NEW ANTIBACTERIAL COMPOUND ISOLATED FROM PSILOXYLON MAURITIANUM AND ITS DERIVATIVES

Abstract

Aspidin BB derivatives, compositions containing them, and methods of making and using them.

Claims

1-24. (canceled)

25. A method of treating a microbial infection, the method comprising administrating a composition comprising an aspidin BB derivative of formula (I) ##STR00014## wherein, n is from 0 to 16; R2 and R3 are selected from the group consisting of hydrogen, OH, Oalkyl, Oaryl, NH.sub.2, NHalkyl, NHaryl, N(alkyl).sub.2, N(aryl).sub.2, and alkyl; R1 and R4 are selected from the group consisting of, OH, Oalkyl, Oaryl, NH.sub.2, NHalkyl, NHaryl, N(alkyl).sub.2, N(aryl).sub.2, alkyl, SH, Salkyl, and SO.sub.2H; G1 and G2 are selected from the group consisting of OH, Oalkyl, NH.sub.2, NHalkyl, NHaryl, N(alkyl).sub.2, N(aryl).sub.2, and SH; and G3, G4n G5, and G6 are selected from the group consisting of hydrogen, CI, BR, F, I, NO2, CN, NH.sub.2, NHalkyl, NHaryl, N(alkyl).sub.2, N(aryl).sub.2, SH, Salkyl, and sulfate, to a patient in need thereof.

26. The method according to claim 25, wherein said aspidin is a compound of formula (II) ##STR00015##

27. The method according to claim 25, wherein said microbial infection is a bacterial infection.

28. The method according to claim 27, wherein said bacterial infection is an infection by a gram-positive bacterial strain.

29. The method according to claim 28, wherein said gram-positive bacterial strain is chosen from Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus argenteus, Staphylococcus haemolyticus, Staphylococcus warnieri, Staphylococcus lugdunensis, Corynebacterium diphteriae, Corynebacterium minutissimurn, Corynebacterium acnes (Propionibacterium acnes), Corynebacterium sp., Bacillus cereus, Bacillus subtilis, Bacillus anthracis, Bacillus sp., Enterococcus faecium, Enterococcus faecalis, Enterococcus sp., Nocardia abscessus, Nocardia farcinica, Nocardia asteroides, Nocardia cyriacigeorgica, Nocardia brasiliensis, Nocardia brevicatena, Nocardia paucivorans, Nocardia nova, Nocardia transvalensis, Nocardia sp., Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus mutans, and Streptococcus sp.

30. The method according to claim 29, wherein said Staphylococcus aureus strain is a methicillin-resistant Staphylococcus aureus or a vancomycin-resistant Staphyloccocus aureus.

31. The method according to claim 27, wherein the bacterial strain is a gram-negative bacterial strain.

32. The method according to claim 21, wherein said gram-negative bacterial strain is chosen from Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.

33. The method according to claim 31, further comprising administering at least one outer membrane permeabilizer.

34. The method of claim 33, wherein the at least one outer membrane permeabilizer is a polymyxin.

35. The method of claim 34, wherein the polymyxin is colistin

36. An antimicrobial composition against Gram-negative bacteria, comprising an aspidin compound of formula (I) in combination with an outer membrane permeabilizer.

37. The antimicrobial compound according to claim 36, wherein the Gram-negative bacteria is selected from the group consisting of Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.

38. The antimicrobial composition according to claim 36, wherein the outer membrane permeabilizer is a polymyxin.

39. The antimicrobial composition according to claim 38, wherein the polymyxin is colistin.

40. The method according to claim 25, wherein the microbial infection is a fungal infection selected from the group consisting of Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Cryptococcus neoformans, Cryptococcus gatti, Pneumocystis jirovecii, Aspergillus fumigatus, Aspergillus flavus, Aspergillus nidulans, Aspergillus versicolor, Aspergillus niger, Aspergillus terreus, Histoplasma capsulatum, H. capsulatum duboisii, and Torulopsis glabrata.

