Effects of Ethanol Extract of Ocimium Gratissimum (Scent Leaves)on Liver Indices in Alloxan–induced Diabetes in Albino Rats

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Effects of Ethanol Extract of Ocimium Gratissimum (Scent Leaves)on Liver Indices in Alloxan–induced Diabetes in Albino Rats

Diabetes mellitus (DM) is a metabolic disease characterized by a persistent elevation of fasting blood glucose level (FBGL) above 200 mg/dl, due to insufficient, or Diabetes mellitus, a syndrome of disordered metabolism is usually caused by a combinationof hereditary and environmental effects, resulting in abnormally high blood sugar levels (Tierney et al 2002).

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In diabetes mellitus, the blood glucose level becomes high due to defects in either insulin secretion or insulin action in the body or both (Rother, 2007). It has been estimated that World-wide prevalence of diabetes mellitus in 2008 was more than 347 million with varying prevalence among different ethnic groups and it is expected that in 2025the number will rise to 500 million (Hamden et al., 2011 and Banu et al., 2012). Cases of type 2 diabetes mellitus have been increasing in contrast of type 1 diabetes mellitus; cure and prevention of type 2 diabetes mellitus have become important concern in developed countries and in the same way, due to urbanization and life style changes toward “Western style” diet, cure and prevention of type 2 diabetes mellitus supposed to become a more serious problem in developing countries (Deguchi and Miyazaki 2010).
Diabetes mellitus is a chronic disease that can be managed effectively through modified life styles such as monitoring ones weight, diet, exercise to long term use of oral hypoglycemia drugs (Arthur et al., 2000) Treatment can also be achieved by the use of synthetic drugs such as sulphonylureas (Diribe et al., 2004).
Ocimum gratissimum, also known as sent leaf is a species of Ocimum. It is native to Africa, Madagascar, southern Asia, and naturalized in Polynesia, Hawaii, Mexico, Panama, West Indies, Brazil, and Bolivia The essential oil of Ocimum gratissimum contains eugenol and shows some evidence of antibacterial activity (Oliveira et al., 2009 and Martinset al., 2008).

Traditional medicine is a popular form therapy in developing countries and its use is widely documented in various literatures (Akinyemi et al., 2000). Due to the huge potential of plant herbs in the discovery of novel therapeutic molecules, various national governments have placed traditional medicine under the supervision of health ministries (Nwaeze and Eze 2009). In many rural areas of Nigeria the use of herbal plants as treatments for various bacterial infections still thrives. Some of these plants have been analysed and shown to be effective against well known pathogens such as Staphylococcus aureus and Salmonella typhimurium (Adebolu and Oladimeji 2005).
Ocimum gratissimum L. is a shrub belonging to the family Lamiaceae. It is commonly known as Scent leaf or Clove basil and is found in many tropical countries. Africa and Asia are however, the two continents where most variants of the plant exists (Matasyohet al., 2000 and Mann 2012). O. gratissimum is found in the tropical and warm temperature regions such as India and Nigeria (Nwinyiet al., 2012). Some of the vernacular names in Nigeria include: (Ncho-anwu, Ahuji) Igbo, (Efinrin,) Yoruba, (Aramogbo) Edo and (Daidoya) Hausa Kocheet al., (2012) . O. gratissimum has been described to have other species in the flora of tropical West Africa. These include: Ocimum viride Linn, Ocimum suave Linn, Ocimum basilicum Linn and Ocimum canum Sims (Kocheet al., 2012).
Mshana et al., (2000) reported their numerous medical uses. The Ocimum oil has been described to be active against several species of bacteria and fungi. These include Listeria monocytogenes, Shigella, Salmonella and Proteus, for fungi Trichophyton rubrum, and Candida albicans (Lopez et al., 2005). From recent findings, O. gratissimum has proved to be useful in the medication for people living with Human Immunodeficiency Virus (HIV), and Acquired Immuno Deficiency Syndrome virus AIDs (Nwinyiet al., 2009) In Congo, O. gratissimum decoction is used for gonorrheal infection, vaginal douches for metritis and vaginitis and used in treatment of mental illness (Nwinyiet al., 2009).
Though a lot of literature on the hepatoprotective action of the plant is available, little information is known about its anti diabetic effect. Anti diabetic study on Ocimum gratissimum is rare in Nigeria and hence this study seeks to justify the traditional uses of the plant in Ebonyi State. Thus, this study was carried out to examine the anti diabetic and hepatoprotective effect of Ocimum gratissimum on alloxan induced diabetic rats.

