Method of inhibiting propionibacterium acnes

Abstract

The invention discloses the anti-acne potential of and pyranocycloartobiloxanthone A and Artonine E, isolated from the stem bark of Artocarpus hirsutus by inhibiting the growth of Propionibacterium acnes.

Claims

1. A method of inhibiting Propionibacterium acnes, said method comprising step of bringing into contact Propionibacterium acnes with effective concentration of Pyranocycloartobiloxanthone A as represented in STR #1 or Artonine E as represented in STR #2, isolated from the stem bark of Artocarpus hirsutus. ##STR00003##

2. The method as in claim 1, wherein Pyranocycloartobiloxanthone A is present as and anomers in the ratio 90-50:10-50.

3. The method as in claim 2, wherein the ratio of to Pyranocycloartobiloxanthone A is 70:30.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the .sup.1H NMR spectra of and pyranocycloartobiloxanthone A, isolated from stem bark of Artocarpus hirsutus.

(2) FIGS. 2, 2a and 2b show the .sup.13C NMR spectra of and pyranocycloartobiloxanthone A, isolated from stem bark of Artocarpus hirsutus.

(3) FIGS. 3 and 3a show the Atmospheric pressure chemical ionization-Mass Spectrometer (APCI-MS) data of and pyranocycloartobiloxanthone A, isolated from stem bark of Artocarpus hirsutus.

(4) FIG. 4 shows the Fourier transform Infra-red (FTIR) spectrum of and pyranocycloartobiloxanthone A, isolated from stem bark of Artocarpus hirsutus.

(5) FIG. 5 shows the D.sub.2O exchanged .sup.1H NMR spectrum of and pyranocycloartobiloxanthone A, isolated from stem bark of Artocarpus hirsutus.

(6) FIGS. 6 and 6a show the Atmospheric pressure chemical ionization-Mass Spectrometer (APCI-MS) data of Artonine E, isolated from stem bark of Artocarpus hirsutus.

(7) FIG. 7 shows the .sup.1H NMR spectra of Artonine E, isolated from stem bark of Artocarpus hirsutus.

(8) FIG. 8 shows the .sup.13C NMR spectra of Artonine E, isolated from stem bark of Artocarpus hirsutus.

(9) FIG. 9 shows the zone of inhibition of isolated compounds and pyranocycloartobiloxanthone A and Artonine E against P. acnes. A reference standard (Clindamycin) is included as control.

(10) FIG. 10 shows the Graphical representation of the zone of inhibition of compound and pyranocycloartobiloxanthone A and Artonine E against P. acnes. A reference standard (Clindamycin) is included as control.

DESCRIPTION OF THE MOST PREFERRED EMBODIMENTS

(11) In the most preferred embodiment, the present invention discloses a method of isolating natural molecules from the bark of Artocarpus hirsutus, said method comprising steps of: a) Cutting and drying the stem bark of Artocarpus hirsutus and pulverising to coarse powder b) Refluxing the stem bark powder of step a with ethanol (w/v ratio 1:10) thrice for 3 hours each to obtain three separate extracts; c) Combining the extracts of step b and concentrating at reduced pressure between 55-60 C. and suspending in 2 volumes of water, followed by fractionation with n-hexane (>99% v/v, 2 volumes), chloroform (>99% v/v, 2 volumes), and ethyl acetate (>99% v/v, 2 volumes); d) Purifying the chloroform layer of step c on a silica gel column (60-120 mesh) followed by elution with dichloromethane and dichloromethane/Acetone (98:2 to 90:10) and collecting 80 fractions of 250 mL each; e) Comparing the fractions of step d using TLC and combining similar fractions (33-67) followed by concentration and drying under vacuum to obtain enriched material; f) Washing the enriched material obtained in step e with dichloromethane to provide a powder, characterized using spectroscopic techniques as anomeric mixture of and Pyranocycloartobiloxanthone A as represented in STR #1, in the ratio of 90-50:10-50 and filtrate;

(12) ##STR00001## g) Further purification of the filtrate of step f over silica gel and elution with n-hexane-ethyl acetate (70:30) to provide a compound that was characterized using spectroscopic techniques as Artonine E as represented in STR #2.

(13) ##STR00002##

(14) In a related embodiment, Pyranocycloartobiloxanthone A is present as and anomers in the ratio 70:30.