41. An aspidin BB derivative of formula (II) ##STR00016##

42. A method of extracting the aspidin of claim 25 from a plant of a genera selected from the group consisting of Dryopteris, Arachniodes, Elophoglossum, Stigmatopteris, Dryopsis, Polystichum, Rumohro, Nothoperanema, Lastreopsis, Polybotrya, Acrophorus, Ctenitis, Pleocnemia, and Peranema, said method comprising: Drying the plant leaves at room temperature, Crushing the dried leaves, Macerating the crushed plant with an organic solvent, Collecting the organic solvent, and Purifying the aspidin by chromatography

43. A green method of extracting the aspidin of claim 25 from a plant of a genera selected from the group consisting of Dryopteris, Arachniodes, Elaphoglossum, Stigmatopteris, Dryopsis, Polystichum, Rumohra, Nothoperanema, Lastreopsis, Polybotrya, Acrophorus, Ctenitis, Pleocnemia, and Peranema, said method comprising: Drying the plant leaves at room temperature, Grinding the dried leaves, Extracting the dried leave powder with supercritical CO.sub.2 using or not Ethanol as co-solvent, and Collecting the organic phase and removing the residual solvent under reduced pressure.

44. The method according to claim 43, wherein said plant is Psiloxylon mauritianum.

45. A method of preparing a cosmetic composition comprising the steps of mixing (i) at least one preservative agent which is an aspidin BB derivatives of claim 25 (ii) at least one agent chosen among a moisturizing agent, an anti-aging agent, a slimming agent, and a whitening agent.

46. The method according to claim 45, wherein said aspidin is represented by formula (II) ##STR00017##

47. A cosmetic composition, comprising (i) at least one preservative agent, which is an aspidin BB derivative of claim 25; and (ii) at least one agent chosen from the group consisting of a moisturizing agent, an anti-aging agent, a slimming agent, and a whitening agent.

48. The cosmetic composition according to claim 47, wherein at least one preservative agent, which is an aspidin, is represented by formula (II) ##STR00018##

Description

BRIEF DESCRIPTION OF THE FIGURES

[0070] FIG. 1. Structure of the compounds isolated from Psiloxylon mauritianum.

[0071] FIG. 2. Chromatographic profiles SFE extract obtained with 100% CO.sub.2.

EXAMPLES

I - Materials and Methods

[0072] General Experimental Procedures

[0073] Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 500 MHz spectrometer or on a Bruker 700 MHz spectrometer equipped with 5 mm inverse detection Bruker. Chemical shifts (δ) are reported in ppm based on the signal for TMS. Chemical shifts were referenced using the corresponding solvent signals (δ.sub.H 2.05 and δ.sub.C 29.92 for (CD.sub.3).sub.2CO). HRESIMS measurements were performed using a Waters Acquity UHPLC system with a column bypass coupled to a Waters Micromass LCT Premier time-of-flight mass spectrometer equipped with an electrospray interface (ESI). X-ray diffraction data for compound 1 were collected on the PROXIMA 2A (PX2A) beamline at the SOLEIL Synchrotron, Gif-sur-Yvette, Paris, France. They were indexed, integrated with XDS and scaled with AIMLESS, as implemented within the autoProc toolbox. For compound 2, data were collected using redundant ω scans on a Rigaku XtaLabPro single-crystal diffractometer using microfocus Mo Kα radiation and a HPAD PILATUS3 R 200K detector. Its structure was readily solved by intrinsic phasing methods (SHELXT) and by full-matrix least-squares methods on F2 using SHELX-L. The non-hydrogen atoms were refined anisotropically, and hydrogen atoms, identified in difference maps, were positioned geometrically and treated as riding on their parent atoms. Molecular graphics were computed with Mercury 4.3.0. Flash chromatography was performed on a Grace Reveleris system with dual UV and ELSD detection equipped with a 40 g C.sub.18 column. Preparative HPLCs were conducted with a Gilson system equipped with a 322 pumping device, a GX-271 fraction collector, a 171 diode array detector, and a prepELSII detector electrospray nebulizer. The columns used for these experiments included a Phenomenex Kinetex C8 5 μm 4.6 x 250 mm analytical column and Phenomenex Kinetex C8 5 μm 21.2×250 mm preparative column. The flow rate was set to 1 or 21 mL/min, respectively, using a linear gradient of H.sub.2O mixed with an increasing proportion of CH.sub.3CN. Both solvents were of HPLC grade, modified with 0.1% formic acid.

