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Toxicity, Antibacterial, and Phytochemical Analyses of <i>Antrocaryon klaineanum</i> Pierre Extracts

Toxicity, Antibacterial, and Phytochemical Analyses of Antrocaryon klaineanum Pierre Extracts Hindawi Advances in Pharmacological and Pharmaceutical Sciences Volume 2023, Article ID 9304681, 12 pages https://doi.org/10.1155/2023/9304681 Research Article Toxicity, Antibacterial, and Phytochemical Analyses of Antrocaryon klaineanum Pierre Extracts 1,2 1,3 1,2 Ce´dric Sima Obiang , Thiery Ndong Mba, Joseph Privat Ondo, 1,2 1,2 Rick Le´onid Ngoua Meye Misso, Juliette Ornely Orango Bourdette, 4 1 2 Elvis Otogo N’Nang, Joefred Mbogho Abogho, Elvis Jolinom Mbot, 1,2 1 Louis Cle´ment Obame Engonga, and Edouard Nsi Emvo Laboratoire de Recherche en Biochimie (LAREBIO), Facult´e des Sciences, Universit´e des Sciences et Techniques de Masuku, Franceville, Gabon ` ´ Laboratoire de Substances Naturelles et de Syntheses Organometalliques (LASNSOM), Universit´e des Sciences et Techniques de Masuku, Franceville, Gabon Laboratoire de Biologie Mol´eculaire et Cellulaire (LABMC), Facult´e des Sciences Universit´e des Sciences et Techniques de Masuku, Franceville, Gabon Laboratoire de Chimie des Substances Naturelles (LACSN), Institut Supe´rieure d’Agronomie et de Biotechnologie, Universit´e des Sciences et Techniques de Masuku, Franceville, Gabon Correspondence should be addressed to Ce´dric Sima Obiang; simaobiangcedric@gmail.com Received 5 November 2022; Revised 18 March 2023; Accepted 7 April 2023; Published 22 April 2023 Academic Editor: Benedetto Natalini Copyright © 2023 Cedric Sima Obiang et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Medicinal plants are traditionally used in Gabon to treat several types of illnesses. Te study’s purpose was to determine the toxic, antibacterial, and anti-infammatory efects of Antrocaryon klaineanum Pierre extracts and to characterize their phytochemical compounds. Toxicity was evaluated on frog tadpoles (Phrynobatrachus africanus Hallowell). Te microorganism susceptibility test was performed by the difusion method, while minimum inhibitory concentration (MIC) and minimum bactericidal concen- tration (MBC) were evaluated using the microdilution technique. Anti-infammatory activity was tested through protein de- naturation and membrane stabilization methods. Chromatography and molecular network techniques were used to characterize chemical compounds. Te lethality test showed that the lethal concentration (LC ) 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 frst mortalities (12.5%) were observed on the ffth day of exposure. A relative decrease in mature erythrocytes exposed to plant extracts was observed. Te antibacterial activity shows that the Ak F , Ak F , and Ak F fractions (from the water-ethanol crude extract) gave the greatest 2 3 4 antibacterial activities compared to the other extracts. Te water, water-acetone, and water-ethanol extracts showed good in- hibition of denaturation. Te haemolysis test shows that the extracts exhibited good anti-infammatory activities. Phytochemical characterisation revealed four major compounds, including monogallate epicatechin and hydroxy-ergostadian. Te molecular network revealed fve main clusters. Our study shows that A. klaineanum Pierre could be a promising natural product for the isolation of molecules with potential biological activities. Organisation (WHO), more than 80% of the African pop- 1. Introduction ulation regularly uses traditional and herbal medicine for the Traditional medicine is often the only afordable and ac- treatment of various conditions [1]. Plant research can lead cessible source of care in poor countries, especially for to acceptable therapeutic responses and low-cost pricing in the poorest patients. According to the World Health a socioeconomic environment, combining proven scientifc 2 Advances in Pharmacological and Pharmaceutical Sciences efcacy with ideal cultural acceptability. Te scientifc val- Te water-ethanol (30/70, v/v), water-acetone (30/70, idation of traditional medicine should contribute to the v/v), and water (100%) extracts were prepared from the dry development of phytomedicines. powder of A. klaineanum. Each sample (25 g) was mixed Given the scientifc interest in plant-based medicines, with 250 mL of extraction solvents. Te aqueous extracts recent years have seen the therapeutic valorisation of bi- were boiled for 60 minutes; water-ethanol and water-acetone ologically active molecules contained in plant species. Tis extracts were macerated for 24 h. All extracts were fltered, has resulted in the creation of new molecules of natural concentrated, and lyophilized. Te extracts obtained were origin. Despite the large amount of research that has already stored at 4 C until they were used for the various tests. been carried out on plants, the results are still insufcient, given that only a small number of plants have been sys- 2.3. Toxicity of Aqueous Extracts on Frog Tadpoles tematically studied for bioactive compounds [2]. (Phrynobatrachus africanus Hallowell) Many researchers are interested in biologically active compounds isolated from plant species for the elimination of 2.3.1. Tadpole Sampling. Te determination of Phrynoba- pathogenic microorganisms because of the resistance that trachus africanus Hallowell was carried out by the identi- microorganisms have developed against synthetic antibi- fcation guide of amphibians of central and southern Africa otics. Gabon, by virtue of its geographical location, is home [10]. Te tadpoles of this species were captured in June 2021 to a rich and diverse vegetation. in a body of water near a river. Upon their arrival at the Many studies, albeit scattered, have been carried out on laboratory, the tadpoles were placed in glass basins con- A. klaineanum, which shows the importance attached to taining reconstituted water. Te tadpoles selected for the traditional medicine. Antrocaryon klaineanum Pierre study had a mass of 2.00± 1.00 g and were at a stage of (Family: Anacardiaceae) is known by several names in development corresponding to the appearance and pro- Gabon, depending on the ethnic group (Angokong (Fang), gression of the buds of the hind limbs. ˆ ˆ ˆ ` ˆ ˆ Osongongo (Mpongwe); mungongu-bogu (Bapunu), and mungongu-b ˆ ogu ˆ (Banzabi)); in Cameroon its common 2.3.2. Lethal and Sublethal Testing. Lethal and sublethal tests name is Onzabili [3, 4]. It is a very large tree that is common were performed using the method described by Obiang throughout Gabon. It has compound leaves. Te fowers are et al. [11]. 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 2.3.3. Hematological Parameters. After 1, 4, 8, and 16 days of diseases. Te roots treat abdominal and liver diseases [5]. In exposure, the tadpoles were sacrifced according to the Cameroon, its bark is used in traditional medicine to treat concentrations (1.95; 3.9; 7.81 μg/mL) of the extract in order wounds, chlamydia, and female sterility [6]. Phytochemical to collect the biological material necessary for the hema- studies on the isolation of Antrocarins A-F of the ergostane tological analyses. Te tadpoles were frst anesthetized and steroid type and antiplasmodial activities were carried out by 5 μL of blood was collected by cardiac puncture. A smear was Douanla [7]. Other works have shown that A. klaineanum immediately prepared, followed by Leishman staining, then has antioxidant and anti-infammatory properties [8, 9]. the cells were observed under an optical microscope (Motic However, research on toxicity and other biological Digital Microscope) coupled to a computer using Motic properties of A. klaineanum stem barks is limited. image plus 2.0 software, and fnally photos were taken. Te present context aims not only to study the toxicity, antibacterial, and anti-infammatory properties but (1) Relative Proportions of Erythrocytes. To determine the also to characterise the phytochemical constituents of relative proportions of erythrocytes, 500 cells were identifed A. klaineanum. and categorized into three subgroups: mature erythrocytes, erythroblasts, and degenerating erythrocytes. Te erythro- blasts were recognizable, according to the description made 2. Material and Methods by Szubartowska [12], by their rounded shape, basophilic cytoplasm, and prominent nucleus, while degenerating 2.1. Chemicals, Reagents, and Media. Organic solvents (HPLC grade) were obtained from Himedia, India. NaCl erythrocytes were identifed by their wrinkled and stunted appearance. Te percentage of mature erythrocytes, eryth- (Suvchem), sodium chloride (NaCl); Potassium chloride (KCl); Calcium chloride (CaCl ); agar (Biokar); glucose roblasts, and degenerating erythrocytes per 500 counted cells (total erythrocytes) per individual was determined. (Accumix); and other chemicals were obtained from Sigma, USA. All chemicals used were of analytical grade. 