[0001] The present invention relates to the antibacterial proficiency of an isoprenyl pyranoflavone natural scaffold, artocarpin, towards ESKAP pathogens, especially against MDR strains of S. aureus. More particularly, the present invention relates to a synergistic combination of isoprenyl pyranoflavone artocarpin, and an antibiotic drug such as gentamycin. The synergistic combinations of the invention are particularly effective in the treatment of nosocomial infection causing multi drug resistant bacterial strains. The synergistic combinations of the invention are particularly effective in the treatment of infections caused by multi-drug resistant strains of Staphylococcus aureus.

BACKGROUND OF THE INVENTION

[0002] Quite a few natural products are widely used as potential drugs against numerous diseases. Paclitaxel isolated from Taxus brevifolia, Camptothecin isolated from Camptotheca acuminata are some leading examples. In this context, several natural entities can turn up into potential drugs and thus we can further reduce the cost and side effects. In addition, the combinations with the existing drugs can also make promising leads. Artocarpus species are well-recognized for their flavonoid contents. Isoprenyl and geranyl flavonoids are the major components present reported to have numerous medicinal properties. Antimicrobial properties of these are hardly explored including their activity against MDR strains and their synergy studies with the existing drugs.

[0003] Reference is made to Kojima Hiroyuki et al. Jack fruit extract-containing antibacterial and antiseptic agents and cosmetics, JP3501855B2, 1994, a Japanese patent, disclosed the activity of jackfruit extract as an antibacterial, antiseptic, and as skin cosmetic for acne improvement active against Staphylococcus aureus, Bacillus subtilis, acne bacteria, etc. and there is a strong activity against dandruff-causative bacteria also.

[0004] Reference is made to Hiroshi Ando et al, Antimicrobial agent and preservative and cosmetic containing Artocarpin or/and Sophoraflavanone G, JPH0873372A, 1994, another Japanese patent disclosed the preparation of a new antimicrobial agent from Artocarpus genus and Sophora genus.

[0005] Artocarpin along with some standard drugs were tested against methicillin-resistant S. aureus (MRSA) as well as the Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli. Reference is made to Abdi W. S et al, Synergistic Effect of Artocarpin on antibacterial activity of some antibiotics against methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli, Pharmaceutical Biology, 2016, 54.4, 686-691. Time kill assays of the combinations suggest that they are well active against Gram-negative bacteria also.

[0006] Reference is made to Abdi W. S et al, Antibacterial assay-guided isolation of active compounds from Artocarpus heterophyllus heartwood, Pharmaceutical Biology, 2015, 53.11, 1608-1613, which reports isolation of compounds from Artocarpus heterophyllus heartwood and their antibacterial activity was reported by examining their MIC and MBC values against different bacteria like S. mutans, S. pyogenes, B. subtilis, S. aureus, and S. epidermidis. Artocarpin only showed inhibitory activity against Pseudomonas aeruginosa, which is a gram negative bacteria.

[0007] Screening of Thai medicinal plants for various antimicrobial and whitening agents have been of huge research interest. Anti-tyrosinase and antimicrobial properties of some selected Thai medicinal Plants (Dej-adisai Sukanya et al, Anti-tyrosinase and antimicrobial activities from Thai medicinal Plants, Archives of Pharmacal Research, 2014, 37.4, 473-483) were reported. Preliminary screening for antimicrobial activity was done by agar disc diffusion and broth micro-dilution methods, where the isolated compounds artocarpin and cudraflavone C showed the potential of antibacterial activity against S. aureus, S. epidermidis, and P. acnes. [0008] Isolation of Artocarpin from A. hirsutus. and their activity against S. aureus ATCC 29213 was reported in “A study for isolation of new active molecules from A. hirsutus Lam. Moraceae”, University of Mysore, 2018, http://hdl.handle.net/10603/251009.

[0009] It is necessary to develop antibiotics that can overcome the limitations of the conventional antibacterial agents and rather work against both types of bacteria. Since the bacteria is acquiring resistance towards the available antibiotics, it is essential to develop antibacterial drugs that are more effective. The above-mentioned prior arts didn’t provide any scope towards the potentials of the molecule on inhibiting a biomass of bacteria, the in-vivo effects, the scope of antibiotic resistance, post antibiotic effect, etc.

