Title of Invention	AN ANTI BACTERIAL COMPOSITION
Abstract	The invention provides an anti-bacterial composition comprising an effective amount of 3,5-Dihydroxy-4-pent-4'-enoyl-l'-oxymethyl benzoic acid (oenostacin) isolated from Oenothera biennis together with therapeutically acceptable conventional additives, said composition being useful for the treatment of diseases caused by Streptococci and Staphylococci.

Full Text

This invention relates to an anti-bacterial composition . Field of Invention This invention relates to the use of a compound 3,5-Dihydroxy-4-pent-4'-enoyl-l'-oxymethyl benzoic acid (Oenostacin) isolated from the roots of the plant Oenothera biennis. The compound has been identified as an effective antibacterial agent active against bacteria such as staphylocoed, streptococci and the like. The invention also provides pharmaceutical compositions comprising 3,5-Dihydroxy-4-pent-4'-enoyl-l'-oxymethyl benzoic acid (Oenostacin) useful for treatment of skin infections such as endocarditis in humans, caused by Staphylococcus epidermidis j Background of invention The antimicrobial era has reached a point where the emergence of resistant microbes is accelerating while the pace of discovery of new drugs seems decelerating (Science 157:1064-IQfft^ Uiirt* wentiy harfy any new drug or combination arrived in time to overcome t$ks problem of resistance. Few novel chemical entities have been brought to tte arafat dto»g Ae past decwfe but mostly the new drugs are derivatives of older cetepownds. Some of these have increased activity or a broader spectrum of activity or improved pharmacological properties but can only temporarily overcome the problem of resistance. There is need to develop new class of antimicrobial compounds from different sources preferably plants. This need is particularly critical for infections caused during seemingly uncomplicated hospital treatment procedures such as catheterization, insertion of intrauterine contraceptive devices, intravenous injections etc. Inflammation of the endocardium, i.e., the tissue lining of the cavities of the heart is called endocarditis. Infective (infectious) endocarditis may be due to infection by a range of micro-organisms such as Haemophilus sp., Staphylococcus aureus, S.epidermidis (especially in patients with prosthetic valves), Streptococcus faecalis, Neisseria gonorrhae, Candida etc. Infective endocarditis may be acute (e.g, when due to streptococci or gonococci) or subacute when due to viridans streptococci or fungi. Infection occurs via blood stream and organisms may gain access to the blood stream during dental treatment, catheterization or insertion of intrauterine contraceptive devices, intravenous injections etc. The symptoms are fever, malaise, heart murmurs, weight loss, clubbing of fingertips, embolism; late symptoms of subacute bacterial endocarditis include vasculitis, petechial rash, osier's node. Damage to heart valves may lead to heart failure. Endocarditis may also occur as a complication of other infectious diseases. Oenothera biennis (Onagraceae) is a genus of herbs and undershrubs distributed mainly in temperate America together with some species occurring in tropics. Some of the species including O. biennis has been introduced into Indian Gardens (J.Med.Arom Plant Set., 20:1998,432). The oil from seeds of O. biennis (Evening Primrose) is known to be a rich dietary source of r-linolenic acid required for the formation of prostaglandins and related hormones (Z Phytother, 4,1983,531). The seeds are reported to contain fatty acids (J.Am Oil Chem Soc, 61,1984,540) and sterols (Riv Ital Sastanze Grasse, 53, 1976,25) while the leaves contain flavonoids (Phytochemistry, 6,1967,317) and Oenothein A (Chem Pharm Bull,39,1991,1157). However, no compounds have been reported from the routs of G-.hienms. Since the applicants observed specific activity in the root extracts, a systematic activity directed fractionation was followed to isolate pure compounds. This systemic activity directed fractionation of root extracts resulted* in the identification of ten compounds, four of which are novel and reported in the Indian Journal of Chemistry, Vol. 38B, 1999, pages 705-708. One of the compounds identified and reported in the said publication was a chemical entity isolated from the roots of the plant Oenothera biennis (commonly called evening primrose) and this was characterized as 3,5-Dihydroxy-4-pent-4'-enoyl-l'-oxymethyl benzoic acid. Since the compound is obtained from Oenothera biennis, it has been named as 'Oenostacin\ This chemical compound has the molecular formula CnH^Oe and its structural formula is shown in Figs 1 and Fig.2. This compound has not been isolated before from any other source whether by synthetic or other means. Further, the activity of this compound has also not been identified nor detected so far. The applicants accordingly, screened this