Description:PHYTOCHEMICAL COMPOSITION AGAINST VANCOMYCIN-RESISTANT ENTEROCOCCUS FAECIUM
FIELD OF THE INVENTION
[001] The present invention relates to a phytochemical composition capable of providing effective anti-bacterial activity against vancomycin resistant Enterococcus faecium. In particular, the invention pertains to a phytochemical composition comprising Norhormone and 2-methoxy-4-vinylphenol, which exhibit a synergistic anti-bacterial effect against vancomycin-resistant E. faecium.
BACKGROUND OF THE INVENTION
[002] Antimicrobial resistant bacteria are increasingly being identified in hospital settings across various countries, primarily due to their resistance to commonly used antibiotics. There is a growing concern regarding their ability to acquire pan-drug resistance to all known antibiotics through the acquisition of foreign genetic material via plasmid or transposon transformation, or through mutation.
[003] The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus,Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) are the most common cause of nosocomial infections globally. These pathogens are often drug-resistant, posing a significant challenge in clinical treatment. Multidrug resistance is a top global health threat, driven by factors such as prescription drug abuse and improper use of antibiotics. Accordingto the ECDC and WHO, the prevalence of antibiotic-resistant bacteria is rising. In the US, at least 2 million people are infected with antibiotic-resistant bacteria annually, leading to at least 23,000 deaths, with global deaths due to antimicrobial resistance estimated to reach 700,000 each year, potentially rising to 10 million by 2050. Understanding bacterial resistance is crucial for developing new antimicrobial treatments. Enterococci have become significant nosocomial pathogens, being the second most common bacteria in urinary tract and wound infections, and the third leading cause of bacteraemia in the US. They account for 5-10% of hospital-acquired infections and have seen an increase in vancomycin-resistant clinical isolates.
[004] There has been a rise in clinical isolates of vancomycin-resistant Enterococcus (VRE), particularly Enterococcus faecium, a major ESKAPE pathogen causing infections. These organisms persist due to their resistance to commonly used antibiotics and their ability to develop resistance to all known antibiotics through mutation or acquisition of foreign genetic material. Identified in the late 1980s, VRE is now a serious clinical problem, designated as a high priority by the WHO for novel antibiotic development. VRE infections are increasing, with nearly 55,000 cases in the US in 2017, highlighting the need for a better understanding of VRE pathogenesis. Failing antimicrobial medications are leading to increased mortality associated with antimicrobial resistance and tolerance, necessitating a multimodal approach and the development of complex new antibiotics. [005] Hence, it is necessary to develop a composition which combats antimicrobial resistance in vancomycin resistant Enterococcus faecium. The composition should be safe without any side effects.
SUMMARY OF THE INVENTION
[006] The following presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the present invention. It is not intended to identify the key/critical elements of the invention or to delineate the scope of the invention. Its sole purpose is to present some concept of the invention in a simplified form as a prelude to a more detailed description of the invention presented later.

[007] The present invention discloses a combination of phytochemicals that are Norharmane and 2-methoxy-4-vinyl phenol that exhibits synergistic bactericidal activity against vancomycin-resistant Enterococcus faecium.
[008] In particular, the disclosed composition is a synergistic combination of Norharmane, and 2-methoxy-4-vinylphenol with a weight percentage ranging from 0.0005% to 0.02% shows almost 80% of inhibition in vancomycin resistant E. faecium. The composition of Norharmane and 2-methoxy-4-vinylphenol has a synergistic interaction with a weight by weight (w/w) ratio of 1:1 to 0.5:1 and 1:0.5. The specific concentration range of 0.0005% to 0.02% for Norharmane and 0.001% to 0.02% for 2-methoxy-4 vinylphenol demonstrates synergistic effects for vancomycin resistant E. faecium.
[009] The disclosed synergistic combination suppresses anti-microbial resistance genes (AMR ((MarR, TetR, VanR/VicR, Alr, PonA, rfbB, proB, and dltA)) and has a strong bactericidal impact against vancomycin resistant strains of Enterococcus faecium.
[010] The synergistic effects of this phytochemical combination would be an ideal component in wound healing creams, disinfectant/ antiseptic liquids, in addition to its possible applications in cleaning hospital floors, washing medical equipment, surface cleaning, etc.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
[011] The following drawings form a part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.