[0074] Plant Material

[0075] Leaves of Psiloxylon mauritianum Baill. (Myrtaceae) were collected in Les Avirons, Reunion Island in 2016, identified by Raymond Lucas (Association APN, Réunion). A voucher specimen was deposited at ICSN-CNRS.

[0076] Extraction and Isolation

[0077] After collection, plant material was air dried in the shade at room temperature. Crushed dried leaves (135 g) were extracted by maceration with EtOAc (2×0.7L,2×24h) on a rotary shaker (90 rpm). The organic solvent was collected by vacuum filtration and concentrated to dryness under reduced pressure to yield 10.6 g of extract. A portion of the extract (1.2 g) was subjected to reverse phase flash chromatography using a gradient of H.sub.2O mixed with an increasing proportion of CH.sub.3CN, both with 0.1% formic acid, to afford 14 fractions (A to N). A portion of fraction I (10 mg), eluted with 100% CH.sub.3CN, was subjected to preparative HPLC (isocratic elution at 20:80) to afford the mixture ursolic acid 3: oleanic acid 4 (6:4) (2.4 mg, RT =6.0 min), and Aspidin BB 1 (4.3 mg, RT=12.5 min). A portion of fraction J (40 mg) was washed with cold MeOH (0° C.) to afford Aspidin CB 2 (20 mg). After NMR experiments, small crystal needles were observed in samples tubes of Aspidin BB 1 and Aspidin CB 2.

[0078] These crystals were carefully collected and analyzed by X ray crystallography.

[0079] Aspidin BB (1): White amorphous solid or colorless crystal needles; .sup.1H-NMR (500 MHz, (CD.sub.3).sub.2CO: δ.sub.H 3.80 (3H, s, H7), 3.57 (2H, s, H7), 3.18 (2H, dd,J =7.3, 7.3 Hz, H9), 3.15 (2H, dd,J =7.2, 7.2 Hz, 2H), 2.10 (3H, s, H12), 1.70 (4H, m, H10, H10), 1.49 (6H, s, H12, H13), 1.00 (3H, t,J =7.4 Hz, H11), 0.98 (3H, t, J =7.4 Hz, H11); .sup.13C-NMR (125 MHz, (CD.sub.3).sub.2CO: δ.sub.C 208.0 (C8), 207.4 (C8), 199.8 (C4), 188.4 (C2), 172.7 (C6), 163.6 (C6), 161.5 (C4), 160.3 (C2), 113.1 (C5), 111.8 (C1), 110.2 (C1), 109.0 (C3), 108.5 (C3), 62.1 (C7), 45.0 (C5), 44.4 (C9), 43.5 (C9), 25.1 (C12), 25.1 (C13), 18.8 (C10), 18.2 (C10), 17.7 (C7), 14.2 (C11), 14.1 (C11), C12 (9.5); HRESIMS [M +H].sup.+m/z 461.2173 (calc. for C.sub.25H.sub.33O.sub.8, 461.2175).

[0080] Aspidin CB (2): White amorphous solid or colorless crystal needles; .sup.1H-NMR (500 MHz, (CD.sub.3).sub.2CO and .sup.13C-NMR (125 MHz, (CD.sub.3).sub.2CO) see Table 1; HRESIMS [M+H].sup.+ m/z 489.2490 (calculated for C.sub.27H.sub.37O.sub.8, 489.2488).

