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Antiplasmodial, antioxidant and toxicological study of leaves extracts of Dalbergia katangensis Lecheneaud (Fabaceae) from Eastern DR Congo

Dalbergia katangensis is used in traditional medicine in Bukavu in management of malaria. In this study, antiplasmodial, antioxidant activities, and toxicological studies where carried out on aqueous and methanolic extracts of its leaves. The plant was selected following an ethnobotanical survey conducted in DR Congo and focusing on plants used traditionally to treat malaria. Extrac’s phytochemical secondary metabolites were determined using standard procedures and the antiplasmodial activity was evaluated using 4-day suppressive test, while antioxidant activity was evaluated by DPPH assay. In acute toxicity, twenty animals (5/group) were given orally singular 2000 mg of extract/kg body weight (BW) then observed for 14 days. In sub-acute toxicity assay, 200, 400 mg/kg BW/Day were given orally, and animals were observed for 28 days. The total phenolic (0.58-0.61 mg GAEg-1), total flavonoid (0.14-0.15 mg QEg-1) and total terpenoids contents (0.32-0.55 %w/w) were in the same rate in the two extracts as well as the antioxidant activity with IC50 value 1.55 ± 0.012 and 1.71 ± 0.014 μg/mL. At the highest oral dose,400 mg/kg body weight, all extracts produced 73–75% chemo-suppression against P. berghei ANKA and 28 survival days. No deaths were recorded during the acute toxicity assay suggesting the LD50 >2000 mg/kg and no abnormal behavior or variation in toxicity biomarkers were observed during the subacute toxicity assessment. Dalbergia katangensis leaves extracts showed a great antiplasmodial and a very good antioxidant activity. It can be used to prepare antimalarial recipe or isolate antimalarial compounds in the future.


Introduction
Malaria is one of the most prevalent and serious protozoan tropical diseases which causes millions of clinical cases worldwide each year, and approximatively 1 million of death annually [1]. The World Health Organization (WHO) African Region accounted for 93% of all cases in 2018 and more than half of all cases were in six countries: Nigeria (25% of cases), Democratic Republic of Congo, DRC (12%), Uganda (5%), Ivory Coast, Mozambica and Niger, 4% each [2]. DRC is one of the central African country where malaria with Plasmodium falciparum is highly endemic with 97% of prevalence [3], being one of the most important health problems in the country [4].
Many Congolese people do not have access to modern health care and use medical plants, but many plants used in traditional medicine have not been studied [5]. Another element of the antimalarial control strategy would consist in validating the use of antimalarial plants with the hope to discover new compounds or produce improved traditional drugs. Studies have been conducted to search for antimalarial plants both across the world, in America [6,7], in Asia [8,9] as well as in Africa [10,11] and particularly in DRC [12,13].
In Bukavu, among the many plants reported as antimalarials [5,14] we have selected Dalbergia katangensis Lecheneaud (Fabaceae) [4]. It is a tree from which there is not much information in the literature. The plant is locally named Mungobole, Nfuma (shi), Munyereza (fulero), and it's used in the treatment of malaria and other infectious diseases such vaginitis, bacillary dysentery (root bark), cholera, tuberculosis (stem bark) and conjunctivitis (leaves). This plant has already been investigated for in vitro antiplasmodial activity, study which revealed a great activity [4]. However, no information is reported on its phytochemical composition nor on the in vivo antimalarial activity, and toxicity.
This study aims to evaluate the antiplasmodial activity in vivo and antioxidant in vitro of methanolic, and aqueous extracts of the leaves of Dialium angolense. On this same occasion, we evaluate the acute and subacute toxicity on rats, and we look for large phytochemical groups mainly secondary metabolites with antimalarial potentiality.

Plant material and experimental animals
Leaves of Dalbergia katangensis were collected from Bagira (2 ° 28'12.9''S; 28 ° 49'18''E; 2,883.1 m) in june 2015, and was identified at the herbarium of Meise in Belgium with the following voucher number: BR0000018879285. Healthy Mus musculus (21.6 ± 2.1 g) and Mus norvegicus (265.4 ± 0.73 g) male were procured from animals holding unit of Institut National de Recherches Biomédicales (INRB) Kinshasa-DRC. The animals were acclimatized to 28° C one week before the experiment by being subjected to a 12h light-dark cycle, consuming a standard rodent food (MIDEMA/DRC) and drinking ad libitum.