compound and found to their surprise that it exhibited the potential of an antibacterial drug and found to be specifically active against streptococci and staphylococci, especially Staphylococcus epidermidis, which causes skin infections/endocarditis in humans. Hence, the present invention provides a antibiotic principle from plant O. biennis for use against S. epidermidis infection. Objects of the invention The main object of the invention is to provide Oenostacin (3,5-Dihydroxy-4-pent-4'-enoyl-l'-oxymethyl benzoic acid) obtained from the plant Oenothera biennis, which is useful as anti-bacterial agent. Another object is to provide anti-bacterial pharmaceutical compositions comprising 3,5-Dihydroxy-4-pent-4'-enoyl-r-oxymethyl benzoic acid together with conventional additives and useful for treatment of infections caused by bacteria. Stitt another object of the invention is to provide methods for the treatment of bacterial infections, especially diseases caused by bacteria. Summary Accordingly, the invention provides novel compositions comprising an effective amount of Oenostacin (3,5-Dihydroxy-4-pent-4'-enoyl-r-oxymethyl benzoic acid) and useful in the treatment of skin infections caused by bacteria such as Staphylococci and Streptococci. The invention also provides methods for treatment of skin infections using the novel composition/pharmaceutical compositions. Brief description of the accompanying drawings: In the accompanying drawings, Figure 1 represents the structure of Oenostacin Figure 2 represents the IR-Spectral structure of Oenostacin Detailed description of invention Accordingly, the invention provides novel antibacterial composition comprising an effective amount of Oenostacin (3,5-Dihydroxy-4-pent-4'-enoyl-l'-oxymethyl benzoic acid) obtained from Oenothera biennis together with therapeutically acceptable additives and useful for treatment of diseases caused by bacteria in mammals. In an embodiment, the 3,5-Dihydroxy-4-pent-4'-enoyl-l'-oxymethyl benzoic acid (oenostacin) is an extract obtained from the plant Oenothera biennis In another embodiment, the concentration of oenostacin in the composition is in the range of l-200u£/ml. In yet another embodiment, the concentration of oenostacin is in the range of 10-90%. In still another embodiment, the anti-bacterial composition is effective against bacteria selected from streptococci, staphylococci and Pseudomonas aeruginosa. to yet another embodiment, the antibacterial composition may be used for treatment of bacterial »isetisn»eaa8ed by streptococci and staphylococci. In still another embodiment, the antibacterial composition may be used for the treatment of endocarditis in humans. In another embodiment the antibacterial composition may be used for the treatment of infections caused by S. epidennidis. The invention also provides methods for the treatment of bacterial infections, said method comprising the steps of administering an effective amount of 3,5-dihydroxy-4-pent-4'-enoyl-r-oxymethyl benzoic acid to a subject in need thereof. In an embodiment, the applicants have identified now that the compound 3,5-Dihydroxy-4-pent-4'-enoyl-l'-oxymethyl benzoic acid is active against Staphylococcus epidermis. In still another embodiment, the concentration of oenostacin is in the range of 1-200ug/ml in the pharmaceutical composition. The invention also relates to use of Oenostacin (3,5-Dihydroxy-4-pent-4'-enoyl-l'-oxymethyl benzoic acid) for treatment of bacterial infections caused by Streptococci and Staphylococci. Acute endocarditis is an inflammatory disease of the endocardium i.e., the internal lining of the human heart. It is caused by bacteria such as Staphylococci and gonococoi. Among Staphylococci, S.epidermidis is one of the major etiological agents of this disease. The infections occur mainly in-patients with prosthetic valves. In the screening programme aimed at detecting biomolecules from plant sources, which can specifically ac£ against S. epidermidis the applicants found that the ethanolic extract derived from the roots of a plant catted O! biennis contained a compound 3,5-Dihydroxy-4-pent-4*-enoyf-r-ox procedure adopted in the bioactivity-guided fractionation of ethanolic extract is depicted in the following flow chart: ACTIVITY GUIDED FRACTIQNATION OF Oenothera biennis Compound No. CIM 791 found highly active (3,5-Dihydroxy-4-pent-4'-enoyl-l'-oxymethyl benzoic acid) Each extract and fraction or sub fraction and or isolated pure compound was simultaneously tested for its antibacterial property using Sepidermidis culture in disc diffusion assay. Silica gel chromatography of the ethyl acetate fractions of the methanol extractives of the root yielded compounds 7-10. One compound CIM791 had in its IR spectrum, bands for COOH (1692 cm'1), double bond (1612 cm'1), COOCH3(1730 cm'1), PhOH (3512 cm"1). It also showed UV maxima at 215 and 278 nm. An [M+] ion in