[0081] Ursolic acid (3): oleanicacid (4) (6:4 mixture): White amorphous; .sup.1H-NMR (700 MHz, (CD.sub.3).sub.2CO and .sup.13C-NMR (175 MHz, (CD.sub.3).sub.2CO) see Table HRESIMS [M+ H].sup.+ m/z 457.3661 (calculated for C.sub.30H.sub.49O.sub.3, 457.3682).

[0082] Organic Extractions

[0083] Crushed dried leaves (250 g) were extracted by maceration with EtOAc (0.7 L, during 24 h, twice) on a rotary shaker (90 rpm). The organic solvent was collected by vacuum filtration and concentrated to dryness under reduced pressure to yield 10.6 g of EtOAc extract. A successive maceration was realized with MeOH (0.7 L, during 24 h, twice) on a rotary shaker (90 rpm). The organic solvent was collected by vacuum filtration and concentrated to dryness under reduced pressure to yield 42 g of MeOH extract. Crushed dried stems (135 g) were extracted by maceration with the same procedure described above to yield 1.3 g of EtOAc extract and 5.5 g of MeOH extract.

[0084] Aqueous Extractions

[0085] Crushed dried leaves (10 g) were added to 200 ml of deionized water at 100° C. on a rotary shaker (90 rpm) for 15 min. The material was the infused for 2-16 h. The aqueous phase was collected by vacuum filtration and concentrated to dryness under reduced pressure to yield 2.2 g of water extract. Crushed dried stems (1.0 g) were extracted by maceration with the same procedure described above to yield 1.2 g of water extract.

[0086] Fractionation of Methanolic Extract

[0087] A portion of the extract (1.2 g) was subjected to reverse phase flash chromatography using a gradient of H2O mixed with an increasing proportion of CH3CN, both with 0.1% formic acid, to afford 70 fractions (1 to 70).

[0088] Green Extraction: SFE-CO.sub.2 Extracts from Psiloxylon mauritianum

[0089] Supercritical Fluid Extractions (SFE) were performed on a 1260 Infinity Analytical SFC system (Agilent Technologies, Waldbronn, Germany) consisting of an Aurora module and an “LC-like” system. This one was equipped with a pumping system, which allows adding modifier to CO.sub.2. A stainless steel cylindrical extraction cells located in the oven at 50° C. with a 100×4.6 mm (0.5 mL) were used for the SFE extraction. The extraction cell was fully filled with the plant powder. UHPLCs were conducted with a Shimadzu Nexera X2 system equipped, diode array detector and a SEDEX Model 90 LT-ELSD detector. The column used for these experiments was a Phenomenex Kinetex XB-C18 1.7 μm, 150*2.1 mm. The flow rate was set to 0.6 mL/min using a linear gradient of H.sub.2O mixed with an increasing proportion of CH.sub.3CN. Both solvents were of HPLC grade, modified with 0.1% formic acid.

[0090] Determination of Minimal Inhibitory Concentration: Antibacterial Properties

[0091] The crude extract and pure compounds isolated were tested against human pathogenic microorganisms, including the bacterium Staphylococcus aureus (ATCC 29213), MRSA (ATCC 33591), Candida albicans (ATCC 10213), Trichophyton rubrum (SNB-TR1) and P. aeruginosa ATCC 27853 to screen their antibacterial and antifungal activities. All ATCC strains were purchased from the Pasteur Institute. The clinical isolate was provided by Prof. Philippe Loiseau, Université Paris Sud. The ITS sequence was deposited in the NCBI GenBank database under the registry number KC692746 corresponding to SNB-TR1 strain. The tests were conducted in accordance to the reference protocols from the European Committee on Antimicrobial Susceptibility Testing. The standard microdilutions, ranging from 256 to 0.25 μg/mL were made from stock solutions prepared in DMSO (Sigma-Aldrich, France). The microplates were incubated at 35° C., and MIC values were obtained after 48 h for C. albicans, 24 h for S. aureus and 5 days for T. rubrum. The MIC values reported in Table 2 refer to the lowest concentration preventing visible growth in the wells. Vancomycin (Sigma-Aldrich, Saint-Quentin Fallavier, France) and oxacillin (Sigma-Aldrich, Saint-Quentin Fallavier, France) were used as positive controls for bacteria. Fluconazole (Sigma-Aldrich, Saint-Quentin Fallavier, France) and itraconazole (Sigma-Aldrich, Saint-Quentin Fallavier, France) were used as positive controls for fungi. All assays were conducted in duplicate.