Preparation of extracts
Methanolic extracts (ME) were obtained by macerating 350 g of dried leaves powder in 1.5 L of methanol (Sigma-Aldrich, USA). After 72 h, the extract was filtered on paper (Whatman, USA) and the residue was macerated twice in a similar manner. The filtrates were combined, concentrated, and dried using a rotavapor (Büchi R-210, Switzerland) at 40°C under reduced pressure;130-180 mbar (yield, 12.1 %, W/W). Aqueous extracts (AE) were prepared according to the protocol used in traditional medicine by decocting 340 g of the sample in 1.5 L for 25 minutes of local tap water (boiling for 0.5 h in a close recipient and filtration on paper). The extract was lyophilized (yield, 10.5%, W/W) and for alltest, the extract was dissolve in NaCl 0.9%.

Phytochemical screening
The plant extract was analyzed for the presence of some secondary metabolite including alkaloids, coumarins, flavonoids, saponins, steroids, tannins, terpenoids and phenols, using standard procedures in tube reaction [15,16].

Total phenolic, flavonoids and terpenoids contents
The total phenolics content of each sample was measured by a Folin-Ciocalteu method [17] and expressed as milligrams gallic acid equivalents per gram of dry plant extract (mg GAE/g DE) through a calibration curve gallic acid (y = 0.014x + 0.003, R 2 = 0.997 ; linearity range, 0.5 -200 mg. mL -1 ). The total flavonoids content was determined using an aluminum trichloride assay [18] and expressed as milligrams quercetin equivalents per gram of dry plant extract (mg QE/g DE) through the calibration curve of quercetin (y = 0.006 x + 0.004, R 2 = 0.998; linearity range, 0.1 to 150 mg/mL). Regarding the determination of total terpenoids contents, dried plant extract 100mg (A) was taken and soaked in 9mL of ethanol (Sigma-Aldrich, USA) for 24 hours. The extract after filtration was taken up by 10mL of petroleum ether (Sigma-Aldrich, USA) using separating funnel. The ether extract was separated in pre-weighed glass vials and waited for its complete drying (B). Ether was evaporated and the yield (%) of total terpenoids contents (TTC) was measured by the formula: [19]. (Equation 1).

Antioxidant activity-DPPH Assay
DPPH radical scavenging activity of the plant extracts at varying concentrations (1.95-125 µg/mL) were measured in vitro via the DPPH assay [20]. Briefly, 50 μL of extract prepared at different concentrations were interacted with 1950 μL of 0.002% DPPH in a plate 96 wells (Nunc WVR, Germany) giving concentrations of extracts ranging from 0.048 to 3.125 µg/mL. After mixing and incubating in the dark for 30 minutes, the solution was read at 492 nm (Thermo Fisher Scientific Inc., Waltham, USA). The tests were carried out in triplicate. The percentage of antioxidant activity (AOA) was calculated by the formula: Ab = absorbance measured in the presence of the negative control, Ae = absorbance measured in the presence of the extract, and % AAO = Percentage of inhibition. Depending on their IC50 values, extracts were classified as following: (i) very active if IC50 ≤ 5 µg/ mL, (ii) active if 5 µg / mL ≤ IC50 ≤ 15 µg / mL, (iii) moderately active if 15 µg / mL <IC50 <50 µg / mL, (iv) weakly active if IC50 ≥ 50 µg / mL [20].

Antiplasmodial activity-4-day suppressive test
The in vivo antiplasmodial activity of the extracts were evaluated using the 4-day suppressive test against Plasmodium berghei (ANKA MRA 311 supplied by the INRB) infections in mice [21]. Briefly, donor Mus musculus previously infected with Plasmodium berghei and having parasitemia level of 20-30% were used to infect the experimental mice intraperitoneally with 0.2 mL of their infected blood. The infected mice were randomly divided into six groups of 5 each by weight. Three hours after inoculation, each Mus musculus was orally treated with 200 µL of oral dose of the sample (200, 400 mg kg −1 weight) daily for 4 days. A positive control-group received 10 mg/kg BW of quinine per day, while the negative-control group animals were administered 200 µL of the vehicle (NaCl 0.9%). On day 4, thin blood smear was made and stained with 10% Giemsa and examined under the light microscope with 100 times magnification to determine parasitemia level. Percentage of parasitemia was counted based on infected erythrocytes calculated per 1000 erythrocytes: Total Number of RBC Count From day 4 to day 7, thin blood smears were made from the tail of each Mus musculus, Giemsa stained and examined microscopically for assessment of parasitemia. On day 7, the level of parasitemia in each group of mice was determined so that the percentage chemo-suppression (TSP) were calculated as: where A is the parasitemia in the negative-control group and B the parasitemia in the test group. All the mice were kept alive until the 28th day to assess the survival time (TS) [22,23]. In vivo antiplasmodial activity of extracts were classified as moderate, good, and very good if an extract displayed respective percent parasite suppression equal to or greater than 50% at doses of 500, 250, and 100 mg/kg body weight per day, respectively [24].