[0010] Based upon the foregoing there has been found a need to develop a potent antibacterial agent, especially from natural product, which can (a) Effectively inhibit the biomass or biofilm, which are more antibiotic resistant; (b) With less clinical dosage; (c) High antibiotic resistance towards bacteria after prolonged treatment; and (d) High synergy with standard antibiotics. [0011] Advantageously the present invention confers the detailed investigation on the above- mentioned aspects, which can lead to the development of an efficient antibacterial agent. OBJECTIVES OF THE INVENTION

[0012] Since the bacteria is acquiring resistance towards all the available antibiotics, it is essential to develop antibiotics that are more effective. Natural products embrace wide variety of chemical compounds with numerous pharmacological activity.

[0013] The principal objective of the invention is to explore the antibacterial efficacy of the combinations of a natural chemical entity, especially an isoprenyl flavonoid molecule Artocarpin (AH-5) with standard drugs, against different multi drug-resistant strains of gram positive bacteria S. aureus. [0014] Another objective of the invention is to provide a composition comprising therapeutically effective amount of isoprenyl flavonoid molecule Artocarpin and gentamycin. [0015] Another objective is to provide the confirmation of the antibacterial potential of the synergistic combination of the isoprenyl flavonoid with gentamycin by studying its anti biofilm efficiency and time kill kinetics. SUMMARY OF INVENTION

[0016] Accordingly the present invention provides a synergistic antibacterial composition for multi-drug resistant S. aureus infections comprising,

(a) a flavonoid Artocarpin of formula I and

I

(b) antibacterial drug.

[0017] In an embodiment of the present invention the antibacterial drugs in the synergistic antibacterial composition is gentamycin.

[0018] In an embodiment of the present invention the ratio of artocarpin to gentamycin in the synergistic antibacterial composition is 1 : 0.125.

[0019] As a preferred embodiment of the present invention the synergistic antibacterial composition comprises therapeutically effective amount of a flavonoid Artocarpin of formula

I,

gentamycin and pharmaceutically acceptable excipient, wherein the ratio of artocarpin to gentamycin is 1 : 0.125.

[0020] In an embodiment of the present invention the pharmaceutically acceptable excipient is selected from the group consisting of gelatin, stearic acid, parabens, stabilizers like sorbitol, liquid non-volatile non-aqueous materials mainly glycols such as propylene glycol.

[0021] In an embodiment of the present invention the MIC of the composition is in the range of 0.125 to 2 pg/mL.

[0022] In an embodiment of the present invention the synergistic antibacterial composition, has a FIC index of 0.135 pg/mL.

[0023] In an embodiment of the present invention the synergistic antibacterial composition acts as a potent agent against different multi drug-resistant and susceptible strains of S. aureus selected from MRSA, VRSA, and MSSA.

[0024] In an embodiment of the present invention the synergistic antibacterial composition reduces the bacterial biofilm by 9.7%, as compared to the untreated.

[0025] In an embodiment of the present invention the synergistic antibacterial composition is bactericidal in nature and effective in killing the bacteria up to zero CFU/mL in 24h.

[0026] The other features, objects and advantages of the present invention will become more apparent from the detailed description set forth below when taken in conjunction with the drawings.

[0027] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 illustrates biofilm inhibition assay for detecting the action of the synergistic combination towards the biofilm that is more resistant towards antibiotics, in accordance with an implementation of the present disclosure.

[0029] Figure 2 illustrates time-kill kinetic studies of the above-mentioned compound to find whether the compound is bacteriostatic or bactericidal, in accordance with an implementation of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION ABBREVIATIONS USED

ESKAP - Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa

NMR - Nuclear Magnetic Resonance

Hz - Hertz

MHz - Megahertz

HRMS - High Resolution Mass Spectroscopy MDR - Multi Drug Resistant

MRSA - Methicillin Resistant Staphylococcus aureus

MSSA - Methicillin Susceptible Staphylococcus aureus

VRSA - Vancomycin Resistant Staphylococcus aureus

MIC - Minimum Inhibitory Concentration

FIC - Fractional Inhibitory Concentration

CFU - Colony Forming Unit

PAE - Post Antibiotic Effect Assay

[0030] For a better understanding of the invention, a detailed description of the preferred embodiments of the present invention will now be explained with reference to the accompanying tables and drawings. It should be understood that the disclosed examples are merely exemplary of the invention, which may be embodied in various forms. Therefore, the details disclosed herein are not to be interpreted as limiting but merely as the basis for the claims and as a basis for teaching one skilled in the art how to make or use the invention. [0031] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0032] The articles "a", "an" and "the" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0033] The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as "consists of only".