[0092] Cytotoxicity Evaluation

[0093] Human lung fibroblast cells (MRC-5) were purchased from ATCC (Rockville, Md., USA) and cultured as recommended. Cell growth inhibition was determined by an MTS assay according to the manufacturer's instructions (Promega, Madison, Wis., USA). The cells were seeded in 96-well plates containing the growth medium. After 24 h of culture, samples were dissolved in DMSO (Sigma-Aldrich, France), and added to the cells (at 1 and 10 μM final concentrations). After 72 h of incubation, the reagent was added, and the absorbance at 490 nm was recorded using a plate reader. Cell viability was evaluated in comparison with untreated control cultures. Docetaxel (Taxotere) was used as positive control (IC.sub.50: 0.5 nM). All assays were conducted in triplicate.

[0094] Antioxidant Evaluation

[0095] P. mauritianum extracts were investigated for their antioxidant activities with using an ABTS [2,2′- azinobis-(3-ethylbenzothiazoline-6-sulfonate)] assay.

II - Isolation of Compounds in the Antibacterial EtOAc Plant Extract

[0096] Bioassay guided fractionation of the extract of Psiloxylon mauritianum led to isolation of the known molecules Aspidin BB (1), ursolic acid (3) and oleanic acid (4), along with compound 2 that had not previously been isolated or described in the literature (FIG. 1). The known compounds were identified by comparison of .sup.1H and .sup.13C data with values reported in the literature, together with crystallography data for 1. The common triterpenic acids 3 and 4 were isolated as a 6:4 ratio mixture, and the complete .sup.1H and .sup.13C-NMR assignments were deduced from NMR 1D and 2D experiments conducted on a 700 MHz NMR spectrometer.

[0097] Compound 2 was initially obtained as a white amorphous solid. HRESIMS analysis of 2 revealed a molecular formula of C.sub.27H.sub.36O.sub.8 (m/z 489.2490 for [M+H].sup.+), implying 2 C and 4 H more than in Aspidin BB 1. The .sup.1H-NMR spectrum displayed remarkably downfield-shifted singulet signals at δ.sub.H 15.86, 11.41 and, 10.05, which are characteristic of the hydroxyl groups found in acylphloroglucinols Aspidin derivatives. The .sup.1H-NMR data of 2 were very similar to those for 1 except for the presence of a supplementary signal at δ.sub.H 1.39 (m) integrating four protons (H11 and H12), and a two methyl triplet at δ.sub.H 0.98 (J=7.4 Hz) and δ.sub.H 0.93 (J=7.1 Hz), which are the common signals for methyl terminal groups (Table 1).

TABLE-US-00001 TABLE 1 The 1D and 2D NMR data for Aspidin CB (2) in acetone-d.sub.6. Aspidin CB Position δ.sub.c.sup.1 δ.sub.H (J in Hz).sup.2 COSY HMBC ROESY  1 111.9  2 188.4  3 108.9  4 199.9  5 45.1  6 172.7  7 17.7 3.57, s C1, C2, C6, C1′, C2′, C6′  8 207.5  9 41.6 3.19, dd (7.2, 7.2) H8 C3, C8, H11 C10, C11 10 25.3 1.67, m H9, H11 C8, C9, C11, C12 11 32.5 1.39, m H10, H12 C12 H9 12 23.2 1.38, m H11, H13 C11 13 14.3 0.93, t (7.1) H12 C11, C12  1′ 110.2  2′ 160.6  3′ 108.5  4′ 161.5  5′ 113.2  6′ 163.6  7′ 62.1 3.80, s C4′ H9′  8′ 208.0  9′ 44.8 3.15, dd (7.2, 7.2) H10′ C8′, C10′, H7′ C11′ 10′ 18.8 1.72, sex (7.4) H9′, H11′ C8′, C10′, C11′ 11′ 14.2 0.98, t (7.4) H10′ C9′, C10′ H9′ 12′ 9.5 2.10, s C4′, C5′, H7′ C6′ 6-OH 10.05, s C1, C5, H7, 2′-OH, C6 6′-OH 2′-OH 15.86, s C1′, C3′, 6-OH C8′ 6′-OH 11.41, s C1′, C5′, H7, 6-OH C6′

