Antimicrobial and Cytotoxic Activities of Constituents from the Fruit of Albizia lebbeck L. Benth (Fabaceae)

Twenty-two compounds were isolated from the fruit of Albizia lebbeck including one unprecedented, rare amino acid-derived zwitterionic and one new flavone derivative. The isolation was performed on repeated column chromatography over silica gel and their structures were determined by 1D-, 2D-NMR and HR-ESI-MS spectra together with reported data in the literature. The chemophenetic significance is also discussed. Some isolated compounds were reported for the first time to be found in the species. Additionally, compound 2 showed antibacterial activity and compounds 1 and 2 revealed moderate cytotoxic activity against the Raw 264.7 cancer cell line with IC50 values of 37.19 µM and 29.36 µM, respectively. Furthermore, a proposed biosynthetic pathway of compound 1 is described.


Introduction
Albizia lebbeck (or Acacia lebbeck) is a tree found in the Fabaceae family, mostly reported in tropical areas and in rainfall forests and usually grows in lateritic and sandy soils [1]. During our ethnobotanical survey in Northern Cameroon, Albizia lebbeck was claimed by the local population to treat dysentery, asthma, hemorrhoids, bronchitis, eczema, leprosy, human fertility, and diarrhea, meanwhile some constituents of A. lebbeck are reported for their antimicrobial, antioxidant, anti-inflammatory, and toxicity properties [2][3][4][5][6][7]. In light of our study and as part of our ongoing research on bioactive metabolites from Cameroonian medicinal plants [8,9], we isolated a new amino acid-derived zwitterionic (1) and a new flavone flavonoid derivative (2) together with twenty known compounds from the fruit of A. lebbeck. Eugenol (5) is reported for the first time from the Fabaceae family while zwitterionic (1) and a new flavone flavonoid derivative (2) together with twenty known compounds from the fruit of A. lebbeck. Eugenol (5) is reported for the first time from the Fabaceae family while 29-hydroxyhopane (8), spinassterol (16), spinasterol-3-O-β-D-glucopyranoside (17), D-mannitol (20), octacosanoic acid (21), and bis ((S)-2, 3-dihydroxypropyl) (22) are reported for the first time from A. lebbeck. Herein, we report the isolation, structure elucidation, antimicrobial potential, as well as cytotoxic properties of the extracts and isolated compounds.

Biological Assays
The antimicrobial activities were performed on the pod crude extract and several isolated compounds using ketoconazole and ciprofloxacin as antifungal and antibacterial drug references. The interpretation of the result was based on the positive controls [31]. The CH 2 Cl 2 -MeOH (1:1, v/v) crude extract, n-hexane, ethyl acetate, and n-butanol extracts together with compounds 1, 2, 12, and 13 were tested against five microbial strains ( Table 2). All the tested samples showed antimicrobial activity against at least one microbial strain, except the n-butanol extract which did not show any antimicrobial activity. Compounds 1 and 2 showed the most potent antifungal activity with MIC values of 32 µg/mL against Candida albicans while compound 2 further showed similar antibacterial activity against Escherichia coli. Compounds 12 and 13 were the least active in both bacteria and yeast strains. It clearly appears that the antimicrobial activity of Albizia lebbeck pods against Candida albicans and Escherichia coli increased with fractionation, from the CH 2 Cl 2 -MeOH (1:1, v/v) crude extract (MIC values of 512 µg/mL and 256 µg/mL) to the ethyl acetate extract (MIC values of 128 µg/mL) and finally to compounds 1 and 2, which were the most active (MIC values of 32 µg/mL). The above results are in accordance with previous reports of antimicrobial assays on the seeds, pods, and flowers of A. lebbeck [10,32] and could support the local uses of the fruit of this plant for the treatment of child and adult diarrhea. The mouse macrophages raw 264.7 cancer cell line was used to investigate the in vitro cytotoxicity of compounds 1 and 2 using the WST-1 assay. The concentration-response graph ( Figure 4) showed that compounds 1 and 2 inhibited the proliferation of Raw 264.7 cancer cells in a dosedependent manner. The IC 50 values were determined based on the percentage of viable cells less than 50%. The two compounds showed moderate cytotoxic activity against Raw 264.7 with IC 50 values of 37.19 µM and 29.36 µM, respectively. Raw 264.7 cell line has been exhaustively used for the screening of secondary metabolites for their cytotoxic activities [33,34]. However, the high sensitivity of Raw 264.7 compared to other cancer cell lines, such as human leukemia monocyte cell line THP-1 and the human lung adenocarcinoma A549 cell lines, has been previously reported [8]. To the best of the author's knowledge, the anticancer properties of an isolated compound from the seeds of A. lebbeck have never been reported. The obtained activity of chiakine (1) and lebbeckisoetin A (2) on Raw 264.7 cells indicated that these compounds might be potential candidates for new anticancer agents.