2.4. Bacterial Germs Tested. Te bacterial carrier used in our study consisted of six reference bacterial strains and four 2.2. Preparation of Samples. Te stem bark of A. klaineanum clinical strains. (voucher number: AK01-2017) was collected in Oyem (northern Gabon), in June 2017 and identifed at the Na- tional Herbarium by Professor Henry Bourobou (Botanist). 2.5. Antibacterial Activities. Te difusion method was used Te harvested bark was dried, crushed, and used for to study the susceptibility of the microorganisms. Minimum extractions. inhibitory concentrations (MICs) of crude extracts and Advances in Pharmacological and Pharmaceutical Sciences 3 fractions were determined by the microdilution method in aqueous extracts of A. klaineanum decreases proportionally 96-well microplates [13]. Bactericidal antibacterials were to the days. considered to be those with a MBC/MIC ratio of 1 or 2 and Tis LC is 110.03± 1.25 (APB), 63.55± 4.02 (APB), bacteriostatic if the MBC/MIC ratio was 4 or 16 [14]. 29.36± 2.11 (APB), and 15.86± 2.21 (APB) μg/mL after 24, 48, 72, and 96 hours of tadpole exposure, respectively. Te decrease in the value of LC results in the evolution of the 2.6. Anti-Infammatory Activities increasing number of mortalities. After 96 hours of expo- sure, no tadpole survived the other concentration ranges of 2.6.1. Antiprotein Denaturation Test. Denaturation in- 15.62, 31.2, 62.5, 125, 250, 500, and 1000 μg/mL when all hibition of crude extracts and fractions of Antrocaryon were alive in tanks containing only reconstituted water and klaineanum was performed according to the protein de- the lowest concentrations of aqueous extracts of naturation inhibition method described in the reference by A. klaineanum. Ngoua-Meye-Misso et al. [15]. Te references (diclofenac sodium and paracetamol) used were treated under the same conditions as the crude extracts. 3.1.2. Mortalities during Sublethal Tests. Figure 1 presents the mortalities recorded during the sublethal test in tadpoles exposed to extracts of A. klaineanum for 17 days. 2.6.2. Membrane Stabilization Test. Te membrane stabili- Te result shows the frst mortalities (12.5%) of the sation test was evaluated by the human red blood cell (HRM) tadpoles on the ffth day at 7.81 μg/mL of the plant extracts. haemolysis method. Tis haemolysis was induced by heat on On the ninth and thirteenth days, the mortality rate in- the one hand and distilled water on the other [15]. creased 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 2.7. Fractionation, Identifcation, and Molecular Network rate increased by 25%. It was only on the tenth day that the Analysis. Flash chromatography is a method of separation mortality (12.5%) of the tadpoles exposed to the control based on polarity; it has the same process as column (diclofenac sodium) and to the plant extracts (1.95 μg/mL) chromatography. Separation by fash chromatography was was observed. carried out on Armen Instrument, spot liquid chromatog- raphy fash, according to the gradient of increasing polarity. 32 g of the stem bark water-alcohol extract was submitted to 3.1.3. Description and Relative Proportions of Erythrocytes. fash chromatography on a 70–230 mesh silica gel column Erythroblasts were characterized by their rounded shape, (400 g) with stepwise gradient elution by CH Cl /MeOH 2 2 basophilic cytoplasm, and larger nucleus than that of mature mixtures (100 : 0; 98 : 2; 95 : 5; 90 :10; 80 : 20; 0 :100). Te erythrocytes. Besides these cells (Figure 2), degenerating fractions were collected and combined according to their erythrocytes were also observed in some individuals. thin layer chromatography (TLC) profles on precoated Figure 3 presents the relative proportions of erythrocytes silica gel 60 F plates developed with n-hexane/EtOAc and identifed in the blood of tadpoles exposed to extracts of CH Cl /MeOH mixtures to give groups of fractions. Te 2 2 A. klaineanum Pierre and diclofenac sodium for 16 days. In fraction with the major biological activities was selected and Figure 3(a), a relative decrease in mature erythrocytes ex- analysed by high performance liquid chromatography posed to plant extracts and diclofenac sodium from the frst coupled with the mass spectrometry (HPLC/Q tof). Te to the sixteenth day of the study is observed. On the frst day, compounds obtained were identifed by comparisons of the proportions of mature erythrocytes are 72, 78, 84, and their mass spectra to those of compounds registered in 84%, corresponding to the concentrations of 7.81, 3.9, 1.96, Reaxis and DNP 2019 libraries. and 1.96 μg/mL (standard), respectively, whereas on day 16, Molecular networking was carried out as described by these erythrocytes decrease by 60, 68, 74, and 69%. Essono Mintsa et al. [16]. 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 ex- 2.8. Statistical Analysis. Te data were expressed as mean- posure (4 to 5%) to that exposed to diclofenac sodium (4%). ± standard deviation (SD) of triplicate independent ex- On the other hand, these proportions rise from 5 to 8% on periments and analyzed using one-way analysis of variance the fourth day to remain relatively stable until the eighth day (ANOVA) and Student’s t-test using Staplus Build software. and then climb from 6 to 14% on the sixteenth day. 8.03/Corev7.811 (x86_64). Te values of p≤ 0.05 were Among the erythroblasts observed (Figure 3(b)), we note considered statistically signifcant. their low proportions (2 to 4%) on the frst day of exposure to the diferent concentrations of the plant. From the fourth 3. Results to the sixteenth day of exposure to 7.81 μg/mL of plant extract, a 4 to 12% increase in erythroblasts is clearly ob- 3.1. Toxicity of Aqueous Extracts of Antrocaryon klaineanum served. Also, exposure to 3.9 μg/mL of the plant extract Pierre shows a relative increase in erythroblasts (4 to 6%) from the fourth to the sixteenth day of the study. Although the 3.1.1. Lethal Test. Te results of the analyses presented in Table 1 show that the LC for frog tadpoles exposed to proportions of low concentrations do not undergo major 50 4 Advances in Pharmacological and Pharmaceutical Sciences Table 1: Lethal concentration (LC ) of aqueous extract Antrocaryon klaineanum for frog tadpoles (Phrynobatrachus africanus Hallowell). LC (μg/mL) depending on the duration of exposure Species 50 stage of development 24 hours 48 hours 72 hours 96 hours Phrynobatrachus africanus Hallowell 110.03± 1.25 63.55± 4.02 29.36± 2.11 15.86± 2.21 Tadpoles (2± 1 g) APB APB APB APB Rana clamitan 171.37± 9.25 61.99± 5.02 56.25± 5.01 58.25± 7.21 Tadpoles (0.91 g) [17] EP EP REG REG Bufo melanostictu 22.42 19.81 11.91 8.18 Tadpoles (0.1 g) [18] EP EP REG REG APB: appearance and progression of the buds of the hind limbs. -10 0 510 15 20 Exposure period (Days) 7.81 (μg/mL) 1.95 (μg/mL) 3.9 (μg/mL) Standard (Diclofenac sodium 1.95 μg/mL) Figure 1: Mortalities recorded during the sublethal test in tadpoles exposed to aqueous extract of Antrocaryon klaineanum for 17 days. (a) (b) (c) Figure 2: Tadpole frog (Phrynobatrachus africanus Hallowell). (a) Mature erythrocytes; (b) erythroblasts; (c) degenerating erythrocytes. Percent mortality (%) Advances in Pharmacological and Pharmaceutical Sciences 5 90 84 84 14 78 78 73 74 71 12 69 69 6 6 4 4 4 4 3 20 2 2 2 22 2 Day 1 Day 4 Day 8 Day 16 Day 1 Day 4 Day 8 Day 16 7.81µg/mL 1.96 µg/mL 7.81µg/mL 1.96 µg/mL 3.9 µg/mL Standard (Diclofenac sodium 3.9 µg/mL Standard (Diclofenac sodium 1.96 µg/mL) 1.96 µg/mL) (a) (b) 8 8 6 6 6 5 5 5 4 4 4 Day 1 Day 4 Day 8 Jour 16 7.81 µg/mL 1.96 µg/mL 3.9 µg/mL Standard (Diclofénac de sodium 1.96 µg/mL) (c) Figure 3: Proportion of erythrocyte cells in the blood of tadpoles exposed to Antrocaryon klaineanum for 16 days. (a) Mature erythrocytes; (b) erythroblasts; (c) degenerating erythrocytes. changes, it is noted that the proportions of erythroblasts and Te results of the MICs and CMBs are listed in Table 3. erythrocytes in degeneration tend to increase with high Te MICs (0.625 mg/mL) of the water-ethanol extracts concentrations and as a function of time. (Ak WEE) are the lowest, as are the MICs of the Ak F and Ak F fractions. Table 4 shows the antibacterial efects of crude extracts 3.2. Antimicrobial Activity of Antrocaryon klaineanum and fractions of A. klaineanum. Te aqueous extract has Extracts. Te results of susceptibility tests with the extracts a bactericidal efect on E. coli 105182 CIP, Listeria innocua of A. klaineanum gave the following diameters of inhibition LMG 135668 BHI, Enterococcus faecalis 103907 CIP, Bacillus on the bacterial strains (Table 2). Te screening of the three cereus LMG 13569 BHI, and Salmonella typhi strains and has extracts shows that all have antibacterial