[0098] These findings suggest that 2 is an analogue of 1 with different structure of the side chains. Interpretation of COSY and HMBC experiments, especially HMBC correlations observed with the two ketonic carbons at δ.sub.c 208.0 (C8) and δ.sub.c 207.5 (C8), easily revealed the presence of valeryl and butyryl chains. The connection of the valeryl side chain to the acylfilicinic acid moiety was determined with

[0099] HMBC correlation between the protons of the methylene H9 at δ.sub.H 3.19 with the quaternary carbon C3 at δ.sub.c 108.9 ppm. The allocation of the butyryl chain was established with the ROESY experiment (data not shown). In fact, the protons of the methylene H9 at δ.sub.H 3.15 displayed a ROE correlation with the protons of the methoxyl H7 at δ.sub.H 3.80, which exhibited a clear ROE correlation with the toluene methyl H12 at δ.sub.H 2.10.

[0100] The structure determined with the NMR data was confirmed by single-crystal X-ray diffraction analysis (See Tables S9-S16). Crystallographic data were recorded at 150 K to reduce agitation induced by the length of the valeryl side chain. Compound 2 was named Aspidin CB; this is the first time that this compound was isolated and characterized.

III - Isolation of Compounds from SFE-CO.SUB.2 .Extracts from Psiloxylon mauritianum

[0101] A 1.0 g of dry plant powder was weighed and used for extraction. Different co-solvent percentages of ethanol from 0, 1, 2 and 5% and a back pressure of 200 bars were applied for 15 min, with a flow rate of 2 mL.min.sup.−1. Each fraction resulted from the different percentages of ethanol were concentrated to dryness under reduced pressure and the yield was calculated (table 2)

TABLE-US-00002 TABLE 2 SFE conditions and yields for the fractions of P. mauritianum leaves extraction Percentage Mass Yields Solvent (v/v) (%) (mg) (%) CO.sub.2 100 44.3 mg  4.4% CO.sub.2/EtOH 99/1 4.9 mg 0.5% CO.sub.2/EtOH 98/2 3.7 mg 0.4% CO.sub.2/EtOH 95/5 6.0 mg 0.6%

[0102] The fraction obtained with CO.sub.2 100% was subjected to UHPLC analysis and the presence of aspidin BB (RT=7.69 min) and aspidin CB (RT=8.17 min) was determined by comparison of retention times. (Cf FIG. 2)

IV. Antibacterial Properties

[0103] Antibacterial properties of Crude Extracts from Psiloxylon mauritianum

[0104] All Crude extracts obtained from P. mauritianum (leave and stem) have exhibited significant bacterial inhibition, particularly on S. aureus. (Table 3). An alteration of the growth was observed for C. albicans and P. aeruginosa even at low concentration.

TABLE-US-00003 TABLE 3 Antimicrobial results for crude extracts of leaves and stems from P. mauritianum MIC (μg/ml) S. aureus T. rubrum Extract ATCC 29213 SNB-TR1 Leaves AcOEt 16-32 256 MeOH  8-16 128 Water  8-16 128 Stems AcOEt 512-256 128 MeOH 512-256 32 Water  8-16 128

[0105] Antibacterial Properties of Crude Extracts of Leaves from P. mauritianum against Gram Negative Pathogens

[0106] Bacterial growth kinetics of Acinetobacter baummanii 19606, Escherichia coli WT 25922 and Klebsiella pneumoniae WT 700603 incubated with crude extracts of P. mauritianum at 32 μg/ml were followed during a period of 16 hours. Kinetic curves allowed us to establish that the growth of bacteria was inhibited with all extracts, compared to the negative control, at the test concentration. Crude methanolic and water extracts of P. mauritianum have shown a significant inhibition of Acinetobacter baummanii 19606 at 32 μg/ml.

