Description:FIELD OF INVENTION
[001] The present invention relates to an antimicrobial composition for Prolonged Antimicrobial Protection (PAP) effective against bacteria, fungus, virus, drug resistance bacteria and novel viruses including enveloped SARS-COV-2. Particularly, the present invention relates to a synergistic composition comprising a combination of bio-surfactant, an antimicrobial agent and citric acid.

BACKGROUND OF THE INVENTION
[002] The outbreak of coronavirus (COVID 19) caused public health crises resulting in huge demand of antiviral and sterilization products. Due to high occurrence of disease transmission, substantive measures were taken around the globe to reduce COVID 19 infections resulting in huge demand of Advanced Sterilization Products (ASP) products. Presently silver nanoparticles are present in advanced sanitation products as an active ingredient to improve their antimicrobial potential. For instance, BIRAC has supported Silvo Clean, a hand sanitizer product from Weinnovate Biosolutions Pvt. Ltd. based on silver nanoparticles to disinfect the Coronavirus.
[003] Silver is readily available and is known to have antimicrobial properties due to wide antibacterial spectrum of silver ions. Silver nanoparticles (AgNPs) have been developed having much consideration due to their characteristic chemical, optical, electrical and catalytic properties that can be turned with size, surface nature. Depending on the surface area of nanoparticles, silver ions are released with great efficiency. These nanoparticles are effective against enveloped and non-enveloped viruses such as HIV, hepatitis B, herpes simplex, and influenza constituting major issue in the medial field.
[004] However, the AgNPs are limited only to application on limited surfaces and are effective only for a limited duration of time. None of the existing technologies uses or disclose antimicrobial compositions that can be applied to the body as lotions or the like and that can give a long lasting antimicrobial effect and aids in prevention of the spread of illness and infection in an effective manner.
[005] Therefore, there remains a need to provide effective antimicrobial formulations having prolong activity for treatment of a broad spectrum of infections including bacterial infections.

OBJECTIVES OF THE INVENTION
[006] The primary objective of the present invention is to provide an antimicrobial composition/formulation having Prolonged Antimicrobial Protection (PAP).
[007] Yet another objective of the present invention is to provide an antimicrobial composition/formulation effective against bacteria, fungus, virus, drug resistance bacteria and novel viruses including enveloped SARS-COV-2.
[008] Another objective of the present invention is to provide a synergistic composition comprising a complex of bio-surfactant, an antimicrobial agent and citric acid.
[009] Yet another objective of the present invention is to provide an antimicrobial lotion that is effective against a broad spectrum of microbes including enveloped SARS-COV-2.
[010] Other objects and advantages of the present invention will become apparent from the following description taken in connection with the accompanying examples to disclose the aspects of the present invention.

SUMMARY OF THE INVENTION
[011] The present invention relates to an antimicrobial composition for Prolonged Antimicrobial Protection (PAP) effective against bacteria, fungus, virus, drug resistance bacteria and novel viruses including enveloped SARS-COV-2. Particularly, the present invention relates to a synergistic composition comprising a complex of bio-surfactant, an antimicrobial agent and citric acid having significant antimicrobial activity and effective against a broad spectrum of microbes.

