STUDIES ON THE EFFECTS OF METHANOL EXTRACT OF VITEX DONIANA LEAVES ON PLASMODIUM BERGHEI- INFECTED MICE

ABSTRACT

ABSTRACT

Malaria caused by plasmodium parasite is one of the leading infectious diseases in the tropics including Nigeria. Efforts are on to developing more potent antimalarials from plant sources that will be cheaper, without adverse effects, readily available and will be able to replace existing antimalarials that are already facing resistance by plasmodium. Vitex doniana is a medicinal plant used in many part of Nigeria for the treatment of malaria and other diseases.  This work was set out to investigate the in vivo antiplasmodial effects and some biochemical evaluations of the methanol extract of Vitex doniana leaves in mice. The preliminary qualitative and quantitative analyses of the methanol extract of V. doniana leaves revealed the presence of steroids, carbohydrates, glycosides and acidic compound in relative amount while alkaloids, proteins and flavonoids at moderate level. Phenolics, terpenoids and tannin were present in high amount while saponins were not detected. The results of the quantitative phytochemical constituents of the leaves extracts constitute respectively: alkaloids (433.22± 7.51 mg/100g), flavonoids (302.06 ± 6.21 mg/100g), tannins (1039.60± 181.43 mg/100g), steroids (2.65± 0.08 mg/100g), protein (406.19 + 19.97), terpenoids (1055.87 ± 140.44 mg/100g), carbohydrate (29.93 + 7.23 mg/100g), total phenolics (1806.45± 234.18 mg/100g) and glycosides (5.138 + 0.17 mg/100g). The acute toxicity test of the extract showed no toxicity up to 5000 mg/kg body weight. The animals were successfully passaged with  Plasmodium berghei collected from already infected mice. The result of this study revealed that there was a reduction in the percentage parasitaemia of the treated mice when compared to the untreated animals. In this study, the lipid peroxidation product, Malondiadehyde (MDA) concentration of the treated groups decreased significantly (p < 0.05) when compared to the untreated animals. The levels of antioxidants superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) showed a significant (p < 0.05) increase in the treated groups when compared to untreated group (group 2). GSH of the treated groups 4 and 6 was found to  be  significantly  (p<0.05)  higher  than  the  untreated  group  (group  2).  The  results  from  the haematological parameters showed that the extract significantly  (p < 0.05)  increased the RBC count, PCV count and Hb concentration in the treated groups compared to the untreated group (group 2). White blood cell count in the treated groups were found to be significantly (p < 0.05) lower compared to the untreated animals. Activities of liver marker enzymes such as ALP, AST and ALT significantly (p < 0.05) decreased in all the treated groups when compared to the untreated group. The result of this study also revealed that there was a significant (p < 0.05) decrease in total bilirubin at highest dosage compared to the untreated animals (group 2). The kidney function assessment showed that urea was found to be significantly (p < 0.05) lower in the test groups 4 and 6 than untreated group. There was also a significant (p < 0.05) decrease in the creatinine level of the treated animals compared to the untreated animals. Also, the result of serum electrolyte assessment showed that sodium and potassium were found to be significantly (p < 0.05) higher in the test groups compared to the untreated group. There was a significant (p < 0.05) increase in cholesterol, LDL, HDL and triacylglycerol concentrations in the treated groups compared to the untreated animals. The result of this study showed that V. doniana methanol leaf extract possesses antimalarial effects, which supports the claims in different parts of the country and beyond, that the plant is used in the treatment of malaria and may therefore offer a potential drug source for development of a safe, effective and affordable antimalarial. This could be attributed to the presence of some phytochemicals in the plant known to have antimalarial properties. The present study also suggests that the plant possess antioxidant properties which could fight the free radicals in the system and also has hepatoprotective, anti-anaemic and nephroprotective properties.

Malaria caused by plasmodium parasite is one of the leading infectious diseases in the tropics including Nigeria. Efforts are on to developing more potent antimalarials from plant sources that will be cheaper, without adverse effects, readily available and will be able to replace existing antimalarials that are already facing resistance by plasmodium. Vitex doniana is a medicinal plant used in many part of Nigeria for the treatment of malaria and other diseases.  This work was set out to investigate the in vivo antiplasmodial effects and some biochemical evaluations of the methanol extract of Vitex doniana leaves in mice. The preliminary qualitative and quantitative analyses of the methanol extract of V. doniana leaves revealed the presence of steroids, carbohydrates, glycosides and acidic compound in relative amount while alkaloids, proteins and flavonoids at moderate level. Phenolics, terpenoids and tannin were present in high amount while saponins were not detected. The results of the quantitative phytochemical constituents of the leaves extracts constitute respectively: alkaloids (433.22± 7.51 mg/100g), flavonoids (302.06 ± 6.21 mg/100g), tannins (1039.60± 181.43 mg/100g), steroids (2.65± 0.08 mg/100g), protein (406.19 + 19.97), terpenoids (1055.87 ± 140.44 mg/100g), carbohydrate (29.93 + 7.23 mg/100g), total phenolics (1806.45± 234.18 mg/100g) and glycosides (5.138 + 0.17 mg/100g). The acute toxicity test of the extract showed no toxicity up to 5000 mg/kg body weight. The animals were successfully passaged with  Plasmodium berghei collected from already infected mice. The result of this study revealed that there was a reduction in the percentage parasitaemia of the treated mice when compared to the untreated animals. In this study, the lipid peroxidation product, Malondiadehyde (MDA) concentration of the treated groups decreased significantly (p < 0.05) when compared to the untreated animals. The levels of antioxidants superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) showed a significant (p < 0.05) increase in the treated groups when compared to untreated group (group 2). GSH of the treated groups 4 and 6 was found to  be  significantly  (p<0.05)  higher  than  the  untreated  group  (group  2).  The  results  from  the haematological parameters showed that the extract significantly  (p < 0.05)  increased the RBC count, PCV count and Hb concentration in the treated groups compared to the untreated group (group 2). White blood cell count in the treated groups were found to be significantly (p < 0.05) lower compared to the untreated animals. Activities of liver marker enzymes such as ALP, AST and ALT significantly (p < 0.05) decreased in all the treated groups when compared to the untreated group. The result of this study also revealed that there was a significant (p < 0.05) decrease in total bilirubin at highest dosage compared to the untreated animals (group 2). The kidney function assessment showed that urea was found to be significantly (p < 0.05) lower in the test groups 4 and 6 than untreated group. There was also a significant (p < 0.05) decrease in the creatinine level of the treated animals compared to the untreated animals. Also, the result of serum electrolyte assessment showed that sodium and potassium were found to be significantly (p < 0.05) higher in the test groups compared to the untreated group. There was a significant (p < 0.05) increase in cholesterol, LDL, HDL and triacylglycerol concentrations in the treated groups compared to the untreated animals. The result of this study showed that V. doniana methanol leaf extract possesses antimalarial effects, which supports the claims in different parts of the country and beyond, that the plant is used in the treatment of malaria and may therefore offer a potential drug source for development of a safe, effective and affordable antimalarial. This could be attributed to the presence of some phytochemicals in the plant known to have antimalarial properties. The present study also suggests that the plant possess antioxidant properties which could fight the free radicals in the system and also has hepatoprotective, anti-anaemic and nephroprotective properties.

CHAPTER ONE

INTRODUCTION

Plants and other substances of natural origin have been in use throughout the world for human and  animal  health  care  (Akinpela,  2006).  Medicinal  plants  are  plants  that  have  healing substances in one or more organs (root, stem, leaves and flower) which can be used in the production  of  mainstream  pharmaceutical  products  or  used  in  their  natural  occurring  state without being synthesized (Doughari, 2010).   The use of plant in the management and treatment of diseases started with life especially in Africa where large population of people depend on traditional medicine and folkloric use of plant because of their inability to obtain synthetic drugs ( Bongoni et al., 2013).

Malaria is a mosquito-borne infectious disease caused by a eukaryotic protist of the genus Plasmodium and is one of the leading infectious diseases in many tropical and subtropical regions, including parts of the America, Asia, and Africa (Adebayo and Krettli, 2011). More than

300 to 500 million clinical cases are reported annually resulting in at least 1.5 to 2.7 million deaths (WHO, 2009).  At present, malaria threatens almost one-third of the world‘s population where it is considered an endemic disease (Pimenta et al., 2015).  Five Plasmodium spp. cause malaria  in  human  beings:  Plasmodium  falciparum,  P.  vivax,  P.ovale,  P.  malariae  and  P. knowlesi.    The  most  rampant  and  precarious  type  of  malaria  is  caused  by  Plasmodium falciparum.  Plasmodium  vivax  is  a common  cause of malaria in  Latin  America,  Asia,  and Oceania, but not Africa. Plasmodium malariae and P. ovale are much less common (Saifi et al.,

2013). P. knowlesi are found throughout Southeast Asia (Dondorp et al., 2004).

