Toxicity, Antibacterial, and Phytochemical Analyses of Antrocaryon klaineanum Pierre Extracts

Medicinal plants are traditionally used in Gabon to treat several types of illnesses. The study's purpose was to determine the toxic, antibacterial, and anti-inflammatory effects of Antrocaryon klaineanum Pierre extracts and to characterize their phytochemical compounds. Toxicity was evaluated on frog tadpoles (Phrynobatrachus africanus Hallowell). The microorganism susceptibility test was performed by the diffusion method, while minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were evaluated using the microdilution technique. Anti-inflammatory activity was tested through protein denaturation and membrane stabilization methods. Chromatography and molecular network techniques were used to characterize chemical compounds. The lethality test showed that the lethal concentration (LC50) increased from 110.03 ± 1.25 to 15.86 ± 2.21 μg/mL after 24 and 96 hours of exposure. In tadpoles exposed to 7.81 μg/mL extract, the first mortalities (12.5%) were observed on the fifth day of exposure. A relative decrease in mature erythrocytes exposed to plant extracts was observed. The antibacterial activity shows that the Ak F2, Ak F3, and Ak F4 fractions (from the water-ethanol crude extract) gave the greatest antibacterial activities compared to the other extracts. The water, water-acetone, and water-ethanol extracts showed good inhibition of denaturation. The haemolysis test shows that the extracts exhibited good anti-inflammatory activities. Phytochemical characterisation revealed four major compounds, including monogallate epicatechin and hydroxy-ergostadian. The molecular network revealed five main clusters. Our study shows that A. klaineanum Pierre could be a promising natural product for the isolation of molecules with potential biological activities.


Introduction
Traditional medicine is often the only afordable and accessible source of care in poor countries, especially for the poorest patients. According to the World Health Organisation (WHO), more than 80% of the African population regularly uses traditional and herbal medicine for the treatment of various conditions [1]. Plant research can lead to acceptable therapeutic responses and low-cost pricing in a socioeconomic environment, combining proven scientifc efcacy with ideal cultural acceptability. Te scientifc validation of traditional medicine should contribute to the development of phytomedicines.
Given the scientifc interest in plant-based medicines, recent years have seen the therapeutic valorisation of biologically active molecules contained in plant species. Tis has resulted in the creation of new molecules of natural origin. Despite the large amount of research that has already been carried out on plants, the results are still insufcient, given that only a small number of plants have been systematically studied for bioactive compounds [2].
Many researchers are interested in biologically active compounds isolated from plant species for the elimination of pathogenic microorganisms because of the resistance that microorganisms have developed against synthetic antibiotics. Gabon, by virtue of its geographical location, is home to a rich and diverse vegetation.
Many studies, albeit scattered, have been carried out on A. klaineanum, which shows the importance attached to traditional medicine. Antrocaryon klaineanum Pierre (Family: Anacardiaceae) is known by several names in Gabon, depending on the ethnic group (Angokong (Fang), Osôngôngô (Mpongwè); mungôngu-bôgu (Bapunu), and mungôngu-bôgu (Banzabi)); in Cameroon its common name is Onzabili [3,4]. It is a very large tree that is common throughout Gabon. It has compound leaves. Te fowers are yellowish-white. Te fruits are fattened and pentagonal, with a very hard, evenly shaped stone. Te powder of the bark is used against liver diseases [3], it also treats liver diseases. Te roots treat abdominal and liver diseases [5]. In Cameroon, its bark is used in traditional medicine to treat wounds, chlamydia, and female sterility [6]. Phytochemical studies on the isolation of Antrocarins A-F of the ergostane steroid type and antiplasmodial activities were carried out by Douanla [7]. Other works have shown that A. klaineanum has antioxidant and anti-infammatory properties [8,9].
However, research on toxicity and other biological properties of A. klaineanum stem barks is limited. Te present context aims not only to study the toxicity, antibacterial, and anti-infammatory properties but also to characterise the phytochemical constituents of A. klaineanum.

Preparation of Samples.
Te stem bark of A. klaineanum (voucher number: AK01-2017) was collected in Oyem (northern Gabon), in June 2017 and identifed at the National Herbarium by Professor Henry Bourobou (Botanist). Te harvested bark was dried, crushed, and used for extractions.
Te water-ethanol (30/70, v/v), water-acetone (30/70, v/v), and water (100%) extracts were prepared from the dry powder of A. klaineanum. Each sample (25 g) was mixed with 250 mL of extraction solvents. Te aqueous extracts were boiled for 60 minutes; water-ethanol and water-acetone extracts were macerated for 24 h. All extracts were fltered, concentrated, and lyophilized. Te extracts obtained were stored at 4°C until they were used for the various tests.  [10]. Te tadpoles of this species were captured in June 2021 in a body of water near a river. Upon their arrival at the laboratory, the tadpoles were placed in glass basins containing reconstituted water. Te tadpoles selected for the study had a mass of 2.00 ± 1.00 g and were at a stage of development corresponding to the appearance and progression of the buds of the hind limbs. (1) Relative Proportions of Erythrocytes. To determine the relative proportions of erythrocytes, 500 cells were identifed and categorized into three subgroups: mature erythrocytes, erythroblasts, and degenerating erythrocytes. Te erythroblasts were recognizable, according to the description made by Szubartowska [12], by their rounded shape, basophilic cytoplasm, and prominent nucleus, while degenerating erythrocytes were identifed by their wrinkled and stunted appearance. Te percentage of mature erythrocytes, erythroblasts, and degenerating erythrocytes per 500 counted cells (total erythrocytes) per individual was determined.