DETAILED DESCRIPTION OF THE INVENTION
[012] The following description describes various features and functions of the disclosed system and method with reference to the accompanying figures. In the figure, similar symbols identify similar components, unless context dictates otherwise. The illustrative aspects described herein are not meant to be limiting. It may be readily understood that certain aspects of the disclosed system and method can be arranged and combined in a wide variety of different configurations, all of which are contemplated herein.
[013] Accordingly, those of ordinary skill in the art will recognize that various changes and modifications of the embodiments described herein can be made without departing from the scope of the invention. In addition, descriptions of well-known functions and constructions are omitted for clarity and conciseness.
[014] Features that are described and/or illustrated with respect to one embodiment may be used in the same way or in a similar way in one or more other embodiments and/or in combination with or instead of the features of the other embodiments.
[015] The terms and words used in the following description and claims are not limited to the bibliographical meanings but are merely used to enable a clear and consistent understanding of the invention. Accordingly, it should be apparent to those skilled in the art that the following description of exemplary embodiments of the present invention are provided for illustration purpose only and not for the purpose of limiting the invention.
[016] It is to be understood that the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.
[017] It should be emphasized that the term “comprises/comprising” when used in this specification is taken to specify the presence of stated features, integers, steps, or components but does not preclude the presence or addition of one or more other features, integers, steps, components, or groups thereof. The equations used in the specification are only for computation purpose.
[018] The present invention relates to an antimicrobial formulation for Prolonged Antimicrobial Protection (PAP) effective against bacteria, fungus, virus, drug resistance bacteria and novel viruses including enveloped SARS-COV-2. Particularly, the present invention relates to a synergistic composition comprising a bio-surfactant, an antimicrobial agent and citric acid. The present invention also provides a method for preparation of the synergistic composition.
[019] The present invention provides a composition having antibacterial, antifungal and antiviral properties. The formulation according to the present invention comprises:
1. An antimicrobial agent 0.001% to 2.0%
2. a biosurfactant 0.01% to 2%
3. a preservative 0.1% to 1.8%; and
4. Pharmaceutically acceptable compounds selected from excipients, carrier agents 0.001% to 5%; and/or
5. Other additives 0.01 to 40%; and
6. Water (quantity remaining of 100%)
[020] The antimicrobial agent may be metal nanoparticles, preferably transition metals nanoparticles such as silver nanoparticles, copper nanoparticles, zinc nanoparticles and titanium nanoparticles having size in the range of 100 to 500 nm.
[021] The biosurfactant may be selected from Sophorolipids, rhamnolipid, phospholipid and glycolipid etc.
[022] The preservative may be selected from Citric Acid, Benzoic acid, Calcium Sorbate and sodium benzoate or combinations thereof.
[023] The excipients are selected from synthetic thickeners such as carbomers, polyacrylates, a base/ pH regulator, Xanthan Gum, Carbopol 940 or a combination thereof;
[024] Other additives are selected from fragrances, moisturising agents such as aloevera gel, tea tree oil and the like.
[025] The antimicrobial composition of the present invention may be formulated in the form of wide range of products selected from skin care products such as creams, gels, lotions, sprays, sticks, mousse; hair products such as shampoos, conditioners, hair styling products, suncare/aftersun products, bath and shower products, soaps and hand washes, etc. or any conventional formulation, as is well known to those skilled in the art.
[026] In an exemplary embodiment, the antimicrobial lotion formulation according to embodiment of the present invention comprises:
S. No. Ingredients Composition Percentage
1 Sophorolipids 0.01% to 1.0%
2 Silver Nanoparticles 0.001% to 0.004%
3 Citric Acid 0.1% to 0.8%
4 Xanthan Gum 0.01% to 5.0%
5 Carbopol 940 0.001% to 2.0%
6 Tea Tree Oil 0.01% to 4%
7 Aloe vera Gel 2% to 40%
8 Natural Fragrance 0.01% to 2%
9 Distilled water q.s.100 %
10 Sodium Hydroxide pH-7.0