Natural products have played  dominant roles in the discovery of leads for the development of drugs to treat human diseases, and this fact anticipates that new antimalarial leads may certainly emerge from tropical plant sources (Batista et al., 2009). One of these plant sources is Vitex doniana.

Vitex  doniana  (chaste  tree)  is  a  deciduous  evergreen  tree  grown  in  tropical  West  Africa. Different parts of the plant have been reported to possess medicinal properties. In ethnomedicine, hot aqueous extracts of the leaves are used in the treatment of stomach and rheumatic pains, inflammation, diarrhoea and dysentery (Agunu et al., 2005; Iwueke et al., 2006). In Nigeria, preparations from the roots and leaves are used orally to treat nausea, colic and epilepsy and the

young leaves are used as vegetables or sauces and porridge for meals, especially for diabetic patients (Yakubu et al., 2012). In eastern parts of Nigeria, information available from the indigenous traditional healers indicates that, a decoction of the chopped leaves of V. doniana is prepared and taken orally for treatment of malaria and other disease conditions; although these medicinal properties have not been established scientifically.

However, despite the studies on the traditional uses of this plant, there is paucity of information on the antiplasmodial effects of the leaf extract, hence, this study.

1.1 Profile of Vitex doniana

Vitex doniana belongs to Verbenaceae family. Vitex comprise about 150 species and approximately 60 species occur in tropical Africa of which Vitex doniana is most widely spread (Owolabi et al., 2011). Vitex doniana is a perennial shrub and a deciduous evergreen tree, usually 4-8m high, occasionally up to 15m (32 to 49 feet) with a dense rounded crown. Its bark is light grey with numerous vertical fissures. The leaves are long stalked with 5-7 leaflets. Flowers are numerous, white tinged purple, usually borne in short, and stout axilliary cymes on a long stalk; Calyx and pedicels are densely hairy. It‘s fruit, which ripens between May and August, is sweet and tastes like prunes consisting of a thin exocarp, the edible mesocarp (pulp) and a thick woody endocarp (Irvine, 1961; Etta, 1984; Bouquet et al., 1971; Iwu, 1993).  Reproduction is by seed, but farmers also propagate the tree by taking cuttings. There are no known varieties or sub- species. The diagrams below are the pictures of Vitex doniana tree (fig. 1) and dried leaves (fig.

2), respectively.

Figure 1: Diagrammatic representation of Vitex doniana leaves

Figure 2: Dried leaves of Vitex doniana

1.1.1 Taxonomic Classification of Vitex doniana

Kingdom:                   Plantae Subkingdom:              Tracheobionta Superdivision:            spermatophyte Division:                     Magnoliophyta Class:                          Magnoliopsida Subclass:                    Asteridae Order:                         Lamiales Family:                       Verbenaceae Genus:                        Vitex

Species:                      Vitex doniana

Source: (Contu, 2012)

1.1.2 Common Names of Vitex doniana

Vitex  doniana  is  commonly  called  chaste  tree  (English),  dinya  (Hausa),  dinchi  (Gbagyi), uchakoro (Igbo), oriri (Yoruba), ejiji (Igala) and olih (Etsako) (Burkil, 2000), Mfuru or Mgwobe (Tanzania), Munyamazi (Uganda), Dinya, Tinya orTunci (Fulani),(Atawodi et al., 2003). It is commonly called Fon, or Ewe oyi by traditional healers and plants sellers in Bénin.

1.1.3 Distribution, Climate and Soil Type

Vitex doniana of the Verbanaceae family is a perennial shrub widely distributed in West Africa and some East African countries including Uganda, Kenya and Tanzania; and high rainfall areas. It is found in the middle belt of Nigeria particularly Kogi, Benue, and parts of the savannah regions of Kaduna, Sokoto and Kano states (Etta, 1984). It is widely distributed in the eastern and western parts of Nigeria.The most abundant and widespread genus occur in coastal savannah regions and dry, moist, wet lowland (Enzo, 2006). The plant is also distributed through Angola, Botswana,

Ethiopia, Kenya, Namibia, Niger, Senegal, Somalia, South Africa, Sudan and Zambia (Keay,

1989). The plant is also grown throughout the world as ornamental and as sources of wood ( Jabeen et al., 2009).

1.1.4 Benefits of Vitex doniana

All parts of the plant (leaves, stem barks, roots and flowers) are used for the preparation of remedies in the form of decoction, maceration, infusion or powders to treat diseases. The treatments are administered by oral routes and baths (Witabouna et al., 2011)

In Nigeria, information available from the indigenous traditional healers indicates that, a decoction of the chopped stem barks and leaf of V. doniana is prepared and taken orally for treatment of diabetes and other disease conditions. The plant extracts have been used as medication for infertility, liver disease, anodyne, stiffness, hypertension, malaria, cancer, febrifuge, sedative, digestive regulator and treatment of eye troubles, kidney troubles and as supplement for lack of vitamin A and B (Burkill, 2000; Sofowora, 1993).

Vitex doniana tree is used as treatment of several disorders and diseases which include rheumatism, hypertension, anemia, and the plant is used to treat jaundice, leprosy, and dysentery (Orwa et al., 2009). The root is used for gonorrhea and in treatment of rickets, leprosy, diarrhea, dysentery, fevers, malnutrition and venereal diseases (Orwa et al., 2009).

In eastern Sudan, Vitex doniana fruits are roasted and used as substitute for tea. It contains vitamins A and B and can be made into a jam. The fruits are used as remedy for sores at the corners of the mouth and eyes, which are associated with vitamin A and B deficiencies (Leung et.al., 1986). The twigs are used as chewing sticks for teeth cleaning. Young leaves and shoots of Vitex doniana are used as vegetable in some parts of Africa. Leaf sap is used as an eye drop to treat conjunctivitis and other eye complaints. Leaf decoction is applied externally against headache, measles and skin rashes (Orwa et al., 2009).    Vitex doniana Stem bark is used as remedy for leprosy, paralysis, epilepsy and convulsions. It is administered for ailments including diarrhea pulmonary troubles and skin rashes. Bark powder is added to water and taken to treat colic, and a bark extract is used to treat kidney diseases and to control bleeding after child-birth

(Orwa et. al., 2009). In Kano, the bark of V. doniana is used in traditional medicine as anti- epilepsy and against female sterility (Atawodi, 2005).

Hot aqueous extracts of the leaves are used in the treatment of stomach and rheumatic pains, inflammation, diarrhoea and dysentery (Etta, 1984; Irvine, 1961) indicating that the leaves may possess anti-inflammatory and analgesic properties among others.

Hexane, ethyl acetate and methanolic extracts of stem bark were tested in vitro for antimicrobial potential using the well diffusion technique. The extract showed broad spectrum activity with zones of inhibition ranging from 19 to 24mm (Eghareba, 2010).

The petroleum ether extract of the leaves gave the highest activity with a zone of inhibition of

20mm against Salmonella typhi. Other extracts had zones of inhibition ranging from 4mm-14mm against almost all the tested organisms (Dauda et al., 2011).

Vitex doniana extracts showed significant antimicrobial activity against S. typhi (Dawang et al.,

2012).

Laboratory  tests  demonstrated  the  presence  of  high   concentrations   of  progestogen-like compounds in Vitex doniana, thus natural consumption of Vitex doniana was a likely cause of the observed increases in progestogen (James et al., 2007).

Ukwuani et al. (2012) reported that the aqueous leaves extract of Vitex doniana has a significant antidiarrhoeal activity which supports its use in traditional herbal medicine practice.

Owolabi, et al. (2011) suggested that the use of aqueous leaf extract of Vitex doniana in the treatment of diabetes produce a significant (p < 0.05) antidiabetic and hepatoprotective effect. Research report by James et al. (2010) on the antihepatotoxic ability of aqueous leave and stem extract of V. doniana showed that it was effective against carbon tetrachloride induced liver injury in rats.

The anti-hypertensive effect of extract of stem bark of V. doniana has been reported by Olusola et al, (1997), and showed that the extract exhibited a marked dose-related hypotensive effect in both normotensive and hypertensive rats.