activity. Te a bacteriostatic efect on Staphylococcus aureus ATCC 25293 aqueous, water-ethanol, and water-acetone extracts gave the BHI. Te water-ethanol, water-acetone extracts, and the Ak greatest antibacterial activities against Neisseria gonorrhea F fraction have bactericidal actions on all the strains with the inhibition diameters greater than 19 mm. Te studied. Te Ak F , Ak F , and Ak F fractions exhibit 1 3 4 water-acetone extracts show signifcant antibacterial activ- bactericidal efects on the majority of bacteria. ities in the majority of strains with the exception of E. coli 105182 CIP, Salmonella typhi, E. coli, Staphylococcus aureus, Acinetobacter baumannii, Enterobacter aerogenes, and Sal- 3.3. Anti-Infammatory Activity of Antrocaryon klaineanum monella spp. sensitive to the water-ethanol extract and Extracts. Te results of the in vitro anti-infammatory ac- Bacillus cereus LMG 13569 BHI sensitive to the aqueous tivity of the extracts of A. klaineanum Pierre are summarized extract. in Figure 4. Te results also show that the fractions possess the In each column, the assigned values of diferent al- greatest antibacterial activities than the crude extracts. phabetic letters (a, b, c) indicate signifcantly diferent yields Tese fractions are active against both Gram-positive and (P< 0.05). Gram-negative bacteria. Te Ak F , Ak F , and Ak F Tis study shows that the water (IC � 80.20± 9.65 μg/ 2 3 4 50 fractions gave the greatest antibacterial activities against mL), water-ethanol (IC �110.27± 10.02 μg/mL), and the bacterial strains. Te Ak F fraction is active on all water-acetone extracts (IC � 86.22± 9.22 μg/mL) did not 1 50 strains tested with the exception of Listeria innocua LMG show any signifcant diference compared to the standards 135668 BHI, Pseudomonas aeruginosa, and Salmonella (IC � 97.20± 2.37 μg/mL). Te hemolysis test induced by enterica. a hypotonic solution shows that the three extracts studied 6 Advances in Pharmacological and Pharmaceutical Sciences Table 2: Inhibition zone diameters produced by the extracts and fractions from Antrocaryon klaineanum. Inhibition zone diameters (mm) Standards Extracts Fractions Ak water Ak water Ak water Ak F1 Ak F2 Ak F3 Ak F4 Gen Am Te ethanol acetone Bacteria Reference strains Escherichia coli 105182 CIP 8± 1 10± 1 9± 0 16± 2 17± 1 16± 1 12± 1 17± 1 Nd Nd Listeria innocua LMG 135668 BHI 7± 0 9± 1 11± 1 Nd 14± 2 13± 1 10± 0 13± 0 7± 1 14± 0 Staphylococcus aureus ATCC 25293 BHI 9± 0 10± 1 10± 0 10± 0 12± 0 15± 0 11± 1 15± 1 Nd 17± 1 Enterococcus faecalis 103907 CIP 12± 1 11± 2 14± 0 10± 0 12± 0 12± 2 10± 1 30± 0 7± 1 19± 0 Bacillus cereus LMG 13569 BHI 10± 0 9± 1 9± 0 15± 1 16± 0 15± 1 11± 0 13± 1 Nd 18± 1 Shigella dysenteria 5451 CIP 10± 1 10± 1 11± 1 13± 0 15± 1 15± 0 12± 1 24± 1 Nd 16± 0 Clinical isolates Pseudomonas aeruginosa 7± 0 10± 1 12± 1 Nd 12± 1 11± 1 9± 0 20± 0 7± 1 21± 1 Salmonella enterica 8± 0 10± 1 11± 1 Nd 10± 0 10± 1 11± 1 28± 1 7± 1 16± 1 Salmonella typhi 9± 0 11± 1 10± 0 9± 0 12± 1 13± 0 10± 1 20± 1 7± 0 15± 1 Neisseria gonorrhea 19± 1 20± 1 21± 1 21± 1 24± 0 22± 1 18± 2 22± 2 7± 1 10± 1 Nd � not determinated; Gen � gentamicin (10 μg/mL), Te � tetracycline (30 μg/mL), Am � ampicillin (30 μg/mL), Ak � Antrocaryon klaineanum, and F � fraction. Table 3: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC) of crude extracts and fraction of Antrocaryon klaineanum. MIC and MBC (mg/mL) Ak AE Ak WEE Ak WAE Ak F1 Ak F2 Ak F3 Ak F4 MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC Bacteria Reference strains Escherichia coli 105182 CIP 1.25 2.5 1.25 2.5 1.25 2.5 1.25 1.25 0.625 1.25 1.25 2.5 2.5 5 Listeria innocua LMG 135668 BHI 1.25 2.5 1.25 2.5 1.25 2.5 Nd Nd 1.25 2.5 1.25 5 5 5 Staphylococcus aureus ATCC 25293 1.25 5 1.25 1.25 1.25 2.5 2.5 5 2.5 5 1.25 5 2.5 5 BHI Enterococcus faecalis 103907 CIP 1.25 1.25 0.625 0.625 1.25 2.5 5 5 2.5 5 2.5 2.5 5 5 Bacillus cereus LMG 13569 BHI 1.25 1.25 0.625 0.625 1.25 1.25 1.25 1.25 0.625 1.25 0.625 1.25 2.5 5 Shigella dysenteria 5451 CIP 1.25 2.5 0.625 1.25 1.25 1.25 1.25 5 1.25 2.5 0.625 2.5 >5 >5 Clinical isolates Pseudomonas aeruginosa 1.25 2.5 0.625 1.25 0.625 1.25 Nd Nd 2.5 5 2.5 5 5 >5 Salmonella enterica 2.5 5 1.25 1.25 1.25 1.25 Nd Nd 2.5 5 5 5 2.5 5 Salmonella typhi 2.5 2.5 1.25 2.5 1.25 2.5 5 >5 2.5 5 2.5 2.5 2.5 5 Neisseria meningitides 0.625 0.65 0.625 1.25 0.625 1.25 1.25 1.25 0.625 1.25 1.25 1.25 0.625 1.25 Nd � not determinated; Ak � Antrocaryon klaineanum, WAE � water-acetone extract; WEE � water-ethanol extract; AE � aqueous extract; F � fraction. presented good activities with IC ranging from formulas C H O , C H O , C H O , and C H O . 50 18 20 4 24 32 2 27 28 4 26 36 4 62.26± 5.82 to 72.85± 8.00 μg/mL. For heat-induced he- After dereplication of the majority compounds in the molysis, all extracts exhibited very good antihemolytic ac- databases (Reaxis and Dictionary of natural products), tivity with IC s ranging from 70.59± 5.35 to compound 3 (mass ESI-MS m/z [M + H] � 353.245) 75.25± 8.56 μg/mL. Tese results have no signifcant dif- corresponds to monogallate type epicatechin and com- ference with the standard (diclofenac sodium). pound 4 (mass ESI-MS m/z [M + H] � 413.256) corre- sponds to 7α-Hydroxy-4,24(28)-ergostadien-3-one (Table 5). Te other compounds did not show matches in the 3.4. Chromatographic Analyses and Molecular Network MS/ literature. MS of Antrocaryon klaineanum Pierre Crude Extract. Te Te results of the molecular network of the crude extract results in Figure 5 present a set of chromatogram and mass of A. klaineanum reveal that the compounds are grouped spectra of the crude extract of A. klaineanum. Te study according to structural similarities and fve main clusters reveals 4 major peaks whose masses ESI-MS m/z emerged (Figure 6): group A presents twelve nodes having + + [M + H] � 301.144; ESI-MS m/z [M + H] � 353.245; the very close molecular masses, group B includes 6 nodes, ESI-MS m/z [M + H] � 417.202; and ESI-MS m/z two masses of which (m/z 298.145 and 298.146) correspond [M + H] � 413.265 correspond to the respective molecular to Methoxyformonetin. Clusters C (4 nodes), D (3 nodes), Advances in Pharmacological and Pharmaceutical Sciences 7 Table 4: Antimicrobial efects of plants extracts from Antrocaryon klaineanum. Ak AE Ak WEE Ak WAE Ak F Ak F Ak F Ak F 1 2 3 4 MBC/MIC MBC/MIC MBC/MIC MBC/MIC MBC/MIC MBC/MIC MBC/MIC Efect Efect Efect Efect Efect Efect Efect Bacteria Reference strains Escherichia coli 105182 CIP 2 2 2 1 2 2 2 Listeria innocua LMG 135668 BHI 2 2 2 — 2 3.2 1 Staphylococcus aureus ATCC 25293 BHI 4 1 2 2 2 3.2 2 Enterococcus faecalis 103907 CIP 1 1 2 1 2 1 1 Bacillus cereus LMG 13569 BHI 1 1 1 1 2 2 2 Shigella dysenteria 5451 CIP 2 2 1 2 2 2 — Clinical isolates Pseudomonas aeruginosa 2 2 2 — 2 2 — Salmonella enterica 2 1 1 — 2 1 2 Salmonella thyphimurium 1 2 2 — 2 1 2 Neisseria meningitidis 1 2 2 1 2 1 2 MBC/MIC< 4: bactericidal; MBC/MIC≥ 4: bacteriostatic; WAE � water-acetone extract; WEE � water-ethanol extract; AE � aqueous extract. ac 75.25 ± 8.56 ac 66.86 ± 5.10 ac ac 72.85 ± 8.00 70.59 ± 5.35 70.95 ± 5.32 79.56 ± 2.01 c c 62.26 ± 5.82 72.85 ± 8.00 110.27 ± 10.02 97.20 ± 2.37 86.22 ± 9.22 80.20 ± 9.65 Water‐acetone extract Water‐ethanol extract Water extract Diclofenac sodium Protein denaturation test IC50 (μg/mL) Hemolysis by hypotonicity IC50 (μg/mL) Heat hemolysis IC50 (μg/mL) Figure 4: Anti-infammatory activity of Antrocaryon klaineanum. ESI-MS m/z [M + H] = 417.202 ESI-MS m/z [M + H] = 301.144 C H O C H O 27 28 4 18 20 4 + + ESI-MS m/z [M + H] = 353.245 ESI-MS m/z [M + H] = 413.265 C H O C H O 24 32 2 26 36 4 Figure 5: Chromatograms and mass spectra of the ethanol extract of Antrocaryon klaineanum. 