Toxicological study
Acute toxicity was carried out as described previously [25] using 2000 mg/kg by Weight (BW) in single dose (oral administration; 5 animals per group, followed over 14 days). In subacute toxicity, Mus norvegicus (5 each group) received orally for 28 days, 0 (negative control), 200 or 400 mg/kg BW/day. During blood collection and serum preparation for biochemical analysis, validated procedures were followed [25]. The activities of alkaline phosphatase (ALP), aspartate transaminase (AST), alanine transaminase (ALT), and the levels of urea and creatinine were determined by colorimetric assays with Labtest® kits (Minas Gerais, Brazil).

Statistical analysis
Values were analyzed using GraphPad Prism 6 (GraphPad Software, La Jolla, USA). Comparisons between different groups were carried out by analysis of variance, ANOVA; a probability level p < 0.05 was considered significant.

Ethical approval
The principles governing the use of laboratory animals as laid out by Organization for Economic Co-operation and Development: OECD, Minna Committee on Ethics for Medical and Scientific Research and also existing internationally accepted principles for laboratory animal use and care as contained in the Canadian Council on Animal Care Guidelines and Protocol Review [26] were duly observed. The project proposal and procedures were reviewed and approved by the Department of Pharmacology in the faculty of Pharmaceutical Sciences from the University of Lubumbashi, DRC (UNILU/FSP/DPCOL/PT/002/2014).

Phytochemical screening of Dalbergia katangensis extracts
The phytochemical screening of D. katangensis leaves extracts revealed the presence of quinones, flavonoids, polyphenols, steroids, terpenoids, but the absence of alkaloids, coumarins, and saponins (Table 1). The different total phenolics content: TPC values vary between 0.58-0.61 mg GAEg -1 and are four times higher than the Total flavonoids content: TFC values (0.14-0.15 mg QEg -1 ). Total terpenoids content: TTC, vary between 0.3-0.5 (% W / W) and, a difference between aqueous and methanolic extracts is only observed with TTC with p <0.01 (Table 2).

Antioxidant activity
The scavenging ability of the samples tested showed a dependent concentration activity profile. The anti-free radical activity expressed in the form of IC50-1 is respectively 1.55 ± 0.012 (AE) and 1.71 ± 0.014 μg/mL suggesting a very good antioxidant activity of studied extracts according to the previously proposed classification [20]. No statistically significant difference was observed between the two extracts. In contrast, their antioxidant activity is lower (p <0.001) than ascorbic acid used as a positive control (figure 1).

Antiplasmodial activity
The percentage suppression analysis of the extracts showed decrease (p <0.01) in parasitemia at all dose levels as compared to the negative control group. The group received 400 mg/kg WB/day (ME 400) exhibited maximal suppression (74.81 ± 0.05 %); the effect was significantly lower than the group which received quinine (p<0.001). All doses of the extract significantly enhanced the survival time (TS) of the mice in a "not dose dependent manner" as compared to the negative control group (Table 3).

Clinical signs, weight variation, maximum tolerated dose (DMT) and 50% lethal dose of animal (LD50)
When assessing acute toxicity, signs of asthenia, hair straightening and anorexia were observed in the test groups. On the other hand, during the evaluation of the subacute toxicity, no signs of toxicity were observed. No significant variation in weight was observed either during the assessment of acute toxicity or during the assessment of sub-acute toxicity