Extracts of stem bark of V. doniana have also demonstrated some level of in vitro trypanocidal activity against Trypanosoma brucei brucei (Atawodi, 2005).

The  aqueous  and  methanolic  extracts  has  been  reported  to  exhibited  anti-diarrhea  activity

(Agunu et al., 2005).

1.2 Phytochemical Study of Vitex doniana

The qualitative  phytochemical screening of Vitex doniana show that the Vitex doniana leaves contains flavonoids, alkaloids, cardiac glycosides, terpenes and resins (Dawang, 2015). Phytochemicals such as tannins, glycosides, alkaloids, flavonoids, saponins, carbohydrates and proteins have been identified in the Vitex          na stem bark (Iloh et. al., 2015). In the same manner glycosides, flavonoids have also been identified in the Vitex doniana root and shows absence for alkaloid, steroid and tannins (Dauda et al., 2011). Phytochemical studies on Vitex doniana leaves revealed the presence of carbohydrates, steroids, saponins, glycosides, alkaloid, flavonoids  and  coumarins  and  absence  of  tannins  and  anthraquinones  have  been  reported according to Abdel  et al. (2016). In Latifou et al. (2012), results revealed that the leaves of Vitex doniana showed positive test for flavonoid, essential oil, terpenes, triterpenes but negative test for alkaloid, coumarin, anthraquinone, lignans and saponins.

According to Mustapha et al. (2012), the ethanolic stem bark extracts of Vitex doniana revealed the presence of tannins, phlobatannins, saponins, carbohydrates, cardioactive glycoside, flavonoids, steroids and terpenes. Alkaloids and anthracenosides are absent in the extract. The phytochemicals found are implicated to have much medicinal importance.

In Agbafor and Nwachukwu (2011), phytochemical screening of the ethanol and water extract of Vitex doniana leaves showed positive test for alkaloids, saponins, tannins, anthraquinones, terpenoids, flavonoids but negative for cardiac glycosides. Also, in evaluating the antioxidant property of the extract, the extract inhibited 2,2-diphenyl-1-picryl-hydrazyl(DPPH) in a concentration dependent manner and  also produced a significant (p<.05) decrease in liver MDA levels and significantly increased (p<.05) SOD and CAT activities in a CCL4 induced oxidative stress; these observation are indicative of antioxidant property of the extract.

1.3 Phytochemistry

The medicinal value of some medicinal plants has a link with the phytochemicals in them. These phytochemicals are chemical compounds that occur naturally in plants (phyto means “plant” in Greek). Some phytochemicals are responsible for color, smell etc. The term is generally used to refer to those chemicals that may have biological significance. There may be as many as 4,000 different types. Example of such phytochemicals include: alkaloids, flavonoids, saponin, tannins etc (Palermo et al., 2014).

1.3.1 Classification of Phytochemicals

Phytochemicals are classified as primary or secondary constituents, depending on their role in plant  metabolism.  Primary  constituents  include  the  common  sugars,  amino  acids,  proteins, purines and pyrimidines of nucleic acids, chlorophylls etc. Secondary constituents are the remaining plant chemicals such as alkaloids, terpenes, flavonoids, lignans, plant steroids, curcumines, saponins, phenolics, and glucosides (Hahn, 1998).

1.3.2 Alkaloids

Alkaloids are a group of naturally occurring chemical compounds (natural products) that contain mostly basic nitrogen atoms. This group also includes some related compounds with neutral and even weakly acidic properties (Manske, 1965). Some synthetic compounds of similar structures 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 (Manske, 1965).

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. Many alkaloids are toxic to other organisms. They often have pharmacological effects and are used as medications, as recreational drugs, or in entheogenic rituals. Examples are the local anesthetic and stimulant cocaine, the psychedelic psilocin, the stimulant caffeine, nicotine, the analgesic morphine (Raymond et al., 2010), the anticancer compound vincristine, the anti- hypertension agent, reserpine, the anticholinergic agent, atropine, the vasodilator vincamine, the anti-arrhythmia compound quinidine, the anti-asthma therapeutic ephedrine, and the antimalarial drug quinine. Although, alkaloids act on a diversity of metabolic systems in humans and other animals, they almost uniformly invoke a bitter taste (Rhoades, 1979).

The boundary between alkaloids and other nitrogen-containing natural compounds is not clear- cut. Compounds like amino acid peptides, proteins, nucleotides, nucleic acid, amines and antibiotics are usually not called alkaloids (Raj, 2004). Natural compounds containing nitrogen in the exocyclic position (mescaline, serotonin, dopamine, etc.) are usually attributed to amines rather than alkaloids. Some authors, however, consider alkaloids a special case of amines (Raj,

2004).  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). The structures of some naturally occurring alkaloids are represented below:

Fig. 3: Structures of some naturally occurring alkaloids

Source: (Mamta et al., 2013)

1.3.3 Flavonoids

Flavonoids (or bioflavonoids) (from the Latin word flavus meaning yellow that is, their color in nature) are a class of plant secondary metabolites. They were referred to as vitamin P (probably because of the effect they had on the permeability of vascular capillaries) from the mid-1930s to early 50s, but the term has since fallen out of use (Mobh, 1938) .Flavonoids have hydroxyl group (OH). The effect of the hydroxyl moiety of flavonoids on protein targets varies depending on the position and number of the moiety on the flavonoid skeleton (Mobh, 1938). Flavonoids have been

reported to exert multiple biological property including antimicrobial, cytotoxicity, anti- inflammatory as well as anti-tumor activities but the best-described property of almost every group of flavonoids is their capacity to act as powerful antioxidants which can protect the human body from free radicals and reactive oxygen species (Mamta et al., 2013). The position of hydroxyl groups and other features in the chemical structure of flavonoids are important for their antioxidant and free radical scavenging activities. According to Atmani et al. (2009), flavonoids constitute a wide range of substances that play important role in protecting biological systems against the harmful effects of oxidative processes on macromolecules, such as carbohydrates, proteins, lipids and DNA. Chemical structures of some representative flavonoids are shown below:

Fig. 4: Chemical structures of some representative flavonoids

Source: (Atmani et al., 2009)

1.3.4 Tannin

A tannin (also known as vegetable tannin, natural organic tannins or sometimes tannoid, i.e. a type of biomolecule, as opposed to modern synthetic tannin) is an astringent, bitter plant polyphenolic compound that binds to and precipitates proteins and various other organic compounds including amino acids and alkaloids. 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 (Amorati and Valgimigli, 2012). The anti-carcinogenic and anti- mutagenic potentials of tannins may be related to their antioxidant property (Amorati and Valgimigli, 2012). The anti-microbial properties seemed to be associated with the hydrolysis of ester linkage between gallic acid and polyols hydrolyzed after ripening of many edible fruits (Amorati and Valgimigli, 2012).

1.3.5 Total Phenolics

In organic chemistry, phenols, sometimes called phenolics, are a class of chemical compounds consisting of a hydroxyl group (—OH) bonded directly to an aromatic hydrocarbon group. The simplest of the class is phenol, which is also called carbolic acid C6H5OH. Phenolic compounds are classified as simple phenols or polyphenols based on the number of phenol units in the molecule (Amorati, and Valgimigli, 2012).

Phenolic compounds are synthesized industrially; they are also produced by plants and microorganisms,  with  variation  between  and  within  species  (Hättenschwiler  and  Vitousek,

2000). Although, similar to alcohols, phenols have unique properties and are not classified as alcohols (since the hydroxyl group is not bonded to a saturated carbon atom). They have higher acidities due to the aromatic ring’s tight coupling with the oxygen and a relatively loose bond between the oxygen and hydrogen. The acidity of the hydroxyl group in phenols is commonly intermediate  between  that  of  aliphatic  alcohols  and  carboxylic  acids  (their  pKa  is  usually between 10 and 12).

Loss of a positive hydrogen ion (H+) from the hydroxyl group of a phenol forms a corresponding negative phenolate ion or phenoxide ion, and the corresponding salts which are called phenolates or phenoxides. As they are present in food consumed in human diets and in plants used in

traditional medicine of several cultures, their role in human health and disease is a subject of research (Mishra and Tiwari, 2011). Some phenols are germicidal and are used in formulating disinfectants. Others possess estrogenic or endocrine disrupting activities. Typical phenolics that possess antioxidant activity have been characterized as phenolic acids and flavonoids (Mishra and Tiwari, 2011). 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 and 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. In plants, oils are basically monophenolics such as tocopherols, water-soluble polyphenols are more typical in water-soluble products like fruits, vegetables, tea, coffee and wine, among others (Mishra and Tiwari, 2011). Polyphenolic compounds are known to have antioxidant activity. This activity is due to their redox properties which play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides (Mishra and Tiwari, 2011). Phenolic acids form a diverse group that includes the widely distributed hydroxybenzoic and hydroxycinnamic acids. They display a large range of structures and are responsible for the major organoleptic characteristics of plant-derived foods and beverages, particularly color and taste properties. They also contribute to the nutritional qualities of fruits and vegetables.  The structures of hydroxybenzoic and hydroxycinnamic acids are shown below.

