Antiplasmodial, Antioxidant and Cytotoxicity Activity of Ethanol and Aqueous Extracts of Khaya grandifoliola Stem Bark

Background Malaria is a serious public health problem, especially in sub-Saharan Africa. The aim of this study was to scientifically provide baseline information on the use of Khaya grandifoliola stem bark as an antimalaria drug by traditional healers. Method The stem barks of K.grandifoliola were harvested and dried to obtain powder, and fifty grams of the powder were soaked in ethanol and hot distilled water respectively, for the preparation of ethanol and aqueous extracts, then dried in an oven at 40°C for the ethanol extract and 50°C for the aqueous extract. Plasmodium falciparum strains 3D7 sensitive and Dd2 resistant to chloroquine, were used to evaluate in vitro antiplasmodial activity using SYBR Green. The ability of the extracts to prevent oxidative stress was assessed by trapping 2, 2′-diphenyl-1-picrylhydrazyl (DPPH); nitric oxide, hydrogen peroxide and ferric reducing power. The cytotoxicity test of the extracts was carried out on RAW 264.7 cell lines and on erythrocytes. The data obtained were entered in the Excel software, then in Graph pad where the IC50 was calculated and the curves plotted. Results The fifty percent inhibition (IC50) of the antiplasmodial activity of the chloroquine-resistant strain PfDd2 were 54.27 ± 2.41 μg/mL and 31.19 ± 4.06 μg/mL respectively, for the aqueous and ethanol extracts. As for the Chloroquino-sensitive Pf3D7, IC50 of 53.06 μg/mL was obtained for the aqueous extract and 28.03 ± 1.90 μg/mL for ethanol. The DPPH radical scavenging activity presented IC50 of 104 μg/mL for the aqueous and 2.617 μg/mL for the ethanol extract; for the Nitric oxide (NO) presented an IC50 of 301 ± 21 μg/mL for the aqueous extract 140.7 ± 21 μg/mL for the ethanol; for hydrogen peroxide the ethanol and aqueous presented IC50 of 845.1 ± 21 μg/mL and 509.4 ± 21 μg/mL respectively. The cytotoxicity on RAW 264.7 cells presented High CC50 in particular >1000 μg/mL and 467.4 μg/mL respectively for the aqueous and ethanol extract. Conclusion Extracts of Khaya grandifoliola exhibited antiplasmodial activity. The ability to inhibit oxidative stress as well as lower cell toxicity on RAW 264.7 and erythrocytes, is a good indicator. However, in vivo tests remain important in order to confirm the use of this plant for the treatment of malaria.


Background
Malaria is the most important global parasitic endemic disease with approximately 3.3 billion people at risk in the world [1]. In 2020, with the COVID-19 pandemic, there are about 13 million new cases of illness worldwide and more specifcally in Africa and around 69,000 new cases of death [2]. It is the primary medical concern of many countries in sub-Saharan Africa, where it is responsible for 95% of clinical cases and 96% of deaths, 80% of which are children under 5 years old [2].
In Cameroon, although there is an improvement in the malaria monitoring system, the National Malaria Control Program reports an increase in mortality (18.3%) and morbidity (28%) [3]. Te shortage of antimalarial drugs and their relatively high cost partly explain this increase in malaria morbidity and mortality [3]. Added to this is the low efcacy of the antimalarial therapeutic arsenal of drugs and insecticides due to resistance recorded in Cameroon [4]and in some Asian countries [5]. To overcome these problems there is a pressing need to develop vaccines and new antimalarial drugs. More than 95% of drugs authorized on the market are of natural origin (Quinine and Artemisinin) or synthetic(Chloroquine) [6,7]. In some communities in Africa, such as Guinea, Nigeria excess mortality due to malaria has been reduced thanks to the ability of traditional medicine to control the disease [8,9].
According to Nadia et al. [10] and Abdel Azizi et al. [11] when a human host is infected with the Plasmodium parasite, these parasites stimulate an overproduction of free radicals to fght against infection. Tese free radicals are not only toxic to the parasite but equally toxic to the host. So it will be of paramount importance to have a drug that will possess antiplasmodial and antioxidant properties. In Cameroon, more than 200 plant species have been identifed for their antiplasmodial properties. Traditional healers in Western Cameroon use the stem bark of Khaya grandifoliola for the treatment of malaria and salmonellosis. Te work of Kodjio et al. [12] showed good anti-salmonellosis activity.
Previous work in Cameroon has demonstrated the antisalmonellosis efcacy of various Khaya grandifoliola extracts [13,14]. Te aim of this study was to scientifcally provide baseline information on the use of Khaya grandifoliola as an antimalaria drug in order to justify its usage by traditional healers in Cameroon.