Aim and Objectives
Aim: The aim of the project work was to investigate the anti diabetic effect of Ocimum gratissimum extract on alloxan induced diabetic rats.

Specific Objectives
1 To determine the phytochemical and proximate component of Ocimum gratissimum.
2 To determine the acute toxicity test (determination of LD50) of Ocimum gratissimumextract on albino rats.
3 To investigate the effect of Ocimum gratissimumextracts on alloxan induced diabetic rats.

CHAPTER TWO
LITERATURE REVIEW
Ocimum gratissimum L. (Labiatae) is widely distributed in tropical and warm temperature regions. It grows up to 1-2m in height. Since ancient times, this plant has been connected with the traditional treatment of fever, hence its acronyms such as “fever plant”,”fever leaf”,”mosquito plant” as well as “scent leaf” It is commonly grown around houses as a mosquito repellant.
It is commonly grown around houses as a mosquito repellant in Abakaliki, Ebonyi State and used mainly as spices for cooking delicacies due to its unique aromatic taste.

Scientific Classification
Kingdom Plantae – plant
Subkingdom Tracheobionta – vascular plant
Super division Spermatophyte – seed plant
Division Magnoliophyta – flowering plant
Class Magnoliopsida – Dicotyledon
Subclass Asteridae
Order Lamiales
Family Lamiaceae – mint family
Genus Ocimum L. – basil
Specie Ocimum gratissimum L.-Africa

 

Plate 1: Oliveiraet al., (2009)

Uses of Ocimum Gratissimum
In folk medicine, Ocimum gratissimum is extensively used throughout West Africa as a febrifuge, anti- malarial and anti- convulsant. The crushed leaf juice is used in the treatment of convulsion, stomach pain and catarrh. Oil from the leaves have been found to posses antiseptics, antibacterial and antifungal activities (Ezekwesili et al., 2004).
In the coastal area of Nigeria, the plant is used in the treatment of epilepsy, (Osifo, 1992), high fever (Oliver 1980) and diarrhea (Oliver, 1980 and Sofowara, 1993). While in the savannah areas decoctions of the leaves are used to treat mental illness (Abdulrahman, 1992). Ocimum gratissimum is used by the Ibos of southern Nigeria in the management of the baby’s cord. It is believed to keep the baby’s cord and wound surface sterile. It is used in the treatment of fungal infections, fever, cold and catarrh (Iwu, 1986).

Phytochemical Constituents of Ocimum Gratissimum
There are many phytochemicals found in plants, and even animal species. These are generally grouped as follows:

Saponins
Saponins are a class of chemical compounds found in particular abundance in various plant species. More specifically, they are amphipathicglycosides grouped phenomenologically by the soap-like foaming they produce when shaken in aqueous solutions, and structurally by having one or more hydrophilic glycoside moieties combined with a lipophilictriterpene derivative Daniel et al., (2009). The aglycone (glycoside-free) portions of the saponins are termed sapogenins. The number of saccharide chains attached to the sapogenin/aglycone core can vary – giving rise to another dimension of nomenclature (monodesmosidic, bidesmosidic, etc as can the length of each chain. A somewhat dated compilation has the range of saccharide chain lengths being 1–11, with the numbers 2-5 being the most frequent, and with both linear and branched chain saccharides being represented. Dietary monosaccharides such as D-glucose and D-galactose are among the most common components of the attached chains.
The lipophilic aglycone can be any one of a wide variety of polycyclicorganic structures originating from the serial addition of 10-carbon (C10) terpene units to compose a C 30 triterpene skeleton,(Fukuwatari and Shibita, 2008), often with subsequent alteration to produce a C27 steroidal skeleton. The subset of saponins that are steroidal have been termed saraponins, Aglycone derivatives can also incorporate nitrogen, so some saponins also present chemical and pharmacologic characteristics of alkaloid natural products.
Saponins have also been used as adjuvants in vaccines, e.g. Quil A component QS-21, isolated from the bark of Quillaja saponaria Molina, to stimulate both the Th1 immune response and the production of cytotoxic T-lymphocytes (CTLs) against exogenous antigens make them ideal for use in subunit vaccines and vaccines directed against intracellular pathogens as well as for therapeutic cancer vaccines but with the aforementioned side-effects of hemolysis.In the words of Bodoh et al., (1995), their use as adjuvants in the production of vaccines, toxicity associated with sterol complexation remains a major issue for attention and Saponins are used widely for their effects on ammonia emissions in animal feedingAbner et al., (2010).The mode of action seems to be an inhibition of the urease enzyme, which splits up excreted urea in feceas into ammonia and carbon dioxide. Animal trials have shown that a reduced ammonia level in farming operations causes less damages to the respiratory tract of animals, and may help to make them less vulnerable to diseases (Treben, 1998).