Hydroxybe nzoic acid are Benzoic acid (1), Salicyclic acid (2), Vailinilic acid (3), Gallic acid (4) and

Hydroxycinnamic acid are Cinnamic acid (5), Ferulic acid (6), Sinapic acid (7) and Caffeic acid

Fig. 5: Structures of some hydroxybenzoic and hydroxycinnamic acids

Source: (Mamta et al., 2013)

1.3.6 Terpenoids

The  terpenoids  are  a  class  of  natural  products  which  have  been  derived  from  five-carbon isoprene units. Most of the terpenoids have multi-cyclic structures that differ from one another by their functional groups and basic carbon skeletons (Mamta et al., 2013). They play a role in traditional herbal remedies and are under investigation for antibacterial, antineoplastics, and other pharmaceutical functions (Nita et al., 2014).

1.3.7 Saponins

Saponins are a group of secondary metabolites found widely distributed in the plant kingdom. They form a stable foam in aqueous solutions such as soap, hence the name ―saponin‖. Chemically, saponins as a group include compounds that are glycosylated steroids, triterpenoids, and steroid alkaloids (Mamta et al., 2013).

Many saponins are known to be antimicrobial and to protect plants from insect attack. Saponins may be considered a part of plants‘ defence systems, and as such have been included in a large group  of  protective  molecules  found  in  plants  named  phytoanticipins  or  phytoprotectants (Lacaille and Wagner, 2000).

1.4 Overview of Malaria

Malaria is a mosquito-borne infectious disease caused by a eukaryotic protist of the genus Plasmodium which is transmitted via the bites of infected female Anopheles mosquito. It is a complex and deadly disease. It is widespread in tropical and subtropical regions, including parts of the Americas, Asia, and Africa (Rowe, 2006).

In the human body, the parasites multiply in the liver, and then infect red blood cells (Hedrick,

2011). Although there are many species of Plasmodium, only five infect humans and cause malaria. P. falciparum found in tropical areas is the major contributor to deaths from severe malaria. P. vivax, found in Asia and Latin America, has a dormant stage that can cause relapses. P. ovale is found in Africa and in Pacific islands. P. malariae occurs worldwide and can cause a chronic infection. P. knowlesi are found throughout Southeast Asia (Dondorp et al., 2004).

The plasmodium parasite are known as ‘night-biting’ mosquitoes because they most commonly bite between dusk and dawn (Cowman et al., 2012).   If a mosquito bites a person already infected with malaria, it can also become infected and spread the parasite on to other people. However, malaria cannot be spread directly from person to person (Wilcox et al., 2004). Once you are bitten, the parasite enters the bloodstream and travels to the liver. The infection develops in the liver before re-entering the bloodstream and invading the red blood cells (Rayner et al.,

2011). The parasites grow and multiply in the red blood cells. At regular intervals, the infected blood cells burst, releasing more parasites into the blood. Infected red blood cells usually burst every 48-72 hours (Rayner et al., 2011). Each time they burst, the patient experiences a stint of fever, chills and sweating. Malaria can also be transmitted through blood transfusions and the sharing of needles, but this is very rare (Paul et al., 2003). Typically, the time between being infected and when symptoms start (incubation period) is 7 to 18 days (Duval et al., 2010), depending on the specific infecting parasite. However, in some cases it can take up to a year for symptoms to develop.

When  a patient  suffers  from  the most  serious  type  of malaria,  caused  by the  Plasmodium falciparum parasite, there is a risk that he could quickly develop severe and life-threatening complications such as breathing problems and organ failure if not treated promptly (Ogueke et al., 2007).

1.5 Life Cycle of Plasmodium falciparum

The infection starts when a female mosquito injects (in her saliva) “sporozoites” (one form of P. falciparum) into human skin while taking a blood meal (CDC, 2016). A sporozoite travels through the bloodstream into the liver where it invades a liver cell. It matures into a “schizont” (mother cell) which produces 30000–40000 “merozoites” (daughter cells) within six days. The merozoites burst out and invade red blood cells. Within two days one merozoite transforms into a trophozoite, then into a schizont and finally 8–24 new merozoites burst out from the schizont and the red cell as it ruptures. Then the merozoites invade new red cells. Plasmodium falciparum can

prevent an infected red cell from going to the spleen (the organ where old and damaged red cells are destroyed) by sending adhesive proteins to the cell membrane of the red cell. The proteins make the red cell to stick to small blood vessel walls. This poses a threat for the human host since the clustered red cells might create a blockage in the circulation system.

A merozoite can also develop into a “gametocyte” which is the stage that can infect a mosquito. There are two kinds of gametocytes: males (microgametes) and females (macrogametes). They get ingested by a mosquito, when it drinks infected blood. Inside the mosquito’s midgut, male and female gametocytes merge into “zygotes” which then develop into “ookinetes.” The motile ookinetes penetrate the midgut wall and develop into “oocysts.” The cysts eventually release sporozoites, which migrate into the salivary glands where they get injected into humans. The development inside a mosquito takes about two weeks and only after that time can the mosquito transmit the disease.

A peculiarity of P. falciparum is its ability to adhere to venular endothelium (cytoadherence) of erythrocytes infected with maturing parasites. The parasitized erythrocytes remain attached until merozoites are formed that are released to invade other erythrocytes. Thus, the predominant form seen in the peripheral circulation is the ring-infected erythrocyte, the young form of the parasite. (Miller et al., 1994).

Worthy of note is that it is in the food vac        that the malaria parasite digest the host cell‘s haemoglobin to obtain essential amino acids. However, this process releases large amounts of haem, which is toxic to the parasite. To protect itself, the parasite ordinarily polymerizes the haem to haemozine, which is nontoxic, with the use of haem polymerase (Daniel et al., 1990). Some drugs interfere with this process such as chloroquine.

The diagram below shows the life cycle of Plasmodium falciparum.

Fig. 6: Life cycle of Plasmodium falciparium

Source: (Center for Disease Control and Prevention, 2016)

1.6 Symptoms of malaria

The early symptoms of malaria are flu-like and include a high temperature (fever), headache, sweats, chills and vomiting (Ene et al., 2013). These symptoms are frequently mild and can sometimes be difficult to identify as malaria. With some types of malaria, the fever occurs in four to eight hour cycles. During these cycles, the patient feels cold at first with shivering that lasts for up to an hour. He then develops a fever that lasts for two to six hours, accompanied by severe sweating.  Other symptoms of malaria may include: muscle pains, diarrhoea, and general feeling of unwell (Adeneye et al., 2006).

1.7 Antimalarial Drugs

Current treatment of malaria involves one of several different drugs or a combination of these drugs. The antimalarials in common use come from the following classes of compounds: the quinolines (chloroquine, quinine, mefloquine, amodiaquine, primaquine), the antifolates (pyrimethamine, proguanil and sulfadoxine), the artemisinin derivatives (artemisinin, artesunate, artemether, arteether) and hydroxynaphthaquinones (atovaquine) (Saifi et al., 2013). There are also malarone and doxycycline.

In general, antimalarial drugs are classified in terms of the action against the different stages of the life cycle of the parasite:

A) Blood schizonticidal agents: These are used to treat the acute attack. They act on the erythrocytic forms of the plasmodium.   Examples of such drugs include: Artemisinin, Chloroquine and Quinine

B) Tissue schizonticidal agents: Have a radical cure effect by acting on the parasites in the liver, these drugs also destroy gametocytes and thus reduce the spread of infection. A good example is Primaquine.

Malarone   consists   of   the   compounds   atovaquone   and   proguanil   hydrochloride.   These compounds interfere with the parasite’s production of certain nitrogenous bases, which are necessary for the replication of DNA (Marcus, 2004). Quinine, Chloroquine, Mefloquine and Primaquine build up in the food vacuole of the parasite. Once there, they inhibit the enzyme heme polymerase. This enzyme breaks down a compound that is toxic to the parasite, known as heme, which is produced when the parasite attacks hemoglobin within red blood cells. Without the properly-functioning heme polymerase, heme builds up within the protist cell and kills it (Lambert, 2010). Doxycycline stops the parasite from producing certain essential proteins, which weakens it and allows the immune system to fight it off (CDC, 2010).

