EFFECT OF FRACTION I OF ABRUSPRECATORIUS SEED METHANOL EXTRACT ON PARACETAMOL-INDUCED LIVER DAMAGE IN RATS

ABSTRACT

The aim of this study was to investigate the prophylactic and curative effects of an alkaloid-rich fraction of Abrusprecatorius seedchloroform-methanol extract on paracetamol-induced hepatotoxicity in rats. The percentage yield of the methanol extract of Abrusprecatorius seeds was found to be 2.08% w/w. Further purification of the extract using Sephadex gel G15 to get a purer  sample was done.  The  fractions were  spotted on a  TLC plate  and  was spread with Drangendoff’s  reagent  in  which  some  fractions  turned  purple  indicating  the  presence  of alkaloids. The fractions that turned purple were pulled into a beaker and called fraction I which gave a percentage yield of17.75% and was used in this study. The qualitative phytochemical analysis of fraction Irevealed a wide range of phytochemicals such asalkaloids, flavonoids, saponins, glycosides, tannins and carbohydrates, steroids, terpenoids, and peptides which could be physiologically potent in ameliorating several diseases. The quantitative phytochemical analysis of fraction I of Abrusprecatoriusseed methanol extract showed the presence of alkaloids (5480 ± 184 mg/100g), flavonoids (215 ± 97 mg/100g), saponins (2.98 ± 1.33 mg/100g) and tannins (6.4 ± 0.72 mg/100g). Hepatotoxicity was induced using paracetamol (2500 mg/kg b.w.) orally. For prophylactic treatment (hepato-protective), administration of extract was done for 7 days before paracetamol induction and collection of blood was done after 24 hours of administration. Curative treatment (hepato-curative) was done after paracetamol induction at day 0 and treatment was done for 14 days. Blood was collected on days 8 and 15 for the analyses.Prophylactic and  curative treatments with  fraction I  of Abrusprecatorius methanol extract at the dose of 100 and 200mg/kg b.w for group 4 and 5 produced a significant decrease (p˂0.05) in the activities of the liver marker enzymes (ALP, AST, ALT) and bilirubin levels in a dose- and time-dependent manner compared to the paracetamol untreated group 2 (positive control).Groups 3, 4, and 5 treated with 100 mg/kg b.w.silymarin (standard hepato-protective and curative drug),100mg/k.g. b.w. of fraction I and 200mg/k.g. b.w. of fraction I respectively before and after paracetamol induction caused a significant decrease (p˂0.05) in the serum urea and creatinine concentrations of both hepato-protective and hepato-curative groups compared to the positive control. Serum electrolyte concentrations showed a significant increase (p˂0.05) in the treated groups of both hepato-protective and curative when compared to the positive control.The  MDA concentration decreased  significantly (p˂0.05)  in  the  treated  groups and standard groups compared to the positive control after 24 hours (hepato-protective)and at day 8 and 15 (hepato-curative). Serum SOD activity of both protective and curative models, showed adose- and time-dependent significant increase (p˂0.05) in the treated groups compared to the positive control. The haematological parameters of the rats treated with fraction I of Abrusprecatorius methanol extract at various dosesshowed a significant increase (p˂0.05) in the PCV levels, Hb concentration and RBC count compared to the positive control. A dose- and time-dependentsignificant decrease (p˂0.05) was observed in the  WBC count of all treated groups (hepato-protective and hepato-curative) compared to the positive control. The test groups that received fraction I of Abrusprecatoriusin both models showed a dose- and time-dependent effects on the biochemical markers used in the study similar to the standard drug. However,fraction I had more curative effect than protective but silymarinwas more potent.

CHAPTER ONE

1.0 INTRODUCTION

Plants, the first medicine of human being, have played a remarkable role in health care since the ancient times. Traditionally plant-based medicines still exert a great deal of importance to the people living in developing countries and also lead to the discovery of new drugs for a variety of diseases that threatens human health. Plants are the rich sources of organic compounds, many of which have been used for medicinal purposes. Medicinal plants are the plants whose parts (leaves, seeds, stems, roots, fruits, foliage etc), extracts, infusions, decoctions or powders are used in the treatment of different diseases of humans, plants and animals (Nweze et al., 2004). There is a wide spectrum of trees, plants and shrubs whose seeds, roots, barks and leaves are used by humans throughout the globe due to their nutritional or medicinal value (Doughari et al.,

2009). The importance of herbs in the management of human ailments cannot be over emphasized.