[012] Figure 1(a) and Figure 1(b) illustrates the Antibacterial activity of Methanolic Seed Extract (ME) of M. cymbalaria against Vancomycin-resistant Enterococcus faecium, in accordance with an embodiment of the present disclosure.

[013] Figure 2(a) and Figure 2(b), illustrates the Minimum Inhibitory Concentration (MIC) of Norhormane (Referred as Compound I) against Enterococcus Faecium, in accordance with an embodiment of the present disclosure.

[014] Figure 3(a) and Figure 3(b), illustrates the Minimum Inhibitory Concentration (MIC) of 2-methoxy 4 vinyl phenol (Referred as Compound-II) against Enterococcus Faecium, in accordance with an embodiment of the present disclosure.

[015] Figure 4 illustrates the Cytotoxicity Profile of Norharmane and 2-Methoxy-4 Vinylphenol on HEK-293 Cell Lines. Wherein the Figure 4 depicts the cell viability of compound – I (Norharmane) and compound – II (2 Methoxy-4 Vinylphenol) at different concentration and its effect using HEK-293 cell lines. After being incubated for 24 hours with Norharmane, and 2 Methoxy-4 Vinylphenol the treated cells exhibit no discernible morphological changes.
[016] Figure 5(a) and Figure 5(b) illustrates the assay yielded an IC50 value of 317.8 μg/ml and 304 μg/ml, respectively, for the percentage of viable cells at different concentrations of Norharmane and 2-Methoxy-4 Vinylphenol on HEK-293 cells.
[017] Figure 6(a) and Figure 6(b) illustrates the Minimum Biofilm Inhibitory Concentration (MBIC) of Norharmane (Compound-I) and 2-Methoxy-4-Vinylphenol (Compound-II) against Enterococcus faecium, in accordance with an embodiment of the present disclosure.
[018] Figure 7 illustrates the Minimum Inhibitory Concentration MIC value of Norharmane and 2-Methoxy-4-Vinylphenol at all concentrations.

BRIEF DESCRIPTION OF THE INVENTION
[019] The foregoing has outlined rather broadly the features and technical advantages of the present invention so that those skilled in the art may better understand the detailed description of the invention that follows. Additional features and advantages of the invention will be described here in after that form the subject of the claims of the invention. Those skilled in the art should appreciate that they may readily use the conception, and the specific embodiment disclosed as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. Those skilled in the art should also realize that such equivalent constructions do not depart from the spirit and scope of the invention in its broadest form.
Definitions:
[020] Throughout this specification and the claims which follow, 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 member, integer or step or group of members, integers or steps but not the exclusion of any other member, integer or step or group of members, integers or steps.

[021] The terms "a" and "an" and "the" and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
[022] As used herein, the terms “composition(s)” and “formulation(s)” can be used interchangeably depending on the context in which they are used as would be appreciated by a person skilled in the art.

[023] The term “room temperature” as used herein, means 20 °C to 25 °C. In an embodiment it is 20 °C. In an embodiment it is 21 °C. In an embodiment it is 22 °C. In an embodiment it is 23 °C. In an embodiment it is 24 °C. In an embodiment it is 25 °C.
[024] As used herein, the term “gel”, refers, inter alia, to a carrier or formulation or composition that is not flowable at room temperature, such that when subjected to normal gravity at room temperature, it will retain its form.
[025] The term, "pharmaceutically acceptable excipients" as used herein refers to solubiliser, solvents, co-solvents, preservatives, wetting agents, thickening agents, rheology modifiers or thickening agent, antifoaming agents, stabilizers, an antioxidant, a chelating agent, an oil phase, an emulsifier, a penetration enhancer, a pH adjusting agent, a preservative, an antimicrobial agent, an opacifier, a fragrance, a colorant, a gelling agent, a moisturizer, a surfactant and the like. The pharmaceutical compositions of the present invention may be formulated in a conventional manner using one or more pharmaceutically acceptable excipients.
[026] The term "Topical composition" or "pharmaceutical composition" or “dosage form” as used herein synonymously include topical dosage forms such as a solution, a suspension, a cream, an ointment, a lotion and a gel. Preferably, the topical formulation is a solution or a gel. More preferably, the topical formulation is a gel.