Another option for treating malaria is Artemisinin Combination Therapy. Artemisinin comes from Chinese Wormwood and is a natural, safe and effective medicine, but the specifics of how this herb functions are not known. However, it only works temporarily, so it is often used with Chloroquine, Primaquine, Mefloquine, or Quinine. This combination technique is believed to prevent the malaria parasite from developing resistance against drugs (Hildebrand, 2010).

Although various drugs are currently effective at treating malaria, there is always the concern that the parasite will develop immunity to these drugs. The parasite has already become resistant to certain drugs in different parts of the world (Collins, 2012).  Also, some of these drugs are either not readily available or cannot be afforded by those in under-developed regions who forms the majority of the population being affected by the disease.

In the late 1940s, Chloroquine was massively used and accepted worldwide, but resistance has spread to the vast majority of the malaria endemic regions like Africa, South East Asia and East Asia (Sanket and Sarita, 2009). A combination of the antifolate drugs and sulfadoxine pyrimethamine, soon became choiced antimalaria widely used because it was inexpensive. This drug also faced unacceptable levels of therapeutic failure in many countries in South America, Asia and more recently, Africa (Boland, 2001). Resistance to mefloquine has become an issue in Combodia, Myanmar, and some border areas of Thailand. Whereas in some areas like Brazil and South East Asia where quinine and tetracycline are used in combination for treating uncomplicated malaria, sensitivity to quinine is seriously diminishing (Fidock  et al., 2004). Hence, the problem of resistance of plasmodium to antimalarials in the malaria endemic regions of the world has left this region with an unprecedented situation in which the few an affordable treatment options are rapidly losing therapeutic efficacy (Khozirah et al., 2011). Recently, there was a major breakthrough by Chinese researchers in the discovery of the antimalaria, artemisinin an endoperoxide sesquiterpene lactone as the active component of Artemisia annua, a herb remedy used in Chinese folk medicine for over 2000 years (Ene et al., 2009). The use of artemisinin derivatives have been negatively impacted by the observation that high parental doses of such compounds produced a limited, unique selective brain stem neuropathy in laboratory animals (Ridley, 2002).

Although, clinically artemisinin resistance has not been demonstrated, but it is likely to occur since artemisinin resistance has been obtained in laboratory models (Meshnik, 2002; Sanket and Sarita, 2009). Discovering new antimalarial compound is more than ever a priority due to the alarming rate of resistance to available drugs and the limited number of effective antimalarials (Peter and Antoli, 1998). Plants are usually considered to be possible candidates as alternative and rich source of new drugs.

1.8 Reactive oxygen species (ROS)

These are chemically reactive molecules containing oxygen. Examples include oxygen ions and peroxides. Reactive oxygen species are formed as a natural byproduct of the normal metabolism of oxygen and have important roles in cell signaling and homeostasis. However, during times of environmental stress (e.g. UV or heat exposure, ROS levels can increase dramatically (Devasagayam et al., 2004). This may result in significant damage to cell structures. Cumulatively, this is known as oxidative stress. Reactive oxygen species are also generated by exogenous sources such as ionizing radiation. Normally, cells defend themselves against ROS damage with enzymes such as a superoxide dismutases, catalases, lactoperoxidases, glutathione peroxidases and peroxiredoxins. Small molecule antioxidants such as ascorbic acid (vitamin C) and tocopherol (vitamin E) and glutathione also play important roles as cellular antioxidants. In a similar manner, polyphenol antioxidants assist in preventing ROS damage by scavenging free radicals. In contrast, the antioxidant ability of the extracellular space is less, the most important plasma antioxidant in humans is uric acid. Effects of ROS on cell metabolism are well documented in a variety of species. These include not only roles in apoptosis (programmed cell death) but also positive effects such as the induction of host defence genes and mobilisation of ion transport systems (Rada and Leto, 2008). This implicates them in control of cellular function. In particular, platelets involved in wound repair and blood homeostasis release ROS to recruit additional platelets to sites of injury. These also provide a link to the adaptive immune system via the recruitment of leukocytes. Reactive oxygen species are implicated in cellular activity to a variety of inflammatory responses including cardiovascular disease. They may also be involved in hearing impairment via cochlea damage induced by elevated sound levels, in ototoxicity of drugs such as cisplatin, and in congenital deafness in both animals and humans. In general, harmful effects of reactive oxygen species on the cell are most often :

1. damage of DNA

2. oxidations of polyunsaturated fatty acids in lipids (lipid peroxidation)

3. oxidations of amino acids in proteins

4. oxidatively inactivate specific enzymes by oxidation of co-factors

Fig. 7: Schematic representation of ROS generation

Source:  (Susinjan, 2015).

Fig. 8: Structure of reactive oxygen produced in biological systems derived from oxygen. Note the notation used to denote them and the difference between hydroxyl radical and hydroxyl ion, which is not a radical

Source: (Susinjan, 2015).

1.8.1 Classification of  Reactive Oxygen Specie (ROS)

They are exogenous and endogenous.

1.8.2 Exogenous ROS

Exogenous ROS can be produced from external sources such as pollutants: tobacco, smoke, drugs, xenobiotics, or radiation. Ionizing radiation can generate damaging intermediates through the interaction with water, a process termed radiolysis (Lien et al., 2008). Since water comprises

55–60% of the human body, the probability of radiolysis is quite high under the presence of ionizing radiation. In the process, water loses an electron and becomes highly reactive. Then through a three-step chain reaction, water is sequentially converted to hydroxyl radical (-OH), hydrogen peroxide (H2O2), superoxide radical (O2-) and ultimately oxygen (O2). The hydroxyl radical is extremely reactive that immediately removes electrons from any molecule in its path, turning that molecule into a free radical and so propagating a chain reaction. But hydrogen peroxide is actually more damaging to DNA than hydroxyl radical since the lower reactivity of hydrogen peroxide provides enough time for the molecule to travel into the nucleus of the cell, subsequently wreaking havoc on macromolecules such as DNA (Lien et al., 2008).

1.8.3 Endogenous ROS

Reactive  oxygen  species  are  produced  intracellularly  through  multiple  mechanisms  and depending on the cell and tissue types, the major sources being the “professional” producers of ROS NADPH oxidase (NOX) complexes (7 distinct isoforms) in cell membranes, mitochondria, peroxisomes, and endoplasmic reticulum (Muller, 2000). Mitochondria convert energy for the cell into a usable form, adenosine triphosphate (ATP). The process in which ATP is produced, called oxidative phosphorylation, involves the transport of protons (hydrogen ions) across the inner mitochondrial membrane by means of the electron transport chain. In the electron transport chain, electrons are passed through a series of proteins via oxidation-reduction reactions, with each acceptor protein along the chain having a greater reduction potential than the previous. The last destination for an electron along this chain is an oxygen molecule. In normal conditions, the oxygen is reduced to produce water; however, in about 0.1–2% of electrons passing through the chain (this number derives from studies in isolated mitochondria, though, the exact rate in living organisms is yet to be fully agreed upon), oxygen is instead prematurely and incompletely reduced to give the superoxide radical (·O2-), most well documented for Complex I and Complex III (Li et al., 2013). Superoxide is not particularly reactive by itself, but can inactivate specific enzymes or initiate lipid

peroxidation in its protonated form, hydroperoxyl HO2·. The pKa of hydroperoxyl is 4.8.

Thus, at physiological pH, the majority will exist as superoxide anion. If too much damage is present in mitochondria, a cell undergoes apoptosis or programmed cell death. Bcl-2 proteins are layered on the surface of the mitochondria, detect damage, and activate a class of proteins called Bax, which punch holes in the mitochondrial membrane, causing cytochrome C to leak out. This cytochrome C binds to Apaf-1, or apoptotic protease activating factor-1, which is free-floating in the cell’s cytoplasm. Using energy from the ATP in the mitochondrion, the Apaf-1 and cytochrome  C  bind  together  to  form  apoptosomes.  The  apoptosomes  bind  to  and  activate caspase-9, another free-floating protein. The caspase-9 then cleaves the proteins of the mitochondrial membrane, causing it to break down and start a chain reaction of protein denaturation and eventually phagocytosis of the cell (Li et al., 2013).

