of liver function. Although tests that measure the level of serum liver enzymes are commonly referred to as liver function tests, they usually reflect hepatocyte integrity or cholestasis rather than liver function.

Methotrexate is a drug used in the treatment of cancer and rheumatoid arthritis; it is thought to affect cancer and rheumatoid arthritis by two different pathways. For cancer, methotrexate competitively inhibits dihydrofolate reductase. For the treatment of rheumatoid arthritis, multiple mechanisms appear to be involved including: the inhibition of enzymes involved in purine metabolism, leading to accumulation of adenosine; inhibition of T cell activation and suppression of intercellular adhesion molecule expression by T cells (Wessels et al., 2008). Methotrexate hepatotoxicity is a common complication of long-term treatment with methotrexate. It is associated with mild liver enzyme elevation and related to the duration of therapy (Diouf et al., 2001).

The aim of this research is to investigate the effect of ethanol fruit peel extract of Mangifera indica on liver enzyme activity of albino rats treated with methotrexate.

1. Determination for the activity of serum of AST
2. Determination for the activity of serum of ALP
3. Determination for the activity of serum of ALT


Overview of Medicinal Plants
The use of medicinal plants and plant extracts for their healing powers can be traced to earliest of myths, traditions and writings used to codify those plants that can ease pain and treat diseases. The evolution of these plant-based medicine systems, primarily based on plants within a local area, produced the well known traditional medicine systems (Padulos et al., 2002). Large human population in developing countries is dependent on plant resources for healthcare because allopathic medicine can cure a wide range of diseases, but its high prices and occasional side-effects are causing many people to return to medicinal plants which tend to have fewer side effects. In last few decades, traditional knowledge on primary healthcare has been widely acknowledged across the world. It is estimated that 60% of the world population and 80% of the population of developing countries rely on traditional medicine, mostly plant drugs, for their primary health care needs (Shrestha, 2003). It is estimated that there are about 350,000 species of existing plants (including seed plants, bryophytes, and ferns), among which 287,655 species have been identified as of 2004 (Humber, 2002).Medicinal plants, also called botanical medicine, phytomedicine, or phytotherapy, refers to herbs, herbal materials, herbal preparations, and finished herbal products that contain parts of plants or other materials as active ingredients. The plant parts used in herbal therapy include seeds, berries, roots, leaves, fruits, bark, flowers, or even the whole plants.
Although written records about medicinal plants dated back at least 5,000 years to the Sumerians, who described well-established medicinal uses of plants. With the advent of “conventional” medicine over the past century, medicinal plants has been challenged by practitioners of mainstream medicine because of the lack of scientific evidence in the context of contemporary medicine, despite its long history of effective use. In recent years, there has been a resurgence of the use of plants due to the side effects of chemical drugs, lack of curative modern therapies for several chronic diseases, and microbial resistance (Pan et al., 2010).


Description of Mangifera indica
Mango trees grow up to 35–40 m (115–131 ft) tall, with a crown radius of 10 m (33 ft). The trees are long-lived, as some specimens still fruit after 300 years In deep soil, the taproot descends to a depth of 6 m (20 ft), with profuse, wide-spreading feeder roots; the tree also sends down many anchor roots, which penetrate several feet of soil. The leaves are evergreen, alternate, simple, 15–35 cm (5.9–13.8 in) long, and 6–16 cm (2.4–6.3 in) broad; when the leaves are young they are orange-pink, rapidly changing to a dark, glossy red, then dark green as they mature. The flowers are produced in terminal panicles 10–40 cm (3.9–15.7 in) long; each flower is small and white with five petals 5–10 mm (0.20–0.39 in) long, with a mild, sweet odor suggestive of lily of the valley. Over 400 varieties of mangoes are known, many of which ripen in summer, while some give double crop. The fruit takes three to six months to ripen. The ripe fruits varies in size and color. Cultivars are variously yellow, orange, red, or green, and carry a single flat, oblong pit that can be fibrous or hairy on the surface, and which does not separate easily from the pulp. Ripe, unpeeled mangoes give off a distinctive resinous, sweet smell. Inside the pit 1–2 mm (0.039–0.079 in) thick is a thin lining covering a single seed, 4–7 cm (1.6–2.8 in) long. The seed contains the plant embryo. Mangoes have recalcitrant seeds; they do not survive freezing and drying. The large, flattened, kidney-shaped central stone contains one or more large, starchy embryos, and can constitute up to 20% of fruit weight.