8 Advances in Pharmacological and Pharmaceutical Sciences Table 5: HPLC-ESI-QTOF identifcation of major compounds from ethanol stem barks extracts of Antrocaryon klaineanum. m/z Identifcation Identifcation Name Fraction Compound RT Score Formula Reference [M + H] DNP reaxys of compound 1 32.08 301.144 89.31 C H O 10 hits 15 hits Not determined — 18 20 4 2 33.40 417.202 70.40 C H O 06 hits 7 hits Not determined — 27 28 8 AK Te 3 35.64 353.245 69.64 C H O 32 hits 45 hits (Epi) catechin-(epi) catechin monogallate (B-type) [19] 24 32 2 4 36.86 413.265 80.2 C H O 16 hits 56 hits 7a-Hydroxy-4,24(28)-ergostadien-3-one [6] 26 36 4 AK: Antrocaryon klaineanum, Te � total extracts, Rt � retention time, MS � mass spectrometer, DNP � dictionary of natural products. Advances in Pharmacological and Pharmaceutical Sciences 9 Figure 6: Molecular network of the ethanolic crude extract of Antrocaryon klaineanum Pierre. and E (3 nodes) group together compounds not identifed by a reduction in erythrocyte length, and rounding of eryth- rocytes. Several studies presented similar results, which our available databases. performed experiments on hematological efects in the toad Bufo regularis [21]. Tese authors hypothesized that the 4. Discussion appearance of these efects could be associated with an Te purpose of the work was to study toxicity; establish increase in plasma volume; a reduction in blood copper antibacterial and anti-infammatory activities, and charac- concentration, which plays an important role in erythro- terize the phytochemistry of A. klaineanum extracts. Te poiesis; inhibition of hemoglobin synthesis; and iron de- results of plant extracts exposed to frog tadpoles show a low fciency [22]. Te essential function of erythrocytes is to toxicity compared to pharmaceutical drugs (diclofenac so- ensure the supply of oxygen to the tissues, which is carried, out thanks to hemoglobin. Because of this primary role dium). Te study also shows that the toxicity is progressive over time. Tis result corroborates with that of Obiang et al. attributed to hemoglobin, the diagnosis of anemia is only true when there is a decrease in hemoglobin concentration [11], where the authors stipulate that the mortality correlated to the doses used is all the more increased as the exposure of below the lower limit of normality. frog tadpoles is prolonged over time. Te cells observed on Te study shows that over time, a slight increase in the the smears correspond quite closely to the descriptions made proportions of erythroblasts and degenerating erythrocytes in leopard frogs by Jordan [20]. was evident. Te appearance of young erythrocytes Observation of hematological parameters revealed that (erythroblasts) refects a process of renewal of the eryth- exposure of tadpoles to extracts of A. klaineanum causes rocyte population. In parallel, degenerating erythrocytes are mild anemia. Te observed efects were a decrease in the characteristic of red blood cell lysis. In this case, we could be number of mature erythrocytes in the bloodstream, in the presence of a regenerative type anemia caused by 10 Advances in Pharmacological and Pharmaceutical Sciences hyperhemolysis of erythrocytes leading to erythropoietic 417.190 and 353.245. Tese compounds belong to the stimulation. polyphenols of the (Epi) catechin-(epi) catechin monogallate Antibacterial activities of total extracts and fractions of type [19] and to the family of ergostane-type steroids [7]. bark extracts of A. klaineanum were demonstrated in this Polyphenols are characterized by the presence of at least one study. Te results obtained show that each sample tested has benzene ring on which there is at least one hydroxyl group inhibitory efects on the growth of the majority of the engaged with other functions. Tey are known for their bacterial strains tested. Of all the bacteria submitted to our antioxidant efects. Polyphenols afect membrane perme- study, Neisseria gonorrhea showed more sensitivity to the ability, thus inducing the release of intracellular constituents extracts tested. Te Ak F , Ak F , and Ak F fractions proved into the external environment. Moreover, they also interfere 1 2 3 to be more active on several bacteria compared to the other with the respiratory chain by inhibiting the oxidation of extracts. Tese inhibitions may be due to separations and NADH, the synthesis of nucleic acids and proteins, and the purifcations of plant fractions by diferent chromatography activity of certain enzymes [26]. methods [14]. Ethnopharmacological studies have shown In addition, we also performed crude extract analysis that the bark of A. klaineanum is used for the treatment of using the molecular network (MN) approach by GNPS typhoid fever and other liver-related diseases [8]. Tis study website (https://gnps.ucsd.edu) [27]. All obtained HRE- justifes the traditional use of A. klaineanum for the fght SIMS/MS spectra were preprocessed via MZmine2 software against certain infections of bacterial origin. Te sensitivity following the feature-based molecular networking workfow of bacteria like Bacillus cereus LMG 13569 BHI, Shigella [28]. Exploring the relative contributions of peak areas to dysenteria 5451 CIP, and Neisseria gonorrhea to the extracts network nodes revealed that compounds were clustered could direct further research on the therapeutic properties of based on structural similarities. Te presence of the clusters A. klaineanum against gonorrhea, diarrhea, and other types in the molecular network shows that the crude extract of of infections. Tis sensitivity may be due, in part, to their A. klaineanum exhibits several compounds that can be better ability of plant extracts to complex with amino acids of studied. extracellular and soluble proteins resulting in inactivation and/or loss of function. 5. Conclusion Te anti-infammatory activity of A. klaineanum was evaluated by protein denaturation test and membrane sta- Tis work made it possible to evaluate the toxicity, the bilization. Protein denaturation is a process in which pro- biological (antibacterial and anti-infammatory) activities, teins lose their tertiary and secondary structures by the and the chemical characterization of the aqueous extract of application of external stress or compound, such as strong the bark of A. klaineanum. It emerges from this study that acid, base, or by heat, which most proteins lose their bi- the plant extracts exposed to frog tadpoles show a low ological function when denatured [23]. Protein denaturation toxicity compared to pharmaceutical drugs (diclofenac so- is a well-documented cause of infammation [19]. Te results dium). Some extracts exhibit bactericidal efects. However, on inhibition on protein denaturation showed that there is this sensitivity is dose-dependent and varies according to the no signifcant diference between the plant extracts and the germs and the extracts. Moreover, the extracts having the reference (diclofenac sodium). Similar results, showing the good antibacterial properties showed exceptional anti- good inhibitory activities of protein denaturation by me- infammatory activities, which were signifcantly very high dicinal plants have been found in several studies [15, 24]. compared to diclofenac sodium. Phytochemical character- Stabilization of the red blood cell membrane has been ization of the extracts revealed compounds such as monog- used as a method to study anti-infammatory activity in vitro allate type polyphenols and ergostane-type steroids. Tis because the erythrocyte membrane is analogous to the ly- study justifes certain uses of this in traditional medicine. sosomal membrane. Tis method confrms the anti- infammatory activity of A. klaineanum extracts. Te re- Abbreviations sults showed that at diferent concentrations of extracts, erythrocyte membranes were protected against lysis and heat AK: Antrocaryon klaineanum induced by hypotonic solution. Studies have shown that CM: % corrected mortality certain plants of the family A. klaineanum act at the level of DNP: Dictionary of natural products many mechanisms such as the regulation of anti- EP: Embryonic phase infammatory activity. Te activation of NF-κB plays HPLC: High performance liquid chromatography a key role in the infammatory response. Tese extracts have IPHAMETRA: Institut de Pharmacopee ´ et de Medecine ´ been shown to reduce the level of production of in- Traditionennelle fammation mediators such as cyclooxygenase-2 or TNF-α M2: % mortality in the treated population [25]. Te results of our work confrm the traditional use of M1: % mortality in the control population A. klaineanum in traditional medicine for the treatment of MBC: Minimum bactericial concentrations various pathologies [8]. MIC: Minimum inhibitory concentrations Chromatograms and spectra of A. klaineanum show that MS: Mass spectrometer several major compounds are not part of the dictionary of REG: Regression of external gills natural products and other databases consulted with the Rt: Retention time exception of compounds with masses m/z [M + H] equal to TLC: Tin layer chromatography. Advances in Pharmacological and Pharmaceutical Sciences 11 [12] E. Szubartowska, “Changes in the blood of the frog (Rana Data Availability temporaria) after diferent doses of ekatin,” Annales Uni- versitatis Mariae Curie-Sklodowska, vol. 45, pp. 67–78, 1990. Te data used to support the fndings of this study are in- [13] S. O. Cedric, ´ O. Joseph-Privat, O. E. Louis-Clement ´ et al., cluded within the article. “Phytochemical screening, evaluation of antioxidant and antimicrobial properties of Erythrophleum ivorense A. Chev Conflicts of Interest (Leguminosae) and Megaphrynium macrostachyum Benth (Marantaceae), medicinal plants from Gabon,” International Te authors declare that there are no conficts of interest Journal of Biosciences, vol. 8, pp. 43–53, 2016. regarding the publication of this paper. [14] C. S. Obiang, R. L. N. M. Misso, E. O. N’Nang et al., “Bi- ological properties and phytochemical analysis of fractions and total extracts of Cylicodiscus gabunensis Harms and Acknowledgments Morinda lucida Benth,” Journal of Advances in Microbiology, vol. 21, pp. 29–39, 2021. Te authors are very grateful to Shell Gabon for the fnancial [15] R. L. Ngoua-Meye-Misso, C. Sima-Obiang, J. D. L. C. Ndong, support of the equipment at the USTM Biochemistry Re- J. P. Ondo, F. Ovono Abessolo, and L. C. Obame-Engonga, search Laboratory (SG/CIS/SDM/SA/sa grant no 77). “Phytochemical screening, antioxidant, anti-infammatory and antiangiogenic activities of Lophira procera A. Chev.(Ochnaceae) medicinal plant from Gabon,” Egyptian References Journal of Basic and Applied Sciences, vol. 5, no. 1, pp. 80–86, [1] World Health Organization, “World Health Organization [16] M. Essono Mintsa, E. Otogo N’nang, E. 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Toxicity, Antibacterial, and Phytochemical Analyses of <i>Antrocaryon klaineanum</i> Pierre Extracts