Bacterial Germs Tested.
Te bacterial carrier used in our study consisted of six reference bacterial strains and four clinical strains.

Antibacterial Activities.
Te difusion method was used to study the susceptibility of the microorganisms. Minimum inhibitory concentrations (MICs) of crude extracts and fractions were determined by the microdilution method in 96-well microplates [13]. Bactericidal antibacterials were considered to be those with a MBC/MIC ratio of 1 or 2 and bacteriostatic if the MBC/MIC ratio was 4 or 16 [14].
2.6. Anti-Infammatory Activities 2.6.1. Antiprotein Denaturation Test. Denaturation inhibition of crude extracts and fractions of Antrocaryon klaineanum was performed according to the protein denaturation inhibition method described in the reference by Ngoua-Meye-Misso et al. [15]. Te references (diclofenac sodium and paracetamol) used were treated under the same conditions as the crude extracts.

Membrane Stabilization Test.
Te membrane stabilisation test was evaluated by the human red blood cell (HRM) haemolysis method. Tis haemolysis was induced by heat on the one hand and distilled water on the other [15].

Fractionation, Identifcation, and Molecular Network
Analysis. Flash chromatography is a method of separation based on polarity; it has the same process as column chromatography. Separation by fash chromatography was carried out on Armen Instrument, spot liquid chromatography fash, according to the gradient of increasing polarity. 32 g of the stem bark water-alcohol extract was submitted to fash chromatography on a 70-230 mesh silica gel column (400 g) with stepwise gradient elution by CH 2 Cl 2 /MeOH mixtures (100 : 0; 98 : 2; 95 : 5; 90 : 10; 80 : 20; 0 : 100). Te fractions were collected and combined according to their thin layer chromatography (TLC) profles on precoated silica gel 60 F 254 plates developed with n-hexane/EtOAc and CH 2 Cl 2 /MeOH mixtures to give groups of fractions. Te fraction with the major biological activities was selected and analysed by high performance liquid chromatography coupled with the mass spectrometry (HPLC/Q tof ). Te compounds obtained were identifed by comparisons of their mass spectra to those of compounds registered in Reaxis and DNP 2019 libraries.
Molecular networking was carried out as described by Essono Mintsa et al. [16].

Statistical Analysis.
Te data were expressed as mean-± standard deviation (SD) of triplicate independent experiments and analyzed using one-way analysis of variance (ANOVA) and Student's t-test using Staplus Build software. 8.03/Corev7.811 (x86_64). Te values of p ≤ 0.05 were considered statistically signifcant.  Figure 1 presents the mortalities recorded during the sublethal test in tadpoles exposed to extracts of A. klaineanum for 17 days.

Toxicity of Aqueous Extracts of Antrocaryon klaineanum
Te result shows the frst mortalities (12.5%) of the tadpoles on the ffth day at 7.81 μg/mL of the plant extracts. On the ninth and thirteenth days, the mortality rate increased by 25% and 37.5%, respectively. Te species exposed to 3.9 μg/mL of extracts were exhibited from the ninth day (12.5% of mortality); on the fourteenth day, the mortality rate increased by 25%. It was only on the tenth day that the mortality (12.5%) of the tadpoles exposed to the control (diclofenac sodium) and to the plant extracts (1.95 μg/mL) was observed.