[0034] Throughout this specification, unless the context requires otherwise the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0035] Before describing the various aspects and details of the present invention it will be useful to describe the context in which the invention is used. Consequently, the conventional way of drug discovery is to synthesis complex chemical structures by hectic synthetic steps and using hazardous chemicals, which may have many side effects. In addition, the resistance acquired by the bacterial strains towards the available antibiotics demands more drug candidates that are effective in resisting the MDR class of bacteria.

[0036] The present invention introduces a potential antibacterial natural compound Artocarpin (AH-5) against MDR strains of S. aureus. There is also provided a synergistic combination of the compound with standard drugs gentamycin in 1 : 0.125 ratio that can be utilized as another powerful drug candidates against S. aureus ATCC 29213.

[0037] The present development discloses the antibacterial efficacy of the combination of a natural isoprenyl flavonoid with standard drug gentamycin, against different multi drug- resistant strains of gram-positive bacteria S. aureus.

[0038] The very first aspect of the invention provides the antibacterial efficacy of the synergistic combination of a natural isoprenyl flavonoid Artocarpin (AH-5) represented by general formula I, isolated from the stem bark of Artocarpus hirsutus Lam. and a standard drug gentamycin. I

[0039] Another embodiment of the invention provides the synergistic combination studies of the mentioned compound with other stereotyped drugs such as Ceftazidime, Daptomycin, Gentamycin, Levofloxacin, Linezolid, Meropenem, Minocycline, Rifampicin, Vancomycin etc. using checkerboard assay and the analysis of Fractional Inhibitory Concentration (FIC) index to find out the best combinations.

[0040] A further embodiment of the present invention discloses the in-vitro antibacterial activity of the combination of artocarpin and gentamycin against different multi drug-resistant and susceptible strains of gram-positive bacteria Staphylococcus aureus.

[0041] Another embodiment of the present invention discloses a pharmaceutical composition, comprising therapeutically effective amount of a flavonoid Artocarpin of formula I as disclosed herein with gentamycin and pharmaceutically acceptable excipient, wherein the ratio of artocarpin to gentamycin is 1 : 0.125.

[0042] Yet another embodiment of the present invention discloses a pharmaceutical composition, comprising therapeutically effective amount of a flavonoid Artocarpin of formula I as disclosed herein with gentamycin and pharmaceutically acceptable excipient selected from the group consisting of gelatin, stearic acid, parabens, stabilizers like sorbitol, liquid non volatile non-aqueous materials, glycols, and propylene glycol, wherein the ratio of artocarpin to gentamycin is 1 : 0.125.

[0043] Further embodiment of the present invention discloses a composition comprising a flavonoid Artocarpin of formula I as disclosed herein with gentamycin and pharmaceutically acceptable excipient, wherein the ratio of artocarpin to gentamycin is 1 : 0.125 and MIC of the composition is in the range of 0.125 to 2 pg/mL.

[0044] Another embodiment of the present invention discloses a composition comprising a flavonoid Artocarpin of formula I as disclosed herein with gentamycin, wherein the ratio of artocarpin to gentamycin is 1 : 0.125 and the composition has a FIC index of 0.135 pg/mL. [0045] One another embodiment of the present invention discloses a composition comprising a flavonoid Artocarpin of formula I as disclosed herein with gentamycin, wherein the composition acts as a potent agent against different multi drug-resistant and susceptible strains of S. aureus selected from MRSA, VRSA, and MSSA.

[0046] Further embodiment of the present invention discloses a composition comprising a flavonoid Artocarpin of formula I as disclosed herein with gentamycin and pharmaceutically acceptable excipient, wherein the composition acts as a potent agent against different multi drug-resistant and susceptible strains of S. aureus selected from MRSA, VRSA, and MSSA. [0047] Further another embodiment of the present invention discloses a composition comprising a flavonoid Artocarpin of formula I as disclosed herein with gentamycin, wherein the composition reduces the bacterial biofilm by 9.7%.