1.9 Oxidative damage

In aerobic organisms the energy needed to fuel biological functions is produced in the mitochondria via the electron transport chain. In addition to energy, reactive oxygen species (ROS) with the potential to cause cellular damage are produced. Reactive oxygen species can damage DNA, RNA, and proteins, which, in theory, contributes to the physiology of ageing (Patel et al., 1999).

Reactive oxygen species are produced as a normal product of cellular metabolism. In particular, one major contributor to oxidative damage is hydrogen peroxide (H2O       ), which is converted from superoxide that leaks from the mitochondria. Catalase and superoxide dismutase ameliorate the damaging effects of hydrogen peroxide and superoxide, respectively, by converting these compounds into oxygen and hydrogen peroxide (which is later converted to water), resulting in the production of benign molecules. However, this conversion is not 100% efficient, and residual peroxides persist in the cell. While ROS are produced as products of normal cellular functioning, excessive amounts can cause deleterious effects (Patel et al., 1999). Memory capabilities decline with age, evident in human degenerative diseases such as Alzheimer’s disease, which is accompanied by an accumulation of oxidative damage. Current studies demonstrate that the accumulation of ROS can decrease an organism’s fitness because oxidative damage is a contributor to senescence. In particular, the accumulation of oxidative damage may lead to

cognitive dysfunction, as demonstrated in a study in which old rats were given mitochondrial metabolites and then given cognitive tests.

The accumulation of oxidative damage and its implications for aging depends on the particular tissue type where the damage is occurring. Additional experimental results suggest that oxidative damage is responsible for age-related decline in brain functioning. Older gerbils were found to have higher levels of oxidized protein in comparison to younger gerbils. Treatment of old and young mice with a spin trapping compound caused a decrease in the level of oxidized proteins in older gerbils but did not have an effect on younger gerbils. In addition, older gerbils performed cognitive tasks better during treatment but ceased functional capacity when treatment was discontinued, causing oxidized protein levels to increase. This led researchers to conclude that oxidation of cellular proteins is potentially important for brain function (Carney et al., 1991).

1.10 Lipid peroxidation

Chemically, it involves the formation and propagation of lipid radicals, the uptake of molecular oxygen and arrangement of double bonds in the unsaturated lipids and eventually their destruction, with subsequent production of a variety of breakdown products, including alcohol, ketones, alkanes, aldehydes and ethers (Soumen, 2014). Lipid peroxidation (LPO) product accumulation in human tissues is a major cause of tissular and cellular dysfunction that plays a major role in ageing and most age-related and oxidative stress-related diseases (Anne et al.,

2010).

Lipid peroxidation involves three different stages, which include initiation, progression and termination. It is basically a chain reaction which is initiated by hydrogen abstraction or addition of an oxygen radical, resulting in oxidative breakdown of membrane-associated polyunsaturated fatty acid (PUFA) (Soumen, 2014). Fig. 9 below represents the chemistry of lipid peroxidation.

Figure 9: Chemistry of MLPO showing initiation, progression and termination steps and mechanism of re -initiation of LPO by redox cycling of metal ions

Source: (Soumen, 2014)

1.11.    Malondialdehyde

The assessment of lipid peroxidation is usually performed by analyzing secondary oxidation products such as malondialdehyde (MDA) (Jasna et al., 2008). The condensation of MDA with two molecules of 2-thiobarbituric acid (TBA) has been widely used to measure the extent of oxidative deterioration  of lipids in biological  and food systems (Gray,  1978). The level of oxidative stress in plasma and tissue usually correlates with MDA concentration. The absorbance of the complex is usually measured by spectrophotometry or spectrofluorometry (Angulo et al.,

1998). Malondialdehyde (MDA) is a reliable and commonly used marker of overall lipid peroxidation levels and the presence of oxidative stress (Kayar et al., 2015).

1.12 Antioxidant

An antioxidant is a molecule that inhibits the oxidation of other molecules. Oxidation is a chemical reaction that transfers electrons or hydrogen from a substance to an oxidizing agent. Oxidation reactions can produce free radicals. In turn, these radicals can start chain reactions. When the chain reaction occurs in a cell, it can cause damage or death to the cell. Antioxidants terminate  these  chain  reactions  by  removing  free  radical  intermediates,  and  inhibit  other oxidation  reactions.  They  do  this  by  being  oxidized  themselves,  so  antioxidants  are  often reducing agents (Sies, 1997). Although, oxidation reactions are crucial for life, they can also be damaging; plants and animals maintain complex systems of multiple types of antioxidants, such as antioxidant vitamins,: vitamin C, vitamin E etc as well as antioxidant enzymes such as catalase, superoxide dismutase and various peroxidases. Insufficient levels of antioxidants, or inhibition of the antioxidant enzymes, cause oxidative stress and may damage or kill cells. Oxidative stress is damage to cell structure and cell function by overly reactive oxygen- containing molecules and chronic excessive inflammation. Oxidative stress seems to play a significant role in many human diseases, including cancers. The use of antioxidants in pharmacology is intensively studied, particularly as treatments for stroke and neurodegenerative diseases. For these reasons, oxidative stress can be considered to be both the cause and the consequence of some diseases (Lien et al., 2008).

Antioxidant vitamins are widely used in dietary supplements and have been investigated for the prevention  of  diseases  such  as  cancer,  coronary  heart  disease  and  even  altitude  sickness. Although initial studies suggested that antioxidant supplements might promote health, later large clinical trials with a limited number of antioxidants detected no benefit and even suggested that excess supplementation with certain putative antioxidants may be harmful (Jha et al., 1995).

The  antioxidant  systems  are  classified  into  two  major  groups,  protective  or  enzymatic antioxidants and non-enzymatic antioxidants.

1.12.1  Enzymatic Antioxidants

Enzymatic antioxidants involved in the elimination of ROS include superoxide dismutase (SOD), catalase,  and  glutathione  peroxidase  (GPx)  (Mates,  2000).     The  enzymes,  catalase,  and glutathione peroxidase help to remove hydrogen peroxide. Catalase is an iron-containing enzyme found primarily in the small membrane-enclosed cell components called peroxisomes and detoxifies  hydrogen  peroxide  by  catalyzing  a  reaction  between  two  hydrogen  peroxide

molecules,  resulting  in  the  formation  of  water  and  O2   (Susinjan,  2015).  The  glutathione peroxidase  system  consists  of  several  components,  including  the  enzymes  –  glutathione peroxidase  and  glutathione  reductase  and  the  cofactors  monomeric  glutathione  (GSH)  and reduced nicotinamide adenosine dinucleotide phosphate (NADPH) (Schafer and Buettner, 2001).

1.12.2  Nonenzymatic Antioxidants

In addition to GSH and NADPH, numerous other non-enzymatic antioxidants are present in the cells, most prominently vitamin E (α-tocopherol) and vitamin C (ascorbic acid). Glutathione, an important water-soluble antioxidant, is synthesized from the amino acids glycine, glutamate, and cysteine. Glutathione directly quenches ROS such as lipid peroxides, and also plays a major role in  xenobiotic  metabolism.  Vitamin  E  is  a  major  antioxidant  found  in  the  lipid  phase  of membranes and, like other chemically related molecules, acts as a powerful terminator of lipid peroxidation. During the reaction between vitamin E and a lipid radical, the vitamin E radical is formed, from which vitamin E can be regenerated in a reaction involving GSH and ascorbate.

1.13 Catalase

Catalase is a common antioxidant enzyme found in nearly all living organisms exposed to oxygen  (such  as  vegetables,  fruit  or  animals).  It  catalyzes  the  decomposition  of  hydrogen peroxide to water and oxygen (Chelikani et al., 2004).

It is a very important enzyme in protecting the cell from oxidative damage by reactive oxygen species (ROS). Likewise, catalase has one of the highest turnover numbers of all enzymes; one catalase molecule can convert millions of molecules of hydrogen peroxide to water and oxygen each second (Goodsell, 2004).

1.14 Superoxide Dismutase

Superoxide dismutases (SOD) are antioxidant enzymes that protect the cells from superoxide radical (O2-), Under the action of SOD, O2- is transformed into hydrogen peroxide (H2O2) and O2 (McCord and Fridovich, 1988). Moreover, because of its ability to scavenge O2-, SOD protect cells against the single oxygen (1O2) and hydroxyl radical (OH-), the products of the reaction between O2- and H2O2, which are even more reactive and cytotoxic than either O2- or H2O2 (Liu

et al., 2001).  SOD1 is located in the cytosol and nucleus, SOD2 is present in the mitochondria, and SOD3 is located in the extra-cellular matrix of tissues (Fridovich, 1995).