Herbs play a major role in the management of various liver disorders along with other system associated diseases (Ebenyi et  al., 2012). Medicinal plants such as Aloe vera,Eclipta alba, Phyllanthus niruri, Solanum Indicum, Maytenus emerginata and Aegle mameloes are well known for their hepato-protective effects (Parmar et al., 2010). Abrus precatoriusLinn is a leguminous plant of the Fabaceae family. Its seeds, roots and leaves are widely used for medicinal purposes in Africa and Asia (Yadava and Reddy, 2002). In Nigeria, the Igbos use the aqueous decoction of the seeds to treat a wide range of conditions including ulcer, infections, hypertension, diarrhoea, infarct and ogbanje (Nwodo and Alumanah, 1991).

1.1.General Description of Abrus precatorius Linn

Abrus precatorius, which belongs to the family of fabeceae is a plant that originated from Southeast Asia and now can be found in subtropical climate areas such as India, Sri Laka, Thailand, the Philippine Islands, South China, Tropical Africa and the West Indies (Vavaprasad and Varahalarao, 2009). It is a slender, perennial climber that twines around trees, shrubs, and hedges. The leaves are pinnate and glabrous, with many leaflets (12 or more) arranged in pairs. Flowers are small and pale violet in colour with a short stalk, arranged in clusters. The plant is best known for its seeds, which are used as beads and in percussion instruments, and which are

toxic due to the presence of abrin. The plant is native to Indonesia and grows in tropical and subtropical areas of the world where it has been introduced. It has a tendency to become weedy and invasive where it has been introduced.

1.1.1 An Overview ofAbrus precatorius Seed

The name Abrus, meaning beautiful or graceful, used to describe the appearance of the seed. The seed is found in a variety of colours such as black, brown, white and most commonly, red with a glossy  appearance  with  the  black  band  at  the  end  that  attaches  to  the  plant.  The  Abrus precatorius seed is known by a  variety of names that  include jequirity (India), Crab’s eye (Guam), Rosary  pea (Egypt), Precatory peabean(USA), Indian Liquorice (Nigeria) and Giddee Giddee or Jumbie bead in Trinidad and Tobago (Hartley,  2010). The seeds of Abrus precatorius are much valued in native jewery for their bright coloration. Most beans are black and red, suggesting a ladybug, though other colors are available. The Tamils use Abrus seeds of different colors. The red variety with black eye is the most common, but there are black, white and green varieties as well. The seeds of Abrus precatorius are very consistent in weight. Formerly Indians used these seeds to weigh gold using a measure called a Ratti, where 8 Ratti = 1 Masha; 12

Masha = 1 Tola (11.6 Grams) (Hartley, 2010).

Figure 1: Abrus precatoriusseeds

(Source: Ali and Malek, 1996)

1.1.2Taxonomyof Abrus precatorius Linn

Table 1: Taxonomic Hierarchy of Abrus precatorius Kingdom                Plantae – plantes, planta, vegetal, plants Subkingdom           Viridaeplantae – green plants Infrakingdom         Streptophyta – land plants

Division                 Tracheophyta – vascular plants, tracheophytes

Subdivision            Spermatophytina –  spermatophytes, seed plants, phenerogames

Infradivision          Angiospermae – flowering plants, angiosperms

Class                       Magnoliopsida

Superorder             Rosanae

Order                      Fabales

Family                    Fabaceae – peas, legumes

Subfamily               Faboideae

Genus                     Abrus

Specie                     Abrus precatorius Linn – rosarypea, crab’s eye, jeguerity,    precatory bean, giddee giddee, Indian liquorice.