Tannin
Tannin (or tannoid) is an astringent, polyphenolicbiomolecule that binds to and precipitatesproteins and various other organic compounds including amino acids and alkaloids. However, the term “tannin” by extension is widely applied to any large polyphenolic compound containing sufficient hydroxyls and other suitable groups (such as carboxyls) to form strong complexes with various macromoleculesKim et al.,( 2010).
The tannin compounds are widely distributed in many species of plants, where they play a role in protection from predation, and perhaps also as pesticides, and in plant growth regulation (Kimet al., 2010). The astringency from the tannins is what causes the dry and puckery feeling in the mouth following the consumption of unripened fruit or red wine or tea (Said and Mohammed,2006). Likewise, the destruction or modification of tannins with time plays an important role when determining harvesting times (Treben, 1998). Tannins have molecular weights ranging from 500 to over 3, 000 (gallic acidesters) and up to 20,000 (proanthocyanidins) Bate and Swain (1962). Shown below are the base units or monomer of the tannin. Particularly in the flavone-derived tannins, the base shown must be (additionally) heavily hydroxylated and polymerized in order to give the high molecular weight polyphenol motif that characterizes tannins. Typically, tannin molecules require at least 12 hydroxyl groups and at least five phenyl groups to function as protein binders(Pemberton, 2006). Pseudo tannins are low molecular weight compounds associated with other compounds. They do not change color during the Goldbeater’s skin test, unlike hydrolysable and condensed tannins, and cannot be used as tanning compounds. In addition to the alpha acids extracted from hops to provide bitterness in beer, condensed tannins are also present. These originate both from the malt and hops. Especially in Germany, trained brew masters consider the presence of tannins as a flaw. In some styles, the presence of this astringency is acceptable or even desired, as, for example, in a Flanders red ale(Iyengar, 1995).

Figure 1: Oligostilbenoids (oligo- or polystilbenes) are oligomeric forms of stilbenoids and constitute a class of tannins Abneret al., (1998).

Cardiac Glycoside
Cardiac glycosides are organic compounds containing a glycoside that act on the contractile force of the cardiac muscle. Because of their potency in disrupting the function of the heart, most are extremely toxic Collins et al., (2001).Cardiac glycosides are a diverse family of naturally derived compounds that bind to and inhibit Na+/K+-ATPase. Members of this family have been in clinical use for many years for the treatment of heart failure and atrial arrhythmia, and the mechanism of their positive inotropic effect is well characterized. Exciting recent findings have suggested additional signaling modes of action of Na+/K+-ATPase, implicating cardiac glycosides in the regulation of several important cellular processes and highlighting potential new therapeutic roles for these compounds in various diseases. Perhaps most notably, the increased susceptibility of cancer cells to these compounds supports their potential use as cancer therapies, and the first generation of glycoside-based anticancer drugs are currently in clinical trials Eventoff et al., (1977).
Cardiac glycosides are organic compounds containing a glycoside that act on the contractile force of the cardiac muscle. Because of their potency in disrupting the function of the heart, most are extremely toxic Collins et al., (2001).Cardiac glycosides are found in a diverse group of plants including Digitalis purpurea and Digitalis lanata (foxgloves), Nerium oleander (common oleander), Thevetia peruviana (yellow oleander), Convallaria majalis (lily of the valley), Urginea maritima and Urginea indica (squill), Strophanthus gratus (ouabain), Apocynum cannabinum (dogbane), and Cheiranthus cheiri (wallflower). In addition, the venom gland of cane toad (Bufo marinus) contains large quantities of a purported aphrodisiac substance that has resulted in cardiac glycoside poisoning Boon et al., (2007). Therapeutic use of herbal cardiac glycosides continues to be a source of toxicity today. Goodsell 2007 reported from his findings that when Digtalis lanata was mistakenly substituted for plantain in herbal products marketed to cleanse the bowel; human toxicity resulted. Cardiac glycosides have been also found in Asian herbal products and have been a source of human toxicity. Toxicity may occur after consuming teas brewed from plant parts or after consuming leaves, flowers, or seeds from plants containing cardiac glycosides. Significant toxicity usually is a result of suicide attempt or inappropriate self-administration for the therapeutic purposesCollins et al., (2001).