[027] The term "stable" as used herein refers to formulations that substantially retain the label amount of the therapeutically active ingredient during storage for commercially relevant times, and the drug-related impurity contents in the formulations remain within acceptable limits.
[028] Before undertaking the detailed description of the invention below it may be advantageous to set forth definitions of certain words and phrases used throughout this patent document: the terms “include” and “comprise ” as well as derivatives thereof mean inclusion without limitation; the term “or ” is inclusive meaning and/or; the phrases “associated with” and “associated therewith ” as well as derivatives thereof may mean to include be included within interconnect with contain be contained within connect to or with couple to or with be communicable with cooperate with interleave juxtapose be proximate to be bound to or with have a property of or the like; and the term.
[029] In an embodiment, the invention discloses a pharmaceutical composition comprising a combination of Norharmane and 2-methoxy-4-vinylphenol, effective in inhibiting the growth of vancomycin-resistant Enterococcus faecium.
[030] In the above-mentioned embodiment, the composition suppresses antimicrobial resistant proteins such as MarR, TetR, VanR/VicR, Alr, PonA, rfbB, proB, and dltA. This composition demonstrates a significant increase in the susceptibility of E. faecium cells to conventional antibiotics.
[031] In another embodiment, the invention discloses a synergistic combination of Norharmane and 2-methoxy-4-vinylphenol with a weight percentage ranging from 0.0005% to 0.02% and 0.001% to 0.02% to treat vancomycin-resistant Enterococcus faecium infections. This combination exhibits almost 80% inhibition of vancomycin-resistant E. faecium and has a synergistic interaction with ratios ranging from 1:1 to 0.5:1 and 1:0.5.
[032] In yet another embodiment, the invention discloses a wound-healing cream containing a synergistic combination of Norharmane and 2-methoxy-4-vinylphenol. This cream provides bactericidal activity against vancomycin-resistant Enterococcus faecium, as evidenced by a full log reduction in the E. faecium population.
[033] In another embodiment the invention discloses a method for controlling the growth of vancomycin-resistant Enterococcus faecium by administering a composition containing Norharmane and 2-methoxy-4-vinylphenol. The composition down regulates proteins connected to the pathogenicity pathways of E.faecium, including dltA, secA, ffh, and glnA, and enhances the effectiveness of conventional antibiotics.
[034] The synergistic effects of this phytochemical combination would be an ideal component in wound healing creams, disinfectant/ antiseptic liquids, in addition to its possible applications in cleaning hospital floors, washing medical equipment, surface cleaning, etc.
[035] In an embodiment the invention discloses a wound healing cream comprising Norhormane and 2-methoxy vinyl phenol, a vehicle, an emulsifier, a binder, and preservative.
[036] In the above-mentioned embodiment, the vehicle is Virgin coconut oil and emulsifier are bee’s wax.
[037] In yet another embodiment, the binder is guar gum and preservative in honey.
[038] In a further embodiment of the invention disclose a composition comprising;
a)Norhormane and 2-methoxy-4 vinyl phenol in a concentration range of 0.002% to 0.02% and 0.02%-0.001% (w/w);
b)Virgin coconut oil in a concentration range of 15% to 30% (w/w);
c)Bees wax at a concentration of about 4% to 16% (w/w);
d)Guar gum and Gum Arabica at a concentration of about 0.5 % to 2% (w/w) and;
e)Honey at a concentration of 1% to 4% (w/w) and Water.
EXAMPLES
Example 1: Preparation of phytochemical composition from Momordica cymbalaria
[039] Ripened Momordica cymbalaria fruits were collected from Virudhunagar, Tamil Nadu, India, during the harvest season. The cleaned seeds were dried at room temperature for a week, powdered using an electric blender, and stored in airtight containers. For crude bioactive compound extraction, 10 g of powdered seeds were packed in filter paper and extracted with 250 ml of methanol. The extracts were concentrated to remove excess solvents, dried in a hot air oven, and yielded 0.6g of thick crude, stored in an airtight glass holder for further studies.