[0107] Antibacterial Properties Aspidin CB Against Gram-Positive Pathogens

[0108] Aspidin CB exhibited strong antibacterial activity against standard and methicillin-resistant S. aureus strains, with a minimal inhibition concentration (MIC) of 0.25 μg/mL, and no cytotoxicity was observed at 10.sup.−5 M in MRC5 cells. Similar Anti-SARM activities were also observed against SARM clinical strains. Our results suggested also potent antibacterial activity against other Gram-positive pathogens. (Table 4)

[0109] Furthermore, similar antibacterial properties were observed for different clinical strains of Staphylococcus, such as Staphylococcus hominis, Staphylococcus argenteus, Staphylococcus haemolyticus, Staphylococcus warnieri, Staphylococcus lugdunensis.

TABLE-US-00004 TABLE 4 Examples of antimicrobial results for Aspidin CB againt Gram-positive bacteria MIC MIC Bacteria (μg/ml) Bacteria (μg/ml) S. epidermis 0.5 SARM G + 82* 0.5 MSF G + 15* SARM NCTC 12493 0.5 SARM G + 86* 0.5 S. aureus 0.5 Enterococcus 8 ATCC25923 faecalis 29212 SARM G + 78* 0.5 Enterococcus 0.5 faecium van B* SARM G + 79* 1 S. epidermis 2 MSF G + 16* Bacillus 1 Enterococcus 8 sp. L4 210321* faecium SI299* Corynebacterium 1 sp. L2 220321* *Clinical strain

[0110] Antibacterial Properties of Aspidin CB Against Gram-Negative Pathogens

[0111] Gram-negative bacteria possess a unique outer membrane that makes them resistant to several antimicrobial agents. Permeabilizers are compounds that weaken the outer membrane by enhance the permeability of bacterial cells to antimicrobial agents. [Farrag, H.A.; Abdallah, N.; Shehata, M.M.K.; Awad, E. M. J Biomed Sci, 2019, 26:69] Colistin a.k.a. polymyxin E were extensively used in clinical practice for Gram-negative organisms, but gradually withdrawn from the market due to reports of significant nephrotoxicity and neurotoxicity. [Mohamed, Y. F., Abou-Shleib, H. M., Khalil, A. M., El-Guink, N. M., & El-Nakeeb, M. A. Braz J Microbiol., 2016, 47, 381-388] In combinaison with various antibiotics, colistin is known to destabilize the outer membrane enhancing the antibacterial activity. In our study, we evaluated a combinaison of colistin at a sub-inhibitory concentration and Aspidin CB. The results have showed that Aspidin CB was unambiguously more active against A. baumanii and E. coli in a combination with colistin. (Table 5)

TABLE-US-00005 TABLE 5 Antimicrobial results for Aspidin CB in combination with colistin againt Gram-negative bacteria MIC (μg/ml) Acinetobacter Escherichia coli baummanii WT 25922 19606 Aspidin CB >64 >64 Colistin 1 4 Aspidin CB+ colistine 16 >64, altered growth 0.25 μg/ml Aspidin CB+ colistine n.d. 32 0.50 μg/ml Negative control (DMSO) >64 >64 n.d. not determined

[0112] Other experiments performed with both EtOAc crude extract and isolated compounds are presented in Table 6 and confirm previous results.