Collection and Identifcation of Plants.
Te leaves, fowers, fruits of Khaya grandifoliola (used for the identifcation of the plant) and stem barks (used for the preparation of the diferent extracts) were collected in June 2021 in the city of Foumbot, West Region of Cameroon and identifed at the National Herbarium in Cameroon. A voucher specimen was deposited with the identifcation number 52658/HNC.

Preparation of Ethanol and Aqueous Extracts.
Ethanol solvent was used for the extraction because during the survey traditional practitioner use fermented palm wine (ethanol) or infusion to prepare this remedy.
Tese extracts were prepared according to the method described by Wabo et al. [15]. Te stem bark of Khaya grandifoliola was air-dried at room temperature under shade and pulverized using an electrical grinder under strict hygienic conditions. For the ethanol extract, 100 g of the powder was introduced into one litre of 95% ethanol and homogenised. Te mixture was stirred daily for 72 h. Te homogenate was fltered using cotton and whatman paper number 1. Te fltrate obtained was dried in an oven at 40°C to obtain the ethanol extract. For the aqueous extract, distilled water was heated at 100°C and one litre was introduced into 100 g of powder and the mixture was allowed to cool and flter using cotton and whatman paper number 1. Te fltrate was then dried at 45°C in order to obtain the dried aqueous extract.

In vitro Evaluation of Antiplasmodial Activity
2.3.1. Plasmodium falciparum Cultured. Plasmodium falciparum strains were cultured using the Trager et Jensen Method [16] with slight modifcations. Briefy, the multiresistant PfDd2 and chloroquine-sensitive Pf3D7 strains were cultured in fresh human red blood cells of group O + at 4% hematocrit in complete RPMI medium (Gibco, UK) supplemented with 25 mM HEPES (Gibco, UK), 0.50% Albumax I (Gibco, USA), 1X hypoxanthine (Gibco, USA) and 20 μg/mL gentamicin (Gibco, China)] and incubated at 37°C in a humidifed incubator consisting of 92% N 2 , 5% CO 2 and 3% O 2 . Tis medium was replaced daily to facilitate growth of the parasites in the culture.

Synchronization of Cultures.
Te parasite culture was synchronised at the same evolutionary stage (Trophozoite) by using 5% D. sorbitol, before evaluation of the antiplasmodial efect of Khaya grandifoliola.

In vitro Antiplasmodial Activity Using SYBR Green.
Te in vitro antiplasmodial activity was evaluated based on the method described by Smilkstein et al. [17] with slight modifcation. Briefy, 10 μL of various plant extract concentrations were introduced into a 96 cell microtiter plate. A parasite suspension of 90 μL was added to each well to give a fnal extract concentration of 200-0.001280 μg/mL. Artemisinin and chloroquine were used as positive control while 0.1% DMSO was used as the negative control. Te plates were then incubated for 72 h in a CO 2 incubator. After 72 h of incubation, 200 μL of SYBR green were added and then incubated for 1 hour at 37°C in darkness. Te fuorescence was measured at an excitation and emission wavelength of 485 and 538 nm respectively, using a Tecan Infnite M200 microplate reader. Te IC 50 for each extract was determined. Te Resistance Index [RI] was calculated using the formula: Te experiment was conducted in triplicate. Te radical scavenging activity was calculated as follows:

In vitro Antioxidants Activity of Aqueous and Ethanol
RSA � Absorbance of control − Absorbance of sample Absorbance of control x100. (2)