Description and Relative Proportions of Erythrocytes.
Erythroblasts were characterized by their rounded shape, basophilic cytoplasm, and larger nucleus than that of mature erythrocytes. Besides these cells (Figure 2), degenerating erythrocytes were also observed in some individuals. Figure 3 presents the relative proportions of erythrocytes identifed in the blood of tadpoles exposed to extracts of A. klaineanum Pierre and diclofenac sodium for 16 days. In Figure 3(a), a relative decrease in mature erythrocytes exposed to plant extracts and diclofenac sodium from the frst to the sixteenth day of the study is observed. On the frst day, the proportions of mature erythrocytes are 72, 78, 84, and 84%, corresponding to the concentrations of 7.81, 3.9, 1.96, and 1.96 μg/mL (standard), respectively, whereas on day 16, these erythrocytes decrease by 60, 68, 74, and 69%.
Te proportions of degenerating erythrocytes in the bloodstream of tadpoles exposed to 7.81, 3.9, and 1.96 μg/mL (Figure 3(c)) are relatively similar, on the frst day of exposure (4 to 5%) to that exposed to diclofenac sodium (4%). On the other hand, these proportions rise from 5 to 8% on the fourth day to remain relatively stable until the eighth day and then climb from 6 to 14% on the sixteenth day.
Among the erythroblasts observed (Figure 3(b)), we note their low proportions (2 to 4%) on the frst day of exposure to the diferent concentrations of the plant. From the fourth to the sixteenth day of exposure to 7.81 μg/mL of plant extract, a 4 to 12% increase in erythroblasts is clearly observed. Also, exposure to 3.9 μg/mL of the plant extract shows a relative increase in erythroblasts (4 to 6%) from the fourth to the sixteenth day of the study. Although the proportions of low concentrations do not undergo major   changes, it is noted that the proportions of erythroblasts and erythrocytes in degeneration tend to increase with high concentrations and as a function of time.

Antimicrobial Activity of Antrocaryon klaineanum
Extracts. Te results of susceptibility tests with the extracts of A. klaineanum gave the following diameters of inhibition on the bacterial strains ( Te results also show that the fractions possess the greatest antibacterial activities than the crude extracts. Tese fractions are active against both Gram-positive and Gram-negative bacteria. Te Ak F 2 , Ak F 3 , and Ak F 4 fractions gave the greatest antibacterial activities against the bacterial strains. Te Ak F 1 fraction is active on all strains tested with the exception of Listeria innocua LMG 135668 BHI, Pseudomonas aeruginosa, and Salmonella enterica. Te results of the MICs and CMBs are listed in Table 3. Te MICs (0.625 mg/mL) of the water-ethanol extracts (Ak WEE) are the lowest, as are the MICs of the Ak F 2 and Ak F 3 fractions. Table 4 shows the antibacterial efects of crude extracts and fractions of A. klaineanum. Te aqueous extract has a bactericidal efect on E. coli 105182 CIP, Listeria innocua LMG 135668 BHI, Enterococcus faecalis 103907 CIP, Bacillus cereus LMG 13569 BHI, and Salmonella typhi strains and has a bacteriostatic efect on Staphylococcus aureus ATCC 25293 BHI. Te water-ethanol, water-acetone extracts, and the Ak F 2 fraction have bactericidal actions on all the strains studied. Te Ak F 1 , Ak F 3 , and Ak F 4 fractions exhibit bactericidal efects on the majority of bacteria.

Anti-Infammatory Activity of Antrocaryon klaineanum
Extracts. Te results of the in vitro anti-infammatory activity of the extracts of A. klaineanum Pierre are summarized in Figure 4.
In each column, the assigned values of diferent alphabetic letters (a, b, c)    Te results of the molecular network of the crude extract of A. klaineanum reveal that the compounds are grouped according to structural similarities and fve main clusters emerged ( Figure 6): group A presents twelve nodes having the very close molecular masses, group B includes 6 nodes, two masses of which (m/z 298.145 and 298.146) correspond to Methoxyformonetin. Clusters C (4 nodes), D (3 nodes),