(15) In a preferred embodiment, the invention discloses a method of inhibiting Propionibacterium acnes, said method comprising step of bringing into contact Propionibacterium acnes with effective concentration of Pyranocycloartobiloxanthone A, isolated from the stem bark of Artocarpus hirsutus. In a related embodiment, Pyranocycloartobiloxanthone A is present as and anomers in the ratio 90-50:10-50 (STR #1). More specifically, Pyranocycloartobiloxanthone A is present as and anomers in the ratio 70:30. In yet another preferred embodiment, the invention discloses a method of inhibiting Propionibacterium acnes, said method comprising step of bringing into contact Propionibacterium acnes with effective concentration of Artonine E, isolated from the stem bark of Artocarpus hirsutus.

(16) The specific examples included herein below illustrate the most preferred embodiments of the present invention.

Example I: Isolation of Bioactive Molecules from Stem Bark of Artocarpus hirsutus

(17) Collection of Plant Materials

(18) The stem bark of Artocarpus hirsutus, was collected from Udupi district, Karnataka, India. All samples were authenticated by botanist and sample voucher was kept in herbarium (RD/HAR-AH/11). The stem bark was cut into small pieces and dried under shade. The dried materials were pulverized to coarse powder and stored in air tight containers.

(19) Preparation of Extracts

(20) The stem bark powder (3 kg) of Artocarpus hirsutus was refluxed with ethanol (w/v ratio 1:10), three times for three hours each. The extracts were combined and concentrated at reduced pressure at 55-60 C. The ethanolic extract (yield: 138 g) was filtered and dried completely under vacuum before storing in air tight containers.

(21) Analytical Methods

(22) Normal phase TLC was performed on pre-coated silica gel F.sub.254 plates (Merck Specialties Private Ltd., Mumbai, India) and the products spot were visualized either by UV (UV-254/366 nm) or by iodine vapours. Liquid chromatography mass spectrometry (LC-MS) analysis was carried out on Finnigan LCQ Advantage Max (Thermo, LAM 10234). .sup.1H NMR (300 MHz) and .sup.13C NMR (75 MHz) spectra were recorded on VARIAN NMR spectrometer. Chemical shifts ( values) are reported in ppm (parts per million) with respect to TMS as internal standard, DMSO-d.sub.6 was used as solvent. Column chromatography was performed over silica gel (mesh 60-120). Infra red spectra were recorded on Perkin Elmer FTIR Spectrometer.

(23) Isolation of Active Molecules

(24) The ethanol extract was suspended in water and fractionated with hexane, chloroform and ethyl acetate. The chloroform extract was further purified on silica gel column chromatography. The column was loaded with silica gel (60-120 mesh) in dichloromethane (CH.sub.2Cl.sub.2), eluted with CH.sub.2Cl.sub.2 and CH.sub.2Cl.sub.2/Acetone and collected 80 fractions of 250 mL each. Similar fractions were combined after verifying the TLC analysis. Fractions 33 to 67 were combined, concentrated and dried under vacuum. The material obtained was washed with CH.sub.2Cl.sub.2 to provide Pyranocycloartobiloxanthone A as yellow powder (STR #1). The filtrate was further chromatographed over silica gel and elution with hexane-ethyl acetate (70:30) afforded Artonine E (STR #2) as yellow solid.

(25) Extensive fractionation and purification of the ethanolic extract of Artocarpus hirsutus stem bark on silica gel lead to the isolation of two compounds, characterized as i) a xanthone derivative, Pyrancycloartobiloxanthone A (STR #1) and a flavonoid, Artonine E (STR #2). Pyranocycloartobiloxanthone A was obtained as yellow powder with the melting point 268-270 C. (dec.). The .sup.1H (FIG. 1) and .sup.13C NMR spectra (FIGS. 2, 2a and 2b) of pyranocycloartobiloxanthone A clearly demonstrate the occurrence of two isomers in the ratio of 70 and 30 and were assigned as two anomeric conformers, (70%) and (30%) constituted owing to the presence of carbohydrate moiety in the molecule. In consequences, other signals in .sup.1H (FIG. 1) and .sup.13C NMR (FIGS. 2, 2a and 2b) spectra clearly established the ratio of the anomers to be 70:30.

(26) The APCI-MS (FIGS. 3 and 3a) of the pyranocycloartobiloxanthone A corresponds to the molecular ion peak in positive mode at m/z 451.20 (M+H).sup.+ and in negative mode at m/z 449.09 (MH).sup. representing the molecular mass of the molecule with 450. The FTIR spectrum (FIG. 4, Table 1) showed the presence of hydroxyl and conjugated carbonyl moieties with absorption at 3387 and 1659 cm.sup.1, respectively.