Alkaloids
Alkaloids are a group of naturally occurringchemical compounds that mostly contain basicnitrogen atoms (treben, 1998). This group also includes some related compounds with neutral and even weakly acidic properties. Some synthetic compounds of similar structure are also termed alkaloids. In addition to carbon, hydrogen and nitrogen, alkaloids may also contain oxygen, sulfur and, more rarely, other elements such as chlorine, bromine, and phosphorus (Dzobo and Nark,2013).
Alkaloids are produced by a large variety of organisms including bacteria, fungi, plants, and animals. They can be purified from crude extracts of these organisms by acid-base extraction. Alkaloids have a wide range of pharmacological activities including anti-malarial (e.g. quinine), antiasthma (e.g. ephedrine), anticancer (e.g. homoharringtonine) as reported by (treben,1998).Cholinomimetic (e.g. galantamine) (Eluwa et al., (2010).Vasodilatory (e.g. vincamine), antiarrhythmic (e.g. quinidine), analgesic (e.g. morphine), (Amole et al., (2010).Antibacterial (e.g. cheler ythrine). And antihyperglycemic activities (e.g. piperine) (Dzobo and Nark,2013).
Photochemical evaluation of this plant (Ocimum gratissimum) has shown that it is rich in alkaloid, tannis, phytates, flavonoids and oligosaccharides. It has tolerable cyanogenic content (Ijeh et al., 2004). Many have found use in traditional or modern medicine, or as starting points for drug discovery. Other alkaloids possess psychotropic (e.g. psilocin) and stimulant activities (e.g. cocaine, caffeine, nicotine, theobromine), and have been used in entheogenic rituals or as recreational drugs. Alkaloids can be toxic too (e.g. atropine, tubocurarine), although alkaloids act on a diversity of metabolic systems in humans and other animals, they almost uniformly evoke a bitter taste (Rohde and Rabelo, 2007).
Most alkaloids are weak bases, but some, such as theobromine and theophylline, are amphoteric. Many alkaloids dissolve poorly in water but readily dissolve in organic solvents, such as diethyl ether, chloroform or 1,2-dichloroethane. Caffeine, cocaine, codeine and nicotine are slightly soluble in water (with a solubility of ≥1g/L), whereas others, including morphine and yohimbine are very slightly water-soluble (0.1–1 g/L). Alkaloids and acids form salts of various strengths. These salts are usually freely soluble in water and ethanol and poorly soluble in most organic solvents (Dzobo and Nark,2013).

Figure 2: Isoquinoline derivatives and related alkaloids (Miester, 1998)