[0048] Further embodiment of the present invention discloses a composition comprising a flavonoid Artocarpin of formula I as disclosed herein with gentamycin and pharmaceutically acceptable excipient, wherein the composition reduces the bacterial biofilm by 9.7%.

[0049] Yet another embodiment of the present invention discloses a composition comprising a flavonoid Artocarpin of formula I as disclosed herein with gentamycin, wherein the composition is bactericidal in nature and effective in killing the bacteria up to zero CFU/mL in 24 h.

[0050] Further embodiment of the present invention discloses a composition comprising a flavonoid Artocarpin of formula I as disclosed herein with gentamycin and pharmaceutically acceptable excipient, wherein the composition is bactericidal in nature and effective in killing the bacteria up to zero CFU/mL in 24 h.

[0051] Further embodiment of the present invention discloses a method of treating or preventing a disease or a condition associated with S. aureus, the method comprising administering the composition as disclosed herein to a subject in need thereof.

[0052] Another embodiment of the present invention discloses a method of treating or preventing a disease or a condition associated with S. aureus, the method comprising administering the composition as disclosed herein to a subject in need thereof, wherein the disease or condition is selected from.

[0053] Yet another embodiment of the present invention discloses a composition as disclosed herein for use in treating or preventing a bacterial infection caused by S. aureus.

[0054] Another embodiment of the present invention provides a composition as disclosed herein for use in inhibiting or killing the growth of a microorganism selected from the group consisting of bacteria, virus, fungi and protozoa, wherein the microorganism is S. aureus. [0055] Yet another embodiment of the present invention provides a composition as disclosed herein for use as a medicament.

[0056] Yet another embodiment of the present invention provides use of the composition as disclosed herein for the manufacture of a medicament for treating bacterial infection caused by S. aureus. [0057] Another embodiment of the present invention provides the biofilm inhibition activity of the disclosed combination against complex bacterial communities, which are adaptively resistant to antibiotic treatment.

[0058] Further aspect of the invention provides the time-kill kinetics of the above-mentioned synergy combination. The study aims to explore the bactericidal properties of the combinations.

EXAMPLES

[0059] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices, and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may apply.

[0060] The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention. [0061] According to a preferred embodiment of the invention, it establishes the activity of the combination of a natural chemical entity with a stereotyped antibacterial drug gentamycin against deadly gram-positive bacteria, the process comprising the steps of: (a) Checker board assay to find out the best combination of artocarpin with standard drugs in 1 : 0.125 ratio, in inhibiting the antibacterial activity of MDR S. aureus; (b) In-vitro activity of the combination of artocarpin with gentamycin against methicillin and vancomycin resistant strains of S. aureus; (c) Biofilm inhibition assay of the combination to find the activity against highly resistant bacterial biofilm; and (d) Time kill kinetic assay of the combination to find whether the compound is bacteriostatic or bactericidal.

[0062] For a better understanding of the invention, the present invention will now be explained with reference to the accompanying tables and drawings.

Determination of synergy with FDA approved drugs [0063] Checkerboard method was used to determine synergy between compound and the antibiotics that included Ceftazidime, Daptomycin, Gentamycin, Levofloxacin, Linezolid, Meropenem, Minocycline, Rifampicin, Vancomycin etc. against a panel of MRSA and VRSA strains. The results showed that the compound is in good synergy with gentamycin with an FIC index of 0.135 pg/mL, where AH-5 and gentamycin have MIC values 2 and 0.5 pg/mL respectively.

In-vitro antibacterial analysis

[0064] The minimum inhibitory concentration (MIC) was determined by performing antibiotic susceptibility testing on the compound in accordance with the standard CLSI (clinical and laboratory standards institute) guidelines. The in-vitro antibacterial analysis of the disclosed compound showed efficacy against S. aureus with a MIC of 2 pg/mL. To determine their spectrum of activity against multiple strains of MDR S. aureus, the MIC assay of the combination of AH-5 and Gentamycin in 1:0.125 ratio was performed against various well- defined and characterized clinical strains of MRSA and VRSA. Levofloxacin, Meropenem, and Vancomycin were used as reference standards. The MIC of the combination found far better than the individuals within a range of 0.125 - 2 pg/mL.

