FIELD OF THE INV ENTION

[I'ara000l] The invention in general relates to natural molecules of Artocarpus hirsntus More specifically, the invention relates to a method of isolating natural molecules from Artocarpus hirsntus and their use m therapeutic management of Acne vulgaris.

BACKGROUND OF THE INVENTION

[Para0G02] DESC RIPTION OF PRIOR ART

[Para0003] Acne vulgaris, one of the major diseases of the skin, affects the pilosebaceous unit (hair follicles associated with oil glands), it is caused due to altered follicular keratinisation. hormonal imbalance, immune hypersensitivity, and bacterial iPropionibactcrn ^' im acnes or P. acnes } resulting in pustules, nodules, small bumps (papules) and comedones either with blackheads (open) or with whiteheads in the skin { Williams et al.. (2012) Acne vulgaris. Lancet, 379:361 -372). Many topical and oral medications are being marketed commercially for controlling P. acnes, with limited validity owing to their synthetic nature and profound side effects Pharmaceutical actives possessing remarkable potency; minimal toxicity and capacity to target only the affected sites are in great demand. Therefore, phytochemicals that possess increased safety, toierability and efficacy against /'. acnes would be very effective for the treatment of Acne vulgaris. Artocarpus hirsntus, belonging to the Moraceae family, is reported to have wide range of therapeutic applications. The bark of the plant is known for treating pimples and skin cracks (Artocarpus hirsntus: EN VIS Centre on Conservation of Medicinal Plants, Ministry of Environment, Forest and Climate change. Government of India) This indicates the wound healing and anti-inilamrnatoiy potential of Artocarpus hirsntus bark. However, the antimicrobial effect of Artocarpus hirsntus bark against Prophnibacterinm acnes has never been anticipated or is obvious. The present invention solves tins technical problem by disclosing a method for isolating the bioactive molecules from Artocarpus hirsntus and evaluating their antimicrobial activity against /'. acnes.

[Para0004] It is the principle objective of the invention to disclose a method for isolating and identifying the natural molecules present in Artocarpus hirsntus. [Para0005] it is yet another objective of the present invention to disclose the anti-acne potential of the molecules isolated from stern bark, of Artocarpus hirsutm by inhibiting the growth of h opionihacfeviuin acnes.

[Para0006] The present invention fulfils the aforesaid objectives and provides further related advantages.

SUMMARY OF THE INVENTION

[Para0007] The present invention discloses a method for the isolation and characterisation of natural biologically active molecules from stem bark of ^' Artocarpus hinufns The present invention also discloses the anti-acne potential of isolated molecules of Artocarpus hirsutm by their ability to inhibit the growth of ^' Propiombacferinm acnes

[Para0008] Other features and advantages of the present invention will become apparent from the following more detailed description, taken in conjunction with the accompanying images., which illustrate, by way of example, the principle of the invention.

BRIEF DESC RIPTION OF THE DRAWINGS

[Para0009] Fig. I shows the 1H NMR spectra of a and β pyranocycioartobiioxanthone A, isolated from stem bark of Artocarpus hinufns

[Para0010] Figs. 2, 2a and 2b show the ^U C NMR spectra of a and β pyranocycioartobiioxanthone A. isolated from stem bark of Artocarpus hirsutm.

[Para0011 ] Figs. 3 and 3a show the Atmospheric pressure chemical ionization Mass Spectrometer ( APCT-MS) data of α and β pyranocycioartobiioxanthone A, isolated from stem bark of Artocarpus hirsutm.

[Para0012] Fig. 4 shows the Fourier transform Infra-red (FUR) spectrum of a and β pyranocycioartobiioxanthone A. isolated from stem bark, of Artocarpus hirsutm.

[Para0013] Fig. 5 shows the D:0 exchanged Ή NMR spectrum of a and β pyranocycioartobiioxanthone A. isolated from stem bark of Artocarpus hinufns

[Para0014] Figs. 6 and 6a show the Atmospheric pressure chemical ionization - Mass Spectrometer { APCI-MS) data of Artonine B, isolated from stem bark of Artocarpus hinufns.

[Para0015] Fig. 7 shows the ^! H NMR spectra of Artonine E„ isolated from stem bark of Artocarpus hirsuts. [Para0016] Fig,8 shows the ¹³ C NMR spectra of Artonine £\ isolated from stem bark of Artocarpm htrsnlns.