Flavonoids
Flavonoids are widely distributed in plants, fulfilling many functions. Flavonoids are the most important plant pigments for flower coloration, producing yellow or red/blue pigmentation in petals designed to attract pollinator animals. In higher plants, flavonoids are involved in UV filtration, symbiotic nitrogen fixation and floral pigmentation Millset al., (2013). They may also act as chemical messengers, physiological regulators, and cell cycle inhibitors. Flavonoids secreted by the root of their host plant help Rhizobia in the infection stage of their symbiotic relationship with legumes like peas, beans, clover, and soy. Rhizobia living in soil are able to sense the flavonoids and this triggers the secretion of Nod factors, which in turn are recognized by the host plant and can lead to root hair deformation and several cellular responses such as ion fluxes and the formation of a root nodule. In addition, some flavonoids have inhibitory activity against organisms that cause plant diseases, e.g. Fusarium oxysporum (Collins et al., 2001).
Flavonoids (specifically flavanoids such as the catechins) are “the most common group of polyphenolic compounds in the human diet and are found ubiquitously in plants” (Treben,1998) Flavonols, the original bioflavonoids such as quercetin, are also found ubiquitously, but in lesser quantities. The widespread distribution of flavonoids, their variety and their relatively low toxicity compared to other active plant compounds (for instance alkaloids) mean that many animals, including humans, ingest significant quantities in their diet. Foods with a high flavonoid content include parsley, onions, blueberries and other berries, black tea, green tea and oolong tea,bananas, Behall et al., (1987), all citrus fruits, Ginkgo biloba, red wine, sea-buckthorns, and dark chocolate (with a cocoa content of 70% or greater). Flavonoids exist naturally in cocoa, but because they can be bitter, they are often removed from chocolate, even dark chocolate.
Flavonoids have been shown to have a wide range of biological and pharmacological activities in in vitro studies. Examples include anti-allergic, olyaemi et al., (2011)anti-inflammatory, antioxidant,anti-microbial (antibacterial,antifungal, and antiviral, anti-cancer and anti-diarrheal activities Serifin et al., (2003). Flavonoids have also been shown to inhibit topoisomerase enzymes and to induce DNAmutations in the mixed-lineage leukemia (MLL) genein vitro studies. However, in most of the above cases no follow up in vivo or clinical research has been performed, leaving it impossible to say if these activities have any beneficial or detrimental effect on human health flavonoids are poorly absorbed in the human body (less than 5%), with most of what is absorbed being quickly metabolized and excreted. Serifin et al., (2003) These findings suggest that flavonoids have negligible systemic antioxidant activity, and that the increase in antioxidant capacity of blood seen after consumption of flavonoid-rich foods is not caused directly by flavonoids, but is due to production of uric acid resulting from flavonoid depolymerization and excretion.

Figure 3: Flavylium skeleton of anthocyanidins and Flavan structure (Serifin et al., 2003).

Diabetes Mellitus
Diabetes mellitus is a type of metabolic disorder that is characterized by hyperglycemia and alterations in carbohydrate, fat and protein metabolisms associated with absolute or relative deficiencies in insulin secretion and/or insulin action. Diabetes mellitus (DM), commonly referred to as diabetes, is a group of metabolic diseases in which there are high blood sugar levels over a prolonged period. Symptoms of high blood sugar include frequent urination, increased thirst, and increased hunger. If left untreated, diabetes can cause many complications. Acute complications can include diabetic ketoacidosis, nonketotic hyperosmolar coma, or death. Serious long-term complications include heart disease, stroke, chronic kidney failure, foot ulcers, and damage to the eyes (Petzoldet al., 2015).

Approximately one-third of patients with type 1 diabetes will develop related complications. The causes are not completely understood, but a more sedentary lifestyle, the consumption of an energy rich diet, obesity, a longer life span (Petzoldet al., 2015) long-term diabetes, poor glycemic control, hypertension and dyslipidemia are linked to diabetes-related vascular complications. Diabetes is characterized by a high incidence of cardiovascular disease. There has been increasing evidence recently that postprandial diabetes and hyperglycemia are important contributory factors in atherosclerosis (Phillipset al., 2016).In diabetes, the postprandial phase is characterized by a massive rapid increase in blood glucose levels where alteration in the sensitivity or reactivity of vascular smooth muscle to neurotransmitters and circulating hormones may cause or contribute to diabetic vessel complications.
Diabetes is due to either the pancreas not producing enough insulin or the cells of the body not responding properly to the insulin produced. There are three main types of diabetes mellitus:
• Type 1 DM results from the pancreas’s failure to produce enough insulin. This form was previously referred to as “insulin-dependent diabetes mellitus” (IDDM) or “juvenile diabetes”. The cause is unknown.
• Type 2 DM begins with insulin resistance, a condition in which cells fail to respond to insulin properly. As the disease progresses a lack of insulin may also developCooke and Plotnick (2008). This form was previously referred to as “non insulin-dependent diabetes mellitus” (NIDDM) or “adult-onset diabetes”. The primary cause is excessive body weight and not enough exercise.
• Gestational diabetes is the third main form and occurs when pregnant women without a previous history of diabetes develop high blood-sugar levels (Phillipset al., 2016).