Fig 1: Ripe and unripe mango fruits (Meskin and Mark, 2014)

Scientific Classification of Mangifera indica
Kingdom Plantae
Subkingdom Trachebionta
Superdivision Spermatophyta
Division Magnoliophyta
Class Magnoliopsida
Subclass Rosidae
Order Sapindales
Family Anacardiaceae
Genus Mangifera
Species Indica
(Natural plant Database, 2004)

Common Name of Mangifera Indica
Mangifera indica has its common name as mango in English, manga in Arabics, mangue in French, mangobaum in Germany and Pabo in Philippines.

For thousands of years in South Asia mangoes have been cultivated and reached Southeast Asia between the fifth and fourth centuries BCE. The mango is now cultivated in most frost-free tropical and warmer subtropical climates; almost half of the world’s mangoes are cultivated in India alone, with the second-largest source being China. Mangoes are also grown in Andalusia, Spain (mainly in Málaga province), as its coastal subtropical climate is one of the few places in mainland Europe that permits the growth of tropical plants and fruit trees. The Canary Islands are another notable Spanish producer of the fruit. Other cultivators include North America (in South Florida and California’s Coachella Valley), South and Central America, the Caribbean, Hawai’i, South, West, and Central Africa, Australia, China, South Korea, Pakistan, Bangladesh, and Southeast Asia. Though India is the largest producer of mangoes, it accounts for less than 1% of the international mango trade; India consumes most of its own production. In 2013, world production of mangoes (data including mangosteens and guavas) was nearly 43 million tonnes.

Medicinal Uses of Mangifera Indica
The juice of fresh bark has a marked action on mucous membranes, in menorrhea, leucorrhoea, bleeding piles and diarrhea. The leaves are also useful in vitiated conditions of cough, hiccup, hyperdipsia, burning sensation, hemorrhages, haemoptysis, haemorrhoids wounds, ulcers, diarrhea, dysentery, pharyngopathy, scorpion string (Nuñez-Selles, 2005). The unripe fruits are acidic, acrid, refrigerant, digestive and carminative. They are useful in dysentery ophthalmia, eruptions, urethrorrhoea and vaginopathy. The ripe fruits are refrigerant, sweet, laxative, cardiotonic and haemostatic. They are also used in anorexia, dyspepsia, cardiopathy, haemoptysis, haemorrhages from uterus, lungs and intestine emaciation, and anemia (Godfrey, 2007). The pharmacological uses of Mangifera indica includes antibacterial, anti malaria, osteoporosis prevention, recognition of memory, cardio protective, immunoregulator and brochodilatory (Gbeassor et al., 2005).

Liver Parameters
Liver, the largest organ of human body is saddled with the responsibility of detoxifying chemicals and other xenobiotics by inactivating and metabolizing those substances (Izunya et al., 2010).
Liver enzymes are used to assess liver functions or liver injury. An initial step in detecting liver damage is a simple blood test to determine the level of certain liver enzymes in the blood. Elevated liver enzymes may indicate inflammation or damage to cells in the liver (Dufour et al., 2000). Inflamed or injured liver cells leak higher than normal amount of chemicals including liver enzymes, into the blood stream which can result in elevated liver enzymes on blood test. Elevated liver enzymes may be discovered during routine blood testing. It can be mild or temporarily elevated. Analyses of these liver enzymes activities in blood serum give diagnostic information for several disease conditions. Alanine aminotransferase and aspartate aminotransferase are important in the diagnosis of heart and liver damage caused by heart attack drug toxicity, or infection. After a heart attack, a variety of enzymes, including these aminotransferases leak from injured heart cells into the bloodstream (Nelsonand Cox, 2005).


Aspartate transaminase
Aspartate transaminase (AST) or aspartate aminotransferase is a pyridoxal phosphate (PLP)-dependent transaminase enzyme (EC AST catalyzes the reversible transfer of an a-amino group between aspartate and glutamate and, as such, is an important enzyme in amino acid metabolism. AST is found in the liver, heart, skeletal muscle, kidneys, brain, and red blood cells. Serum AST level, serum ALT (alanine transaminase) level, and their ratio (AST/ALT ratio) are commonly measured clinically as biomarkers for liver health.

Functions of Aspartate Transaminase
Aspartate transaminase catalyzes the interconversion of aspartate and a ketoglutarate to oxaloacetate and glutamate.
Aspartate (Asp) + a-ketoglutarate ? oxaloacetate + glutamate (Glu) as a prototypical transaminase AST relies on PLP (Vitamin B6) as a cofactor to transfer the amino group from aspartate or glutamate to the corresponding ketoacid. In the process, the cofactor shuttles between PLP and the pyridoxamine phosphate (PMP) form (Kirsch et al., 1984).
The amino group transfer catalyzed by this enzyme is crucial in both amino acid degradation and biosynthesis. In amino acid degradation, following the conversion of a-ketoglutarate to glutamate, glutamate subsequently undergoes oxidative deamination to form ammonium ions, which are excreted as urea. In the reverse reaction, aspartate may be synthesized from oxaloacetate, which is a key intermediate in the citric acid cycle (Berg et al., 2006).