Biofilm inhibition assay

[0065] It has been demonstrated that bacteria form a biofilm under certain stress conditions to protect themselves, often leading to therapeutic failure. Incidentally, most of the approved drugs have very limited activity against biofilm protected pathogens, thus it is imperative to determine the anti-biofilm activity of molecules in drug development. The disclosed synergistic combination reduced the biofilm by 9.7%, as compared to the untreated, which is far better than the existing drugs levofloxacin IX MIC (6.8%) and Vancomycin IX MIC (6.3%).

Time kill kinetic assay

[0066] The bactericidal and bacteriostatic activities were assessed by the time-kill method. Kill curves were constructed by counting the colonies from plates and plotting the CFU/mL of surviving bacteria at each time point in the presence and absence of compound. IX MIC combination of both AH-5 and gentamycin showed excellent bactericidal property, where it reduces the CFU/mL to zero in 24 h of treatment, proving its concentration-dependent bactericidal property.

Example 1

Isolation of Artocarpin [0067] The stem bark of Artocarpus hirsutus Lam. was collected from the CSIR-National Institute for Interdisciplinary Science and Technology (NIIST) campus, Pappanamcode, Thiruvananthapuram District, Kerala, India. Artocarpin, which is an isoprenyl flavonoid, was isolated from the stem bark of Artocarpus hirsutus Lam. by the solvent extraction method using acetone as solvent, which was further purified by silica gel column chromatographic method. The compound was well characterized by spectroscopic analysis.

[0068] *H NMR (500 MHz, CDCb, TMS) : d 13.98 (brs, 1H, OH), 8.83 (brs, 2H, OH), 7.22 (d, 7=8.0 Hz, 1H), 6.76-6.71 (m, 1H), 6.61 (dd, 7i=16.5 Hz, 7 ₂ =1.0 Hz 1H), 6.58 (d, 7=2.0 Hz, 1H),6.56 (s, 1H), 6.53 (dd, 7i=8.0 Hz, 7 ₂ =2.0 Hz 1H), 5.15-5.12 (m, 1H), 3.97 (s, 3H, -OCH ), 3.14 (d, 7=7.0 Hz, 2H), 2.48-2.41 (m, 1H), 1.58 (d, 7=1.0 Hz, 3H), 1.44 (d, 7=2.5 Hz, 3H), 1.11 (s, 3H), 1.09 (s, 3H) ppm.; ¹³ C NMR (125 MHz, CDCb, TMS): d 182.4, 163.0, 161.6, 160.7, 159.0, 156.6, 156.4, 141.3, 131.4, 131.3, 121.7, 121.0, 116.1, 112.0, 108.9, 107.2, 104.7, 103.0, 89.6, 55.7, 33.1, 24.9, 23.8, 22.3, 16.8 ppm.; HRMS (ESI): m/z Calcd for C ₂₅ H ₂₆ 0 ₅ (M+H) ⁺ : 437.1964; Found : 437.1962.

Example 2

Synergy studies with FDA approved standard drugs

[0069] Checkerboard method was used to determine synergy between compound and the antibiotics that included Ceftazidime, Daptomycin, Gentamycin, Levofloxacin, Linezolid, Meropenem, Minocycline, Rifampicin, Vancomycin etc. against a panel of MRSA and VRSA strains. According to the recommendations of CLSI, serial two-fold dilutions of each drug to at least double the MIC were freshly prepared prior to testing. The compounds were serially diluted along the ordinate ranged from 0.03 to 4 pg/mL while the antibiotics were serially diluted as shown along the abscissa ranged from 0.03 to 64 pg/mL /ml in 96 well microliter plate. An inoculum equal to 10 ⁵ or 10 ⁶ CFU/mL was prepared from each MRSA and VRSA isolate in MHB. Each microliter well was inoculated with 100 pL of a bacterial inoculum of 10 ⁵ or 10 ⁶ CFU/mL, and plates were incubated at 37 °C for 24 h under aerobic conditions. According to the CLSI guidelines for broth micro dilution, the MIC was defined as the lowest concentration of antibiotic that completely inhibited the growth of the organism as detected with the naked eye. The åFICs (fractional inhibitory concentrations) were calculated as follows: åFIC=FIC A + FIC B where, FIC A is the MIC of drug A in the combination/MIC of drug A alone, and FIC B is the MIC of drug B in the combination/MIC of drug B alone. The combination of 1:1 ratio of AH- 5 and Gentamycin is considered synergistic when the åFIC is <0.5, indifferent when the åFIC is >0.5 to 4, and antagonistic when the åFIC is >4.