[Para0017] Fig. 9 shows the /one of inhibition of isolated compounds a and β pyranocycloartobiloxanthone A and Artonine E against P. acnes. A reference standard (Clindamycin) is included as control

[Para00I 8] Fig. 10 shows the Graphical representation of the zone of inhibition of compound a and β pyranocycloartobiloxanthone A and Artonine E against P. nates. A reference standard (Clindamycin) is included as control

DESCRIPTION OF THE MOST PREFERRED EMBODIMENTS

[Para00I 9] In the most preferred embodiment, the present invention discloses a method of isolating natural molecules from the bark of Artocarpm hirsntits, said method comprising steps of: a) (l ulling and drying the stem bark of Anocarpns hirsutw and pulverising to coarse powder b) Refluxing the stem bark, powder of step a with etbanol (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% s'/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 TI..C 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 a and β Pyranocycloartobiloxanthone A as represented in STR #1. in the ratio of 90-50: 10-50 and filtrate,

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

[Para0020] In a related embodiment, Pyranocycloartobiloxanthone A is present as a and β anomers in the ratio 70.30.

[Para0021] 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 a and β anomers in the ratio 90-50.10-50 (STR#1 ). More specifically, Pyranocycloartobiloxanthone A is present as a and β anomers in the ratio 70:30. In yet another preferred embodiment, the invention discloses a method of inhibitittg Propionibacterium acnes, said method comprising step of bringing into contact Propionibacterium acnes with effective concentration of Artonine E, isolated from the stem bark οΐ Artocarpus hirsutus. |Para0022| The specific examples included herein below illustrate the most preferred embodiments of the present: invention.

[Para0023] Example I: Isolation of bioactive molecules from stem bark of Artocarpus hirsutus

[Para0024] Collection of plant materials

[Para0025] The stem hark of Artocarpus htrsutus, was collected from Udupi district. Karnataka, India. All samples were authenticated by botanist and sample voucher was kept in herbarium (RD/HAR-AH/1 1 ). The stem bark was cut into small pieces and dried under shade. The dried materials were pulverized to coarse powder and stored m air tight containers.

[Para0026] Preparation of extracts

(Para0027) The stern bar k powder (3kg > 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 ethanoiic extract (yield: 138 g) was filtered and dried completely under vacuum before storing in air tight containers.

[Para0028] Analytical methods

[Para0029] Normal phase TLC was performed on pre-coated silica gel plates (Merck Specialities 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 Finnigau LCQ Advantage Max (Thermo, LAM 10234). ^l l l NMR {300 MHz) and ¹³ C NMR {75 MHz) spectra were recorded on VARIAN NMR spectrometer. Chemical shifts ίδ values) are reported in ppm (pans per million) with respect to TMS as internal standard, DMSO-d* was used as solvent. Column chromatography was performed over silica gel (mesh 60-120). Infra red spectra were recorded on Perkin Elmer FTIR Spectrometer.

[Para0030] Isolation of active molecules

[Para0031] 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 diehloromethane ((CH ₂ Cl ₂ ) ehtted with CH ₂ Cl ₂ and CH ₂ CL ₂ /Acetone and collected 80 fractions of 250 niL 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 Cl i;Cl; to provide Pyranoeyeloartobiloxanthone A as yellow powder (S I R #1 ). The filtrate was further chromatographed over silica gel and ehition with hexane-ethyl acetate (70:30) afforded Anonine E (STR #2) as yellow solid.

[Para0032] Extensive fractionation and purification of the ethanoiic extract of .4 rfocarpm hirsntits stem bark on silica gel lead to the isolation of two compounds, characterized as i) a xanthone derivative.. Pyrancycloartobiloxanthone A (STR#J ) and a fiavonoid, Artonine E (STR&2). Pynmocycloartobiloxamhone A was obtained as yellow powder with the melting point 268 - 270 °C (dec.). The Ή (Fig. 1) and ^K *C NMR spectra (Figs. 2, 2a and 2b) of pyranoeyeloartobiloxanthone A clearly demonstrate the occurrence of two isomers in the ratio of 70 and 30 and were assigned as two anomeric conformers. a (70%) and β (30%) constituted owing to the presence of carbohydrate moiety in the molecule. In consequences, other signals in ^! H (Fig, I ) and ¹³ C NMR (Figs. 2, 2a and 2b) spectra clearly established the ratio of the anomers to be 70:30.