Discussion
Te purpose of the work was to study toxicity; establish antibacterial and anti-infammatory activities, and characterize the phytochemistry of A. klaineanum extracts. Te results of plant extracts exposed to frog tadpoles show a low toxicity compared to pharmaceutical drugs (diclofenac sodium). Te study also shows that the toxicity is progressive over time. Tis result corroborates with that of Obiang et al. [11], where the authors stipulate that the mortality correlated to the doses used is all the more increased as the exposure of frog tadpoles is prolonged over time. Te cells observed on the smears correspond quite closely to the descriptions made in leopard frogs by Jordan [20]. Observation of hematological parameters revealed that exposure of tadpoles to extracts of A. klaineanum causes mild anemia. Te observed efects were a decrease in the number of mature erythrocytes in the bloodstream, a reduction in erythrocyte length, and rounding of erythrocytes. Several studies presented similar results, which performed experiments on hematological efects in the toad Bufo regularis [21]. Tese authors hypothesized that the appearance of these efects could be associated with an increase in plasma volume; a reduction in blood copper concentration, which plays an important role in erythropoiesis; inhibition of hemoglobin synthesis; and iron defciency [22]. Te essential function of erythrocytes is to ensure the supply of oxygen to the tissues, which is carried, out thanks to hemoglobin. Because of this primary role attributed to hemoglobin, the diagnosis of anemia is only true when there is a decrease in hemoglobin concentration below the lower limit of normality.
Te study shows that over time, a slight increase in the proportions of erythroblasts and degenerating erythrocytes was evident. Te appearance of young erythrocytes (erythroblasts) refects a process of renewal of the erythrocyte population. In parallel, degenerating erythrocytes are characteristic of red blood cell lysis. In this case, we could be in the presence of a regenerative type anemia caused by Antibacterial activities of total extracts and fractions of bark extracts of A. klaineanum were demonstrated in this study. Te results obtained show that each sample tested has inhibitory efects on the growth of the majority of the bacterial strains tested. Of all the bacteria submitted to our study, Neisseria gonorrhea showed more sensitivity to the extracts tested. Te Ak F 1 , Ak F 2 , and Ak F 3 fractions proved to be more active on several bacteria compared to the other extracts. Tese inhibitions may be due to separations and purifcations of plant fractions by diferent chromatography methods [14]. Ethnopharmacological studies have shown that the bark of A. klaineanum is used for the treatment of typhoid fever and other liver-related diseases [8]. Tis study justifes the traditional use of A. klaineanum for the fght against certain infections of bacterial origin. Te sensitivity of bacteria like Bacillus cereus LMG 13569 BHI, Shigella dysenteria 5451 CIP, and Neisseria gonorrhea to the extracts could direct further research on the therapeutic properties of A. klaineanum against gonorrhea, diarrhea, and other types of infections. Tis sensitivity may be due, in part, to their ability of plant extracts to complex with amino acids of extracellular and soluble proteins resulting in inactivation and/or loss of function.
Te anti-infammatory activity of A. klaineanum was evaluated by protein denaturation test and membrane stabilization. Protein denaturation is a process in which proteins lose their tertiary and secondary structures by the application of external stress or compound, such as strong acid, base, or by heat, which most proteins lose their biological function when denatured [23]. Protein denaturation is a well-documented cause of infammation [19]. Te results on inhibition on protein denaturation showed that there is no signifcant diference between the plant extracts and the reference (diclofenac sodium). Similar results, showing the good inhibitory activities of protein denaturation by medicinal plants have been found in several studies [15,24].
Stabilization of the red blood cell membrane has been used as a method to study anti-infammatory activity in vitro because the erythrocyte membrane is analogous to the lysosomal membrane. Tis method confrms the antiinfammatory activity of A. klaineanum extracts. Te results showed that at diferent concentrations of extracts, erythrocyte membranes were protected against lysis and heat induced by hypotonic solution. Studies have shown that certain plants of the family A. klaineanum act at the level of many mechanisms such as the regulation of antiinfammatory activity. Te activation of NF-κB plays a key role in the infammatory response. Tese extracts have been shown to reduce the level of production of infammation mediators such as cyclooxygenase-2 or TNF-α [25]. Te results of our work confrm the traditional use of A. klaineanum in traditional medicine for the treatment of various pathologies [8].
Chromatograms and spectra of A. klaineanum show that several major compounds are not part of the dictionary of natural products and other databases consulted with the exception of compounds with masses m/z [M + H] + equal to 417.190 and 353.245. Tese compounds belong to the polyphenols of the (Epi) catechin-(epi) catechin monogallate type [19] and to the family of ergostane-type steroids [7]. Polyphenols are characterized by the presence of at least one benzene ring on which there is at least one hydroxyl group engaged with other functions. Tey are known for their antioxidant efects. Polyphenols afect membrane permeability, thus inducing the release of intracellular constituents into the external environment. Moreover, they also interfere with the respiratory chain by inhibiting the oxidation of NADH, the synthesis of nucleic acids and proteins, and the activity of certain enzymes [26].
In addition, we also performed crude extract analysis using the molecular network (MN) approach by GNPS website (https://gnps.ucsd.edu) [27]. All obtained HRE-SIMS/MS spectra were preprocessed via MZmine2 software following the feature-based molecular networking workfow [28]. Exploring the relative contributions of peak areas to network nodes revealed that compounds were clustered based on structural similarities. Te presence of the clusters in the molecular network shows that the crude extract of A. klaineanum exhibits several compounds that can be better studied.

Conclusion
Tis work made it possible to evaluate the toxicity, the biological (antibacterial and anti-infammatory) activities, and the chemical characterization of the aqueous extract of the bark of A. klaineanum. It emerges from this study that the plant extracts exposed to frog tadpoles show a low toxicity compared to pharmaceutical drugs (diclofenac sodium). Some extracts exhibit bactericidal efects. However, this sensitivity is dose-dependent and varies according to the germs and the extracts. Moreover, the extracts having the good antibacterial properties showed exceptional antiinfammatory activities, which were signifcantly very high compared to diclofenac sodium. Phytochemical characterization of the extracts revealed compounds such as monogallate type polyphenols and ergostane-type steroids. Tis study justifes certain uses of this in traditional medicine.