[0070] Combination of AH-5 and Gentamycin (ratio of 1:0.125) increases the inhibitory activity, which is a decrease in inhibitory concentration confirming the synergistic effect. FIC index is found l/4 ^th of the MIC of gentamycin as shown in Table 1.

Table 1: Synergy studies and calculation of FIC index

Example 3

Activity against MDR strains of S. aureus [0071] The activity of the combination of 1 : 0.125 ratio of AH-5 (IX MIC) and gentamycin

(IX MIC) against MRS A, MSS A, and VRSA were checked in comparison with the existing drugs and found that the compound do not change their MIC against different bacterial strains as shown in Tables 2, 3 and 4 below. The MIC values were found to be excellent for the combination around 0.125 and 2 pg/mL, which can be considered as the efficiency of the molecule to inhibit the MDR strains. Table 2: MIC of combination against MSS A panel

Table 3: MIC of combination against VRSA panel

Table 4: MIC of combination against MRS A panel Example 4

Biofilm inhibition assay

[0072] Bacteria within the biofilm are adaptively resistant to antibiotic treatment and it can take up to 1000 times more antibiotics to kill cells within the biofilm when compared to planktonic bacterial cells. The disclosed synergistic combination of AH-5 and Gentamycin reduced the biofilm by 9.7%, as compared to the untreated, which is far better than the existing drugs levofloxacin IX MIC (6.8%) and Vancomycin IX MIC (6.3%) (Figure 1).

Example 5

Time kill kinetic assay

[0073] The bactericidal activity was assessed by the time-kill method. S. aureus ATCC 29213 cells were diluted up to ~10 ⁵ CFU/mL and treated with compound for concentrations corresponding to IX and 10X of MIC of a compound and vancomycin in MHB in triplicate and incubated at 37 °C. 0.1 mL samples were collected after time intervals of 0, 1, 6 and 24 h, serially diluted in PBS and plated on TSA followed by incubation at 37 °C for 18-20 h. Kill curves were constructed by counting the colonies from plates and plotting the CFU/mL of surviving bacteria at each time point in the presence and absence of compound. The combination of AH-5 and Gentamycin showed excellent bactericidal property and can reduce the CFU/mL to zero in 24 h, proving its concentration-dependent bactericidal property (Figure 2).

Example 6 Pharmaceutical composition

[0074] The pharmaceutical composition was prepared by mixing artocarpin and gentamycin in 1:0.125 ratio, using excipients like gelatin, stearic acid, parabens, stabilizers like sorbitol, liquid non-volatile non-aqueous materials mainly glycols such as propylene glycol.

ADVANTAGES OF THE PRESENT INVENTION

[0075] High synergy combination of the disclosed compound with gentamycin (1:0.125), lead to the excellent antibacterial property against MDR strains of S. aureus like MRSA, VRSA. [0076] The combination is bactericidal in nature, which means it is inhibiting the growth of bacteria, represented in CFU/mL.

[0077] The combination is also capable of inhibiting the biofilm, which is more resistant to antibiotic treatment revealing the efficiency of the disclosed compound as an antibacterial agent. [0078] Cost-effective: Easy to isolate from the stem bark of Artocarpus hirsutus Lam. by simple acetone extraction.

[0079] The combination is also proved to be bactericidal in nature, effective in killing the bacteria up to zero CFU/mL in 24 h, which is a much-needed feature for a drug lead candidate. It also proves the concentration-dependent bactericidal nature of the combination and its capability to resist the growth of colony forming units of bacteria in a static manner. This bactericidal nature of these combination makes it more efficient towards MDR bacteria. In short, the disclosed combination of AH-5 and gentamycin is highly active against the MDR bacterial strains of S. aureus, one of the current threats in public medicine. In-vitro analysis and the synergistic studies also suggesting that the combination could be converted into an effective antibacterial drug clinically.

[0080] The foregoing description of the preferred embodiments is provided to enable any person skilled in the art to make or use the present invention. The various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without the use of the inventive faculty. Thus the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein. Various changes and modifications may be made therein without departing from the spirit of the invention. Such changes and modifications are to be understood as included within the scope of the present invention as defined by the appended claims unless they depart there from.