(Para0033] The A PC I -MS (Figs.3 and 3a) of die pyranoeyeloartobiloxanthone A corresponds to the molecular ion peak in positive mode at m/z 451 .20 {M+HV and in negative mode at m/z 449.09 (M-H) ^. representing the molecular mass of the molecule with 450 The FOR spectrum (Fig. 4, Table I ) showed the presence of hydroxy! and conjugated carbonyi moieties with absorption at 33S7 and 1659 cm ^"1 , respectively.

[Para0034] Table 1: FTIR values for pyranocycioartobiloxanthone A

[Para0035] The solubility of the compound was relatively poor m most of the solvents except in DMSO and thus Ή NMR and ^! C NMR spectra were recorded in DMSCW6. In 'B NMR spectrum, the phenolic Oil (5-01 i » adjacent to the carbonyi was assigned from the downfield signal at 6 13.36 ppm {70%) and 13.33 ppm (30%) and these two signals appeared due lo the presence of the two coriformers a and ji in about 70.30 ratio The assignment of two doublets at ό 7.15 ppm ⁽ -30%) and 6.94 ppm ( '70%) was crucial in Ή NMR spectrum. Nonetheless the disappearance of these two signals in D20 exchanged ^! I I NMR spectrum (Fig. 5) ascertained thai 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-Η). Thus the signals at 6 7.15 ppm (d, -30%. β-ΟΗ, J = 7.5 Hz) and at 6.94 ppm id. •70%. ct-OH, J ^::: 4.2 Hz) were assigned corresponding to the anoinerie 14-011. Similarly, though the anomerie proton ( i 4-11) 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 δ 5.32 ppm (dd, -70%. 14-u-H, .1 - 4 2 Hz, 2 1 I-LEJ and another one as triplet at 4.87 ppm (t. -30%. 14-β- H. J = 7.8 Hz). The coupling interferences in 14-α/β-Η due to 14-OH was disappeared in D2G exchanged Ή NMR spectrum (Fig. 5) and signals were clearly appeared as doublets at δ 5.28 (d, -70%, 14-a-H. J = 1.2 Hz) and 4.81 ppm (d, -30%, 14-β-Η. J = 8.7 Hz). As expected the signals ratio -70:30 were reflected in the ¹ C NMR spectrum also, ft was observed that carbonyl (C-4) peak appeared at δ 179.2 { -70%} and 179.2 (-30%) ppm. Most importantly characteristic anomerie C-14 signal was noticed in two positions at 6 97.6 { -30%) and at 93.2 ( -70%) ppm which correspond to C- 14β and C-14cx anomer respectively. The presence of other 1haracteristic signals both in ^! H and ^{S %} C NMR spectra confirms the compound as a mixture of a and β-pyranocycloartobiloxanthone A in approximately 70:30 ratio (Table 2). The analytical data of pyranocycloartobiloxamhone A was comparable with that of pyranoeyeioartobiioxanthone A reported by Hasima el (2010), Two new xanthones from Artocarpus ohtitsits, J Asian Nat Prod Res, 12(2): 106- 1 12. However, the reported structure was established as single conibrmer whereas the instant invention reports this molecule as a mixture of two conformers (a and β). Notwithstanding the illustrative ratio of 70.30 for a mixture of a and p-pyranoeyeloartobiloxanthorie 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 m the ratio between u and β-pyranocycioartobiioxanthone A are envisioned and encompassed in this patent application. The Ή NMR and "C NMR spectra of a. and β-pyranoeycloartobiioxantone A is specified in table 2 with following data:

[Para0036[ « and β-pyranocycloartobiloxantone A: Yellow powder with melting point:

[Para0036] Table 2: Spectral data of a. and β-pyranocycloartobiloxantone A

[Para0037] Artonine B (STR#2) was isolated as yellow powder. The mass spectrum (APCI- MS) (Fig.6 and 6a) showed the molecular ion peak in positive mode at m/z 437 09 (M÷H) and in negative mode 435.05 (M-I I ) ^" corresponding to the molecular mass of compound 2 as 436. in the Ή NMR spectrum (Fig.7), three singlets at o 6.68, 6.46 and 6.21 correspond to three aromatic protons of ( i 1-3', I l-6'and 116) whereas the signal at o ^* 13.2 ppm represents the chelated phenolic proton (5-OH). The presence of prenyl side chain was con tinned from its the corresponding characteristic signals at 6 5.05 (t. i H. .1=6.9 Hz, H- i 0). 3.03 (d. 2H, J= 6.9 Hz, 11-9), 1 56 (s, ?H, 11-13) and 1.41 (s, 3H, H-12). Similarly, signals at δ 6.52 ( I H d, J-9.9 Hz, H-14 ⁾ . δ 5.69 ( I H. d. J=9.9 Hz, H-15). δ 1.41 ⁽ 6H, s, H-17 and H- 18) ascertained the presence of 2,3-dimethylpyran ring. The presence of characteristic signals in ^{i ¾} C NMR spectrum (Fig. 8) also confirms the structure. It was noticed that the analytical values were correlated well with Srituiarak et al, (2010) New 2-Arylbenzofurans from the Root Bark of Arfocarpm lakoocha. Molecules, 15:6548-6558, and consequently was identified as Artonine E with the following spectral data.

[Para0038] Artonine E: Yellow powder with melting point: 242-246 ^C C; 1R (KBr) v,.. _3N 3426,

I6h 29.6 (C-17), 27.7 (C-18). 25.5 (C- 13), 23.7 (C-9). 17.4 (C-12) (Fig. 8); APCl-MS m/z 4.37.09 (Μ ΗΓ ) and 435.05 (Μ-Ι-Γ), (C^H^O _? requires 436.4539) (Fig.6 and 6a)

[Para0039| Example II: Antiacne potential of pyranoevcioartobiioxantone A and Artonine E

[Para0040] /// vitro ami-acne activity of isolated compounds 1 and 2 from the stem bark extract of Artocmpus hinntns were evaluated against acne causing bacterium, Propionibacteriitm nates. 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.

[Para0041] Microorganisms: Acne causing bacterium Propionibuaerium acnes (ATCC i 1827) was procured from American type culture collection Rockvilie. USA.

|Para0042| Media: Reinforced Clostridial Agar (Hi Media. M l 54) and Reinforced Clostridial broth (Hi Media: M443) were used in the experiments.

[Para0043] Determination of antibacterial activity

|Para0044] The antibacterial activities of isolated compounds were performed by agar well diffusion method. The samples were dissolved in DM SO to obtain a concentration in the range of 0 5 - 100 mg/mL. Propionibacteriitm acnes was cultured on Reinforced Clostridial Agar ( RCA) M i 54 procured from Hi Media and incubated at 37 °C for 48 h in an anaerobic chamber providing gas mixture containing S0% nitrogen, 10% carbon dioxide and 10% hydrogen. The bacterial culture was suspended in sterile normal saline and adjusted to 1 .0x 108 CRJ/mL (CLSL M02-A1 I ; Vol.32 No.l ). 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 7mm 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 /one of inhibition around the wells were measured and recorded.

[Para0045| The zone of inhibition of isolated compounds: pyranocycloartobiloxantone A and Artonine Έ around the wells displayed good inhibition The inhibition was detected in ail tested concentrations (10 - 1.25 pg/mL) (Fig. 9 and Fig. 10).

[Para0046] Determination of Minimum Inhibitory Concentration (MIC) |Para0G47] The minimum inhibitory concentrations (MIC) of the isolated compounds against aaias were determined by broth micro dilution method. Two-fold dilutions of the isolated compounds were prepared m Reinforced Clostridial broth (RCB) to obtain concentrations in the range of0 .1-2000 pg/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 1 x 10 ⁵ CFU (CLSf, M l 1-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. aaies was considered as minimum inhibitory concentration (MIC).

[Para0048] It was observed that the isolated compounds exhibited highly potent anti-acne activity against P. acnes with MIC values 2 pg/mL each (Table 3).

[Para0049] Table 3: MIC of pyrauocycloartobiloxantone A and Artoninc E against P. acnes

[Para0050] To summarise, fractionation of ethanolic extract from the stem bark of A. hmnfits yielded a mixture of a xanthoue derivative. Pyranocycloartobiloxantlione A constituting a and p conformers in almost 70:30 ratio. The conformers were confirmed from its spectral analysis and reported for the first time. Artonine B was the second molecule isolated from the stem bark and both the compounds exhibited significant anti-acne activity with MIC value of 2 ug/mL each and comparable with antibiotic Clindamycin (M1C ^::: 0.03 Hg/mL).

[Para0051] 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.