Phytochemical Screening and Antioxidant Activities of Some Selected Medicinal Plants Used for Malaria Therapy in Southwestern Nigeria

Purpose : Oxidative stress has been shown to play an important role in the development of anaemia in malaria. Indeed, increase in total antioxidant status has been shown to be important in recovery from malaria. The antioxidant activities of four medicinal plants traditionally used in the treatment of malaria in southwestern Nigeria were determined. Methods : The ethanolic extracts of the leaves of Carica papaya Linn. [Caricaceae] , stem bark of Magnifera indica Linn. [Anacardiaceae], leaves of Psidium guajava Linn. [Myrtaceae] and the leaves of Vernonia amygdalina Del. [Compositae], were used in the present study. The plant parts commonly used in the locality in malaria therapy were employed in this study. The plants were screened for the presence of phytochemicals and, their effect on 2,2-Diphenyl-1-picryl-hydrazyl radical (DPPH) was used to determine their free radical scavenging activity. Results : Phytochemical screening of the plants showed the presence of flavonoids, terpenoids, saponins, tannins and reducing sugars. M. indica did not contain cardiac glycosides and alkaloids while, P. guajava also showed the absence of alkaloids and anthraquinones. Anthraquinones was similarly absent from V. amygdalina. Concentrations of the plant extracts required for 50% inhibition of DPPH radical scavenging effect (IC 50 ) were recorded as 0.04 mg/ml, 0.313 mg/ml, 0.58 mg/ml, 2.30 mg/ml and 0.054 mg/ml for P. guajava, M. Indica, C. papaya, V. amygdalina and Vitamin C, respectively. Conclusion : All the plants showed potent inhibition of DPPH radical scavenging activity, P. guajava being the most potent. The free radical scavenging (antioxidant) activities of these plants probably contribute to the effectiveness of the above plants in malaria therapy.


INTRODUCTION
Malaria is a global disease prevalent in the tropics caused by Plasmodium species. It is one of the oldest and greatest health challenges affecting 40% of the world's population. It affects 300-500 million people and kills 1.5-2.7 million people annually 1 . High mortality rate is reported in children and pregnant women, also the disease has a negative impact on the economy of prevalent countries 2 . In Nigeria, malaria is endemic throughout the country.
World health Organization (WHO) has estimated malaria mortality rate for children under five in Nigeria at 729 per 100,000 3 Nigeria's Ministry of Health reported in April 2004 that malaria is responsible for one out of ten deaths in pregnant women and costs the Federal Government of Nigeria over one billion naira annually 3 .
One of the major reasons for the development of anaemia in malaria seems to be oxidative stress 4-6 . The immune system of the body is activated by infections, including malaria, thereby causing the release of reactive oxygen species.
In addition to this, the malaria parasite also stimulates certain cells to produce reactive oxygen species thereby resulting in haemoglobin degradation 5,7 . Indeed, depressed level of plasma antioxidants has been shown in Plasmodium falciparum-infected children and it has been suggested as a possible contributor to the morbidity and mortality of malaria

Extraction of plant materials
The plant materials (leaves of C. papaya, P. guajava, V. amygdalina and the stem bark of M. indica) were air-dried at room temperature (26˚C) for 2 weeks, after which it was grinded to a uniform powder. The ethanol extracts were prepared by soaking 100 g each of the dry powdered plant materials in 1 L of ethanol at room temperature for 48 h. The extracts were filtered after 48 h, first through a Whatmann filter paper No. 42 (125mm) and then through cotton wool. The extracts were concentrated using a rotary evaporator with the water bath set at 40˚C. The percentage yield of extracts ranged from 7-19%w/w.

Test for reducing sugars (Fehling's test)
The aqueous ethanol extract (0.5 g in 5 ml of water) was added to boiling Fehling's solution (A and B) in a test tube. The solution was observed for a colour reaction.
Test for anthraquinones 0.5 g of the extract was boiled with 10 ml of sulphuric acid (H 2 SO 4 ) and filtered while hot. The filtrate was shaken with 5 ml of chloroform. The chloroform layer was pipette into another test tube and 1 ml of dilute ammonia was added. The resulting solution was observed for colour changes.

