PHYTOCHEMICAL ANTIOXIDANT AND TOXICOLOGICAL PROPERTIES OF METHANOL EXTRACT OF PHYLLANTHUS AMARUS

ABSTRACT

The use of medicinal plants for the treatment of many diseases is associated with folk medicine from different parts of the World. However, information on the toxicology of these plants part used in Nigeria folk medicine is rare. Thus, this work is aimed at revealing a range of phytochemicals in the Phyllanthus amarus plant, the antioxidant constituents and its toxicologic effects on some important biochemical parameters in male albino rats. The potent bioactive agents in the leaves of Phyllanthus amarus plant were extracted and the antioxidant and toxicological potentials for in vitro analyses of the crude plant extract were evaluated using in vitro methods and male white albino rats as the model. The results showed that methanol extract scavenged 1,1- diphenyl-2- picryhydrazyl radical (DPPH) in a concentration – dependent  manner  with  a  correlation  coefficient  (R2)  of  0.989,  indicating  antioxidant  activity  with effective concentration that inhibits fifty percent of the radical (EC50) of 6.93µg/ml compared to ascorbic acid  standard  with  EC50   of  4.69µg/ml.  Superoxide  radical  scavenging  activity  was  concentration dependent with an EC50 of 5.01µg/ml compared with ascorbic acid standard with EC50 of 4.49µg/ml. The crude extract also showed hydroxyl radical scavenging activity with an EC50 of 6.47µg/ml compared to α – tocopherol standard with EC50 of 5.73µg/ml. The methanol extract, also scavenged nitric oxide radical in a concentration – dependent manner with 600µg/ml being more potent than 600µg/ml of α – tocopherol standard. Comparison of the anti-radical power (ARP) of DPPH (0.144), superoxide radical (0.199) and hydroxyl radical (0.175) of the extract revealed that the ARP of the extract against superoxide radical was most efficacious. The antioxidant vitamin contents of the extract showed that vitamin C was significantly higher (p ˂ 0.05), 1.65mg/100g when compared to vitamin A (1.52mg/100g) and vitamin E (0.89mg/100. Acute toxicity test was conducted using mice and there was no death recorded in the mean lethal dose (LD50) investigation. The 100, 200 and 400 mg/kgbw fed to rats showed significantly higher activity of catalase (p ˂ 0.05) at week two and week four. The aspartate aminotransferase (AST) showed non- significantly lower activity (p > 0.05) in group 3 of week one and four, while group 3 of week two was significantly higher (p ˂ 0.05)  in week four. The alanine aminotransferase (ALT) indicated a relatively lower activity of ALT from week one to three while there was relative elevation of ALT activity in the test group of week four. The serum alkaline phosphatase (ALP) was significantly lower (p > 0.05) in the test group when compared to the control group 1 in week one. At week two and three, there were higher activities of ALP in all groups, though non- significant while in week four, there was a non- significantly lower activity of the enzyme in all groups. The serum urea concentration showed a significantly higher (p

˂ 0.05) level in all groups except group four in week one. In week two and three, there was a significantly higher level (p ˂ 0.05) while week four exhibited a non-significant increase in serum urea concentration in all groups. The creatinine concentration indicated a significantly higher level (p ˂ 0.05) in groups 2, 3 and 4 in week one. At week two, there was a significantly lower level (p > 0.05) in group two and four. In week three, there was a significantly higher concentration (p ˂ 0.05) in group two and four, while in week four; there was a non- significant difference in the concentration of serum creatinine in all groups. The Packed cell volume (PCV) and haemoglobin count were significantly higher (p ˂ 0.05) in all groups in week one. In week two, there was no significant increase (p > 0.05) in group three. In week three, there was a significantly higher level of PCV and Hb respectively (p ˂ 0.05). Week four indicated a non- significant decrease in all groups. White blood cell count showed a significantly higher level in group 3 and 4 (p ˂ 0.05) except group two in week one. In week two and three, there was an increase in group three while others showed no significant difference. In week four, there was a non – significant decrease in all groups. Histological analysis showed some level of  toxicity in 100, 200 and 400mg/kgbw at beyond

CHAPTER ONE

INTRODUCTION

Plants have been the basis of many traditional medicine systems throughout the World for thousands of years and still remain as the main new source of structurally important chemical substances that lead to the development of innovative drugs (Fabricant and Farnsworth, 2001; Jachak and Saklani, 2007). The use of medicinal plants for the treatment of many diseases is associated with folk medicine from different parts of the World (Harvey, 2000 ; Bakhotmah and Alzahrani,  2010).  Man  therefore  has  a  high  dependency  on  plants  which  leads  to  its incorporation into their various ways of maintaining survival and livelihood including healthcare. For these reasons, the health of an average African depends more on his flora environment than the services of the orthodox physician located at a substantial geometrical separation from him (Ajibade et al., 2004).