Example 2:
Antibacterial Analysis of phytochemical composition by Agar Well Diffusion Method
[040] The in-vitro antibacterial activity of methanol-extracted crude bioactive compounds was tested on a 0.4 to 0.5 optical density (OD) culture of Enterococcus faecium for 6-8 hours. The Agar well diffusion method measured the zones of 10 inhibition. The diluted culture was spread on LB agar plates, dried, and 6 mm wells were made. The crude extract was loaded in descending concentrations (20 mg, 10 mg, 5 mg, 2 mg) with methanol as a positive control. Plates were incubated for 18 hours at 37°C. The inhibition zones were observed and measured using an antibiotic zone scale. The findings indicated that Enterococcus faecium is susceptible to M. cymbalaria's methanolic seed extract (shown in Figure 1a) at 20 mg, exhibiting a 17 mm zone of inhibition. The experimental data supporting this analysis is shown in the Table-1.
[041] Table 1: Plant crude extract shows Zone of inhibition against 20 vancomycin resistant Enterococcus faecium
Plant crude extract shows Zone of inhibition against vancomycin resistant Enterococcus faecium
Concentration (mg)
Zone of inhibition (mm)
2
9.6±0.57
5
11.6 ±0.57
10
13.3±1.15
20
16.6± 0.57
11
[042] The methanolic seed extract of M. cymbalaria from example-1 was further subjected to GC-MS analysis to analyse active biocomponents. The Figure 1b displays the GCMS results for the methanolic seed crude extract of M. cymbalaria, along with the peaks obtained in relation to retention time. The peak represents the substance's concentration, while the retention time reveals the kind of compound which is responsible for the peak. There were 26 different compounds identified in this extract through various peaks. These compounds were identified as alkaloids, flavones, flavonoids, phenols, fatty acids, and hydrocarbons. Further investigations were conducted on the identified active compounds. (Refer the below given Data in Table 2)
[043] Table 2: List of Compounds Identified from Methanol Seed Extract of Momordica cymbalaria through GC-MS analysis
12
13
Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of Norharmane and 2-Methoxy-4-Vinylphenol against Enteococcus faecium -JJSBBC EF

[044] Materials and Method:
The phytochemicals such as Norharmane and 2-Methoxy-4-Vinylphenol used in the composition of the present disclosure were procured from SIGMA-ALDRICH. The potential growth inhibitory effects of Norharmane and 2-Methoxy-4-10 Vinylphenol against Enterococcus faecium were assessed using a micro broth dilution assay in 24-well polystyrene plates following CLSI guidelines (CLSI, 2006). Briefly, 1% of a bacterial culture with 108 CFU/ml of E. faecium was used to inoculate 1 mL of LB broth with 1% glucose. Serial dilutions of various concentrations, ranging from 5000 μg to 9.7 μg, were prepared to determine their inhibitory concentrations. Negative and vehicle controls were included in parallel. After 24 hours of incubation at 37°C, growth changes were observed and cell density was measured using a spectrophotometer at 600 nm. The percentage of growth inhibition was calculated using the formula:
% of growth inhibition = (Control OD -Test OD / Control OD) × 100
[045] In this context, "Test" refers to LB with phytocompounds and inoculum, "Vehicle Control" refers to LB/10% DMSO with bacterial inoculum, and "Negative Control" refers to LB alone. The determined MIC values are presented in Table 1, and the results are depicted in Figure (2a, 2b, 3a, 3b). MBC was determined by seeding LB agar plates with 100 μl aliquots of bacterial culture treated with 25 phytocompounds, followed by an 18-hour incubation at 37°C.

[046] Table 3. Concentration of phytocompounds (Norharmane and 2-Methoxy-4-Vinylphenol) showed ≥ 80% and ≥ 70% of growth inhibition against Enterococcus faecium.