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Hindawi Advances in Pharmacological and Pharmaceutical Sciences Volume 2023, Article ID 9304681, 12 pages https://doi.org/10.1155/2023/9304681 Research Article Toxicity, Antibacterial, and Phytochemical Analyses of Antrocaryon klaineanum Pierre Extracts 1,2 1,3 1,2 Ce´dric Sima Obiang , Thiery Ndong Mba, Joseph Privat Ondo, 1,2 1,2 Rick Le´onid Ngoua Meye Misso, Juliette Ornely Orango Bourdette, 4 1 2 Elvis Otogo N’Nang, Joefred Mbogho Abogho, Elvis Jolinom Mbot, 1,2 1 Louis Cle´ment Obame Engonga, and Edouard Nsi Emvo Laboratoire de Recherche en Biochimie (LAREBIO), Facult´e des Sciences, Universit´e des Sciences et Techniques de Masuku, Franceville, Gabon ` ´ Laboratoire de Substances Naturelles et de Syntheses Organometalliques (LASNSOM), Universit´e des Sciences et Techniques de Masuku, Franceville, Gabon Laboratoire de Biologie Mol´eculaire et Cellulaire (LABMC), Facult´e des Sciences Universit´e des Sciences et Techniques de Masuku, Franceville, Gabon Laboratoire de Chimie des Substances Naturelles (LACSN), Institut Supe´rieure d’Agronomie et de Biotechnologie, Universit´e des Sciences et Techniques de Masuku, Franceville, Gabon Correspondence should be addressed to Ce´dric Sima Obiang; simaobiangcedric@gmail.com Received 5 November 2022; Revised 18 March 2023; Accepted 7 April 2023; Published 22 April 2023 Academic Editor: Benedetto Natalini Copyright © 2023 Cedric Sima Obiang et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Medicinal plants are traditionally used in Gabon to treat several types of illnesses. Te study’s purpose was to determine the toxic, antibacterial, and anti-infammatory efects of Antrocaryon klaineanum Pierre extracts and to characterize their phytochemical compounds. Toxicity was evaluated on frog tadpoles (Phrynobatrachus africanus Hallowell). Te microorganism susceptibility test was performed by the difusion method, while minimum inhibitory concentration (MIC) and minimum bactericidal concen- tration (MBC) were evaluated using the microdilution technique. Anti-infammatory activity was tested through protein de- naturation and membrane stabilization methods. Chromatography and molecular network techniques were used to characterize chemical compounds. Te lethality test showed that the lethal concentration (LC ) 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 frst mortalities (12.5%) were observed on the ffth day of exposure. A relative decrease in mature erythrocytes exposed to plant extracts was observed. Te antibacterial activity shows that the Ak F , Ak F , and Ak F fractions (from the water-ethanol crude extract) gave the greatest 2 3 4 antibacterial activities compared to the other extracts. Te water, water-acetone, and water-ethanol extracts showed good in- hibition of denaturation. Te haemolysis test shows that the extracts exhibited good anti-infammatory activities. Phytochemical characterisation revealed four major compounds, including monogallate epicatechin and hydroxy-ergostadian. Te molecular network revealed fve main clusters. Our study shows that A. klaineanum Pierre could be a promising natural product for the isolation of molecules with potential biological activities. Organisation (WHO), more than 80% of the African pop- 1. Introduction ulation regularly uses traditional and herbal medicine for the Traditional medicine is often the only afordable and ac- treatment of various conditions [1]. Plant research can lead cessible source of care in poor countries, especially for to acceptable therapeutic responses and low-cost pricing in the poorest patients. According to the World Health a socioeconomic environment, combining proven scientifc 2 Advances in Pharmacological and Pharmaceutical Sciences efcacy with ideal cultural acceptability. Te scientifc val- Te water-ethanol (30/70, v/v), water-acetone (30/70, idation of traditional medicine should contribute to the v/v), and water (100%) extracts were prepared from the dry development of phytomedicines. powder of A. klaineanum. Each sample (25 g) was mixed Given the scientifc interest in plant-based medicines, with 250 mL of extraction solvents. Te aqueous extracts recent years have seen the therapeutic valorisation of bi- were boiled for 60 minutes; water-ethanol and water-acetone ologically active molecules contained in plant species. Tis extracts were macerated for 24 h. All extracts were fltered, has resulted in the creation of new molecules of natural concentrated, and lyophilized. Te extracts obtained were origin. Despite the large amount of research that has already stored at 4 C until they were used for the various tests. been carried out on plants, the results are still insufcient, given that only a small number of plants have been sys- 2.3. Toxicity of Aqueous Extracts on Frog Tadpoles tematically studied for bioactive compounds [2]. (Phrynobatrachus africanus Hallowell) Many researchers are interested in biologically active compounds isolated from plant species for the elimination of 2.3.1. Tadpole Sampling. Te determination of Phrynoba- pathogenic microorganisms because of the resistance that trachus africanus Hallowell was carried out by the identi- microorganisms have developed against synthetic antibi- fcation guide of amphibians of central and southern Africa otics. Gabon, by virtue of its geographical location, is home [10]. Te tadpoles of this species were captured in June 2021 to a rich and diverse vegetation. in a body of water near a river. Upon their arrival at the Many studies, albeit scattered, have been carried out on laboratory, the tadpoles were placed in glass basins con- A. klaineanum, which shows the importance attached to taining reconstituted water. Te tadpoles selected for the traditional medicine. Antrocaryon klaineanum Pierre study had a mass of 2.00± 1.00 g and were at a stage of (Family: Anacardiaceae) is known by several names in development corresponding to the appearance and pro- Gabon, depending on the ethnic group (Angokong (Fang), gression of the buds of the hind limbs. ˆ ˆ ˆ ` ˆ ˆ Osongongo (Mpongwe); mungongu-bogu (Bapunu), and mungongu-b ˆ ogu ˆ (Banzabi)); in Cameroon its common 2.3.2. Lethal and Sublethal Testing. Lethal and sublethal tests name is Onzabili [3, 4]. It is a very large tree that is common were performed using the method described by Obiang throughout Gabon. It has compound leaves. Te fowers are et al. [11]. 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 2.3.3. Hematological Parameters. After 1, 4, 8, and 16 days of diseases. Te roots treat abdominal and liver diseases [5]. In exposure, the tadpoles were sacrifced according to the Cameroon, its bark is used in traditional medicine to treat concentrations (1.95; 3.9; 7.81 μg/mL) of the extract in order wounds, chlamydia, and female sterility [6]. Phytochemical to collect the biological material necessary for the hema- studies on the isolation of Antrocarins A-F of the ergostane tological analyses. Te tadpoles were frst anesthetized and steroid type and antiplasmodial activities were carried out by 5 μL of blood was collected by cardiac puncture. A smear was Douanla [7]. Other works have shown that A. klaineanum immediately prepared, followed by Leishman staining, then has antioxidant and anti-infammatory properties [8, 9]. the cells were observed under an optical microscope (Motic However, research on toxicity and other biological Digital Microscope) coupled to a computer using Motic properties of A. klaineanum stem barks is limited. image plus 2.0 software, and fnally photos were taken. Te present context aims not only to study the toxicity, antibacterial, and anti-infammatory properties but (1) Relative Proportions of Erythrocytes. To determine the also to characterise the phytochemical constituents of relative proportions of erythrocytes, 500 cells were identifed A. klaineanum. and categorized into three subgroups: mature erythrocytes, erythroblasts, and degenerating erythrocytes. Te erythro- blasts were recognizable, according to the description made 2. Material and Methods by Szubartowska [12], by their rounded shape, basophilic cytoplasm, and prominent nucleus, while degenerating 2.1. Chemicals, Reagents, and Media. Organic solvents (HPLC grade) were obtained from Himedia, India. NaCl erythrocytes were identifed by their wrinkled and stunted appearance. Te percentage of mature erythrocytes, eryth- (Suvchem), sodium chloride (NaCl); Potassium chloride (KCl); Calcium chloride (CaCl ); agar (Biokar); glucose roblasts, and degenerating erythrocytes per 500 counted cells (total erythrocytes) per individual was determined. (Accumix); and other chemicals were obtained from Sigma, USA. All chemicals used were of analytical grade. 