Nitric
Oxide Radical Scavenging Assay. Te Nitric oxide radical scavenging activity was evaluated using the method described by Cheraf, [19]. Te aqueous and ethanol extracts were dissolved in 3.53 mL of phosphate bufered saline (ph � 7.4). A volume of 1520 μL of sodium nitroprusside was added respectively, into each sample containing 180 μL of extracts and vitamin C (at the various fnal concentrations of 1, 3, 10, 30, 100, 300 μg/mL). An equivalent volume of 1% sulfanilamide was added to 500 μL of each sample and incubated for 5 min at room temperature in darkness. Naphthyl Ethylene Diamine (NED, 0.1%) was introduced into the mixture and incubated in darkness for another 5 minutes. Te absorbance was read 30 minutes later with a spectrophotometer (BIOBASE-BK-D560) at a wavelength of 530 nm. Te radical scavenging activity (RSA) expressed as a percentage was calculated as follows:   Figure 3 shows hydrogen peroxide (H 2 O 2 ) scavenging activity. It appears that the aqueous and ethanol extract presented IC 50 of 845.1 µg/mL and 509.4 µg/mL respectively. Tese extracts had scavenging activity which was greater than Ascorbic acid (∼9291 µg/mL). Figure 4 expresses the ability of the aqueous and ethanol extract of Khaya grandifoliola to reduce iron. It appears that the aqueous extract (156.2 µg/ mL) and ethanol (71.77 µg/mL) have a reducing power of iron, which is much lower than that of Ascorbic acid (6.672 µg/mL). Figure 5 shows the efect of the aqueous and ethanol extract of Khaya grandifoliola on human erythrocytes. It follows from the analysis of this Figure that for all doses, the aqueous extract showed lower haemolytic activity than that of the ethanol extract. Te highest haemolysis percentage was observed for the ethanol extract at a dose of 1000 (μg/mL). Table 2 shows the efects of aqueous and ethanol extracts on RAW 264. 7 cells. It appears that the CC 50 of the aqueous and ethanol extract were >1000 μg/mL and 467.4 μg/mL respectively. Table 3 shows the phytochemical screening of the aqueous and ethanol extracts of Khaya grandifoliola sterm bark. It follows from the analysis of this table that the aqueous extract contains alkaloids, saponins, triterpenoids, anthocyanins, and anthraquinone. Similarly, the ethanolic extract contains the same compounds except saponins. Figure 6 shows the amount of favonoid present in each extract. It appears from this fgure that the ethanol extract (448.9 ± 68.85 mg/g) content more favonoids than the aqueous extract (162.2 ± 48.20 mg/g). Figure 7 shows the total phenolic content of the aqueous and ethanol extracts. Similarly, more phenolic compounds where found in the ethanol extract (372.4 ± 7.328 mg/g) compared to the aqueous extract (631.9 ± 16.44 mg/g).

Discussion
Antiplasmodial activity on the PfDd2 chloroquine-resistant strain was 54.27 ± 2.41 μg/mL and 31.19 ± 4.06 μg/mL respectively for the aqueous and ethanol extracts. As for the Chloroquino-sensitive Pf3D7 an IC 50 of 53.06 μg/mL for the aqueous extract and 28.03 ± 1.90 μg/mL for ethanol were obtained. According to the classifcation criteria established by Kumari et al. [28] which states that a plant extract with IC50 < 5 μg/mL is declared very active; between 5 and 50 μg/ mL is declared active; 50-100 μg/mL moderate and >100 inactive. Tese results show that the ethanol extract was active while the aqueous extract had moderate activity both for 3D7 sensitive and Dd2 resistant to strain. Similar results were observed by Abdel Azizi et al. [11] when evaluating the In Vitro Antiplasmodial Cytotoxicity and Antioxidant Activities of Lophira lanceolata (Ochnaceae): A Cameroonian Plant Commonly Used to Treat Malaria. Tis could be explained by the nature of the solvent used for the extraction. Furthermore, the ethanol extracts content more phytochemical constituent compared to the aqueous extract as demonstrated in our results. According Koagne et al. [29] favonoids could lead to more useful derivatives for the development of an antiplasmodial agent. Te work of Azebaze et al. [30] demonstrated the antiplasmodial activity of isolated phenolic compounds. Te stem bark of Khaya grandifoliola is rich in compounds such as Alkaloids, Flavonoid, Phenol, Terpenoids, Anthocyanins and Anthraquinone [31]. One of these compounds such as phenols or favonoid have antiplasmodial properties [32]. Te high capacity of ethanol to extract these phytochemical constituents [33] could explain the better activity obtained with the ethanol extract. A similar study carried out in Nigeria on the Chloroquino-resistant W2 clone of Plasmodium falciparum showed an active activity of the aqueous extract of Khaya