S.no
Phyto compounds
Test Organism
Growth inhibition against the test strain
MIC values of phytocompounds (μg/mL)
1
Norharmane
Enterococcus faecium
MIC-80%
78.125
2
2-Methoxy-4-Vinylphenol
Enterococcus faecium
MIC-70%
78.125 [047] Specifically, at the value of 78.125 μg/mL of Norharmane and 2-Methoxy-4-Vinylphenol, showed growth inhibition of ≥ 80% and ≥ 70% against the test strain, respectively, were found to be at the minimum inhibitory concentration (MIC) of E.faecium (Table 3). The percentage of inhibition for each concentration was computed (figure 2b, 3b). The identified compounds' bactericidal properties were confirmed by the observation of 99.4% and 99.1% inhibition at higher concentrations. After MIC the suspension from each well was inoculated in LB agar 10 (1% glucose) and allowed to incubate for a full day. The findings indicate that various doses of substances (ranging from 5000 to 9.7 μg/mL) strongly reduced the development of E. faecium. In comparison to the control, a significant log reduction was seen in both samples at all concentrations (Figure 2a, 3a). [048] The bactericidal efficacy of the identified compounds was demonstrated by achieving 99.4% and 99.1% inhibition at elevated concentrations. Minimum Inhibitory Concentrations (MIC) of 80% and 70% were recorded at 78.125 μg for Norharmane and 2-Methoxy-4-Vinylphenol, respectively. Consequently, these concentrations were utilized as reference points in the experimental examples outlined in the present invention.Evaluation of Cytotoxicity Profile of Norharmane and 2-Methoxy-4 Vinylphenol on HEK-293 Cell Lines
[049] The MTT assay, as described by Mosmann (1983), was used to assess the cytotoxicity profile of Norharmane and 2-Methoxy-4 Vinylphenol on HEK-293 cell lines (ATCC). Initially, 200 ml of DMEM low-glucose media with a suspension of 5 HEK-293 cells (approximately 50,000 cells per well) was seeded into a 96-well plate. The cells were allowed to grow for 24 hours. Subsequently, the cells were exposed to various concentrations of 2-Methoxy-4 Vinylphenol and Norharmane, ranging from 20 to 150 μg/ml. Wells containing only medium and medium with cell seeds served as blank and control, respectively.
[050] The plate was incubated for 24 hours at 37°C in a 5% CO2 environment. After incubation, the spent medium was removed, and the wells were washed twice with phosphate-buffered saline (pH 7.2). MTT reagent [3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; # 4060 HiMedia] was added to achieve a final concentration of 0.5 mg/mL. The plates were covered with aluminum foil and incubated at room temperature for three hours. Following this, the MTT reagent was removed, and 100 μL of DMSO was added to each well to dissolve the MTT formazan crystals. The absorbance of the eluted formazan was measured at 570 nm. The percentage of cell viability was calculated using the formula:

[051] The IC50 value was determined using the linear regression equation y = mx+cy = mx + c, as shown in Figure 5. After the 24-hour incubation, both control and treated HEK-293 cells were observed under a microscope.
Acridine orange/Ethidium bromide (AO/EtBr) Fluorescent Staining:
16
[052] The cell morphology was evaluated using Acridine Orange/Ethidium Bromide (AO/EtBr) staining as described by Gunaseelan et al. (2017). Approximately 4000 cells were seeded in each well of a microtiter plate and incubated with Norharmane and 2-Methoxy-4 Vinylphenol for 24 hours. The cells were stained with 10 μL of AO/EtBr solution (100 μg/mL) and visualized under a fluorescent microscope (Olympus CKX-53). The results of the study are given in Figure 4 (a) and (b). Checkerboard Assay to Determine the Synergism Between Norharmane (Compound - I) and 2-Methoxy-4-Vinylphenol (Compound - II)
[053] The checkerboard assay is a micro-dilution assay performed in vitro to evaluate the potential synergy between drug candidates when used together. This assay compares the combined potency of the interacting molecules to their individual activities, demonstrating that synergy can be achieved at lower concentrations than those of individual drug candidates.
[054] To evaluate the interactions between the drugs, the fractional inhibitory concentration index (FICI) values were calculated. In vitro two-dimensional checkerboard experiments were conducted using a 96-well microtitre plate. Active lead (A) at its minimum inhibitory concentration (MIC) was added to the first column, with each subsequent column containing concentrations two times lower. Similarly, active lead (B) at its MIC was added to the first row, with each subsequent row's concentration halved.
[055] To analyze synergism, 25 combinations of Norharmane (Compound - I) and 2-Methoxy-4-Vinylphenol (Compound - II) at different concentrations were evaluated in this study. This process created six distinct combinations of active leads, applied to five adjacent columns and rows, and was replicated in triplicate. The final test concentration was set at 0 μg/mL to evaluate the activity of individual 25 drug candidates in combination. Blank, negative control, and appropriate controls were included. The plates were incubated for 24 hours at 37°C, and absorbance was measured at 600 and the percentage of growth inhibition was calculated using the formula:
17
[056] To evaluate the synergistic effect between Norharmane (Compound - I) and 2-Methoxy-4-Vinylphenol (Compound - II), fractional inhibitory concentration index (FICI) values were calculated. The FICI values were interpreted as follows:

[057] Table: 4 Concentration of Norharmane (Compound - I) and 2-Methoxy-4-Vinylphenol (compound - II) used for Checker Board Assay.