the mass spectrum of 3 at m/z 266 was in agreement with the molecular formula C13H14O6. The further identification properties and the NMR spectral data of this compound is described in Indian J Chem., 38B, 1999, 705. Once the bioactive pure compound was obtained the minimal inhibitory concentration, which is a measure of, the growth inhibitory potential of the compound was determined against 5". epidermidis by two-fold broth dilution technique. The invention is further explained by the following examples and should not be construed to limit the scope of the invention. EXAMPLE-1 Extraction of Oenostacin The air-dried and powdered roots (4.7kg) of O. biennis were extracted with MeOH (7x8.5L) and the combined extract concentrated in vacua to 500 mL. Water was added and the extract then fractionated successively into n-hexane (5x600mL,65g); EtOAc (5x600mL,52g) and n-BuOH (5x600mL,41g). The MeOH extract and the various solvent fractions formed the initial material from which bioactivity guided fractionation was performed. EXAMPLE Each of the solvent fractions obtained from the powdered roots of O. biennis were tested for their antibacterial property. This was performed by single disc diffusion assay as per Bauer et al., 1966, (American Journal of Clinical Pathology 45: 493-496) with slight modifications. The discs were prepared (5 mm diameter made of Whatman#3 filter paper) by impregnating 8ul of test compound and placing them on pre-inoculated agar surface. A disc containing only the solvent was used as the control. As evident from the table-1 the EtOAc fraction showed significant antibacterial activity against Staphylococcus epidermidis and Streptococcus mutans. This indicated the presence of antibacterial substance in the EtOAc fraction of O.biennis roots. These results formed the basis of further experimentation. Table-1 Antibacterial activity of solvent extracts from roots ofO.biennis. (Table Removed) EXAMPLE-3 Tests of anti-bacterial activity: TQ isolate Hie antibacterial principle from the Et€)&c fraction, a portion (19g) of this fraction wa* d»omategt»pfaett m a- cotomn over stlica get (SQflg), eluting with varying prorjettroiw of CHCL? and Me€Wf to afford compounds- 7-10. In each case lOOmL fractions collected were monitored by TLC. Compound 10 (CIM 791) was identified as 3,5-Dihydroxy-4-pent-4'-enoyl-l'-oxymethyl benzoic acid based on NMR spectral data mentioned in Indian J Chem., 38B, 1999, 705. Table-2 indicates that the EtOAc fraction-3 & 4 possessed the expected antibacterial activity against the test bacterial strains. The EtOAc fraction-4 exhibited growth inhibitory activity against Pseudomonas aeruginosa indicating the presence of a specific chemical compound in this fraction. Table-2 Antibacterial activity of different fractions of EtOAc extract of roots of Obiennis. (Table Removed) EXAMPLE-4 In order to identify the antibacterial chemical entities present in the EtOAc fraction 3&4 of O.biennis roots the fractions were further run on silica gel column chromatography again eluting with varying proportions of Chloroform and MeOH. Aliquots of eluate were run on TLC to check for the desired purity of the compound. The pure compounds thus obtained were analyzed for their antibacterial properties by two-fold broth dilution method to determine the minimal inhibitory concentration (MIC). The results indicated (Table-3) that one compound CIM791 had strong antibacterial activity against Staphylococcus epidermidis since the MIC was found to be 31.5 (ig/ml. Yet another compound CIM-790 was found to possess low inhibitory activity against Streptococcus mutans (MIC= 625fag/ml). The compounds CIM791 and 790 were identified as 3,5-Dihydroxy-4-pent-4'-enoyl-l'-oxymethyl benzoic acid and as gallic acid respectively using NMR specteal'&te; Tie structure of CIM 791 is depicted in Figs. 1 & 2. Table-3 The minimal inhibitory concentration of Oenostacin against bacterial strains. (Table Removed) We Claim: 1. An antibacterial composition useful for treatment of diseases caused by bacteria in mammals, said composition characterized in that it comprises an effective amount of Oenostacin (3,5-Dihydroxy-4-pent-4'-enoyl-V-oxymethl benzoic acid) obtained from Oenothera biennis together with conventional therapeutically acceptable additives wherein the concentration of oenostacin is in the range of 10-90% and the rest therapeutically acceptable additives. 2. An anti-bacterial composition useful for treatment of diseases caused by bacteria in mammals substantially as herein described with reference to the examples.

Documents:

1195-del-2001-abstract.pdf

1195-del-2001-Claims-(11-07-2007).pdf

1195-del-2001-claims.pdf

1195-del-2001-correspondence-others.pdf

1195-del-2001-correspondence-po.pdf

1195-del-2001-description (complete).pdf

1195-del-2001-drawings.pdf

1195-del-2001-form-18.pdf

1195-del-2001-form-2.pdf

1195-del-2001-form-3.pdf