2.4. Bacterial Germs Tested. Te bacterial carrier used in our study consisted of six reference bacterial strains and four 2.2. Preparation of Samples. Te stem bark of A. klaineanum clinical strains. (voucher number: AK01-2017) was collected in Oyem (northern Gabon), in June 2017 and identifed at the Na- tional Herbarium by Professor Henry Bourobou (Botanist). 2.5. Antibacterial Activities. Te difusion method was used Te harvested bark was dried, crushed, and used for to study the susceptibility of the microorganisms. Minimum extractions. inhibitory concentrations (MICs) of crude extracts and Advances in Pharmacological and Pharmaceutical Sciences 3 fractions were determined by the microdilution method in aqueous extracts of A. klaineanum decreases proportionally 96-well microplates [13]. Bactericidal antibacterials were to the days. considered to be those with a MBC/MIC ratio of 1 or 2 and Tis LC is 110.03± 1.25 (APB), 63.55± 4.02 (APB), bacteriostatic if the MBC/MIC ratio was 4 or 16 [14]. 29.36± 2.11 (APB), and 15.86± 2.21 (APB) μg/mL after 24, 48, 72, and 96 hours of tadpole exposure, respectively. Te decrease in the value of LC results in the evolution of the 2.6. Anti-Infammatory Activities increasing number of mortalities. After 96 hours of expo- sure, no tadpole survived the other concentration ranges of 2.6.1. Antiprotein Denaturation Test. Denaturation in- 15.62, 31.2, 62.5, 125, 250, 500, and 1000 μg/mL when all hibition of crude extracts and fractions of Antrocaryon were alive in tanks containing only reconstituted water and klaineanum was performed according to the protein de- the lowest concentrations of aqueous extracts of naturation inhibition method described in the reference by A. klaineanum. Ngoua-Meye-Misso et al. [15]. Te references (diclofenac sodium and paracetamol) used were treated under the same conditions as the crude extracts. 3.1.2. Mortalities during Sublethal Tests. Figure 1 presents the mortalities recorded during the sublethal test in tadpoles exposed to extracts of A. klaineanum for 17 days. 2.6.2. Membrane Stabilization Test. Te membrane stabili- Te result shows the frst mortalities (12.5%) of the sation test was evaluated by the human red blood cell (HRM) tadpoles on the ffth day at 7.81 μg/mL of the plant extracts. haemolysis method. Tis haemolysis was induced by heat on On the ninth and thirteenth days, the mortality rate in- the one hand and distilled water on the other [15]. creased 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 2.7. Fractionation, Identifcation, and Molecular Network rate increased by 25%. It was only on the tenth day that the Analysis. Flash chromatography is a method of separation mortality (12.5%) of the tadpoles exposed to the control based on polarity; it has the same process as column (diclofenac sodium) and to the plant extracts (1.95 μg/mL) chromatography. Separation by fash chromatography was was observed. carried out on Armen Instrument, spot liquid chromatog- raphy fash, according to the gradient of increasing polarity. 32 g of the stem bark water-alcohol extract was submitted to 3.1.3. Description and Relative Proportions of Erythrocytes. fash chromatography on a 70–230 mesh silica gel column Erythroblasts were characterized by their rounded shape, (400 g) with stepwise gradient elution by CH Cl /MeOH 2 2 basophilic cytoplasm, and larger nucleus than that of mature mixtures (100 : 0; 98 : 2; 95 : 5; 90 :10; 80 : 20; 0 :100). Te erythrocytes. Besides these cells (Figure 2), degenerating fractions were collected and combined according to their erythrocytes were also observed in some individuals. thin layer chromatography (TLC) profles on precoated Figure 3 presents the relative proportions of erythrocytes silica gel 60 F plates developed with n-hexane/EtOAc and identifed in the blood of tadpoles exposed to extracts of CH Cl /MeOH mixtures to give groups of fractions. Te 2 2 A. klaineanum Pierre and diclofenac sodium for 16 days. In fraction with the major biological activities was selected and Figure 3(a), a relative decrease in mature erythrocytes ex- analysed by high performance liquid chromatography posed to plant extracts and diclofenac sodium from the frst coupled with the mass spectrometry (HPLC/Q tof). Te to the sixteenth day of the study is observed. On the frst day, compounds obtained were identifed by comparisons of the proportions of mature erythrocytes are 72, 78, 84, and their mass spectra to those of compounds registered in 84%, corresponding to the concentrations of 7.81, 3.9, 1.96, Reaxis and DNP 2019 libraries. and 1.96 μg/mL (standard), respectively, whereas on day 16, Molecular networking was carried out as described by these erythrocytes decrease by 60, 68, 74, and 69%. Essono Mintsa et al. [16]. 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 ex- 2.8. Statistical Analysis. Te data were expressed as mean- posure (4 to 5%) to that exposed to diclofenac sodium (4%). ± standard deviation (SD) of triplicate independent ex- On the other hand, these proportions rise from 5 to 8% on periments and analyzed using one-way analysis of variance the fourth day to remain relatively stable until the eighth day (ANOVA) and Student’s t-test using Staplus Build software. and then climb from 6 to 14% on the sixteenth day. 8.03/Corev7.811 (x86_64). Te values of p≤ 0.05 were Among the erythroblasts observed (Figure 3(b)), we note considered statistically signifcant. their low proportions (2 to 4%) on the frst day of exposure to the diferent concentrations of the plant. From the fourth 3. Results to the sixteenth day of exposure to 7.81 μg/mL of plant extract, a 4 to 12% increase in erythroblasts is clearly ob- 3.1. Toxicity of Aqueous Extracts of Antrocaryon klaineanum served. Also, exposure to 3.9 μg/mL of the plant extract Pierre shows a relative increase in erythroblasts (4 to 6%) from the fourth to the sixteenth day of the study. Although the 3.1.1. Lethal Test. Te results of the analyses presented in Table 1 show that the LC for frog tadpoles exposed to proportions of low concentrations do not undergo major 50 4 Advances in Pharmacological and Pharmaceutical Sciences Table 1: Lethal concentration (LC ) of aqueous extract Antrocaryon klaineanum for frog tadpoles (Phrynobatrachus africanus Hallowell). LC (μg/mL) depending on the duration of exposure Species 50 stage of development 24 hours 48 hours 72 hours 96 hours Phrynobatrachus africanus Hallowell 110.03± 1.25 63.55± 4.02 29.36± 2.11 15.86± 2.21 Tadpoles (2± 1 g) APB APB APB APB Rana clamitan 171.37± 9.25 61.99± 5.02 56.25± 5.01 58.25± 7.21 Tadpoles (0.91 g) [17] EP EP REG REG Bufo melanostictu 22.42 19.81 11.91 8.18 Tadpoles (0.1 g) [18] EP EP REG REG APB: appearance and progression of the buds of the hind limbs. -10 0 510 15 20 Exposure period (Days) 7.81 (μg/mL) 1.95 (μg/mL) 3.9 (μg/mL) Standard (Diclofenac sodium 1.95 μg/mL) Figure 1: Mortalities recorded during the sublethal test in tadpoles exposed to aqueous extract of Antrocaryon klaineanum for 17 days. (a) (b) (c) Figure 2: Tadpole frog (Phrynobatrachus africanus Hallowell). (a) Mature erythrocytes; (b) erythroblasts; (c) degenerating erythrocytes. Percent mortality (%) Advances in Pharmacological and Pharmaceutical Sciences 5 90 84 84 14 78 78 73 74 71 12 69 69 6 6 4 4 4 4 3 20 2 2 2 22 2 Day 1 Day 4 Day 8 Day 16 Day 1 Day 4 Day 8 Day 16 7.81µg/mL 1.96 µg/mL 7.81µg/mL 1.96 µg/mL 3.9 µg/mL Standard (Diclofenac sodium 3.9 µg/mL Standard (Diclofenac sodium 1.96 µg/mL) 1.96 µg/mL) (a) (b) 8 8 6 6 6 5 5 5 4 4 4 Day 1 Day 4 Day 8 Jour 16 7.81 µg/mL 1.96 µg/mL 3.9 µg/mL Standard (Diclofénac de sodium 1.96 µg/mL) (c) Figure 3: Proportion of erythrocyte cells in the blood of tadpoles exposed to Antrocaryon klaineanum for 16 days. (a) Mature erythrocytes; (b) erythroblasts; (c) degenerating erythrocytes. changes, it is noted that the proportions of erythroblasts and Te results of the MICs and CMBs are listed in Table 3. erythrocytes