18
[058] Since Norharmane and 2-Methoxy-4-Vinylphenol at concentration of 78.125 μg/mL each, respectively showed ≥ 80% and ≥ 70% of growth inhibition against E. faecium. This particular concentration was further used for assay studies. (Compound - I -MIC 80% - 78.125 (ROW) Compound - II MIC 70% - 78.125 (COLOUM)
[059] Table 5: Determination of synergistic activity of Norharmane (compound - I) and 2-Methoxy-4 Vinylphenol (compound - II) combination against Enterococcus.faecium
[061] Here, ⁎ denotes growth inhibition ≥ 80% are active working synergistic concentrations. numerical values denote percentages of inhibition and FICI.
[062] As shown in Table 5, the antibacterial activity was observed at most of the 15 combinations, but the synergistic effect of Norharmane and 2-Methoxy-4-Vinylphenol was observed at combinations of (Norharmane + 2-Methoxy-4-Vinylphenol (i.e C-I+C-II): 19.5 μg/mL + 19.5 μg/mL, 19.5 μg/mL + 9.75 μg/mL and 9.75 μg/mL + 19.5 μg/mL with fractional inhibitory concentration index (FICI) value as 0.49, 0.37 and 0.37 respectively shows almost ≥ 80% of growth inhibition against E. faecium (refer to Figure 7). Further, the weight percentages of Norharmane and 2-Methoxy-4 Vinylphenol 0.002% to 0.0005% shows almost 80%, of inhibition in vancomycin resistant E. faecium. The composition of Norharmane and 2-methoxy-4-vinylphenol has a synergistic interaction with a w/w ratio of 1:1 to 0.5:1 and 1:0.5. (19.5:19.5, 9.75:19.5 and 19.5:9.75 μg).
[063] Further, it was observed that the remaining 22 combination of Norharmane and 2-Methoxy-4-Vinylphenol used at various did not show the synergistic anti-bacterial activity against vancomycin resistant E. faecium, having FICI values greater than 0.5 and lesser than 2. Also, Norharmane and 2-Methoxy-4-Vinylphenol as individual component at a higher concentration of 78.125 μg/mL each, respectively did not show the synergistic anti-bacterial activity against vancomycin resistant E. faecium.
[064] Therefore, it can be inferred that a total of three synergistic combinations of of Norharmane + 2-Methoxy-4-Vinylphenol (i.e C-I+C-II): 19.5 μg/mL + 19.5 μg/mL, 19.5 μg/mL + 9.75 μg/mL and 9.75 μg/mL + 19.5 μg/mL were considered 20 as the working examples, whereas, remaining 22 combination of Norharmane + 2-Methoxy-4-Vinylphenol used at various concentrations (see Table5) and as individual component (see Table 3) were considered as the non-working examples. The three working synergistic combinations of compositions of Norharmane + 2-Methoxy-4-Vinylphenol with their FICI values and Growth inhibition against test strain vancomycin resistant E. faecium are tabulated in Table 6.
[065] Table 6: Working synergistic concentrations of Norharmane (C-I) and 2-Methoxy-4 Vinylphenol (C-II):
30
20
Minimum Biofilm Inhibitory Concentration (MBIC) of Norharmane and 2-Methoxy-4-Vinylphenol against Enterococcus faecium
[066] The MBIC of Norharmane and 2-Methoxy-4-Vinylphenol was determined using a crystal violet staining assay, with slight modifications based on the method reported by Mathimaran et al. (2024). In brief, varying concentrations of the phytocompounds (5000 μg/ml to 9.7 μg/ml) were prepared via serial dilution and added to LB broth inoculated with 10^8 CFU/ml of Enterococcus faecium. The cultures were incubated for 24 hours at 37°C. After incubation, planktonic cells were discarded, the wells were rinsed thrice with water, air-dried for 10 minutes, and then stained with 0.4% crystal violet for 30 minutes at 37°C. Excess crystal violet stain was washed off with distilled water, and 30% glacial acetic acid (GAA) was added to the wells to solubilize the crystal violet. The intensity of the blue color of the GAA solution, which correlates with biofilm matrix density, was measured by spectrophotometry at 570 nm. The MBIC (percentage of inhibition) was calculated using the following formula:
21
[067] Wherein, Test OD indicates inoculated LB broth containing phytocompounds and Control OD indicates inoculated LB with DMSO alone.