(Source; Ali and Malek, 1996)

1.1.3 Importanceof Abrus precatorius Linnin Traditional Medicine

In the Ayurvedic medicine, leaves of Abrus precatorius are used as laxative, expectorant and aphrodisiac  medicines known as Coq’s eye.  Seeds are  said  to  be  purgative, emetic,  tonic, antiphlogistic, aphrodisiac and antiopthalmic. For the indigenous people they are potent phytomedicines, many of them in mixture with other plants. Their toxicity is underestimated (Anant and Maitreyi, 2012)

In  Tanzania,  traditional  healers  claim  the  competence  in  the  treatment  of epilepsy.  Abrus precatorius can be found between 60 plants commonly used against this illness (Moshi et al.,

2005). Also in Zimbabwe, extracts of 58 plants popularly known to be effective against schistosomiasis were tested in vitro against excysted cysticercoids. Extracts of stem and root of Abrus precatorius were under the ten most effective samples (Ndamba and Nyazema, 1994).

In China, the herb of Abrus precatorius is used as a folk-medicine for the treatment of bronchitis, laryngitis and hepatitis. Because of their platelet inhibiting activity, abruquinones are suspected to be the active substances (Kuo et al., 1995).

The leaf of Abrus precatorius has been used in Nigeria for the treatment of myriad of diseases including malaria, typhoid, cough, respiratory tract infections and hepatitis (Saganuwan and Onyeyili, 2010).The leaves are also considered useful in biliousness and in leucoderma, itching and other skin diseases. Its juice is employed as a cure for hoarseness, mixed with oil and applied to painful swellings. Dried leaves paste are used as a germicide to wounds in cattle. The seeds are deadly poisonous but it has been reported that the toxic form of abrin gets converted to mitogenic form upon long refrigerated storage. Usually seeds are of two types one is scarlet with black spot and the other variety is pure white and traditionally used againstleucoderma, wounds, alopecia, asthma, tubercular glands, leprosy, fever, ulcer and tumor. Seeds of Abrus precatorius Linn are applied locally in sciatica, stiffness or shoulder joint and paralysis. It  is useful in dysentery and skin diseases (Anant and Maitreyi, 2012).

Roots of Abrus precatorius Linn are used as diuretics and in the preparations prescribed for gonorrhoea, jaundice and haemoglobinuric bile. Some of the parts are used in night blindness,

inflamed gums, muscular pain and convulsion. It is also used for pain relief in groins, mucus in urine and grave land bone fracture in cattle (Anant and Maitreyi, 2012).

1.1.4Pharmacolical Uses of Abrus precatorius

.The seed extract of abrus precatorius have also been shown to possess several pharmacological properties. It has been shown to have antifertility effect (Rao, 2007); ureterotonic effect and antidiarrhoeal effect (Nwodo, 1991). Aqueous extract of A. precatorius seeds has also been shown to exert antimicrobial activities (Desai and Sirsi, 1966).

It has been reported that Methanol extracts of Abrus precatorius exhibited antibacterial activity towards almost all the bacterial microorganisms (Klebsilla pneumonia, staphylococcus aureus, streptococcus mitis and Micrococcus luteus) used in the study (Varaprasad and Varahalarao,

2009). Also,Abrus precatoriushas Abortifacient effect (Sethiet al., 1990); Agglutinin activity

(Lin et al., 1981); Cytotoxic activity (Desai et al., 1966); Anti-inflamatory activity (Anam,

2001); Insecticidal activity (Hartzell and Wilcoxon, 1941); Hemagglutinin activity (Khan  et al.,

1966); Spermicidal effect (Rajeshwari, 2011); Uterine relaxation effect and Uterine stimulant effect amongst many others (Nwodo and Botting, 1983). Although,Abrus precatorius has been shown to have so many pharmacological activities, the presence of toxic lectins in its seed limits its pharmacological utility.

1.1.5 Toxicity of Abrus precatoriusLinn

Abrus precatoriusbeans are known as one of the most toxic plant parts worldwide. The human fatal dose is estimated as 0.1-1 µg/kg(Monago and Alumanah, 2005). The toxicity of the Abrus seed is associated with the presence of the toxic component, Abrin (a type of toxalalbumin) which is a mixture of at least five lectins, abrin A – D, and Abrus-agglutinin(Chaudhari et al.,

2012). The abrins consist of two peptide chains connected by a disulfide bridge.