[027] Method of preparation of formulation
(i) Dissolving a thickener with a moisturizer in a ratio in the range of 1:1 at room temperature and stirring at about 400 to about 1000 rpm;
(ii) Mixing a biosurfactant, a preservative acid and silver nanoparticles in a ratio in the range of 1:1:1 by sonication for about 30 to about 60 minutes in a solvent;
(iii) adding a natural emulsifier and natural gel to the mixture of step (ii) and mixing at a temperature range of about 50°C -60°C and stirring at about 1500 to about 2000 rpm;
(iv)adding the mixture of step (i) to the mixture of step (iii) at a temperature of about 50°C-60°C and stirring at about 1500 rpm to about 2000 rpm;
(v) Adding a natural fragrance to the stirred mixture of step (iv); and
(vi) Adjusting pH of the mixture of step (v) using a pH regulator and volume using the solvent.
[028] The formulation of the present invention is prepared based on the Prolonged Antiviral Protection (PAP) technology, which results in the formation of a protective layer around the skin. The accelerated nanoparticles in the product are engineered to provide long-lasting antimicrobial/anti-viral protection by slowly releasing the reactive oxygen species (ROS) on the skin surface. The released ROS generated through Silver nanoparticles and bio surfactant interferes with the transmembrane protein, outer lipid layer of the virus and helps in degradation of the genetic material of the virus whenever it comes in contact with the skin/hand/body and makes them permanently inactive, ultimately preventing the spread of infection and illness.
[029] The advantageous technical effects of the antimicrobial lotion formulation are attributed to the interplay of the triplet complex of silver nanoparticles, sophorolipids, and the preservative acid component which interferes with the trans-membrane protein and outer lipid layer of the microorganism rendering them functionally inactive and ultimately prevent the spread of infection and illness. This interplay of the three components provides long-lasting antimicrobial protection by slowly releasing the reactive oxygen species (ROS) on the skin surface. Therefore, if there are microbes in droplets or suspensions of microbes including virus on the skin, the microbes will interact with the triplet complex of the lotion formulation applied on the skin and render the virus inactive.
[030] In an exemplary embodiment, the triplet complex of the silver nanoparticles, sophorolipid and citric acid bonded together to form a strong chemical bond that interferes with the transmembrane protein and outer lipid layer of the microorganism and makes them permanently inactive and ultimately preventing the spread of infection and illness. This triplet complex formulation once applied to the skin will provide long-lasting antimicrobial protection by slowly releasing the reactive oxygen species (ROS) on the skin surface. Therefore, if there is a virus droplet on the skin it will interact with the triplet complex applied on the skin in the form of lotion and thus make the virus functionally inactive.

[031] EFFICACY OF THE COMPOSITION OF THE EXEMPLARY COMPOSITION OF THE PRESENT INVENTION

Sr. No. Ingredients Unit Formula (Qty. per 100ml)
1. Sophorolipids 0.1g
2. Silver nanoparticles 0.002g
3. Tea tree oil 0.5ml
4. Citric acid 0.6g
5. Aloe Vera gel 10g
6. Xanthan gum 0.5g
7. Carbopol/Carbomer 1g
8. Sodium hydroxide Adjusted using 10M Stock solution
9. Natural fragrance 0.06
10. Purified Water q.s.100ml

[032] The following tests were performed for determining efficacy of the composition of the present invention:
1. Antiviral Test using Test Standard: American Society for Testing & Materials (ASTM)-E-1052-20
2. Cell Cytotoxicity & Skin Safety test using Regional Center For Biotechnology, India approved protocol
3. Antimicrobial activity using test Standard: American Society for Testing & Materials (ASTM)-E-2315-16
[033] The formulation of the present invention was tested against the MS2 Bacteriophage, a commonly used surrogate for SARS-CoV (Coronavirus). MS2 Bacteriophage is used as a standard to study molecular biology processes of the enveloped and non-enveloped viruses. It includes viral RNA replication, translation method, and the psychology of infected cells. MS2 RNA coding for viral polypeptides includes protein A, coat protein, and RNA replicase complex. The structure of the MS2 virus consists of Protein A and coat protein makeup. The protein composition and molecular biology process of the MS2 virus is similar to the Coronavirus and these attributes make the MS2 virus as a Model for in-vitro virucidal efficacy. The formulation of the present invention was shown to reduce the virus by 99.99% (including Coronavirus).
[034] The lotion of the present invention was tested for cytotoxicity by an assay method as follows:
• The assay was done in a 96-well plate format in 3 wells for each sample.
• 1x10e4 VeroE6 cells were plated per well and incubated at 37-degree C overnight for the monolayer formation.
• Next day, cells were incubated with the test substance (TS) at the indicated concentration. The cells without test substance was the control for water soluble compounds.
• 24 and 48 hours later, cells were stained with Hoechst 33342 and Sytox orange dye.
• Images were taken at 10X, 16 images per well, which covers 90% of well area using ImageXpress Microconfocal (Molecular Devices).
• Hoechst 33342 nucleic acid stain is a popular cell-permeant nuclear counterstain that emits blue fluorescence when bound to dsDNA. It stains all the live and dead cells.
• Sytox orange dye stains nucleic acids in cells with compromised membranes. This stain is an indicator of cell death.
• First, the software counted total number of cells in the Hoechst image.
• In the Sytox image, it counted, among Hoechst positive cells, how many cells were positive for sytox.
[035] The lotion of the present invention was tested for anti-viral screening by an assay method as follows:
• The assay was done in a 96-well plate format in 3 wells for each sample.
• 1x10e4 VeroE6 cells were plated per well and incubated at 37-degree C overnight for the monolayer formation.
• Next day, cells were incubated with the test substance (TS) at the indicated concentration. The cells without test substance was the control for water soluble compounds.
• The cells were infected with SARS-CoV2 at a MOI of 0.01.
• 24 and 48 hours later, viral RNA was extracted from 100 µl culture supernatant and subjected to qRT-PCR (in duplicates) where Ct values for N and E gene sequence were determined.
• Inhibition of virus replication was determined based on the fold change in the Ct value in TS-treated cells compared to the control.
• Remdesivir was used as a positive control for viral inhibition.
[036] Table 1 shows the results of the cytotoxicity and antiviral testing:

