In Vitro Phytochemistry and Antiplasmodial Activity of Leaf Extract and Fractions of Nauclea diderrichii

The current investigation deals with phytochemical screening and in vitro antiplasmodial activity of crude ethanol leaf extract and three fractions of crude ethanol leaf-extract of Nauclea diderrichii. Phytochemical test to screen bioactive compounds was carried out via standard protocols which uncovered the presence of alkaloids, saponins, steroids, phenols, tannins, flavonoids, glycosides and carbohydrates, extraction was done using absolute ethanol to afford the crude extract (Nd-ET) while maceration was done using solvents of different polarity gradient (petroleum ether, chloroform and ethyl acetate) to afford the remaining fractions (Nd-F1, Nd-F2 and Nd-F3). The antiplasmodial activity of the crude-extract and those of crude-extract-fractions against plasmodium falciparum unveil promising percentage elimination at all concentrations, with ethanol crude extract (Nd-ET) and ethyl acetate fraction (Nd-F3) having the highest, with 75.50% and 72.65% at 625μg/ml, and 87.83% and 86.33% at 5000μg/ml, respectively. These results clearly indicated that the active compounds present in the crude leaf extract/fractions of Nauclea diderrichii are highly potent eliminators of plasmodium falciparum and validate their popular usage in folk medicine in Gusau Local Government, Zamfara State, Nigeria, for the treatment of malaria.


Introduction
From time immemorial, medicinal plants have been used to flavour and conserve 334 food, treat health disorders and to prevent diseases including epidemics and infectious disease with increasing number of reports on pathogenic microorganisms resistant to antimicrobials in clinical used (Singh [16]). Assurance of the safety, quality, and efficacy of medicinal plants and herbal products has now become a key issue in industrialized and in developing countries (Singh [16] and Khond et al. [21]). The knowledge of their healing effects has been transmitted over the centuries within and among human communities. This healing effect is due to active compounds produced by the medicinal plants during secondary metabolism (Singh [16] and Dar et al. [12]). Secondary metabolites produced either as a single compound or in combination as a group of related compounds within the natural products (extract/fraction) are usually responsible for the biological activity of medicinal plants and as potentials for drug discovery (Soderberg [22]).
Natural environment is furnished with plants and plant's products which have been found to be of great medicinal and nutritional importance. There are many experimental pieces of evidence indicating the use of plants for medicinal purposes, hence, the plant kingdom has become a target for the search of biologically active lead compounds for the complementary/alternative management of human ailments (Osigwe et al. [11]).
In folkloric medicine in Nigeria and other part of the world, particularly West Africa, Nauclea diderrichii is used in the management of a wide range of ailments ranging from endocrine disorders, infections, wound healing, anti-malaria, anemia, stomach ache, indigestion, fever and jaundice. In Nigeria, bark preparations are used against fever and malaria, and as antiperiodic, appetizer and others (Orwa et al. [10]), it is also used for its anti-diabetic activity with good toxicological profile (Theophine et al. [17]). The bark and leaf decoctions of the plant are taken in Sierra Leone and Ghana against stomach ache, diarrhea, malaria, as a wash for measles and as a foot wash after long walks. In Côte d'Ivoire, the bark is sometimes used to treat bacterial infection. In Gabon, a bark infusion is drunk against fever. In Congo, bark decoctions are taken or the leaf pulp is rubbed in for the treatment of fever, stomach problems, gonorrhea, hepatitis and menstruation problems, while a bark infusion is taken as a vermifuge. In various 335 countries leaf infusions, leaf decoctions and the leaf pulp are drunk or used in washings, baths or embrocation to treat fever. In Guinea leaf preparations are applied on tumours. The ripe infructescence is also eaten as a cough medicine in Sierra Leone (Nauclea diderrichii (PROTA) [7]).
The aim of this investigation is to evaluate the phytochemical contents and to carry out anti-plasmodial test on the crude ethanol leaf-extract (Nd-ET) and three fractions (Nd-F 1 , Nd-F 2 and Nd-F 3 ) of the crude ethanol extract of Nauclea diderrichii at different concentrations.

Botanic description of the plant (Nauclea diderrichii)
Nauclea diderrichii is a plant with English name (African peach) and trade name (Opepe). It is an evergreen tree that reaches a height of 30-40 m and a diameter of 0.9-1.

Traditional description of the plant (Nauclea diderrichii)
The plant, Nauclea diderrichii is commonly known locally as Opepe (Nigeria), It is a medium sized tree up to 30 m tall with bole up to 80 cm in diameter. The Heartwood is orange or golden yellow, darkening on exposure; sapwood whitish or pale yellow, clearly defined. Texture rather coarse; grain usually interlocked or irregular; lustrous; without characteristic odor or taste (Nauclea diderrichii (PROTA) [7]). It is well known as "Tuwon Biri" in Hausa land, Northwestern, Nigeria.

Sample collection and authentication
The fresh leaves of Nauclea diderrichii were plucked directly from the plant at Mada district, Gusau Local Government Area, Zamfara State, Nigeria. It was identified and authenticated at the Department of Plant Biology, Bayero University, Kano, Nigeria, with Herbarium Accession Number 0434.

Sample preparation and extraction
The fresh leaves of Nauclea diderrichii were washed with clean water and air dried under shade at ambient temperature for about a week in the absence of any form of contaminant. It was then pulverized mechanically (using mortar and pestle) to form a fine powder. After pulverization the powdered sample was stored in an air tight container, in cool and dry place away from light and was later subjected to ethanol extraction (Bargah [13]).