Abrin A consists of an A-chain with N-glycosidase activity, which inhibits protein synthesis, and lectin-like B-chain responsible for binding with cell-surface receptors and penetrating of abrin-A molecule into the cell (Ohba and Morowaki, 2004). The relative molecular weight of abrin A and

C are around 64.000Da, that of two agglutinins 128.000Da (Hegde et al., 1991). For furtheridentification, the crystal structure was investigated. The abrin A crystals belong to the monoclinic space group P 2 (Tahirov, 1994). The sequence of amino acids of the B-chain in both abrin-A and abrin-B were elucidated by enzymatic digestion with trypsin. They consist of 268 amino acids and share 256 identical residues (Komira et al., 1993). This chemical structure is assumed to be responsible for its toxic effects.

Abrins disarrange the proteinbiosynthesis by interfering with the 60 S-ribosomes of animal cells irreversibly. The toxicity of these abrins is variable, but they are the most potent toxins of the world, comparable with the botulinus toxin. The fifth of them, Abrus agglutinin, is not very toxic against cells, but it exhibits agglutination toward animal erythrocytes. Also it has been shown that Abrus agglutinin causes a total haemolysis in blood groups followed by a haemorrhagic gastroenteritis (Khanet al., 1966).

Fatal incidents have been reported following ingestion of well-chewed seeds of Abrus precatorius. It has also been reported that poisoning has been experienced through a finger prick when stringing the seed. Symptoms of seed poisoning include severe gastroenteritis with pronounced nausea and vomiting, muscular weakness, tachycardia, cold sweat, bloody diarrhoea, dyspnoea, dehydration, loss of condition and recumbence (Anant and Maitreyi, 2012).

There is no physiological antidote. The treatment is essentially symptomatic. Since there is a long latent period associated with abrin poisoning, little value can be placed on induction of emesis or gastric lavage; these measures are useful only if ingestion has just occurred. Bismuth trisilicate may be given during poisoning by Abrus precatorius to reduce the level of gastrointestinal damage. If the emesis and/or diarrhoea become excessive, replacement fluids and electrolytes are advocated. If haemorrhage occurs, blood transfusion may be necessary(Khan et al., 1966).

1.2.0 PHYTOCHEMICALS

Phytochemicals (from the Greek word phyto, meaning plant) are biologically active, naturally occurring chemical compounds  found  in  plants,  which provide  health  benefits  for  humans (Mamta et al.,2013).They protect plants from disease and damage and contribute to the plant’s color,  aroma  and  flavor.  In  general,  the  plant  chemicals  that  protect  plant  cells  from

environmental hazards such as pollution, stress, drought, UV exposure and pathogenic attack are called as phytochemicals (Narasinga, 2003). Phytochemistry is the study of natural bioactive products found in plants that work with nutrients and dietary fibre to protect against diseases (Doughari et al., 2009). Recently, it is clearly known that they have roles in the protection of human health, when their dietary intake is significant. In wide-ranging dietary phytochemicals are found in fruits, vegetables, legumes, whole grains, nuts, seeds, fungi, herbs and spices. Broccoli, cabbage, carrots, onions, garlic, whole wheat bread, tomatoes, grapes, cherries, strawberries, raspberries, beans, legumes, and soy foods are common sources (Mathai, 2000).

These phytochemicals are present in a variety of plants utilized as important components of both human and animal diets, and they are found in different parts of the plant which include; fruits, flower, bark seeds, root and stem(Tiwari et al.,2011). They are chemical compounds formed during the plant normal metabolic processes. These chemicals are often referred to as ‘secondary metabolites’ of which there are several classes including alkaloids, flavonoids, glycosides, gums, coumarins, polysaccharides, phenols, tannins, terpenes and terpenoids .

1.2.1  Alkaloids

Alkaloids are natural products that contains heterocyclic nitrogen atoms, are basic in character. The name of alkaloids derives from the “alkaline” and it was used to describe any nitrogen- containing base (Mueller-Harvey and McAllan, 1992). These are the largest group of secondary chemical constituents made largely of ammonia compounds comprising basically of nitrogen bases synthesized from amino acid building blocks with various radicals replacing one or more of the hydrogen atoms in the peptide ring, most containing oxygen.