Compound Name Concentration (µM)
Cells Viability (%) % Inhibition of virus replication
24 hr post infection 48 hr post infection
24 hr 48 hr E N E N
Remdesivir 10 µM 99.23 94.37 82.38 80.38 99.8 99.89
Lotion of the present invention-Sample 1 1 µl added from 49.3 mg/ml solution in 200 µl 113.95 95.20 30.32 43.3 32.45 44.43
Lotion Sample of the present invention- (Swab) Sample 2 1 µl added from the 10 times diluted solution in 200 µl 114.45 96.57 30.43 44.2 35.3 45.42

[037] Cell cytotoxicity and skin safety: The assay was done in a 96-well plate format in 3 wells for each sample. 1x10e4 VeroE6 cells were plated per well and incubated at 37-degree C overnight for the monolayer formation. Next day, cells were incubated with the formulation of Example 1(test substance) at the indicated concentration. The cells without the test substance were the control for 5 water-soluble compounds. 24 and 48 hours later, cells were stained with Hoechst 33342 and Sytox orange dye. Images were taken at 10X, 16 images per well, which covers 90% of well area using ImageXpress Microconfocal (Molecular Devices). Hoechst 33342 nucleic acid stain is a popular cell-permanent nuclear counter stain that emits blue florescence when bound to dsDNA. It stains all live 10 and dead cells. Sytox orange dye stains nucleic acids in cells with compromised membranes. This stain is an indicator of cell death. First, the software counts total number of cells in the Hoechst image. In the Sytox image, it will count, among Hoechst positive cells, how many cells are positive for Sytox. Results obtained from the tests are provided in Table 1 above The above results show that the lotion Sample of the present invention shows inhibition to virus replication.
[038] The Cell Cytotoxicity of the COVID-19 Protection lotion was conducted at different interval of times and found to be 100% safe for skin.
[039] The lotion of the present invention was tested for its antimicrobial activity against various microbes using time kill assay method. The results of such testing is provided in Table 2 and Table 3.
[040] Table 2: Antimicrobial activity by time kill assay-ASTM-E 2315-16
S.No. Organism Initial Microbial Count
(cfu/ml) Log Value Contact time Final Microbial Count
(cfu/ml) Log Value Log reduction (LR) % Reduction
1 Candida albicans (MTCC 227) 4.8x107 7.68 1 min 40000 4.60 3.08 99.92
2 S.aureus (ATCC 6538) 5.9x107 7.77 1 min 70000 4.65 2.92 99.88
3 Staph epidermidis (ATCC 12228) 2.9x107 7.46 1 min 41000 4.61 2.85 99.86
4 Escherichia coli(ATCC 8739) 1.6x107 7.20 1 min 15000 4.18 3.02 99.91
5 Listeria monocytogenes(ATCC 19111) 5.5x107 7.74 1 min 61000 4.79 2.95 99.89
6 Salmonella abony (NCTC6017) 5.7x107 7.76 1 min 3600 3.56 4.20 99.99
7 P.aeruginosa (ATCC 9027) 3.7x107 7.57 1 min 38000 4.58 2.99 99.90
8 Aspergillus brasilensis (ATCC 16404) 3.3x107 7.52 1 min 45000 4.65 2.87 99.86
Percentage reduction (%)=100 x (1-10 –LR)

