In Vitro Antiplasmodial Activity, Cytotoxicity, Antioxidant Action and GC-FID Analysis of Allanblackia Floribunda Extracts

Background: Malaria is a disease that is caused by Plasmodium parasite that has resulted in death of so many persons in the world especially children below 5 years. Plasmodium falciparum is one of the most widespread etiological agent for human malaria and has become increasingly resistant to standard antimalarial drugs. This study was therefore aimed at evaluating the in vitro antiplasmodial ecacy and cytotoxicity of Allanbackia oribunda stem bark, leaf and oil. Methods: Trager and Jensen method was used to culture Plasmodium falciparum and maintained in fresh O + human erythrocytes at 3% hematocrit in complete medium (Roswell Park Memorial Institute-RPMI 1640). Mark III test developed by WHO was used to assess the antiplasmodial activity of the plant’s crude extract and fractions against the ring stage of P. falciparum strains Pf3D7. Cytotoxicity was determined against Vero cell line using microculture tetrazolium (MTT). GC-FID was employed to identify bioactive constituents in the most active fraction. Results: The plant extracts showed varied degrees of inhibition of parasitaemia with IC 50Pf3D7 values ranging between 4.0 to 1000 μg/ mL. The methanol stem bark extract of A. oribunda exhibited very active antiplasmodial activity (IC 50Pf3D7 = 4.3 ± 0.17μg / mL), the leaf extract showed active antiplasmodial activity (IC 50Pf3D7 = 8.0 ± 0.28 μg / mL) while the oil extract was inactive against the parasite (IC 50 > 100 μg / mL). Both the leaf and stem bark extracts were found to be non-cytotoxic in contrast to the standard cytotoxic drug, doxorubicin. The selectivity indices (S.I.) of the extracts against the parasites were 13.32 and 8.18 for the stem bark and leaf, respectively. Among the stem bark fractions, dichloromethane (DCM) had the best inhibition against the P. falciparum parasite (DCM IC 50Pf3D7 of 1.51


Background
Malaria is a disease that is caused by Plasmodium parasite. The symptoms and clinical manifestations of this protozoan infection is severe in children, pregnant women and individuals from malaria free regions when compared to subjects who have been pre-exposed to the infection. Artemisinin-base combination therapy (ACT) recommended by WHO for the treatment of uncomplicated malaria has been effective in combating this malady. However, the scourge of the disease is still erce in sub-Saharan Africa because of the mortality rate on a yearly basis. [1]. For example, there are approximately 216,000,000 cases of malaria infection worldwide resulting in 445, 000 death with 91% of the mortality in Africa [1]. It has been impossible to eliminate malaria from under-developing regions such as Africa due to the existence of hard-to-reach communities, inaccessibility of antimalarial drugs, exposure to fake antimalarial drugs, drug resistance, lack of information ow about effective antiplasmodial agents and individuals' low income status. Despite the death toll resulting from parasite plaque in Nigeria, some indigenous people are able to control the ravaging effect to some extent by using plant-based products.
Medicinal plants are known to be rich in bioactive substances with varied pharmacological properties on living systems [2]. However, some plant constituents may cause toxicity to man. Toxicity is the relative ability of a substance to cause adverse effects in living organisms [3]. The concept of toxicity as opined by Paracelsus is with respect to dose, i.e. the amount of substance determines whether it effects will be toxic, nontoxic or bene cial. Information on the therapeutic e cacy and health safety of medicinal plants has been based on oral communication through successive generations among indigenous people for many years. However, the scienti c validation of their therapeutic properties still remain unproven.. Recent reports in eastern [4], northern and southern [5,6] regions of Nigeria suggest possible cases of parasite resistance and recrudescence to the currently used combined antimalarial therapies. Hence, the necessity to investigate and nd possible replacement for the rst line combination therapies commonly used in Nigeria. In order to contribute to the scienti c data of A. oribunda and develop effective antimalarial drug, its antiplasmodial and cytotoxic activities were investigated.

Collection of Plant Stem bark, Leaf and Fruit
The plant parts used in this study were selected based on ethnopharmacological information as antimalarial plant used in southern part of Nigeria. Allanblackia oribunda leaf, stem bark and seed were collected during the period of January and July, 2016, from a forest area at Ohogua community, Ovia East Local Government, Benin City, Edo Sate, Nigeria. The plant was authenticated by a Botanist at the Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Benin, Nigeria and voucher specimen of the sample (UBHA 361 ) was deposited at the herbarium of the same department.
Preparation Of Extract 100 g of the macerated A. oribunda stem bark and leaf were soaked in 1000 mL of absolute methanol for 72 h with occasional stirring. The extracts were then ltered using double layered muslin cloth and the ltrate concentrated to dryness by using a rotary evaporator at reduced pressure while the oil was obtained from about 200 g of the powdered seed using hot water oatation method. The extracts obtained were stored at 4 C until used.