Alkaloids are  significant  for  the  protecting  andsurvival of plant  because  they ensure  their survival against micro-organisms (antibacterial and antifungal activities), insects and herbivores (feeding deterrens) and also against other plants by means of allopathically active chemicals (Molyneuxet al., 1996). The useof alkaloids containing plants as dyes, spices, drugs or poisons can be traced back almost to the beginning of civilization. Alkaloids have many pharmacological activities including antihypertensive effects (many indole alkaloids), antiarrhythmic effect (quinidine, sardine), antimalarial activity (quinine), andanticancer actions (dimeric indoles, vincristine,  and  vinblastine).  These  are  just  a  few  examplesillustrating the  great  economic

importanceof this group of plant constituents (Wink et al.,1998). Some alkaloids have stimulant property as  caffeine  and  nicotine,  morphine are  used  as  the  analgesic  and  quinine  as  the antimalarial drug (Rao et al.,1978).

1.2.2  Flavonoids

Flavonoids are important group of polyphenols widely distributed among the plant flora. Structurally, they are made of more than one benzene ring in its structure (a range of C15 aromatic compounds) and numerous reports support their use as antioxidants or free radical scavengers (Kar, 2007). The compounds are derived from parent compounds known as flavans. They are organic compounds that have no direct involvement with the growth or development of plants, they are plant nutrients that when consumed in fruits and vegetables pose no toxic effect on humans, and are also beneficial to the human body. Flavonoids are poly-phenolic compounds that are ubiquitous in nature (Harborne and Baxter, 1999). More than 4,000 flavonoids have been recognized, many of which occur in vegetables, fruits and beverages like tea, coffee and fruit drinks (Pridham, 1960).

Flavonoids can be classified into five major sub groups, these include; flavones, flavonoids, flavanones, flavonols and anthocyanidines (Nijveldt et al.,2001). Flavones are characterized by a planar structure because of a double bond in the central aromatic ring. Quercetin, one of the best described, is a member of this group. Quercetin is found in abundance in onions, apples, broccoli and berries. Flavonones are mainly found in citrus fruit, an example is narigin. Flavonoid is involved in scavenging of oxygen derived free radicals (Harborne and Baxter, 1999). It has been identified as a potent hypolipidemic agent in a number of studies (Tapas et al., 2008). It has also been established that flavonoids from medicinal plants possess a high antioxidant potential due to their hydroxyl groups and protect more efficiently against free radical related diseases like arteriosclerosis (Kris-Etherton et al.,2002).

1.2.3 Tannins

Tannins are polymerized phenols with defensive properties. Their name comes from their use in tanning, rawhides to produce leather. In tanning, collagen proteins are bound together with phenolic groups to increase the hide’s resistance to water, microbes and heat (Hans-Walter and Fiona,  2005).  Two  categories  of  tannins  that  are  of  importance  are  the  condensed  and

hydrolysable tannins. Though widely distributed, their highest concentration is in the bark and galls of oaks (Hans-Walter and Fiona, 2005). They are phenolic compounds of high molecular weight. Tannins are soluble in water and alcohol and are found in the root, bark, stem and outer layers of plant tissue. They are acidic in reaction and the acidic reaction is attributed to the presence of phenolics or carboxylic group (Kar, 2007).

Many human physiological activities, such as stimulation of phagocytic cells, host-mediated tumour  activity,  and  a  wide range of anti-infective actions,  have  been assigned to  tannins (Haslam, 1996). One of their biological actions is to compete with proteins through non-specific forces such as hydrogen bonding and hydrophobic interactions, as well as by covalent bond formation (Haslam, 1996). Thus, their mode of antimicrobial action may be related to their ability to inactivate microbial adhesions, enzymes, cell envelope, transport proteins etc.

1.2.4 Steroids

Sterols are triterpenes which are based on the cyclopentane hydrophenanthrene ring system (Harborne, 1998). Sterols were at one time considered to be animal substances (similar to sex hormones, bile acids, etc) but in recent years, an increasing number of such compounds have been detected in plant tissues. Sterols have essential functions in all eukaryotes. For example, free sterols are integral components of the membrane lipid bilayer where they play an important role in the regulation of membrane fluidity and permeability (Galm and Shen, 2007). While cholesterol is the major sterol in animals, a mixture of various sterols is present in higher plants, with sitosterol usually predominating. Sterols in plants are generally described as phytosterols with three known types occurring in higher plants: sitosterol (formerly known as ß-sitosterol), stigmasterol and campesterol (Harborne, 1998).