1.15     Gluthathione

It is a cysteine-containing peptide found in most forms of aerobic life, that are not required in the diet and instead synthesized in the cells from the constituent amino acids (Meister and Anderson,

1983). It has antioxidant property since the thiol group in its cysteine moiety is a reducing agent. Due to its high concentration and central role in maintaining the cells redox state, gluthathione is one of the most important cellular antioxidants (Matill, 1947).

The redox-active tripeptide glutathione is an endogenous reducing agent that is found in abundance and throughout the cell (Christian, 2011). Glutathione, abbreviated as GSH is the body’s essential health AID – Antioxidant, Immune booster and Detoxifier. Glutathione is a ubiquitous low molecular weight compound synthesized in the cytosol. It consists of the three amino  acids  glutamate,  cysteine  and  glycine  that  are  linearly linked  by  a  ƴ-peptide  and  a conventional α-peptide bond, respectively. Owing to the sulfhydryl (–SH) group of the cysteine residue, reduced glutathione (GSH) is a redox-active molecule that can participate in a variety of antioxidant reactions. It can either act as an electron donor in the reduction of peroxides, which is catalyzed by glutathione peroxidases, or that of disulfides, a reaction catalyzed by glutaredoxins (Christian, 2011). The reduced and oxidized forms of glutathione (GSH and GSSG) act in concert with other redox-active compounds (e.g., NAD(P)H) to regulate and maintain cellular redox status (Jones et al., 2011). Other functions include (i) maintaining the essential thiol status of proteins and other molecules; (ii) storage of cysteine reserves both in the cell and for inter- organ   transfer;   (iii)   involvement   in   the   metabolism   of   estrogens,   leukotrienes,   and prostaglandins; (iv) participation in the reduction of ribonucleotides to deoxyribonucleotides; (v) participation in the maturation of iron-sulfur clusters in proteins; (vi) copper and iron transfer; (vii) signal transduction from the environment to cellular transcription machinery (Christian, 2011).

1.16 Liver function tests (LFTs)

Liver function tests (LFTs) are groups of blood tests that give information about the state of a patient’s liver (Om and Tej, 2009). Liver transaminases (AST or SGOT and ALT or SGPT) are useful biomarkers of liver injury in a patient with some degree of intact liver function (Johnston,

1999).  Most  liver diseases  cause only mild  symptoms  initially,  but  these diseases  must  be detected early. Hepatic (liver) involvement in some diseases can be of crucial importance. This testing is performed on a patient’s blood sample. Some tests are associated with functionality (e.g., albumin), some with cellular integrity (e.g., transaminase), and some with conditions linked to the biliary tract (gamma-glutamyl transferase and alkaline phosphatase). Several biochemical tests are useful in the evaluation and management of patients with hepatic dysfunction. These tests can be used to detect the presence of liver disease, distinguish among different types of liver disorders, gauge the extent of known liver damage, and follow the response to treatment. Some or all of these measurements are also carried out (usually about twice a year for routine cases) on those individuals taking certain medications, such as anticonvulsants, to ensure the medications are not damaging the person’s liver (Johnston, 1999). The liver function tests include alkaline phosphatase (ALP), Alanine and Aspartate transaminases (ALT and AST), total and direct bilirubin, Gamma-glutamyl transferase (GGT), albumin, and prothrombin time tests (Kamath,

1996). ALT and AST are notably elevated in liver damage caused by liver cell disease. However, in intrahepatic obstructive disease which may be caused by some drugs or billiarry cirrhosis, the alkaline phosphatase is most abnormal.

1.16.1 Alkaline phosphatase (ALP)

Alkaline phosphatase is an enzyme found in many tissues with the highest concentrations in the liver, biliary tract and bone. It is used to assess liver lesions that may cause biliary obstruction such  as  tumors  or  abscesses  (Janis,  2002).  Serum  alkaline  phosphatase  levels  may  greatly increase with liver tumors and lesions, and may show a moderate increase with diseases such as hepatitis.  In  the liver,  the enzyme is  localized  to  the microvilli  of the bile  canaliculi,  and therefore it serves as a great marker of extrahepaticbiliary obstruction,such as a stone in the common bile duct, or in intrahepatic cholestasis, such as drug cholestasis or primary biliary cirrhosis.

1.16.2 Alanine aminotransferase (ALT)

Alanine aminotransferase was formerly called serum glutamic-pyruvic transaminase (GPT or

SGPT).It is found in many tissues like cardiac tissue but highest concentration is in the liver. It is

considered as the most liver-specific enzyme of the transferases. This enzyme usually rises higher than aspartate aminotransferase in liver disease, with moderate increases (up to 10 times normal) in cirrhosis, up to 100 times normal in viral or toxic hepatitis (Barbara et al., 2000). Clinical applications of ALT assays are confined mainly to evaluation of hepatic disorders. Higher elevations are found in hepatocellular disorders than in extrahepatic or intrahepatic obstructive  disorders.  In  acute  inflammatory  conditions  of  the  liver,  ALT  elevations  are frequently higher than those of AST and tend to remain elevated longer as a result of the longer half-life of ALT in serum(16 and 24 hours, respectively).

1.16.3 Aspartate aminotransferase (AST)

Aspartate aminotransferase was formerly called serum glutamic oxaloacetic transferase (GOT or SGOT).The highest concentrations are found in cardiac tissue, liver, and skeletal muscle, with smaller amounts found in the kidney, pancreas, and erythrocytes. It is elevated after myocardial infarction, as well as in liver disease. In liver disease, the AST increase is usually less than ALT increase, but in liver disease caused by alcohol use, the AST increase may be two or three times greater than ALT increase (Vozarova et al, 2002)

1.17 Bilirubin

Bilirubin   is   formed   from   the   lysis   of   red   cells   (the   haem   component)   within   the reticuloendothelial system. Unconjugated bilirubin is transported to the liver loosely bound to albumin. It is water insoluble and therefore cannot be excreted in urine. Conjugated bilirubin is water  soluble  and  appears  in  urine  (Limdi  and  Hyde,  2003).  Within  the  liver,  conjugated bilirubin is conjugated to bilirubin glucoronide and subsequently secreted into bile and the gut respectively. Intestinal flora breaks it down into urobilinogen, some of which is reabsorbed and either excreted via the kidney into urine or excreted by the liver into the gastro-intestinal tract. The remainder is excreted in the stool as stercobilinogen giving stool its brown colour (Limdi and Hyde, 2003). Serum bilirubin is                  mainly in an unconjugated form reflecting a balance between production and hepatobiliary excretion. Bilirubin production increases in haemolysis, ineffective erythropoiesis, resorption of a haematoma, and rarely in muscle injury. In all these cases the bilirubin is mainly in an unconjugated form.

1.18 Renal Function tests

Renal function is an indication of the state of the kidney and its role in renal physiology (Stevens et al., 2006). There are different methods in checking the integrity of the kidney. Basically, this could        be        blood        test        or        urine        test.        Blood        test        encompasses stimated Glomerular Filtration Rate (eGFR),  creatinine  and  urea,  while  urine  test  include albumin/creatinine ratio and urinalysis.

1.18.1 Creatinine

Creatinine is a waste product of creatine phosphate, a substance stored in muscle and used for energy. Creatinine is excreted by the kidney. When renal function is impaired, blood creatinine levels rise, but more than 50% of kidney function must be lost before this happens. Creatinine levels are not affected by diet or hormone levels. Increases occur when there is impairment of urine

formation  or  excretion,  which  occurs  in  renal  disease,  shock,  water  imbalance,  or  ureter blockage.

1.18.2 Urea

Urea, also known as carbamide, is an organic compound with the chemical formula CO(NH2)2. This amide has two –NH2  groups joined by a carbonyl (C=O) functional group. Urea is the largest circulating pool of nitrogen, excluding nitrogen in circulating proteins and its production changes      in      parallel      to      the      degradation      of      dietary      and      endogenous proteins. High urea levels suggest decreased kidney function (Weiner et al., 2014).