in degeneration tend to increase with high Te MICs (0.625 mg/mL) of the water-ethanol extracts concentrations and as a function of time. (Ak WEE) are the lowest, as are the MICs of the Ak F and Ak F fractions. Table 4 shows the antibacterial efects of crude extracts 3.2. Antimicrobial Activity of Antrocaryon klaineanum and fractions of A. klaineanum. Te aqueous extract has Extracts. Te results of susceptibility tests with the extracts a bactericidal efect on E. coli 105182 CIP, Listeria innocua of A. klaineanum gave the following diameters of inhibition LMG 135668 BHI, Enterococcus faecalis 103907 CIP, Bacillus on the bacterial strains (Table 2). Te screening of the three cereus LMG 13569 BHI, and Salmonella typhi strains and has extracts shows that all have antibacterial activity. Te a bacteriostatic efect on Staphylococcus aureus ATCC 25293 aqueous, water-ethanol, and water-acetone extracts gave the BHI. Te water-ethanol, water-acetone extracts, and the Ak greatest antibacterial activities against Neisseria gonorrhea F fraction have bactericidal actions on all the strains with the inhibition diameters greater than 19 mm. Te studied. Te Ak F , Ak F , and Ak F fractions exhibit 1 3 4 water-acetone extracts show signifcant antibacterial activ- bactericidal efects on the majority of bacteria. ities in the majority of strains with the exception of E. coli 105182 CIP, Salmonella typhi, E. coli, Staphylococcus aureus, Acinetobacter baumannii, Enterobacter aerogenes, and Sal- 3.3. Anti-Infammatory Activity of Antrocaryon klaineanum monella spp. sensitive to the water-ethanol extract and Extracts. Te results of the in vitro anti-infammatory ac- Bacillus cereus LMG 13569 BHI sensitive to the aqueous tivity of the extracts of A. klaineanum Pierre are summarized extract. in Figure 4. Te results also show that the fractions possess the In each column, the assigned values of diferent al- greatest antibacterial activities than the crude extracts. phabetic letters (a, b, c) indicate signifcantly diferent yields Tese fractions are active against both Gram-positive and (P< 0.05). Gram-negative bacteria. Te Ak F , Ak F , and Ak F Tis study shows that the water (IC � 80.20± 9.65 μg/ 2 3 4 50 fractions gave the greatest antibacterial activities against mL), water-ethanol (IC �110.27± 10.02 μg/mL), and the bacterial strains. Te Ak F fraction is active on all water-acetone extracts (IC � 86.22± 9.22 μg/mL) did not 1 50 strains tested with the exception of Listeria innocua LMG show any signifcant diference compared to the standards 135668 BHI, Pseudomonas aeruginosa, and Salmonella (IC � 97.20± 2.37 μg/mL). Te hemolysis test induced by enterica. a hypotonic solution shows that the three extracts studied 6 Advances in Pharmacological and Pharmaceutical Sciences Table 2: Inhibition zone diameters produced by the extracts and fractions from Antrocaryon klaineanum. Inhibition zone diameters (mm) Standards Extracts Fractions Ak water Ak water Ak water Ak F1 Ak F2 Ak F3 Ak F4 Gen Am Te ethanol acetone Bacteria Reference strains Escherichia coli 105182 CIP 8± 1 10± 1 9± 0 16± 2 17± 1 16± 1 12± 1 17± 1 Nd Nd Listeria innocua LMG 135668 BHI 7± 0 9± 1 11± 1 Nd 14± 2 13± 1 10± 0 13± 0 7± 1 14± 0 Staphylococcus aureus ATCC 25293 BHI 9± 0 10± 1 10± 0 10± 0 12± 0 15± 0 11± 1 15± 1 Nd 17± 1 Enterococcus faecalis 103907 CIP 12± 1 11± 2 14± 0 10± 0 12± 0 12± 2 10± 1 30± 0 7± 1 19± 0 Bacillus cereus LMG 13569 BHI 10± 0 9± 1 9± 0 15± 1 16± 0 15± 1 11± 0 13± 1 Nd 18± 1 Shigella dysenteria 5451 CIP 10± 1 10± 1 11± 1 13± 0 15± 1 15± 0 12± 1 24± 1 Nd 16± 0 Clinical isolates Pseudomonas aeruginosa 7± 0 10± 1 12± 1 Nd 12± 1 11± 1 9± 0 20± 0 7± 1 21± 1 Salmonella enterica 8± 0 10± 1 11± 1 Nd 10± 0 10± 1 11± 1 28± 1 7± 1 16± 1 Salmonella typhi 9± 0 11± 1 10± 0 9± 0 12± 1 13± 0 10± 1 20± 1 7± 0 15± 1 Neisseria gonorrhea 19± 1 20± 1 21± 1 21± 1 24± 0 22± 1 18± 2 22± 2 7± 1 10± 1 Nd � not determinated; Gen � gentamicin (10 μg/mL), Te � tetracycline (30 μg/mL), Am � ampicillin (30 μg/mL), Ak � Antrocaryon klaineanum, and F � fraction. Table 3: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) or minimum fungicidal concentration (MFC) of crude extracts and fraction of Antrocaryon klaineanum. MIC and MBC (mg/mL) Ak AE Ak WEE Ak WAE Ak F1 Ak F2 Ak F3 Ak F4 MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC Bacteria Reference strains Escherichia coli 105182 CIP 1.25 2.5 1.25 2.5 1.25 2.5 1.25 1.25 0.625 1.25 1.25 2.5 2.5 5 Listeria innocua LMG 135668 BHI 1.25 2.5 1.25 2.5 1.25 2.5 Nd Nd 1.25 2.5 1.25 5 5 5 Staphylococcus aureus ATCC 25293 1.25 5 1.25 1.25 1.25 2.5 2.5 5 2.5 5 1.25 5 2.5 5 BHI Enterococcus faecalis 103907 CIP 1.25 1.25 0.625 0.625 1.25 2.5 5 5 2.5 5 2.5 2.5 5 5 Bacillus cereus LMG 13569 BHI 1.25 1.25 0.625 0.625 1.25 1.25 1.25 1.25 0.625 1.25 0.625 1.25 2.5 5 Shigella dysenteria 5451 CIP 1.25 2.5 0.625 1.25 1.25 1.25 1.25 5 1.25 2.5 0.625 2.5 >5 >5 Clinical isolates Pseudomonas aeruginosa 1.25 2.5 0.625 1.25 0.625 1.25 Nd Nd 2.5 5 2.5 5 5 >5 Salmonella enterica 2.5 5 1.25 1.25 1.25 1.25 Nd Nd 2.5 5 5 5 2.5 5 Salmonella typhi 2.5 2.5 1.25 2.5 1.25 2.5 5 >5 2.5 5 2.5 2.5 2.5 5 Neisseria meningitides 0.625 0.65 0.625 1.25 0.625 1.25 1.25 1.25 0.625 1.25 1.25 1.25 0.625 1.25 Nd � not determinated; Ak � Antrocaryon klaineanum, WAE � water-acetone extract; WEE � water-ethanol extract; AE � aqueous extract; F � fraction. presented good activities with IC ranging from formulas C H O , C H O , C H O , and C H O . 50 18 20 4 24 32 2 27 28 4 26 36 4 62.26± 5.82 to 72.85± 8.00 μg/mL. For heat-induced he- After dereplication of the majority compounds in the molysis, all extracts exhibited very good antihemolytic ac- databases (Reaxis and Dictionary of natural products), tivity with IC s ranging from 70.59± 5.35 to compound 3 (mass ESI-MS m/z [M + H] � 353.245) 75.25± 8.56 μg/mL. Tese results have no signifcant dif- corresponds to monogallate type epicatechin and com- ference with the standard (diclofenac sodium). pound 4 (mass ESI-MS m/z [M + H] � 413.256) corre- sponds to 7α-Hydroxy-4,24(28)-ergostadien-3-one (Table 5). Te other compounds did not show matches in the 3.4. Chromatographic Analyses and Molecular Network MS/ literature. MS of Antrocaryon klaineanum Pierre Crude Extract. Te Te results of the molecular network of the crude extract results in Figure 5 present a set of chromatogram and mass of A. klaineanum reveal that the compounds are grouped spectra of the crude extract of A. klaineanum. Te study according to structural similarities and fve main clusters reveals 4 major peaks whose masses ESI-MS m/z emerged (Figure 6): group A presents twelve nodes having + + [M + H] � 301.144; ESI-MS m/z [M + H] � 353.245; the very close molecular masses, group B includes 6 nodes, ESI-MS m/z [M + H] � 417.202; and ESI-MS m/z two masses of which (m/z 298.145 and 298.146) correspond [M + H] � 413.265 correspond to the respective molecular to Methoxyformonetin. Clusters C (4 nodes), D (3 nodes), Advances in Pharmacological and Pharmaceutical Sciences 7 Table 4: Antimicrobial efects of plants extracts from Antrocaryon klaineanum. Ak AE Ak WEE Ak WAE Ak F Ak F Ak F Ak F 1 2 3 4 MBC/MIC MBC/MIC MBC/MIC MBC/MIC MBC/MIC MBC/MIC MBC/MIC Efect Efect Efect Efect Efect Efect Efect Bacteria Reference strains Escherichia coli 105182 CIP 2 2 2 1 2 2 2 Listeria innocua LMG 135668 BHI 2 2 2 — 2 3.2 1 Staphylococcus aureus ATCC 25293 BHI 4 1 2 2 2 3.2 2 Enterococcus faecalis 103907 CIP 1 1 2 1 2 1 1 Bacillus cereus LMG 13569 BHI 1 1 1 1 2 2 2 Shigella dysenteria 5451 CIP 2 2 1 2 2 2 — Clinical isolates Pseudomonas aeruginosa 2 2 2 — 2 2 — Salmonella enterica 2 1 1 — 2 1 2 Salmonella thyphimurium 1 2 2 — 2 1 2 Neisseria meningitidis 1 2 2 1 2 1 2 MBC/MIC< 4: bactericidal; MBC/MIC≥ 4: bacteriostatic; WAE � water-acetone extract; WEE � water-ethanol extract; AE � aqueous extract. ac 75.25 ± 8.56 ac 66.86 ± 5.10 ac ac 72.85 ± 8.00 70.59 ± 5.35 70.95 ± 5.32 79.56 ± 2.01 c c 62.26 ± 5.82 72.85 ± 8.00 110.27 ± 10.02 97.20 ± 2.37 86.22 ± 9.22 80.20 ± 9.65 Water‐acetone extract Water‐ethanol extract Water extract Diclofenac sodium Protein denaturation test IC50 (μg/mL) Hemolysis by hypotonicity IC50 (μg/mL) Heat hemolysis IC50 (μg/mL) Figure 4: Anti-infammatory activity of Antrocaryon klaineanum. ESI-MS m/z [M + H] = 417.202 ESI-MS m/z [M + H] = 301.144 C H O C H O 27 28 4 18 20 4 + + ESI-MS m/z [M + H] = 353.245 ESI-MS m/z [M + H] = 413.265 C H O C H O 24 32 2 26 36 4 Figure 5: Chromatograms and mass spectra of the ethanol extract of Antrocaryon klaineanum. 