Discussion
Twenty-two (1-22) compounds were isolated using chromatographic techniques from the crude extracts of the fruit of Albizia lebbeck L. Benth, including a new carbamide (1) and flavone (2) derivatives together with twenty known compounds grouped into two flavonoids (3,4), three alkylphenols (5-7), four triterpenoids (8)(9)(10)(11), three sugars (12,13,20), three sterols (14,16,18), three saponins (15,17,18), and two fatty acids (21,22). To the best of our knowledge, compounds (1, 2, 5, 8, 16, 17, 20, 21, and 22) are herein reported for the first time from A. lebbeck. This is the first report of compound 5 in either the Mimosaceae subfamily or the Fabaceae family. Compound 5 is considered a chemomarker of Syzygium aromaticum, well known as "Clou de Girolf " [3]. However, compounds 1 and 2 were found to be new and are reported for the first time from the Fabaceae family as their derivatives were reported from A. lebbeck [10,35]. This is the first report of compounds 8, 16, 17, 20, 21, and 22 from Albizia lebbeck, although they have already been reported from the Albizia genus as 8 (Albizia chinensis) [36], 16-17 (Albizia dealbata and Albizia melanoxylon) [28] and 20-22 (Albizia Amara and Albizia julibrissin) [37,38]. This paper has then strengthened the chemophenetic survey on the Albizia genus. These observations give new insights into the occurrence of flavonoids and saponins in the genus Albizia and the family Fabaceae. The flavone flavonoids might represent a new significant chemophenetic finding in the Albizia genus, specifically in the Albizia lebbeck plant. However, saponins in general, could be considered chemotaxonomic markers for the genus Albizia due to the soapy aspect of all the species. The study of the fruit of A. lebbeck L. Benth led to the isolation of a chemical constituent which enriches the information on the phytochemistry of the plant and provides further knowledge in regard to the possible chemotaxonomic markers present in the A. lebbeck species, the Albizia genus, as well as the Fabaceae family. It is notable that compounds 1, 2, and 5 have not yet been reported either from the Albizia genus or the Fabaceae family and suggest a new observation of chemotaxonomic knowledge of the Albizia and more diverse chemical constituents from A. lebbeck L. Benth.

General Experimental Procedures
The melting points were measured on a Gallenkamp melting point apparatus. Optical rotations were recorded with a JASCO DIP-360 polarimeter. The IR spectrum was measured in MeOH on a JASCO A-302 spectrophotometer. The proton and carbon (1D and 2D)-NMR (600/500 MHz) spectra were measured on a Bruker AMX machine. The chemical shifts of proton and carbon were recorded based on the internal reference TMS (Tetramethylsilane) in δ (ppm). Moreover, coupling constants (J) were measured in Hz. The ESIMS was recorded on a double-focusing mass spectrometer (Varian MAT 311A), while HREIMS was recorded on a JEOL HX 110 mass spectrometer. Silica gel 60 (0.2-0.5 mm and 0.2-0.063 mm) (70-230 and 240-300 mesh sizes, E. Merck) was used as a stationary phase for CC (Column Chromatography). The purity of compounds and the monitoring of fractions were based on precoated silica gel TLC (Thin Layer Chromatography) plates supported on either plastic or aluminum sheets (E. Merck, F 254 ). Spots were visualized on TLC with UV light (254 nm and 365 nm) on a CN-6 UV spectrometer (made in France) then sprayed with ceric sulphate and heated at about 90 • C.

Plant Material
The fruit of A. lebbeck was harvested on 20 April 2018 in Maroua town, headquarters of the Far-North Region of Cameroon and identified by Mr Tadjouteu Flubert by comparing it with a specimen available at the National Herbarium at Yaoundé with a Voucher specimen 58964/NHC.

Antimicrobial Assay
Five microorganisms from American Type Culture Collection including four bacterial (two Gram-positive and two Gram-negative) and one fungal strain were used: Candida albicans ATCC 9028, Staphylococcus aureus (ATCC 1026), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 74117). The antibacterial activity of the crude extract, fractions, and compounds was assessed by determining the minimum inhibitory concentration (MIC) and minimum bactericidal and fungicidal concentration (CBC and MFC) using the broth microdilution method, as previously described [39]. Briefly, the MIC was performed by broth microdilution method, with Mueller-Hinton broth for bacteria and Sabouraud dextrose broth for yeast. Stock solutions of samples were prepared in 100% dimethyl sulfoxide and twofold serial dilutions were prepared in media in amounts of 100 µL per well in a 96-well. Microbial suspensions were prepared in culture media, and 100 µL of this inoculum was added to each well of the plate, resulting in a final inoculum of 1.5 × 10 6 CFU/mL for bacteria and 2 × 10 4 CFU/mL for yeast. The final concentration of samples ranged from 2 µg/mL to 256 µg/mL and from 8 µg/mL to 1024 µg/mL for extracts. The medium without the agents was used as a growth control and the blank control used contained only the medium. Ciprofloxacin and ketoconazole served as the standard drug controls. The MIC of samples was detected after 24 h (for bacteria) and 48 h (for fungi) of incubation at 37 • C, following an addition (40 µL) of 0.2 mg/mL of p-iodonitrotetrazolium (INT) chloride and incubation at 37 • C for 30 min, as the lowest sample concentration that prevented the color change of the medium and exhibited complete inhibition of microbial growth. The MBC or MFC was determined by adding 50 µL aliquots of the preparations, which did not show any growth after incubation during MIC assays to 150 µL of adequate broth. These preparations were incubated at 37 • C for 48 h. The MBC or MFC was regarded as the lowest concentration of a sample which did not produce a color change after an addition of INT, as mentioned above.

Cytotoxicity Assay
The cytotoxicity of unprecedented carbamide (1) and flavone (2) derivatives were evaluated on the Raw 264.7 cells (ATCC) cancer cell line. Cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) culture media supplemented with 10% fetal calf serum and 1% antibiotics (100 IU/mL penicillin and 100 µL/mL streptomycin) and maintained at 37 • C in a humidified atmosphere containing 5% CO 2 . The WST-1 assay was used to quantify the cell viability and the cytotoxicity was evaluated by determining the concentration inhibiting 50% of viable cells (IC 50 ), as previously described [8].

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The raw data supporting the conclusions of this article will be made available by the authors without undue reservation to any qualified researcher.