(27) TABLE-US-00001 TABLE 1 FT1R values for pyranocycloartobiloxanthone A List of Peak area/height Peak No. X(cm1) Y (% T) 1 3280.50 92.04 2 1659.32 88.49 3 1601.71 89.38 4 1548.85 78.42 5 1481.99 73.64 6 1361.64 84.01 7 1336.70 85.19 8 1320.99 85.00 9 1273.18 77.84 10 1228.39 88.51 11 1162.21 84.41 12 1114.23 86.93 13 1085.20 88.56 14 1061.45 90.16 15 1032.17 88.66 16 987.50 88.88 17 966.93 85.70 18 834.18 84.95 19 803.05 88.92 20 782.94 90.60 21 759.87 90.95 22 731.53 91.54 23 712.43 91.60 24 666.65 91.61 25 614.41 89.94

(28) The solubility of the compound was relatively poor in most of the solvents except in DMSO and thus .sup.1H NMR and .sup.13C NMR spectra were recorded in DMSO-d6. In .sup.1H NMR spectrum, the phenolic OH (5-OH) adjacent to the carbonyl was assigned from the downfield signal at 13.36 ppm (70%) and 13.33 ppm (30%) and these two signals appeared due to the presence of the two conformers and in about 70:30 ratio. The assignment of two doublets at 7.15 ppm (30%) and 6.94 ppm (70%) was crucial in .sup.1H NMR spectrum. Nonetheless the disappearance of these two signals in D2O exchanged .sup.1H NMR spectrum (FIG. 5) ascertained that these two doublet signals are nothing but appeared due to the presence of the hydroxyl group (14-OH) sustaining powerful coupling with anomeric proton (14-H). Thus the signals at 7.15 ppm (d, 30%, -OH, J=7.5 Hz) and at 6.94 ppm (d, 70%, -OH, J=4.2 Hz) were assigned corresponding to the anomeric 14-OH. Similarly, though the anomeric proton (14-H) was expected to appear as a doublet however owing to further coupling with 14-OH, it appeared as two signals one as double doublets at 5.32 ppm (dd, 70%, 14--H, J=4.2 Hz, 2.1 Hz) and another one as triplet at 4.87 ppm (t, 30%, 14--H, J=7.8 Hz). The coupling interferences in 14-/-H due to 14-OH was disappeared in D2O exchanged .sup.1H NMR spectrum (FIG. 5) and signals were clearly appeared as doublets at 5.28 (d, 70%, 14--H, J=1.2 Hz) and 4.81 ppm (d, 30%, 14--H, J=8.7 Hz). As expected the signals ratio 70:30 were reflected in the .sup.13C NMR spectrum also. It was observed that carbonyl (C-4) peak appeared at 179.2 (70%) and 179.2 (30%) ppm. Most importantly characteristic anomeric C-14 signal was noticed in two positions at 97.6 (30%) and at 93.2 (70%) ppm which correspond to C-14 and C-14 anomer respectively. The presence of other characteristic signals both in .sup.1H and .sup.13C NMR spectra confirms the compound as a mixture of and -pyranocycloartobiloxanthone A in approximately 70:30 ratio (Table 2). The analytical data of pyranocycloartobiloxanthone A was comparable with that of pyranocycloartobiloxanthone A reported by Hasima et al., (2010), Two new xanthones from Artocarpus obtusus, J Asian Nat Prod Res, 12 (2):106-112. However, the reported structure was established as single conformer whereas the instant invention reports this molecule as a mixture of two conformers ( and ). Notwithstanding the illustrative ratio of 70:30 for a mixture of and -pyranocycloartobiloxanthone A as elucidated herein above, it is obvious to a person of ordinary skill in the art that the ratio may vary depending on the raw material, seasonal and climatic variation and geographical origin of the raw material. Thus all exemplary variations in the ratio between and -pyranocycloartobiloxanthone A are envisioned and encompassed in this patent application. The .sup.1H NMR and .sup.13C NMR spectra of and -pyranocycloartobiloxantone A is specified in table 2 with following data:

(29) and -pyranocycloartobiloxantone A: Yellow powder with melting point: 268-270 C. (dec.); IR (KBr) .sub.max. (Table 1) .sup.1H NMR (DMSO, d-6, 300 MHz) (Table 2); .sup.13C NMR (DMSO-d6, 75 MHz) (Table 2); APCI-MS m/z 451.20 (M+H.sup.+) and 449.09 (MH.sup.) (C.sub.25H.sub.22O.sub.8 requires 450.4373) (FIGS. 3 and 3a).