1.2.5 Glycosides

Glycosides in general, are defined as the condensation products of sugars (including polysaccharides) with  a  host  of different  varieties of organic  hydroxyl (occasionally thiol) compounds (invariably monohydrate in character), in such a manner that the hemi-acetal entity of the carbohydrate must essentially take part in the condensation. Glycosides are colorless, crystalline carbon, hydrogen and oxygen-containing (some contain nitrogen and sulfur) water- soluble phyto-constituents, found in the cell sap. Chemically, glycosides contain a carbohydrate

(glucose) and a non-carbohydrate part (aglycone or genin) (Kar, 2007). Glycosides are neutral in reaction and can be readily hydrolyzed into its components with ferments or mineral acids. Glycosides are classified on the basis of type of sugar component, chemical nature of aglycone or pharmacological action (Firn, 2010).

1.3Acetaminophen (Paracetamol)

Acetaminophen chemically known as N-acetyl-p-aminophenol, is a widely usedanalgesic and antipyretic agent with little anti-inflammatory effect (McDaid et al.,2010).Acetaminophen is a white, odorless, crystalline powderwith a slightly bitter taste. It has a molecular formula of C8H9NO2and molecular weight of 151.16 g.It is the most widely used drug for pain relief. Paracetamol is  the  International Non-proprietary Name  (INN)  and  British Approved Name (BAN), while acetaminophen is the United States Adopted Name (USAN) and Japanese Adopted Name (JAN)(Macintyreet al., 2008).

Paracetamol is classified as a mild analgesic. It is commonly used for the relief of headaches and other minor aches and pains and is a major ingredient in numerous cold and flu remedies. In combination with opioid analgesics, paracetamol can also be used in the management of more severe pain such as post-surgical pain and providing palliative care in advanced cancer patients. Though paracetamol is used to treat  inflammatory pain, it  is  not generally classified as an NSAID because it exhibits only weak anti-inflammatory activity(Macintyreet al., 2008).

1.3.1 Pharmacokinetics

In order for increase effectiveness, paracetamol canbe administered rectally, orally and intravenously. While all three mode of administration can achieve adequate plasma concentrations, there are differences in absorption and time to reach the plasma peak levels. With rectal administration, absorption can be unpredictable with bioavailability ranging from 24% to

98% varying with factors such as formulation of suppositories number used and the particle size of the paracetamol (McDaid et al.,2010). Oral bioavailability is dose dependant: with larger doses, the  hepatic first  pass effect  is reduced due to overwhelming of the  liver enzymatic capacity; and therefore, bioavailability is increased. In this case, bioavailability is inconsistent and in overall reduced, due to incomplete dissolution of the suppository in the rectum. The absorption rate through this route of administration is elongated.

The analgesic activity is attributable to the small fraction that penetrates into the brain(McDaid et al.,2010). Paracetamol given at therapeutic doses binds to plasma proteins at less than 20%. In case of intoxication, this proportion may increase to up to 50%(Huber et al., 2009). Paracetamol is essentially metabolized in the liver by conjugation with glucuronic acid (55%) and sulfuric acid (35%). Hepatotoxic metabolites are produced in small amounts by the cytochrome P450 (isoenzyme  CYP2E1).  In  the  therapeutic  plasma  concentration  range,  this  metabolite  is detoxified by conjugation with glutathione(Macintyreet al., 2008). In case of intoxication the amount of this toxic metabolite increases and outweighs the amount of available glutathione, which can lead to hepatic failure and renal tubular necrosis. Metabolites are excreted through the kidneys in the urine. Only 2-5% of the dose is excreted in an unchanged form in the urine. As a consequence of its short elimination half-life (1-3h), 24 hours after the ingestion of a single dose of paracetamol, 98% of the dose is eliminated(McDaid et al.,2010).

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