Test for terpenoids (Salkowski test)
To 0.5 g each of the extract was added 2 ml of chloroform. Concentrated H 2 S0 4 (3 ml) was carefully added to form a layer. A reddish brown colouration of the interface indicates the presence of terpenoids.

Test for flavonoids
Three methods were used to test for flavonoids. First, dilute ammonia (5 ml) was added to a portion of an aqueous filtrate of the extract. Concentrated sulphuric acid (1 ml) was added.
A yellow colouration that disappear on standing indicates the presence of flavonoids. Second, a few drops of 1% aluminium solution were added to a portion of the filtrate. A yellow colouration indicates the presence of flavonoids. Third, a portion of the extract was heated with 10 ml of ethyl acetate over a steam bath for 3 min. The mixture was filtered and 4 ml of the filtrate was shaken with 1 ml of dilute ammonia solution. A yellow colouration indicates the presence of flavonoids.

Test for saponins
To 0.5 g of extract was added 5 ml of distilled water in a test tube. The solution was shaken vigourously.and observed for a stable persistent froth. The frothing was mixed with 3 drops of olive oil and shaken vigourously after which it was observed for the formation of an emulsion.

Test for tannins
About 0.5 g of the extract was boiled in 10 ml of water in a test tube and then filtered. A few drops of 0.1% ferric chloride was added and observed for brownish green or a blue-black colouration Test for alkaloids 0.5 g of extract was diluted to 10 ml with acid alcohol, boiled and filtered. To 5 ml of the filtrate was added 2 ml of dilute ammonia. 5 ml of chloroform was added and shaken gently to extract the alkaloidal base. The chloroform layer was extracted with 10 ml of acetic acid.
This was divided into two portions. Mayer's reagent was added to one portion and Draggendorff's reagent to the other. The formation of a cream (with Mayer's reagent) or reddish brown precipitate (with Draggendorff's reagent) was regarded as positive for the presence of alkaloids.

Test for cardiac glycosides (Keller-Killiani test)
To 0.5 g of extract diluted to 5 ml in water was added 2 ml of glacial acetic acid containing one drop of ferric chloride solution. This was underlayed with 1 ml of concentrated sulphuric acid. A brown ring at the interface indicated the presence of a deoxysugar characteristic of cardenolides. A violet ring may appear below the brown ring, while in the acetic acid layer a greenish ring may form just above the brown ring and gradually spread throughout this layer.

Determination of antioxidant activity
The radical scavenging activities of the plant extracts against 2,2-Diphenyl-1-picryl hydrazyl radical (Sigma-Aldrich) were determined by UV spectrophotometry at 517 nm. Radical scavenging activity was measured by a slightly modified method previously described 12,13 . The following concentrations of the extracts were prepared, 0.05, 0.1, 0.5, 1.0, 2.0 and 5 mg/ml in methanol (Analar grade).
Vitamins C was used as the antioxidant standard at concentrations of 0.02, 0.05, 0.1, 0.2, 0.5 and 0.75 mg/ml. I ml of the extract was placed in a test tube, and 3 ml of methanol was added followed by 0.5 ml of 1 mM DPPH in methanol. A blank solution was prepared containing the same amount of methanol and DPPH. The radical scavenging activity was calculated using the following formula: where A b is the absorption of the blank sample and A a is the absorption of the extract

Phytochemical screening of plant materials
The phytochemical screening of the plants studied showed the presence of flavonoids terpenoids, saponins and tannins (Table 1) (Table 1). The presence of flavonoids and tannins in all the plants is likely to be responsible for the free radical scavenging effects observed. Flavonoids and tannins are phenolic compounds and plant phenolics are a major group of compounds that act as primary antioxidants or free radical scavengers 14 .

Radical scavenging (antioxidant) activity
The DPPH test provides information on the reactivity of the test compounds with a stable free radical. DPPH gives a strong absorption band at 517nm in visible region. When the odd electron becomes paired off in the presence of a free radical scavenger, the absorption reduces and the DPPH solution is decolourised as the colour changes from deep violet to light yellow. The degree of reduction in absorbance measurement is indicative of the radical scavenging (antioxidant) power of the extract. The crude extract of P. guajava appeared to be as potent as Vitamin C with a maximum inhibition of 91% at 0.5mg/ml which is comparable to 95% for vitamin C at the