TABLE-US-00006 TABLE 6 Antimicrobial and cytotoxic results for EtOAc crude extract and isolated compounds. MIC (μg/mL) C. albicans T. rubrum S. aureus MRSA MRC5 Cell Viability (%) Compounds ATCC 10213 SNB-TR1 ATCC 29213 ATCC 33591 10.sup.−5 M 10.sup.−6 M 1 >256 >256 2 1 86 ± 3 104 ± 1 2 >256 256 0.25 0.25 99 ± 2 105 ± 2 Crude extract 8 256 8 nd 96 ± 2 100 ± 3 Fluconazole.sup.1 1 4 nd nd nd nd Itraconazole.sup.1 <0.5 <0.5 nd nd nd nd Oxacillin.sup.1 nd.sup.2 nd 0.25 nd nd nd Vancomycin.sup.1 nd.sup.  nd nd 4 nd nd .sup.1Positive control. .sup.2Not determined.

[0113] Compounds 1 and 2 exhibited antibacterial activity against S. aureus and MRSA higher than the positive control, with MICs of 0.25 μg/mL for 2 and 2 and 1 μg/m L for 1, respectively, against these two pathogens. Our results indicated that compound 2 has the same MIC as oxacillin against S. aureus and was 16-fold more potent than the standard antibiotic vancomycin against MRSA. Interestingly, Aspidin CB (2) showed higher activity than compound 1 against both bacteria. Furthermore, no cytotoxicity was observed for compound 2 at concentrations up to 10.sup.−5 M (Table 2). In our assays, Aspidin CB (2) was more active against MRSA and slightly less toxic than 1, which is known to exhibit no toxicity when S. aureus was killed .

[0114] In accordance with the literature, Aspidin BB (1) was strongly active against both S. aureus and MRSA but was inactive against the human pathogenic fungi. Aspidin BB (1) is known to exert strong antibacterial activity against Gram-positive bacteria, like S. aureus, S. epidermis or Propionibacterium acnes Li et al. identified the relationship between antibacterial activity and increase levels of reactive oxygen species in S. aureus cells. Moreover, the authors demonstrated that 1 induced peroxidation of membranes, DNA damage and protein degradation in S. aureus. By comparing the effects of compounds 1 and 2 on S. aureus strains, our results demonstrated that a longer carbon chain (2 additional carbons) on the acylfilinic acid moiety is correlated with 4-to 8-fold stronger activity (Table 2). Compared to Aspidin BB (1), the antibacterial potency of Aspidin CB (2) may result from better cell wall penetration due to a longer alkyl chain inducing improved lipophilicity.

IV. Cosmetic Applications of AcOEt and MeOH extracts of P. mauritianum

[0115] Antioxidant Properties

[0116] It was found that AcOEt and MeOH extracts of P. mauritianum leaves and stems possessed significant antioxidant capacities compared to positive controls (trolox and tocopherol). (Table 7) HPLC chromatograms of water and MeOH extracts of P. mauritianum (leaves and stems) were very similar and suggested a similar composition.

TABLE-US-00007 TABLE 7 Radical scavenging capacity of P. Mauritianum extracts at 50 μg/ml. % ABTS inhibition EtOAC leaves extract 77.5 MeOH leaves extract 97.6 EtOAC stems extract 83.2 MeOH stems extract 97.8 Tocopherol 97.6 Trolox 98.0

[0117] Antibacterial Properties

[0118] The water extract of P. mauritianum leaves has clearly showed a preservative efficacy in a cleansing milk. (table 8)

TABLE-US-00008 TABLE 8 Challenge test results for cleansing milk formulated with 3% of P. mauritianum leave water extract. Log reduction at (days) Organisms 0 2/5 7/12 14/19 28/29 Staphylococcus aureus 6.00 >4.00 >4.00 >4.00 >4.00 Escherichia coli 6.38 0.57 >3.38 >4.38 >4.38 Pseudomonas aeruginosa 6.59 >4.57 >4.57 >4.57 >4.57

[0119] A part of the description of the invention, especially regarding experimental part, is also available in the article: Potent and Non-Cytotoxic Antibacterial Compounds against Methicillin-Resistant Staphylococcus aureus Isolated from Psiloxylon mauritianum, A Medicinal Plant from Reunion Island published in Molecules in August 2020. DOI: 10.3390/molecules25163565