Fractionation of A. oribunda stem bark extract
Fractionation of the stem bark extract was done using solvent of increasing polarity as described by Hassan et al. (2012). The methanol stem bark extract of A. oribunda was partitioned into fractions of nhexane, dichloromethane, ethyl acetate and hydromethanol. The fractions obtained were concentrated using a rotary evaporator (RE 300, Bibby Scienti c, UK) with reduced pressure at 45˚C and nal concentrate was obtained using silica gel.
Qualitative Phytochemical Screening and antioxidant activity of A. oribunda stem bark Fractions The qualitative test for phytochemicals present in fractions were carried out using standard procedures [7,8]. The free radical scavenging capacity of the fractions against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was determined by a modi ed method of Brand-Williams et al. [9]. Phosphomolybdate reduction capacity was estimated using the method described by Prieto et al. [10].

Cultivation Of Parasites
Trager and Jensen procedure was used to culture P. falciparum [11,12]. The Plasmodium strain (Pf3D7) used was chloroquine sensitive isolated from Nigeria and India, respectively. The parasites were cultured in O + RBC as host cells and maintain in RPMI 1640 medium supplemented with gentamicin solution 0.01 mg/mL, 25 mM HEPES buffer, 25 mM NaHCO 3 and 1% Albumax II maintained in 5% CO 2 and incubated at 37 °C. Parasitaemia was determined using light microscopy (Giemsa stain).

In Vitro Antiplasmodial Activity
The growth inhibition of chloroquine-sensitive Plasmodium falciparum strain (3D7) by the plant extracts was evaluated by means of the Mark III test, as developed by the WHO [13]. Filter sterilized extracts (25,12 The antiplasmodial activities of the extracts were expressed using IC 50 . The IC 50 is the inhibitory concentrations of the drug/extracts that induced 50% reduction in parasitaemia compared to control (100% parasitaemia). It was determined using SPSS statistical tool pack based on interpolation from the parasite growth inhibition curve generated from each plant extract. Each sample was tested in duplicate and the IC 50 obtained were pooled and expressed as geometric means and standard deviations. The independent sample t-test was used to compare mean IC 50 of antimalarial activity between plant extracts using STATA version 13 (Software Stata Corp, College Station, TX, USA). Finally, the in vitro antiplasmodial activity was rated in accordance with the system of antiplasmodial activity of Rasoanaivo et al. [14].

Cytotoxicity Test (cell Viability Assay)
Monkey kidney epithelial cell (LLC MK2) were used to assess the cytotoxicity of the most active plant extract. Vero cells were maintained in Dulbecco's Modifed Eagle's medium (DMEM) supplemented with 10% fetal bovine serum FBS, glutamine (2 mM), penicillin (100 units / mL) and streptomycin (100 µg / mL). The cells were seeded in 96 well plates at 10,000 cells per well in 100 µL culture medium. The cells were incubated at 37 C in a humidi ed 5% CO 2 atmosphere. After 48 h, the medium was discarded by inversion of the microtitre plate and thereafter replaced with 100 µL of fresh culture medium followed by 100 µL of crude plant extract at a concentration of 1000 µg / mL in row H of the 96 well plate and serially diluted twofold to give concentrations ranging from 500 to 7.81 µg / mL. Row A. of the 96 well plate serve as control wells, whereby the negative control contained culture medium and the LLC-MK2 cells without plant extract (Maximal cell growth). The cells were maintained at 37 in a CO 2 incubator before determining their viability by using MTT assay as described by Mosmann [15]. The absorbance for each well was measured between 490-630 nm in a micro-titre plate reader and the percentage cell viability (CV) was calculated using the formula:

Statistical Analysis
The statistical analyses of the results were carried out. The various results obtained from this study were expressed as mean ± SEM. One way analysis of variance (ANOVA) followed by Tukey's HSD (honest signi cant difference) test was used to determine signi cance differences between the groups. Statistical signi cance was declared when P value was less than 0.05. The statistical analysis was performed using the statistical package for social science (SPSS) for windows, version 16.0.