Journal of Tropical Medicine
grandifoliola with an IC 50 of 15.2 μg/mL [34]. Tis diference observed between the antiplasmodial activity of the two aqueous extracts could be linked to the diference in the Plasmodium strains on which the extracts were evaluated, and also to the chemical composition of the plant which may vary according to certain characteristics such as climatic and edaphic factors [35].
Te aqueous and ethanol extracts displayed less free radical scavenging ability than ascorbic acid in the DPPH and NO scavenging tests. Similar results on the Meliaceae plant Entandrophragma cylindricum revealed lower free radical scavenging ability of the methanol extract than Ascorbic acid [36]. Our fndings, however, do not support those of Kodjio et al. [12] who demonstrated a more efective DPPH scavenging activity than Ascorbic acid. Te low concentration of phenolic compounds and favonoids that can release H+ to assist the scavenging of DPPH may account for this discrepancy.
As for the hydrogen peroxide scavenging test, the aqueous extract (IC 50 : 845.1 and ethanol (IC 50 : 509.4) presented high scavenging activity compared to that of ascorbic acid (∼9291). Tis means that these extracts may have a greater power of hydrogen peroxide inhibition. Tis is an advantage for the host organism because although hydrogen peroxide, plays a role in the body's defense, it is often toxic [37].     Te aqueous and ethanol extract presented a ferric reducing power lower than that of ascorbic acid. Tis result indicates that these extracts promote the production of Fe 2+ . Te ethanol extract reduces the synthesis of Fe 2+ more than the aqueous extract. Our results corroborate those obtained by Kodjio et al. [12] where the ferric reducing power of the ethanol extract was greater than the aqueous extract on the evaluation of the antioxidant activity of the stem bark of K. grandifoliola. Tis high antioxidant power could be due to the strong presence of favonoids and phenolic compounds [11,12,38].
Te cytotoxicity test on the erythrocytes revealed a negligible lysis of the erythrocytes at all concentrations. Similarly, a study carried out on aqueous leaf extracts of Aerva lanata, Calotropis gigantea, and Elaeocarpus ganitrus alone and in combination showed low cytotoxicity [39]. On the other hand, the work carried out on Allium stracheyi Baker showed strong erythrocyte lysis [40]. Tese diferences could be justifed by the low capacity of the aqueous and ethanol extract of Khaya grandifoliola to reduce iron.
Tese cytotoxicity tests on RWAN264.7 had CC 50 of >1000 μg/mL for the aqueous and 467.4 for the ethanol extract. According to the American National Cancer Institute, any extract considered cytotoxic has a CC 50 < 30 μg/mL [41]. Tese extracts are therefore considered non-toxic. Similar research by EL Souda et al. [42] showed that the volatile extract of Khaya senegalensis had moderate cytotoxicity on MCF7 cells with a IC 50 of 79.7 μg/mL. On the other hand, the same study found that Khaya grandifoliola's volatile extract showed a high level of cytotoxicity against MCF7 cells, with an IC 50 of 21.8 μg/mL. [42]. Tis would be supported by the high concentration of sesquiterpenes, particularly isocaryophyllene and humulene (caryophyllene), in the volatile extract of K. grandifoliola, which could be infuenced by both the plant's collection site(region) and the parts of the plant used to prepare the extract.

Conclusion
Te ethanol and aqueous extract of Khaya grandifoliola exhibited antiplasmodial activity against Plasmodium falciparum 3D7 sensitive and Dd2 resistant to chloroquine. Tese extracts presented good scavenging against most of the free radicals, and were non cytotoxic. Tis refects a possible use of these extracts to avoid oxidative stress related to Plasmodium infection in the treatment of malaria. However, further in vitro studies are required to ascertain their antiplasmodial activities.

Data Availability
All data generated and analysed are included in this research Article.

Conflicts of Interest
Te authors declare that no conficts of interest exist.