[041] Table 2 shows that the formulation of the present invention was found to be having 99.9% antimicrobial activity against the eight microorganisms with the specified condition.
[042] Table 3: Antimicrobial activity by time kill assay-ASTM-E 1052-20
S.No. Organism Initial Microbial Count
(cfu/ml) Log Value Contact time Final Microbial Count
(cfu/ml) Log Value Log reduction (LR) % Reduction
1 E.Coli bacteriophage MS2 virus (ATCC 15597-B1) 5.0 x107 7.70 1 min 43000 4.63 3.067 99.9142
12 hrs 7200 3.86 3.843 99.9856
Percentage reduction (%)=100 x (1-10 –LR)

[043] Table 3 shows that the formulation of the present invention was found to be having 99.9% antimicrobial activity against the abovementioned MS2 bacteriophage with this specified conditions.
, Claims:WE CLAIM:
1. An antimicrobial composition comprising:
a) An antimicrobial agent 0.001% to 2.0%;
b) a biosurfactant 0.01% to 2%;
c) a preservative 0.1% to 1.8%;
d) Pharmaceutically acceptable compounds selected from excipients, carrier agents 0.001% to 5%; etc. and/or
e) Other additives 0.01 to 40%
f) Water (quantity remaining of 100%)
2. The antimicrobial composition as claimed in claim 1, wherein the antimicrobial agent is selected from the group of transition metal nanoparticles having size in the range of 100-500nm.
3. The antimicrobial composition as claimed in claim 2, wherein the transition metal nanoparticles are selected from the group of silver nanoparticles, copper nanoparticles, zinc nanoparticles, titanium nanoparticles or combination thereof.
4. The antimicrobial composition as claimed in claim 1, wherein the biosurfactant may be selected from Sophorolipids, rhamnolipid, phospholipid, glycolipid or a combination thereof.
5. The antimicrobial composition as claimed in claim 1, wherein the preservative is selected from the group of Citric Acid, Benzoic acid, Calcium Sorbate, sodium benzoate or a combination thereof.
6. The antimicrobial composition as claimed in claim 1, wherein the excipients are selected from the group of synthetic thickeners such as carbomers, polyacrylates, a base/pH regulator, Xanthan Gum, Carbopol 940 or a combination thereof.
7. The antimicrobial composition as claimed in claim 1, wherein the other additives are selected from the group of fragrances, moisturising agents such as aloevera gel, tea tree oil and the like.
8. The antimicrobial composition as claimed in claim 1, wherein the reduction in antimicrobial count is 99.9%.
9. A method of preparation of an antimicrobial composition as claimed in claim 1, comprising:
i. Dissolving a thickener with a moisturizer in a ratio in the range of 1:1 at room temperature and stirring at about 400 to about 1000 rpm;
ii. Mixing a biosurfactant, a preservative acid and silver nanoparticles in a ratio in the range of 1:1:1 by sonication for about 30 to about 60 minutes in a solvent;
iii. adding a natural emulsifier and natural gel to the mixture of step (ii) and mixing at a temperature range of about 50°C-60°C and stirring at about 1500 to about 2000 rpm;
iv. adding the mixture of step (i) to the mixture of step (iii) at a temperature of about 50°C-60°C and stirring at about 1500 rpm to about 2000 rpm;
v. Adding a natural fragrance to the stirred mixture of step (iv); and
vi. Adjusting pH of the mixture of step (v) using a pH regulator and volume using the solvent.
10. The method as claimed in claim 9, wherein the solvent is purified water.