1.19 Serum Electrolytes

1.19.1 Sodium

Sodium is the dominant extracellular cation (positive ion) and cannot freely cross from the interstitial space through the cell membrane into the cell. Its homeostasis (stability of concentration) inside the cell is vital to the normal function of any cell. Hyponatremia is low sodium concentration in the serum. Exercise can induce hyponatremia. When sodium levels in the blood become excessively low, excess water enters the brain cells and the cells swell. This can lead to headache, nausea, vomiting and seizures (Moritz and Ayus, 2003). The main source of body sodium is sodium chloride contained in ingested foods (Terri and Sesin, 1958). Hyponatremia is  found  in  a variety of conditions  including the following:  severe polyuria,

metabolic acidosis, Addison‘s disease, diarrhoea and renal tubular disease. Hypernatremia (increased serum sodium level) is found in the following conditions: hyperadrenalism, severe dehydration, diabetic coma after therapy with insulin, excess treatment with sodium salts (Maruna, 1958).

1.19.2 Potassium

Potassium, a metallic inorganic ion is the most abundant cation in the body. The vast majority of potassium is in the intracellular compartment with a small amount in the extracellular space. Total body potassium is approximately 55mEq/Kg body weight. The intracellular potassium concentration is on average 150 mEq/L. The ratio of intracellular to extracellular K+ (K1: K2) is the major determinant of the resting membrane potential and plays a crucial role in the normal functioning of all cells, especially those with inherent excitability (Arruda et al., 1981). Elevated Potassium (Hyperkalemia) is often associated with renal failure, dehydration, shock or adrenal insufficiency. Decreased Potassium concentration in the plasma (Hypokalemia) are associated with malnutrition, negative nitrogen balance, gastro-intestinal fluid losses and hyperactivity of adrenal cortex (Terri and Sesin, 1958).

1.20 Haematology

Haematology is the study of blood, the blood-forming organs, and blood diseases (Sidell and Kristin, 2006). Haematology includes the study of etiology, diagnosis, treatment, prognosis, and prevention of blood diseases that affect the production of blood and its components, such as blood cells, haemoglobin, blood proteins, and the mechanism of coagulation.

1.20.1 Packed Cell Volume (PCV)

Packed Cell Volume (PCV) or Erythrocyte Volume Fraction (EVF), is the volume percentage of red blood cells in the body. It is considered to be an integral part of a person‘s complete blood count results, along with haemoglobin (Hb) concentration, white blood cell (WBC) count and platelet count. Low level of red blood cells points to a problem with the integrity of the kidney cells (Jelkmann, 2004).

1.20.2 Haemoglobin

Haemoglobin is the iron-containing oxygen- transport metalloprotein in the blood cells of almost the vertebrates. It carries oxygen from the lungs to body parts for the metabolism of glucose in order to generate energy (Sidell and Kristin, 2006). If haemoglobin is low, it signifies anaemia. Anaemia is a condition in which the number of red blood cells is insufficient to meet the body‘s needs (WHO, 2001). Sickle cell anaemia is the most important haemoglobinopathy (Murray et al.,  2006).  The  functions  of  blood  are  many  and  varied.   Besides  providing  material nourishments, blood also provides the necessary moisture needed by the internal organs to function properly. Insufficient blood or blood deficiencies can cause many problems ranging from weakness, inability to concentrate, hot flushes, increased susceptibility to infection, shortness of breath, fatigue, dizziness, palpitation, anxiety, depression, insomnia, nervousness, headache and diminished sex drive. Women in particular, are especially susceptible to blood deficiencies due to their monthly menstrual cycle. In addition, because the life span of the red blood cells is relatively short, the blood needs to be constantly replenished (Murray et al., 2006). In Nigeria, the local people are known for using natural herbs and herbal formulae for addressing various kinds of blood deficiencies. In south-eastern Nigeria, the roots of Vitex doniana among others, are considered excellent natural herbal blood boosters, used especially for debilitating conditions, acute blood loss and blood deficiency diseases (Murray et al., 2006).

1.20.3 Red Blood Cells

Red blood cells  (also referred to as  erythrocytes) are the most common type of  blood cell  and the  vertebrate  organism’s principal means of delivering  oxygen  (O2) to the body tissues via the blood  flow through the circulatory system. They take up oxygen in the lungs or gills and release it while squeezing through the body’s capillaries. In humans, mature red blood cells are flexible biconcave disks that lack a cell nucleus and most organelles. 2.4 million new erythrocytes are produced per second (Kleinbongard et al., 2006).   A red blood cell (RBC) count is typically ordered as part of a complete blood count (CBC) and may be used as part of a health check-up to screen for a variety of conditions. This test may also be used to help diagnose and/or monitor a number of diseases that affect the production or lifespan of red blood cells (Ford, 2013).

1.20.4  White Blood Cells

White blood cells (leukocytes) are an important part of the body‘s defense against infectious organisms and foreign substances. Like all blood cells, white blood cells are produced in the bone marrow. They develop from stem (precursor) cells that mature into one of the five major types of white blood cells: Neutrophils, Lymphocytes, Monocytes, Eosinophils, and Basophils. Too few or too many white blood cells indicate a disorder (Furlan et al., 2014)

1.21     Lipid Profile

A standard lipid  profile includes  measurements  of plasma or serum  concentrations  of total cholesterol,  LDL  cholesterol,  high-density lipoprotein  (HDL)  cholesterol,  and  triacyglycerol (Børge et al., 2016). Cholesterol is a lipid (fat) produced by the liver and is vital for the normal functioning of the body.  It is an important lipid found in human blood. However, when it is in excess, it may cause health problems such as arteriosclerosis (build-up of plague in the arteries) resulting in myocardial infarction (Kurian, 2013). Every cell in the human body has cholesterol in its outer membrane. Cholesterol is carried in the blood by molecules called lipoproteins (Tajoda et al., 2013).   Most of the lipids in the human body and in food are comprised of triglycerol. They are present in the blood plasma. Triglycerides, in association with cholesterol, form the plasma lipids (blood fat) (Tajoda et al., 2013).

1.22 Acute toxicity

Acute toxicity describes the adverse effects of a substances that result either from a single exposure or from multiple exposures in a short space of time (less than 24 hours). Most acute toxicity data come from animal testing or in vitro testing methods (Walum, 1998). The median lethal dose (LD50) is the dose required to kill half the numbers of a tested population after a specified test duration (Lorke, 1983). Investigation of the acute toxicity is the first step in the toxicological investigations of an unknown substance. The index of the acute toxicity is the LD50 (Lorke, 1983). Scientific investigation of previously unknown and known plants is necessary not only because of the need to discover new drugs but to assess the toxicity faced by the users. Besides, it is important that traditionally claimed therapeutic properties of plants be confirmed and its toxicity limit determined (Prohp amd Onoagbe, 2012).

1.23 Aim and Objectives of the Study

1.23.1 Aim of the study

This research is therefore aimed at evaluating the antimalarial effects and some biochemical status of methanol extracts of vitex doniana leaves in plasmodium berghei-infected mice to scientifically validate the claim or prove otherwise the folkloric believe.

1.23.2  Specific Objectives of the Study

The following specific objectives were designed to achieve the above stated aim.

   To determine the qualitative and quantitative phytochemical constituents of methanol extracts of Vitex doniana leaves.

   To determine the median lethal dose (LD50) of methanol extracts of Vitex doniana leaves

   To determine the effect of methanol extracts of Vitex doniana leaves on the percentage parasitemia of P. berghei– infected mice.

   To  determine  the  effect  of  methanol  extracts  of  Vitex  doniana  leaves  on  lipid

peroxidation marker (MDA) of P. berghei-infected mice.

   To determine the effect of methanol extracts of Vitex doniana leaves on Antioxidant status of P. berghei-infected mice such as superoxide dismutase (SOD), catalase (CAT) and Glutathione (GSH).

   To determine the effect of methanol extracts of Vitex doniana leaves on haematological

indices: Red Blood Cell (RBC) count, Packed Cell Volume (PCV), White Blood Cell

(WBC) count and haemoglobin (Hb) ) of P. berghei-infected mice.

   To determine the effect of methanol extract of Vitex doniana leaves on the activities of liver  marker  enzymes  such  as  Alanine  Aminotransferase  (ALT),  Aspartate aminotrasferase (AST), Alkaline phosphatase (ALP) of P. berghei-infected mice.

   To determine the effect of methanol extracts of Vitex doniana leaves on Total Bilirubin concentration and kidney markers such as Urea and Creatinine of P. berghei-infected mice.

   To determine the effect of methanol extracts of Vitex doniana leaves on serum electrolyte such as Sodium and Potassium of P. berghei-infected mice.

   To determine the effect of methanol extracts of Vitex doniana leaves on lipid profile (Total Cholesterol, Low density Lipoprotein (LDL), High density lipoprotein (HDL), Triacylglycerol (TAG)) of P. berghei-infected mice.

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