(30) TABLE-US-00002 TABLE 2 Spectral data of and -pyranocycloartobiloxantone A Compound 1 in DMSO-d6 Position .sup.1H NMR (.sub.H), 300 MHz .sup.13C NMR (.sub.C), 75 MHz 1 2 160.8 3 100.8 4 179.2 (70%) 179.1 (30%) 5 151.31 6 6.17 (s, 1H) 98.7 7 160.7 8 104.23 (70%) 104.18 (30%) 9 157.9 10 103.7 11 1.78-1.92 (m, 1H) 21.6 (70%) 3.10-3.20 (m, 1H) 22.1 (30%) 12 2.51-2.62 (m, 1H) 31.2 13 1.78-1.92 (m, 1H) 35.7 (70%) 37.0 (30%) 14 5.32 (dd, 1H, J = 4.2 Hz, 1.95 Hz) 93.1 (70%) (70%) 4.87 (t, 1H, J = 7.8 Hz) (30%) 97.9 (30%) 15 1.07 (d, 1H, J = 6.9) (70%) 14.7 1.10 (d, 1H, J = 6.9 Hz) (30%) 13.9 16 6.85 (d, 1H, J = 9.9 Hz) (70%) 114.8 6.84 (d, 1H, J = 9.9 Hz) (30%) 17 5.72 (d, 1H, J = 10.2 Hz) 127.3 18 78.0 19, 20 1.44 (s, 3H), 1.41 (s, 3H) 27.7, 28.0 1 111.05 (70%) 110.97 (30%) 2 151.28 (70%) 151.17 (30%) 3 6.42 (s, 1H) 103.1 4 150.8 (70%) 150.7 (30%) 5 132.6 (70%) 134.3 (30%) 6 124.7 (70%) 123.9 (30%) 5-OH 13.36 (s, 1H) (70%) 13.33 (s, 1H) (30%) 14-OH 6.94 (d, 1H, J = 4.2 Hz) (70%) 7.12 (d, 1H, J = 7.5 Hz) (30%) 2-OH 9.93 (s, 1H) (70%) 10.00 (s, 1H) (30%) 4-OH 9.81 (s, 1H) (70%) 9.84 (s, 1H) (30%)

(31) Artonine E (STR #2) was isolated as yellow powder. The mass spectrum (APCI-MS) (FIGS. 6 and 6a) showed the molecular ion peak in positive mode at m/z 437.09 (M+H).sup.+ and in negative mode 435.05 (MH).sup. corresponding to the molecular mass of compound 2 as 436. In the .sup.1H NMR spectrum (FIG. 7), three singlets at 6.68, 6.46 and 6.21 correspond to three aromatic protons of (H-3, H-6 and H6) whereas the signal at 13.2 ppm represents the chelated phenolic proton (5-OH). The presence of prenyl side chain was confirmed from its the corresponding characteristic signals at 5.05 (t, 1H, J=6.9 Hz, H-10), 3.03 (d, 2H, J=6.9 Hz, H-9), 1.56 (s, 3H, H-13) and 1.41 (s, 3H, H-12). Similarly, signals at 6.52 (1H, d, J=9.9 Hz, H-14), 5.69 (1H, d, J=9.9 Hz, H-15), 1.41 (6H, s, H-17 and H-18) ascertained the presence of 2,3-dimethylpyran ring. The presence of characteristic signals in .sup.13C NMR spectrum (FIG. 8) also confirms the structure. It was noticed that the analytical values were correlated well with Sritularak et al, (2010) New 2-Arylbenzofurans from the Root Bark of Artocarpus lakoocha, Molecules, 15:6548-6558, and consequently was identified as Artonine E with the following spectral data.

(32) Artonine E: Yellow powder with melting point: 242-246 C.; IR (KBr) .sub.max 3426, 2982, 1642, 1560, 1523, 1462, 1355, 1154, 828, 767, 698 cm.sup.1. UV (Methanol) max 204, 267.5, 350 nm. .sup.1H NMR (DMSO-d6, 300 MHz): 13.2 (s, 1H, 5-OH), 9.5, 9.3, 8.6 (3s, 3H, 2,4, 5-OH), 6.68 (s, 1H, H-6), 6.52 (d, 1H, J=9.9 Hz, H-14), 6.46 (s, 1H, H-3), 6.21 (s, 1H, H-6), 5.69 (d, 1H, J=9.9 Hz, H-15), 5.05 (t, 1H, J=6.6 Hz, H-10), 3.03 (d, 2H, J=6.6 Hz, H-9), 1.56 (s, 3H, H-13), 1.41 (s, 9H, H-12, H-17 and H-18) (FIG. 7); .sup.13C NMR (DMSO-d6, 75 MHz): 181.8 (C-4), 161.7 (C-2), 160.9 (C-5), 158.5 (C-7), 151.7 (C-8a), 148.8 (C-2), 148.5 (C-4), 138.0 (C-5), 131.4 (C-11), 127.7 (C-15), 121.5 (C-10), 119.9 (C-3), 116.1 (C-6), 114.2 (C-14), 109.3 (C-1), 104.2 (C-4a), 103.9 (C-3), 100.5 (C-8), 98.8 (C-6), 78.1 (C-16), 29.6 (C-17), 27.7 (C-18), 25.5 (C-13), 23.7 (C-9), 17.4 (C-12) (FIG. 8); APCI-MS m/z 437.09 (M+H.sup.+) and 435.05 (MH.sup.), (C.sub.25H.sub.24O.sub.7 requires 436.4539) (FIGS. 6 and 6a).