1.1      Phyllanthus amarus

The Phyllanthus genus of the family Euphorbiaceae was first identified in Central and Southern India in 18th century. It is also found in Kogi State and it is popularly called “Eyin olobe”.  It is commonly called “Carry me seed”, stone-breaker, windbreaker, gulf leaf flower or gala of wind (Bharatiya, 1992).  In  folk medicine  Phyllanthus amarus  has  reportedly been used  to  treat jaundice, diabetes, otitis, diarrhoea, swelling, skin ulcer, gastrointestinal disturbances and blocks DNA polymerase in the case of hepatitis B virus during reproduction (Oluwafemi and Debiri,

2008). In traditional medicine, it is used for its hepatoprotective, anti-diabetic, antihypertensive, analgesic, anti-inflammatory and antimicrobial purposes (Adeneye et al., 2006).  The beneficial medicinal effects of plant materials typically result from the combinations of secondary products present in the plant (Joseph and Raj, 2010). Several compounds including alkaloids, flavonoids, lignans, phenols and terpenes were reported to be found in Phyllanthus amarus.

Phyllanthus amarus leaf flower

1.2     Phytochemistry of Phyllanthus amarus

The  study of  natural products is  called  phytochemistry. These  non-nutrient plant  chemical compounds or bioactive components are often referred to as phytochemicals (‘phyto-‘ from Greek – phyto meaning ‘plant’) or phytoconstituents and are responsible for protecting the plant against microbial infections or infestations by pests (Abo et al., 1991; Liu, 2004; Nweze et al.,

2004; Doughari et al., 2009). Phytochemicals have been isolated and characterized from fruits such as grapes and apples, vegetables such as broccoli and onion, spices such as turmeric, beverages such as green tea and red wine, as well as many other sources (Doughari and Obidah,

2008; Doughari et al., 2009). The significance of medicinal plants is directly linked to the wide

range of chemical compounds synthesized in the various biochemical pathways. These compounds are classified as secondary metabolites (Ameyaw and Duker-Eshun, 2009). The importance of natural molecules in medicine lies not only in their chemotherapeutic effect but also in their roles as template molecules for the production of synthetic drugs. There are three

large classes of secondary metabolites in plants: Nitrogen-containing compounds, terpenoids and phenolics. Some of these phytochemicals are alkaloids, flavonoids, saponin, resins, tannins, among others.

1.2.1   Alkaloids

Alkaloids are a group of naturally occurring chemical compounds that contains mostly basic nitrogen atoms. This group also includes some related compounds with weakly acidic properties (Manske,1965). They have a wide range of pharmacological effects and biological activities such as anti-malarial, anti-microbial, anti-hyperglycemic and anti-inflammatory effect. They are also used as analgesic, antibacterial and  are used as recreational drugs, or in etheogenic rituals (Cushine et al., 2014; Raymond et al., 2010; Tackie and Schiff, 1993). Alkaloids have pharmacological applications as anesthetics and CNS stimulants (Madziga et al., 2010). There is, also the non-basic forms such as quaternary compounds and N-oxides (Ameyaw nd Duker- Eshun, 2009). Alkaloids such as atropine, tubocurarine can be toxic or poisonous to other organisms (Robbers et al., 1996).

1.2.2   Flavonoids

Flavonoids are a large family of compounds synthesized by plants that have a common chemical structure. Flavonoids, carotenoids among others are among the antioxidants produced by plants for their own sustenance (Ali et al., 2010). Flavonoids have hydroxyl group (OH-). The effect of hydroxyl moiety on the flavonoids on protein targets varies depending on position and number of the moiety on the flavonoid skeleton (Cazarolli et al., 2008). The position of the hydroxyl group and  other features are  important to  their antioxidant and  free radical scavenging activities.