Prevention and treatment involve maintaining a healthy diet, regular physical exercise, a normal body weight, and avoiding use of tobacco. Control of blood pressure and maintaining proper foot care are important for people with the disease. Type 1 DM must be managed with insulin injections. Type 2 DM may be treated with medications with or without insulin. Insulin and some oral medications can cause low blood sugar.Rippe, et al., (2010)Weight loss surgery in those with obesity is sometimes an effective measure in those with type 2 DM.Picot et al., (2009). Gestational diabetes usually resolves after the birth of the baby (Cash, 2014).
As of 2015, an estimated 415 million people had diabetes worldwide, with type 2 DM making up about 90% of the cases. Shi, et al., (2014). This represents 8.3% of the adult population, with equal rates in both women and menVos et al. (2012). As of 2014, trends suggested the rate would continue to rise. Diabetes at least doubles a werson’s risk of early death. From 2012 to 2015, approximately 1.5 to 5.0 million deaths each year resulted from diabetes. The global economic cost of diabetes in 2014 was estimated to be US$612 billion. In the United States, diabetes cost $245 billion in 2012 Phillipset al., (2016).

Alloxan
Alloxan, also known as alloxan hydrate, refers to the organic compound with the formula OC(N(H)CO)₂C(OH)₂. Alloxan is a toxic glucose analogue, which selectively destroys insulin producing cells in the pancreas (that is beta cells) when administered to rodents and many other animal’s species. This causes an insulin-dependent diabetics mellitus in these animals, with characteristics similar to type 1 diabetic in humans. However, toxic to the liver and the kidneys in high doses It is classified as a derivative of pyrimidine. The anhydrous derivative CO is also known as well as a dimeric derivative. These are some of the earliest known organic compounds. They also exhibit a variety of biological activities. Alloxan C4H2N2O4 has molacular Formula , Molar mass: 142.07 g/mol, Melting point: 492.8°F (256°C) Loreto and Elina (2009). A first transient hypoglycaemic phase of up to 30 min starts within minutes of alloxan injection. This short-lived hypoglycaemic response is the result of a transient stimulation of insulin secretion, as documented by an increase in the plasma insulin concentration. The underlying mechanism is a temporarily reduced consumption and increased availability of ATP caused by blockade of glucose phosphorylation through glucokinase inhibition Szkudelski (2001). The second phase starts with an increase in the blood glucose concentration, 1 h after administration of the toxins, and a decrease in plasma insulin. This first hyperglycaemic phase, which usually lasts 2–4 h, is caused by inhibition of insulin secretion leading to hypoinsulinaemia. During this phase the beta cells show the following morphological characteristics: intracellular vacuolisation, dilation of the rough endoplasmic reticulum, decreased Golgi area, reduced secretory granules and insulin content, and swollen mitochondria. Tyrberget al., (2001).
In addition to, a hypoglycaemic phase, typically occurs 4–8 h after the injection of the alloxan and lasts several hours. It may be so severe that it causes convulsions, and may even be fatal without glucose administration, in particular when liver glycogen stores are depleted through starvation. This severe transitional hypoglycaemia is produced by the flooding of the circulation with insulin as a result of toxin-induced secretory granule and cell membrane rupture. Pancreatectomy prevents this phase. In addition to the morphological changes seen in the first phase, the beta cell nuclei are pyknotic.
Alloxan is a toxic glucose analogue, which selectively destroys insulin-producing cells in the pancreas (that is beta cells) when administered to rodents and many other animal species. This causes an insulin-dependent diabetes mellitus (called “alloxan diabetes”) in these animals, with characteristics similar to type 1 diabetes in humans Danilova et al., (2014). Alloxan is selectively toxic to insulin-producing pancreatic beta cells because it preferentially accumulates in beta cells through uptake via the GLUT2 glucose transporter. Alloxan, in the presence of intracellular thiols, generates reactive oxygen species (ROS) in a cyclic reaction with its reduction product, dialuric acid. The beta cell toxic action of alloxan is initiated by free radicals formed in this redox reaction. One study suggests that alloxan does not cause diabetes in humans Lenzen(2008). Others found a significant difference in alloxan plasma levels in children with and without diabetes Type 1 (Mrozikiewiczet al., 1994).

 

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