Results
In vitro antiplasmodial activity of A. oribunda stem, leaf and oil extract The inhibition of parasite growth by the plant extracts was dose dependent from 0.05 to 25 µg / mL except for A oribunda stem bark extract which had a higher percentage inhibition (30%) at 0.025 µg / mL compared to 26% at 0.05 µg / mL. At 5 and 25 µg / mL both leaf and stem bark extracts had over 50% parasite inhibition. However, A. oribunda oil extract recorded less than 41% of P. falciparum inhibition even at the highest dose (Fig. 1)  terpenoids, tannins and phenols in all the fractions studied. Majority of the phytochemicals were highly detected in the dichloromethane, ethylacetate and hydromethanol fractions. However, some of these bioactive substances were less detected in the hexane fraction. Quinones were undetected in the hydromethanol fraction. Figure 3 represents the result of the total antioxidant capacity which parallels that of the phytochemical screening of the different stem bark fractions of A. oribunda. The reduction of molybdenum (VI) to molybdenum (V) by the fractions was highest in the hydromethanol (205 µg/ mL) followed by ethylacetate fraction (101.4 µg/ mL) and then the dichloromethane (93.2 µg/ mL). The hexane fraction had the lowest antioxidant activity (82.4 µg/ mL). DPPH radical scavenging activity of the stem bark fractions of A. oribunda (Fig. 4) was over 70% at a concentration range of 20 to 100 µg/ mL. An exception to this is the activity recorded for hexane fraction in which less than 60% DPPH radicals were inhibited at 100 µg/ mL. Table 4 shows the IC 50DPPH of the various fractions as follow: ethylacetate (IC 50DPPH = 5 × 10 − 4 ± 0.0001 µg/ mL), hydromethanol (IC 50DPPH = 8 × 10 − 4 ± 0.0005 µg/ mL) and dichloromethane (IC 50DPPH = 2.89 ± 0.33). Ethylacetate, hydromethanol and dichloromethane fractions were as good as the standard antioxidant compound, vitamin C (IC 50 DPPH = 4 × 10 − 4 ± 0.00 µg/ mL).
However, the antioxidant capacity of the hexane fraction ( IC 50 = 192 ± 14.1 µg/ mL) was low and signi cantly different from others at p < 0.05.
The activity of the various fractions of A. oribunda stem bark with respect to inhibition of parasite growth is shown in Table 4 and Fig. 5. Among the fractions, dichloromethane (DCM) had the best inhibition against the P. falciparum parasite (DCM = IC 50PfD3 1.51 µg/ mL) and was closely followed by the hydromethanol (HMet) fraction (HMet = IC 50 PfD3 5.0 µg/ mL) while that of ethylacetate (EAct) and hexane (Hxn) fractions were almost similar (IC 50 PfD3 = 6 and 6.25 µg/ mL, respectively). In general, the in vitro antiplasmodial activity of the fractions followed the order: DCM > HMet > EAct > Hxn.

Gas chromatography ame ionization detector (GC-FID) analysis on dichloromethane fraction (most active fraction)
According to table 5, avonoid pro le of dichloromethane fraction showed the presence of higher level of kaemferol; other avonoids found in high amount are catechin, naringenin, luteolin, epigallocatechin, quercetin and myricetin (Fig. 6). In Table 6, azulene, α-pinene, pinene-2-ol, γ-terpinene, camphor, 1, 8cineole, borneol, neryl acetate, β-Caryophyllene and humulene were the terpenes found in relatively high amount (Fig. 7). Humulene was signi cantly higher in the terpenes pro le compared to others. Chrysophanol (Fig. 8) was the only signi cant quinone detected in high proportion while others like aloe emodin, rhein and emodin were found in low levels respectively, table 7. In table 8, terpenoids pro le showed the presence of low α-amyr in, and β-amyrin in the DCM fraction but their concentrations were still much higher than others like taraxerol, lupeol, bauerenol acetate (Fig. 9). alkaloids and volatile organic constituents pro les were generally low in phytochemicals, however, alkaloids such as coniine, coniceine, cassine, spectaline and volatile organic constituents like butanoic acid, 2-methyl butenoic acid were detected in relatively higher amount compared to others which were extremely low,