8 Advances in Pharmacological and Pharmaceutical Sciences Table 5: HPLC-ESI-QTOF identifcation of major compounds from ethanol stem barks extracts of Antrocaryon klaineanum. m/z Identifcation Identifcation Name Fraction Compound RT Score Formula Reference [M + H] DNP reaxys of compound 1 32.08 301.144 89.31 C H O 10 hits 15 hits Not determined — 18 20 4 2 33.40 417.202 70.40 C H O 06 hits 7 hits Not determined — 27 28 8 AK Te 3 35.64 353.245 69.64 C H O 32 hits 45 hits (Epi) catechin-(epi) catechin monogallate (B-type) [19] 24 32 2 4 36.86 413.265 80.2 C H O 16 hits 56 hits 7a-Hydroxy-4,24(28)-ergostadien-3-one [6] 26 36 4 AK: Antrocaryon klaineanum, Te � total extracts, Rt � retention time, MS � mass spectrometer, DNP � dictionary of natural products. Advances in Pharmacological and Pharmaceutical Sciences 9 Figure 6: Molecular network of the ethanolic crude extract of Antrocaryon klaineanum Pierre. and E (3 nodes) group together compounds not identifed by a reduction in erythrocyte length, and rounding of eryth- rocytes. Several studies presented similar results, which our available databases. performed experiments on hematological efects in the toad Bufo regularis [21]. Tese authors hypothesized that the 4. Discussion appearance of these efects could be associated with an Te purpose of the work was to study toxicity; establish increase in plasma volume; a reduction in blood copper antibacterial and anti-infammatory activities, and charac- concentration, which plays an important role in erythro- terize the phytochemistry of A. klaineanum extracts. Te poiesis; inhibition of hemoglobin synthesis; and iron de- results of plant extracts exposed to frog tadpoles show a low fciency [22]. Te essential function of erythrocytes is to toxicity compared to pharmaceutical drugs (diclofenac so- ensure the supply of oxygen to the tissues, which is carried, out thanks to hemoglobin. Because of this primary role dium). Te study also shows that the toxicity is progressive over time. Tis result corroborates with that of Obiang et al. attributed to hemoglobin, the diagnosis of anemia is only true when there is a decrease in hemoglobin concentration [11], where the authors stipulate that the mortality correlated to the doses used is all the more increased as the exposure of below the lower limit of normality. frog tadpoles is prolonged over time. Te cells observed on Te study shows that over time, a slight increase in the the smears correspond quite closely to the descriptions made proportions of erythroblasts and degenerating erythrocytes in leopard frogs by Jordan [20]. was evident. Te appearance of young erythrocytes Observation of hematological parameters revealed that (erythroblasts) refects a process of renewal of the eryth- exposure of tadpoles to extracts of A. klaineanum causes rocyte population. In parallel, degenerating erythrocytes are mild anemia. Te observed efects were a decrease in the characteristic of red blood cell lysis. In this case, we could be number of mature erythrocytes in the bloodstream, in the presence of a regenerative type anemia caused by 10 Advances in Pharmacological and Pharmaceutical Sciences hyperhemolysis of erythrocytes leading to erythropoietic 417.190 and 353.245. Tese compounds belong to the stimulation. polyphenols of the (Epi) catechin-(epi) catechin monogallate Antibacterial activities of total extracts and fractions of type [19] and to the family of ergostane-type steroids [7]. bark extracts of A. klaineanum were demonstrated in this Polyphenols are characterized by the presence of at least one study. Te results obtained show that each sample tested has benzene ring on which there is at least one hydroxyl group inhibitory efects on the growth of the majority of the engaged with other functions. Tey are known for their bacterial strains tested. Of all the bacteria submitted to our antioxidant efects. Polyphenols afect membrane perme- study, Neisseria gonorrhea showed more sensitivity to the ability, thus inducing the release of intracellular constituents extracts tested. Te Ak F , Ak F , and Ak F fractions proved into the external environment. Moreover, they also interfere 1 2 3 to be more active on several bacteria compared to the other with the respiratory chain by inhibiting the oxidation of extracts. Tese inhibitions may be due to separations and NADH, the synthesis of nucleic acids and proteins, and the purifcations of plant fractions by diferent chromatography activity of certain enzymes [26]. methods [14]. Ethnopharmacological studies have shown In addition, we also performed crude extract analysis that the bark of A. klaineanum is used for the treatment of using the molecular network (MN) approach by GNPS typhoid fever and other liver-related diseases [8]. Tis study website (https://gnps.ucsd.edu) [27]. All obtained HRE- justifes the traditional use of A. klaineanum for the fght SIMS/MS spectra were preprocessed via MZmine2 software against certain infections of bacterial origin. Te sensitivity following the feature-based molecular networking workfow of bacteria like Bacillus cereus LMG 13569 BHI, Shigella [28]. Exploring the relative contributions of peak areas to dysenteria 5451 CIP, and Neisseria gonorrhea to the extracts network nodes revealed that compounds were clustered could direct further research on the therapeutic properties of based on structural similarities. Te presence of the clusters A. klaineanum against gonorrhea, diarrhea, and other types in the molecular network shows that the crude extract of of infections. Tis sensitivity may be due, in part, to their A. klaineanum exhibits several compounds that can be better ability of plant extracts to complex with amino acids of studied. extracellular and soluble proteins resulting in inactivation and/or loss of function. 5. Conclusion Te anti-infammatory activity of A. klaineanum was evaluated by protein denaturation test and membrane sta- Tis work made it possible to evaluate the toxicity, the bilization. Protein denaturation is a process in which pro- biological (antibacterial and anti-infammatory) activities, teins lose their tertiary and secondary structures by the and the chemical characterization of the aqueous extract of application of external stress or compound, such as strong the bark of A. klaineanum. It emerges from this study that acid, base, or by heat, which most proteins lose their bi- the plant extracts exposed to frog tadpoles show a low ological function when denatured [23]. Protein denaturation toxicity compared to pharmaceutical drugs (diclofenac so- is a well-documented cause of infammation [19]. Te results dium). Some extracts exhibit bactericidal efects. However, on inhibition on protein denaturation showed that there is this sensitivity is dose-dependent and varies according to the no signifcant diference between the plant extracts and the germs and the extracts. Moreover, the extracts having the reference (diclofenac sodium). Similar results, showing the good antibacterial properties showed exceptional anti- good inhibitory activities of protein denaturation by me- infammatory activities, which were signifcantly very high dicinal plants have been found in several studies [15, 24]. compared to diclofenac sodium. Phytochemical character- Stabilization of the red blood cell membrane has been ization of the extracts revealed compounds such as monog- used as a method to study anti-infammatory activity in vitro allate type polyphenols and ergostane-type steroids. Tis because the erythrocyte membrane is analogous to the ly- study justifes certain uses of this in traditional medicine. sosomal membrane. Tis method confrms the anti- infammatory activity of A. klaineanum extracts. Te re- Abbreviations sults showed that at diferent concentrations of extracts, erythrocyte membranes were protected against lysis and heat AK: Antrocaryon klaineanum induced by hypotonic solution. Studies have shown that CM: % corrected mortality certain plants of the family A. klaineanum act at the level of DNP: Dictionary of natural products many mechanisms such as the regulation of anti- EP: Embryonic phase infammatory activity. Te activation of NF-κB plays HPLC: High performance liquid chromatography a key role in the infammatory response. Tese extracts have IPHAMETRA: Institut de Pharmacopee ´ et de Medecine ´ been shown to reduce the level of production of in- Traditionennelle fammation mediators such as cyclooxygenase-2 or TNF-α M2: % mortality in the treated population [25]. Te results of our work confrm the traditional use of M1: % mortality in the control population A. klaineanum in traditional medicine for the treatment of MBC: Minimum bactericial concentrations various pathologies [8]. MIC: Minimum inhibitory concentrations Chromatograms and spectra of A. klaineanum show that MS: Mass spectrometer several major compounds are not part of the dictionary of REG: Regression of external gills natural products and other databases consulted with the Rt: Retention time exception of compounds with masses m/z [M + H] equal to TLC: Tin layer chromatography. 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