Extraction process
About 1000 g of the powdered leaf sample of Nauclea diderrichii was percolated with 3.0 L of absolute ethanol with shaking at regular interval for one week, after which the extract was separated from the debris by filtration. The filtrate was then concentrated using a Rotavapor (R110 at 40°C) and was coded Nd-ET (ethanol crude extract), weighed and kept in a cool and dry place away from any form of contaminant (Sarkar et al. [15]).

Maceration of crude (Nd-ET) extract
The crude ethanol extract (Nd-ET) was then macerated with petroleum ether, chloroform and ethyl acetate to afford the remaining fractions, coded Nd-F 1 , Nd-F 2 and Nd-F 3 respectively. Each fraction obtained was weighed and concentrated at room temperature.

In vitro phytochemical screening
Preliminary phytochemical tests for the screening and identification of bioactive chemical constituents like alkaloid, carbohydrates, flavonoid, glycoside, phenol, saponin, steroid, and tannin present in the crude leaf extract and three extract-fractions of Nauclea diderrichii were carried out using standard procedures adopted from Harborne [4] and Gurav et al. [3].

Preparation of stock solution and other concentrations
The stock solutions of the ethanol-crude-extracts (Nd-ET) and those of crudeextract-fraction (Nd-F 1 , Nd-F 2 and Nd-F 3 ) were prepared by dissolving 10 mg of each extract in 1 mL of dimethyl sulphuroxide (DMSO) to produce 10000 µgmL −1 . Other Lower concentration solutions of 5000, 2500, 1250 and 625 µgmL −1 were obtained from the stock solutions by serial double dilution.

Preparation of culture media (RPMI 1640)
The media was prepared by dissolving 10.4g of the powdered material into 1 Litre of distilled water, 1 mL of gentamicin was added as a stabilizer and then autoclaved at 121°C for 15 minutes.

Anti-plasmodial activity
About 0.1 mL of the test solution, 0.1 mL of healthy rabbit blood, and 0.2 mL of the culture media were all added together in a vial containing 0.1 mL of the parasitemia erythrocytes and mixed thoroughly. The mixture was incubated for 24hrs at 37°C. the incubation was carried out in a glass bell jar containing a lighted candle to ensure the supply of required quantity of CO 2 (about 5%), O 2 (about 2%) and N 2 (g) (about 93%) (Trager and Jensen [23]). Alongside a positive control, 2000 µg/mL Artemether Lumefantrine and a negative control, culture media and positive erythrocytes were also incubated.
After 24hrs incubation, a drop of the incubated sample was smeared on a microscopic slide and stained Giemsa's staining technique. The stained microscopic slides were viewed under light microscope and the numbers of cleared RBC are counted, this was repeated 3 times and the mean number erythrocytes appearing as discoid cells for each concentration were recorded. The activity of the test samples was calculated as percentage elimination using expression below (Mukhtar et al. [20]).
where N=Total number of RBC cleared, Nx=Total number of parasitized RBC, "RBC" =Red Blood Cells.

Fractionation of Nauclea diderrichii leaf extract
The results of fractionation of the crude extract (Nd-ET) are shown in Table 1.

Phytochemical screening
The results of the phytochemical test for the screening of potent compounds imbedded in the crude leaf extract of N. diderrichii and those of fractions can be seen in Table 2. Literatures from previous studies, also confirmed the presence of phytochemical ingredients like alkaloids, cardiac glycosides, saponins, phenolic compounds, tannins, phytosterols, carbohydrates, flavonoids and terpenoids, imbedded in the leaf extract of Nauclea diderrichii (Isa et al. [19] and Ette et al. [18]).

Antiplasmodial assay of Nauclea diderrichii leaf extracts
The anti-plasmodial activities of the crude leaf extract and those of the three fractions (crude leaf extract-fractions) of Nauclea diderrichii uncovered promising percentage elimination of the parasite at all concentrations. Ethanol crude extract (Nd-ET) and ethyl acetate fraction (ND-EA) demonstrated the highest activity with percentage elimination of 75.50% and 72.65 at 625 µgmL −1 , and 87.83% and 86.33% at 5000 µgmL −1 respectively. These results mentioned can be seen in Table 3. The anti-plasmodial activity of Nauclea diderrichii extract (Nd-ET) and fractions (Nd-F 1 , Nd-F 2 and Nd-F 3 ) justifies part of the ethnomedicinal claims on the plant, as effective as conventional medicine in combating pathogenic microorganisms and it is a preliminary scientific validation for the use of the plant, Nauclea diderrichii as antimalaria to promote proper conservation and sustainable use.

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The primitive use of the plant, in folk medicine in Madadistrict, Gusau local government, Zamfara State, Nigeria, represent a cheaper and safer alternatives in the treatment of infectious diseases.

Conclusion
The findings of present study virtually confirmed the phytochemical ingredients and anti-plasmodial potency of bioactive phytochemicals imbedded in Nauclea diderrichii, which validates the primitive use of the plant as a curative agent for malaria and other diseases of clinical concern.
Haudecoeur et al. [14] reported that the sustainability of the plant, N. diderrichii, should be kept in mind to adapt local uses and preparation modes of traditional remedies.
Further studies involving isolation, characterization and toxicological studies in vivo are needed with model animals to elucidate and validate the stability and dose dependent activity of the pure active compounds from the plant, Nauclea diderrichi.