Discussion
Allanblackia oribunda stem bark, leaf and oil extracts were examined for their in vitro antiplasmodial ability and cytotoxic activity; Fractionation of the most active crude extract was done and phytochemical screening, in vitro antioxidant and antiplasmodial assays were further investigated, nally, analysis of the most active fraction was carried out with GC-FID. The result of this investigation showed that the inhibition of parasite growth by the plant extracts in vitro was dose dependent. At 5 and 25 µg / mL both leaf and stem bark had over 50% parasite inhibition. However, A. oribunda oil extract recorded less than 41% of P. falciparum inhibition even at the highest dose. The in vitro antiplasmodial activity was analyzed in accordance with the system of antiplasmodial activity of Rasoanaivo et al. [9]. According to this norm, an extract is regarded to be very active if IC 50 < 5 µg / mL, active if 5 < IC 50 < 50 µg / mL, weakly active 50 µg / mL < IC 50 < 100 µg / mL and inactive IC 50 > 100 µg / ML. IC 50 is the inhibitory concentrations of the drug/extracts that can cause 50% reduction in parasitaemia level. The stem extract of A. oribunda showed excellent antiplasmodial activity followed by the leaf extract. However, the oil extract showed IC 50Pf3D7 value that was over 100 µg/ mL, hence inactive against the parasite in vitro.
Herbert and fellow researchers suggested possible mechanisms of how terpenes inhibit Plasmodium parasite growth. Jomaa et al. [23] reported that isoprenoid biosynthesis in Plasmodium falciparum depends on the DOXP/2-C-methyl-d-erythritol-4-phosphate (MEP) pathway while in humans, isoprenoids are synthesized via the mevalonate pathway [24]. This made isoprenoid biosynthesis a potential target for P. falciparum especially in the presence of phytochemicals such as terpenes. Some malaria researchers [25] opined that terpenes inhibit dolichol biosynthesis in the trophozoite and schizont stages and that this bioactve compound (terpenes) may be acting through inhibition of the isoprenyl diphosphate synthases. This family of enzymes catalyzes consecutive 1′-4 condensations of isopentenyl-PP with the allylic substrate to form the linear backbone for all isoprenoid compounds, including prenylated proteins, prenylated quinones, and dolichol [26]. Due to the structural similarity terpenes have with the substrates required for dolichol synthesis, it might interfere with the parasite's biosynthesis of polyisoprenoids by competing with original substrates in enzyme-substrate reactions or by interfering with the mechanisms of elongation of isoprenic chains [25]. This metabolic interference may cause parasite death as seen in the crude extract of A. oribunda methanol extracts because of the presence of secondary metabolites like terpene (16). Other possible mechanisms of parasite inhibition by the plant extracts could be through prevention of merozoites invasion of erythrocytes, inhibition of haemozoin biocrystallization by the alkaloids, inhibition of P. falciparum fatty acid biosynthesis and protein synthesis disruption by triterpenoids [27,28,29,30]..
The result of the cytotoxicity study showed that the stem and leaf extracts of A. oribunda were non-toxic as against the standard cytotoxic drug, doxorubicin (an anthracycline antibiotic) which was found to be highly cytotoxic". According to the American National Cancer Institute (NCI), the threshold for crude extract toxicity is CC 50 less than 30 µg/ mL [31]. However, a crude plant extract with CC 50 less than 20 µg/ mL is considered highly cytotoxic [32]. In the light of NCI guidelines, A. oribunda extracts at 7.81 to about 125 µg/ mL were considered to be non-cytotoxic because their respective CC 50 were signi cantly greater than 30 µg/ mL. The selectivity indices, a ratio of CC 50 to IC 50Pf3D7 revealed that the extracts were selectively toxic to the parasite and safe to mammalian cells with the stem bark having a higher selectivity for Plasmodium falciparum when compared to leaf. However, worked done by some other researchers [34, 35, 36, and 37] on the hearthwood and the root bark of Allanblackia oribunda reported that the xanthones, benzophenones, and some bi avonoids of A. oribunda exhibited cytotoxic activity. This may be possible due to the fact that the amount of secondary metabolites present in A. orinbunda may be higher in the root and hearthwood compared to their concentrations in the stem bark and leaf. Besides, the time of plant harvest and geographical location may also in uence the levels of plant's bioactive compounds. These two factors may account for the difference in the cytotoxicity result. In this study, the constituents of A. oribunda probably had little or no effect on Vero cell line, hence A. oribunda maybe considered safe to normal cells but toxic to malaria parasite. This nding somewhat, to certain extent, validates the claim of traditional healers about the safety of A. oribunda decoction when administered orally to individuals having fever associated with malaria.

Conclusion
Findings from this study show that A. oribunda methanol stem bark and leaf extracts have some levels of activity against Plasmodium falciparum parasite and are relatively non-toxic to normal cells. The antiplasmodial action is credited to the presence of bioactive avonoids and terpenes that may have acted singly or synergistically. This therefore supports the use of A. oribunda in the treatment of malaria in ethno-medicinal practices. However, the plant extract should be subjected to more investigations to identify the active compounds in its methanol extracts and elucidate their antiplasmodial mechanisms.

Declarations Ethical Approval
The study was approved by the Institutional Ethics Review Committee, University of Benin (LS19114).

Consent for Publication
Not applicable.    Chromatogram showing the names and amount of the various terpenoids present in dichloromethane fraction Figure 10 Chromatogram showing the names and amount of the various alkaloids present in dichloromethane fraction Page 23/23

Figure 11
Chromatogram showing the names and amount of the variousvolatile organic constituents present in dichloromethane fraction

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