Flavonoids have been shown to have a wide range of biological and pharmacological activities such as antioxidant, anti-allergic, anti-inflammatory and anti-diarrheal activities (Cazarolli et al.,

2008; Schuier et al., 2005; Yamamotor and Gaynor, 2001).

1.2.3   Saponin

The term saponin is derived from Saponaria vaccaria (Quillaja saponaria), a plant,which abounds in saponins and was once used as soap.Saponins therefore possess‘soaplike’ behaviour in water, i.e they produce foam. On hydrolysis, an aglycone is produced, which is called sapogenin.

Saponins are also important therapeutic agents shown to have hypolipidemic and anticancer activity. Saponins are also necessary for activity of cardiac glycosides. The two major types of steroidal sapogenin are diosgenin and hecogenin. Steroidal saponins are used in the commercial production  of  sex  hormones  for  clinical  use.  For  example,  progesterone  is  derived  from diosgenin. The most abundant starting material for the synthesis of progesterone is diosgenin isolated from Dioscorea species, formerly supplied from Mexico, and now from China (Sarker and Nahar, 2007). Other steroidal hormones, e.g. cortisone and hydrocortisone, can be prepared from the starting material hecogenin, which can be isolated from Sisal leaves found extensively in East Africa (Sarker and Nahar, 2007).

1.2.4   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 hemiacetal 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 phytoconstituents, found in the cell sap. Chemically, glycosides contain a carbohydrate (glucose) and a non-carbohydrate part (aglycone or genin) (Kar, 2007; Firn, 2010).

1.2.5   Tannins

Tannins are naturally occurring plant polyphenols. They bind and precipitate protein. They form complexes, also with carbohydrates, bacterial cell membranes and enzymes involved in protein and carbohydrate digestion. The tannin phenolic group is an excellent hydrogen donor that forms strong hydrogen bonds with the protein’s carboxyl group (Reed, 1995). They have molecular weights ranging from 500 to over 3,000 gallic acid esters and up to 20,000 proanthocyanidins (Bate-Smith and Swain, 1962). The anti-carcinogenic and mutagenic potentials of tannin may be related to their antioxidant property (Chung et al., 1998). The anti-microbial properties seemed to be associated with the hydrolysis of ester linkage between gallic acid and polyls hydrolyzed after ripening of many edible fruits (Chung et al., 1998).

1.2.6   Essential Oil

Essential oils are the odorous and volatile products of various plant and animal species. Essential oils have a tendency to evaporate on exposure to air even at ambient conditions and are therefore also  referred to  as volatile oils or ethereal oils.  They mostly contribute to  the odoriferous constituents or ‘essences’ of the aromatic plants that are used abundantly in enhancing the aroma of some spices (Martinez et al., 2008). Essential oils can be prepared from various plant sources either by direct steam distillation, expression, extraction or by enzymatic hydrolysis. Chemically, a single volatile oil comprises of more than 200 different chemical components, and mostly the trace constituents are solely responsible for attributing its characteristic flavour and odour (Firn, 2010).

1.2.7   Total Phenolics

Typical phenolics that possess antioxidant activity have been characterized as phenolic acids and flavonoids (Kahkonen et al., 1999).   Polyphenols also characteristically possess a significant binding affinity for proteins, which can lead to the formation of soluble and insoluble protein- polyphenol complexes (Papadopoulou and Frazier, 2004). Antioxidant activity of plant extracts is not limited to phenolics. Activity may also come from the presence of other antioxidant secondary metabolites, such as volatile oils, carotenoids, vitamins A, C and E. Crude extracts of fruits, herbs, vegetables, cereals and other plant materials rich in phenolics are increasingly of interest in the food industry because they retard oxidative degradation of lipids and thereby improve the quality and nutritional value of food (Shahidi et al., 1992). Phenolics are the main antioxidant components of food. While in plants, oils are basically monophenolics such as α- tocopherols; water-soluble polyphenols are more typical in water-soluble products like fruits, vegetables, tea, coffee, wine, among others (D’Archivio et al., 2010). Polyphenolic compounds are known to have antioxidant activity. This activity is believed to be mainly due to their redox properties which play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides (Ali et al., 2010).

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