Example II: Antiacne Potential of Pyranocycloartobiloxantone A and Artonine E

(33) In vitro anti-acne activity of isolated compounds 1 and 2 from the stem bark extract of Artocarpus hirsutus were evaluated against acne causing bacterium, Propionibacterium acnes. The antibacterial activity was determined by agar well diffusion method. The minimum inhibitory concentration (MIC) of the compounds were then ascertained by broth micro dilution method.

(34) Microorganisms: Acne causing bacterium Propionibacterium acnes (ATCC 11827) was procured from American type culture collection Rockville, USA.

(35) Media: Reinforced Clostridial Agar (Hi Media; M154) and Reinforced Clostridial broth (Hi Media; M443) were used in the experiments.

(36) Determination of Antibacterial Activity

(37) The antibacterial activities of isolated compounds were performed by agar well diffusion method. The samples were dissolved in DMSO to obtain a concentration in the range of 0.5-100 mg/mL. Propionibacterium acnes was cultured on Reinforced Clostridial Agar (RCA) M154 procured from Hi Media and incubated at 37 C. for 48 h in an anaerobic chamber providing gas mixture containing 80% nitrogen, 10% carbon dioxide and 10% hydrogen. The bacterial culture was suspended in sterile normal saline and adjusted to 1.010.sup.8 CFU/mL (CLSI, M02-A11; Vol. 32 No. 1). The sterile RCA was seeded with the standardized culture of P. acnes and poured into plates. The agar medium was allowed to solidify. Wells of 7 mm diameter equidistant from each other were punched into the agar surface using a sterile borer. Aliquot of each sample, diluent control (DMSO), and Clindamycin as standard antibiotic were loaded in the wells. The plates were kept at 4-8 C. for 3 h and then incubated in the anaerobic chamber for 48 h. The diameter of zone of inhibition around the wells were measured and recorded.

(38) The zone of inhibition of isolated compounds: pyranocycloartobiloxantone A and Artonine E around the wells displayed good inhibition. The inhibition was detected in all tested concentrations (10-1.25 g/mL) (FIG. 9 and FIG. 10).

(39) Determination of Minimum Inhibitory Concentration (MIC)

(40) The minimum inhibitory concentrations (MIC) of the isolated compounds against P. acnes were determined by broth micro dilution method. Two-fold dilutions of the isolated compounds were prepared in Reinforced Clostridial broth (RCB) to obtain concentrations in the range of 0.1-2000 g/mL. The diluted samples were loaded in 96-well micro titre plates. The samples were inoculated with the test culture of P. acnes so that the final concentration of the bacteria in each well is 110.sup.5 CFU (CLSI, M11-A8; Vol. 32 No. 5). The plates were incubated under anaerobic conditions at 37 C. for 48 h and thereafter observed for inhibition of bacterial growth. The minimum concentration required for inhibiting the growth of P. acnes was considered as minimum inhibitory concentration (MIC).

(41) It was observed that the isolated compounds exhibited highly potent anti-acne activity against P. acnes with MIC values 2 g/mL each (Table 3).

(42) TABLE-US-00003 TABLE 3 MIC of pyranocycloartobiloxantone A and Artonine E against P. acnes Sl. No. Compound MIC (g/mL) 1 pyranocycloartobiloxanthone 2 A ( & ) 2 Artoninc E 2 3 Clindamycin 0.03

(43) To summarise, fractionation of ethanolic extract from the stem bark of A. hirsutus yielded a mixture of a xanthone derivative, Pyranocycloartobiloxanthone A constituting and conformers in almost 70:30 ratio. The conformers were confirmed from its spectral analysis and reported for the first time. Artonine E was the second molecule isolated from the stem bark and both the compounds exhibited significant anti-acne activity with MIC value of 2 g/mL each and comparable with antibiotic Clindamycin (MIC=0.03 g/mL).

(44) While the invention has been described with reference to a preferred embodiment, it is to be clearly understood by those skilled in the art that the invention is not limited thereto. Rather, the scope of the invention is to be interpreted only in conjunction with the appended claims.