Clinical significance of Aspartate transaminase
ALT is a more specific indicator of liver inflammation than AST, as AST may be elevated also in diseases affecting other organs, such as myocardial infarction, acute pancreatitis, acute hemolytic anemia, severe burns, acute renal disease, musculoskeletal diseases, and trauma (Hayashi et al., 2003). AST was defined as a biochemical marker for the diagnosis of acute myocardial infarction in 1954. However, the use of AST for such a diagnosis is now redundant and has been superseded by the cardiac troponins. (Gaze, 2007)AST is commonly measured clinically as a part of diagnostic liver function tests, to determine liver heart. However, it is important to keep in mind that the source of AST (and, to a lesser extent, ALT) in blood tests may reflect pathology in organs other than the liver. In fact, when the AST is higher than ALT, a muscle sourcing of these enzymes should be considered. For example muscle inflammation due to dermatomyositis may cause AST>ALT. This is a good reminder that AST and ALT are not good measures of liver function because they do not reliably reflect the synthetic ability of the liver and they may come from tissues other than liver (such as muscle).Laboratory tests should always be interpreted using the reference range from the laboratory that performed the test. Example of reference ranges is 8-40IU/L for male and 6-34IU/L for female (Keith and John, 2010).

Alanine Transaminase
Alanine aminotransaminase (ALT) is a transaminase enzyme. It is also called alanine aminotransferase (ALAT) and was formerly called serum glutamate pyruvate transaminase (SGPT) or serum glutamicpyruvic transaminase (SGPT). ALT is found in plasma and in various body tissues, but is most common in the liver. It catalyzes the two parts of the alanine cycle. Serum ALT level, serum AST (aspartate transaminase) level, and their ratio (AST/ALT ratio) are commonly measured clinically as biomarkers for liver health. The tests are part of blood panels.

Functions of Alanine Transaminase
ALT catalyzes the transfer of an amino group from L-alanine to a-ketoglutarate, the products of this reversible transamination reaction being pyruvate and L-glutamate.ALT and all transaminases require the coenzyme pyridoxal phosphate, which is converted into pyridoxamine in the first phase of the reaction, when an amino acid is converted into a keto acid (Wang et al., 2012).

Clinical significance of Alanine Transaminase
ALT is commonly measured clinically as a part of a diagnostic evaluation of hepatocellular injury, to determine liver health. When used in diagnostics, it is almost always measured in international units/liter (IU/L) (Ghouri et al., 2010). While sources vary on specific reference range values for patients, 10-40 IU/L is the standard reference range for experimental studies. (Wang et al., 2012).Test results should always be interpreted using the reference range from the laboratory that produced the result. However typical reference intervals for ALT are: Significantly elevated levels of ALT (often suggest the existence of other medical problems such as viral hepatitis, diabetes, congestive heart failure, liver damage, bile duct problems, infectious mononucleosis, or myopathy, so ALT is commonly used as a way of screening for liver problems. Elevated ALT may also be caused by dietary choline deficiency. However, elevated levels of ALT do not automatically mean that medical problems exist. Fluctuation of ALT levels is normal over the course of the day, and they can also increase in response to strenuous physical exercise (Paul et al., 2004).
When elevated ALT levels are found in the blood, the possible underlying causes can be further narrowed down by measuring other enzymes. For example, elevated ALT levels due to hepatocyte damage can be distinguished from bile duct problems by measuring alkaline phosphatase. Also, myopathy-related elevations in ALT should be suspected when the aspartate transaminase (AST) is greater than ALT; the possibility of muscle disease causing elevations in liver tests can be further explored by measuring muscle enzymes, including creatine kinase. Many drugs may elevate ALT levels, including Zileuton, omega-3-acid ethyl esters (Lovaza) (Watkins et al., 2006), anti-inflammatory drugs, antibiotics, cholesterol medications, some antipsychotics such as risperidone, and anticonvulsants. Paracetamol may also elevate ALT levels (Watkins et al., 2006).

Alkaline Phosphatase
ALP is nonspecific enzyme which hydrolyzes aliphatic, aromatic or heterocyclic compounds; the pH optimum for the reaction is from 9 to 10. It is activated by magnesium and manganese. Zinc is a constituent ion of ALP. It is produced by osteoblasts of bone and is associated with the calcification process. It is localized in cell membranes (ecto-enzymes) and is associated with transport mechanisms in liver, kidney and intestinal mucosa (Vasudevan and Sreekumari, 2010).