Discussion
In this study, we provide knowledge on the aqueous and methanolic extracts of the leaves of Dalbergia katangensis regarding the phytochemical composition on secondary metabolites, the pharmacological activity (antiplasmodial in vivo on Plasmodium berghei and antioxidant in vitro on DPPH), the acute, and subacute toxicities in Mus norvegicus model. Water is the solvent used in traditional medicine during the preparation of antimalarial recipe based on D. katangensis leaves [4]. On the other hand, methanol, is the solvent most used during the biological screening of plants because it is easily handled, thanks to its great evaporative power. A concomitant test with the aqueous extract and the methanolic extract consists in varying the possibility of switching from the solvent used in traditional medicine to that used in the experimental laboratory.
This study reports a chemical similarity composition in secondary metabolites between methanolic and aqueous extract of leaves of D. katangensis by the concomitant presence of flavonoids, quinones, terpenoids and overall polyphenols (Table 1) suggesting the possible similarity in pharmacological activities. Several compounds with antioxidant [27][28][29][30][31][32] and antiplasmodial [33][34][35][36][37] activity from different groups identified within the leaves of Dalbergia katangensis during this study were isolated from many natural product, so that these groups are increasingly considered as groups with antimalarial and antioxidant potential.
No phytochemical study has been reported regarding D. katangensis; Nevertheless, other species of the same genus Dalbergia have been studied for phytochemical composition. During this study, we encountered in the two aqueous, and methanolic extracts, polyphenols, particularly flavonoids, quinones, steroids and terpenoids. Several phytochemical groups, including flavonoids, phenols, quinones, steroids, and terpenoids have been isolated from various species of the Dalbergia genus but mainly in the heartwood part. In Leaves theses phytochemical group have been found in D. sissoo DC, D. odorifera T.C.Chen, and D. lanceolaria L.f.; most compound isolated were polyphenols particularly flavonoids [38]. Isoflavones particularly apioglucoside has been proposed as a chemotaxonomic marker of the genus Dalbergia [39].
The methanolic extract from the leaves of D. katangensis (Table 2) contains the TPC tree times higher than that of D. sissoo (0.182 ± 0.007 mg GAEg -1 ) [40]. In contrast, D. sissoo with TFC = 5.5 mgQEg -1 of methanolic extract [41], contains a TFC, 50 times greater than that of D. katangensis. The polyphenols, however abundant they may be in the leaves of D katangensis, are mostly non-flavonoid.
D. katangensis extracts sufficiently reduced parasitemia in the animals on day 7 of observation (Table 3) and these reductions in parasitemia were dose dependent. This situation is observed in plants with antimalarial activity in the rodent model and constitutes one pledge of their activities [49][50][51]. According to the classification previously proposed [52], all extract presented a good antiplasmodial activity in vivo. Some species of the genus Dalbergia have previously led to the isolation of antimalarial compounds. The antimalarial activity of the stem of D. parviflora Roxb. has been attributed to dalparvone, a flavonoid [53], that of D. louvelii R.Vig., was attributed to 4 compounds: (R)-4′′methoxydalbergione(quinone),7,4'Dihydroxy-3-methoxyisoflavone(flavonoid), Obtusafuran (phenol) and isoliquiritigenin (Phenol) [54]. Isoliquiritigenin has also been reported in D. odorifera T.C.Chen [55] and D. cochinchinensis Laness [56]. It is therefore probable to discover within D. katangensis, antimalarial compounds resulting from these phytochemical groups identified in the plant during the present study ( Table 1).
The leaves of D. katangensis exhibited an antiplasmodial activity positioned variously in relation to some antimalarial species from the DRC studied previously. Its activity is lower than the activity of the leaves of Senna occidentalis (L.) Link (Fabaceae): ME, TSP 73% at 200 mg/kg BW [57] and the bark of the roots of Alstonia congensis Engl. (Apocynaceae): AE, TSP: 80.43 ± 0.12 mg/kg BW [1] but it is superior to the leaves of Physalis angulata L (Solanaceae): ME, TSP = 60% at 300 mg/kg BW and Anisopappus chinensis Hook (Asteraceae): AE, TSP: 46.6% at 300 mg/kg [23].
At experimental doses of 200, 400 mg/kg (subacute toxicity) and 2000 mg/kg (acute toxicity), the toxicity of D. katangensis has not been sufficiently established. We can assume, as for other previous studies on other species of the same genus, in particular D. sissoo DC. [58], D. saxatilis Hook.f. [59], D. monetaria L.f. [60], D. candenatensis (Dennst.) Prain [61], that the toxicity of D. katangensis leaves would only occur at higher doses. This information further supports the use of D. katangensis in traditional medicine. However, it should be noted that unlike these aforementioned species, D. katangensis showed some signs of toxicity during the acute toxicity assessment, suggesting that at doses higher than those used during this study, the plant would present some toxicity in vivo. This aspect should be considered in subsequent studies to establish the toxicological profile of this plant.

Conclusion
For the first time, a promising antiplasmodial activity in vivo against P. berghei on Mus musculus model with an interesting toxicological profile on Mus norvegicus is demonstrated for the leaves of Dalbergia katangensis and its antioxidant activity in vitro previously known is confirmed. This plant is particularly interesting for a further investigation as very few is known about its phytochemical composition.

Statement of ethical approval
The project proposal and procedures were reviewed and approved by the Department of Pharmacology in the faculty of Pharmaceutical Sciences from the University of Lubumbashi, DRC (UNILU/FSP/DPCOL/PT/002/2014).