[068] Results of the study is given in Figures 3 (a) to 3 (d). Effect of Norharmane (compound - I) and 2-Methoxy-4-Vinylphenol (compound - II) against biofilm formation of Enterococcus faecium. a, c) Dose dependent reduction in the biofilm formation was observed. b and d) represent the percentages of biofilm inhibition at various concentrations.

Wound Healing Cream Formulation comprising Norharmane and 2-methyl 4 vinyl phenol
[069] Table 7 depicts a formulation table comprising the composition of present disclosure. The formulation as depicted was a wound healing cream formulation have a desired synergistic bactericidal activity against vancomycin-resistant Enterococcus faecium. According to the present disclosure, the formulation was prepared by using the active ingredients i.e., synergistic combination of Norharmane (Compound I) and 2-Methoxy-4-Vinylphenol (Compound II) along with other mentioned ingredients in the desirable quantity and the working example for the formulation as depicted in table 7. The formulation further includes one or more additional components, such as: (a) a vehicle, (b) one or more emulsifiers, (c) one or more binders, and (d) one or more preservatives as specified in the table 7. The detailed composition of three wound healing cream formulations, including the percentage composition (% w/w), functions of each component, and specific concentrations for Cream 1, Cream 2, and Cream 3, are provided in Table 7. These formulations utilize natural ingredients, avoiding the inclusion of harsh chemicals. The preparation involves incorporating the aqueous phase into the oil phase under continuous stirring, as outlined in Table 7.
30
22
[070] Table: 7 Formulation of Wound Healing Cream
[071] A water-in-oil (w/o) cream formulation generally comprises a cream base and active components (in this case, phytochemicals) dispersed in an aqueous phase. The cream base incorporates elements such as a vehicle, emulsifier, binder, and preservative. As detailed in Table 7, three cream formulations were developed for the intended study using natural ingredients and avoiding harsh chemicals. These formulations were prepared by continuously stirring to combine the aqueous phase with the oil phase. Among them, the cream 1 formulation demonstrated effective results with a moderate concentration of the desired active ingredients. , C , Claims:I/We Claim:
1) A composition comprising:
0.0005% to 0.02% (w/w) of Norharmane; and
0.001% to 0.02% (w/w) of 2-methoxy-4-vinylphenol,
Wherein Norharmane to 2-methoxy-4-vinylphenol is in the weight ratio range of 1:1, 0.5:1, and 1:0.5; and
Wherein the said composition exhibits bactericidal effect against vancomycin-resistant Enterococcus faecium.
2) The composition as claimed in claim 1, wherein the composition includes one or more pharmaceutically acceptable excipients, and wherein the said excipients 10 includes solubiliser, solvents, co-solvents, preservatives, wetting agents, thickening agents, rheology modifiers or thickening agent, antifoaming agents, stabilizers, an antioxidant, a chelating agent, an oil phase, an emulsifier, a penetration enhancer, a pH adjusting agent, an opacifier, a fragrance, a colorant, a gelling agent, a moisturizer, or a surfactant.
3). The composition as claimed in claim 1, wherein the composition includes one or more pharmaceutically acceptable excipients, and wherein the said excipients preferably Virgin coconut oils, bees wax, Guar gum, Gum Arabia, Honey and water.
4). The composition as claimed in claim 1, suppresses antimicrobial resistance genes, including MarR, TetR,VanR/VicR, Alr, PonA, rfbB, proB, and dltA.
5) A phytochemical composition comprising;
Norharmane and 2-methoxy-4 vinyl phenol in a concentration range of 0.002% to 0.02% and 0.02% to 0.001% (w/w) respectively;
Virgin Coconut oil in a concentration range of 15% to 30% (w/w);
Bees wax at a concentration of about 4% to 16% (w/w);
Guar gum and Gum Arabica at a concentration of about 0.5 % to 2% (w/w) and;
Honey at a concentration of 1% to 4% (w/w) and Water.