Clinical Significance of Alkaline Phosphatase
Alkaline phosphatase (ALP) is an enzyme in the cells lining the biliary ducts of the liver. ALP levels in plasma rise with large bile duct obstruction, intrahepatic cholestasis, or infiltrative diseases of the liver. ALP is also present in bone and placental tissue, so it is higher in growing children (as their bones are being remodelled) and elderly patients with Paget’s disease. In the third trimester of pregnancy, ALP is about two to three times higher (Nageh et al., 2003).


Mechanism of Action of Methotrexate
Methotrexate, as a folic acid antagonist, which is widely used as a cytotoxic chemotherapeutic agent in the treatment of various malignancies such as acute lymphoblastic leukaemia as well as in the treatment of various inflammatory diseases blocks the synthesis of purines and pyrimidines, suppresses transmethylation reactions with accumulation of polyamines, reduces antigen-dependent T-cell proliferation, and promotes adenosine release by inhibiting several key enzymes :Inhibition of dihydrofolate reductase (DHFR), decreases tetrahydrofolate (THF) levels, and other folate dependent enzymes, such as thymidylate synthetase and 5-aminoimidazole-carboxamide-ribonucleotidetransformylase (AICAR) which results in attenuated DNA/protein/lipid methylation. The effect on purine and pyrimidine biosynthesis is also responsible for much toxicity of methotrexate, including bone marrow suppression, liver toxicity, and stomatitis (Chan et al., 2002). It was recently showed that methotrexate increases adenosine release, inhibits adenosine deaminase and, finally, inhibits neutrophil chemotaxis. This mechanism probably acts through methotrexate’s inhibition of AICAR at nanomolar concentrations. Other potential mechanisms of action include normalization of low interleukin-2 (IL-2) through an effect on polyamine synthesis, reduced IgM- rheumatoid factor (RF) production, decreased interleukin-1 (IL-1) production, secretion or binding, and decreased interleukin-6 (IL-6)activity (Pincus et al., 2003).

Effect of Methotrexate on the Liver Enzymes
Methotrexate used for cancer chemotherapy is well known to produce a toxic side effect in multiple organ systems. Methotrexate exerts its primary toxic effects against the rapidly replicating cells of the bone marrow and gastrointestinal epithelium producing leucopoenia and thrombocytopenia (Kim et al., 1999). It has been reported that liver damage may occur as well in particular high doses or following chronic administration of methotrexate. However, long-term administration of methotrexate is associated with an increase risk of liver damage. Most of the hepatotoxic potential of methetrexate comes from its use in nonmalignant disease, such as psoriasis and rheumatoid arthritis. Both low and high-dose therapy can cause hepatotoxicity. High-dose therapy results in elevated liver enzymes and low dose therapy produces a different type of hepatotoxicity which includes cirrhosis fibrosis of the liver, hypertrophy of the hepatocytes, hepatitis, hepatocellular necrosis and death (Uraz et al., 2008).
Use of methotrexate for neoplastic diseases has been associated with abnormalities of liver biochemical tests. High dose intravenous methotrexate, elevates serum ALT levels to 10- 20 times the upper limit of normal within 12 to 48 hours, AST level increases to 8% and ALP significantly increased(< 0.05), but levels then fall rapidly to normal with only rare instances of jaundice or symptoms of liver injury (Schmajuk et al., 2014).With long term, low-to-moderate dose methotrexate therapy, elevations in serum ALT, or AST values occur in 15 to 50% of patients, but are usually mild and self-limiting. Approximately 5% of patients have elevations greater than twice normal and these abnormalities resolve rapidly with discontinuation or dose modification, but can resolve even with continuation at the same dose level (Wessels et al., 2008). The rate of ALT elevations during therapy varied considerably, perhaps because of differences in frequency of determinations (like in every 3, 6 or 12 months) and due to the timing of the blood sampling (whether just before or soon after the once weekly dose). The mechanism by which methotrexate adversely affects the liver is unclear.It was demonstrated that metotrexate increases the amount of hydrogen peroxide and other free radicals which are released by stimulated polymorphonuclear neutrophils , which may lead to toxicity thus accelerating the rate of cellular damage (Vezmar, 2003) It was known that methotrexate strongly interferes with the metabolism of homocysteine by reducing the levels of 5-methyltetrahydrofolate and as an indirect result, the levels of homocysteine, S-adenosylmethionine (SAM) were also found to be decreased. Hepatic folate stores are depleted by methetroxate in the doses used in cancer treatment and these stores can be repleted by short term administration of oral folinic acid (Kim et al., 1999).

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