Title of Invention

"AN ANTIMICROBIAL SOLUTION FOR WOUND COVERING AND PROCESS FOR PREPARATION THEREOF"

Abstract An antimicrobial solution for wound covering comprising encapsulated active ingredient with film forming adhesive polymeric solution in ratio 1:1-1.2 in presence of 0.05-0.1% sodium salt of ethylenediaminotetra acetic acid and neutral emulsifier.
Full Text FIELD OF INVENTION
The present invention relates to an antimicrobial solution for wound covering which has antimicrobial and biodegradable polymeric composition and the process of preparation of the composition thereof.
PRIOR ART
Commonly used tincture iodine is mere iodine in alcohol solvent, and when it is applied on wound, alcohol evaporates and iodine powder comes out. This is only temporary for 1 or 2 minutes to cover the wound and afterwards the wound is exposed to pathogenic microorganisms. Binding of anti-microbial by polymer is required for prolonged action of wound covering solution.
Polyurethanes (PU) are extensively used in wound healing applications, particularly for occlusive and semi occlusive (barrier dressing). One of the drawbacks of the semi occlusive dressing of PU coating is that it is two pack system in which the components have to be mixed to the correct proportion at the time of dressing and therefore not user friendly. The second limitation is that significant fluid accumulation can occur underneath wound. However, biodegradable natural polymers are preferred in barrier dressing for the following reason:
- it is a single pack system and users friendly, and
- it is biodegradable
The vast majority of work on anti-microbial, is by conventional method of leaching technique. The mechanism used by leaching anti-microbial to poison microorganism. U.S. Patent 6573249 uses antibiotic namely Cromblyn and hyaluronic acid as leachate in anti-microbial formulation. Antibiotics chlorhexidine biopatch for wound healing. U.S. Patents 6844010 and 6248075 uses leaching type microcapsule anti-microbial. U.S. Patent 6503,531 uses

broad-spectrum diiodomethyl - p- tolyl sulphone as release anti-microbial for wound dressing. U.S. Patents 5814,094 and 6087,549, uses Nano silver colloids as anti-microbial for wound covering and dressing. In U.S.Patent 6,087,549; multiplayer conductive silver having wound healing, covering and analgesic properties. Use of antibiotic as leachate is having following drawbacks:
- they can affect normal skin bacteria, cause rashes and other skin irritation,
- they are not eco friendly, and
- their shorter shelf life.
Some of the above mentioned wound covering or dressing contains either antibiotics or silver ion as anti-microbial. These anti-microbial dressing materials, suffers from the disadvantage of having high cost, not eco friendly and does not contain any natural preservatives.
Silicone based wound healing molecularly bonds with skin and gets fused. The disadvantage of this is that it gets bonded to skin and long term effects are not known. U.S. Patent 6379,726, a water resistant composition which may be directly precipitated from a solution under high pressure treatment with carbon dioxide and does not have to be cross linked. This method is less effective and hence limited use as for are micro encapsulation is concerned. Where as anti-microbial can be micro encapsulated in-situ in the polymer matrix of casein and other biopolymer blend by cross linking using reactive compatibilizer namely maleic anhydride. This process is known as Inter penetrating network and is effective in micro-encapsulation where in antimicrobial is tightly held in three dimensional network of polymer matrix there by inhibiting the leaching of anti-microbial and also gives stable emulsion/coating. Non leaching type does not pose any toxilogical problem to the skin.

There is a growing concern among consumers about the level of antibiotics present in the wound covering/healing, which are applied on the skin need. As a alternate technique to encapsulate some of the natural phytochemicals such as neem and turmeric, having anti-microbial properties in a polymer matrix to prevent infection has been attempted in this invention. An anti-microbial with a completely different mode of action than the leaching technologies is to bind the anti-microbial in three dimensional network i.e. micro encapsulation and not allows it to leach and absorbed into the skin or re-absorption.
OBJECT OF INVENTION
The main objective of the present invention is to prepare antimicrobial solution for wound healing.
Another object of the invention is to incorporate biodegradable polymeric matrix in an anti-microbial solution.
Further, object of the invention is to microencapsulate natural phytochemicals.
SUMMARY OF INVENTION:
To obviate drawbacks of the prior art the present invention provides an antimicrobial solution for wound covering comprising encapsulated active ingredient with film forming adhesive polymeric solution in ratio 1:1-1.2 in presence of 0.05-0.1% sodium salt of ethylenediaminotetra acetic acid and neutral emulsifier.
The present invention also provides a process for the preparation of antimicrobial solution for wound covering comprising;

encapsulating aqueous propylene glycol extract of active ingredient with film forming adhesive polymeric solution in ratio 1:1-1.2 by adding 0.05-0.1% sodium salt of ethylene diamino tetra acetic acid and heating at 85-95°C; and
emulsifying with neutral emulsifier to obtain clear antimicrobial solution for wound covering.
DETAILED DESCRIPTION OF THE INVENTION:
In order to achieve the aforementioned objectives present invention provides an antimicrobial solution for wound covering comprising encapsulated active ingredient with film forming adhesive polymeric solution in ratio 1:1-1.2 in presence of 0.05-0.1% sodium salt of ethylenediaminotetra acetic acid and neutral emulsifier.
The active ingredient is natural phytochemical/s such as Azadirachta indica leaves, Curcumin longa either alone or in combination.
The neutral emulsifier is polysorbate.
The invention further provides a process for the preparation of antimicrobial solution for wound covering comprising;
encapsulating aqueous propylene glycol extract of active ingredient with film forming adhesive polymeric solution in ratio 1:1-1.2 by adding 0.05-0.1% sodium salt of ethylene diamino tetra acetic acid and heating at 85-95°C; and
emulsifying with neutral emulsifier to obtain clear antimicrobial solution for wound covering.
The aqueous propylene glycol extract is obtained by mixing and grinding Melia azadirachta leaves (neem) and Curcuma longa (turmeric) in ratio 1:0.6-1.5 to

form paste and heating with 20-50 times of solvent aqueous propylene glycol for 2-3 hours at 85-95°C and filtering to obtain their aqueous propylene glycol extract. The aqueous propylene glycol is mixture of water and propylene glycol in ratio 3-6:1.
The film forming adhesive polymeric solution is obtained by heating mixture of 60-80% dewaxed shellac, 10-20% casein, 10-20% polyvinyl alcohol and 1-2% maleic anhydride to melt and adding 10-20 times absolute alcohol slowly while heating and then adding 1-3 times propylene glycol to obtain clear solution to witch 30% ammonia solution is added to adjust pH 7.8-8.2.
The invention is now described with reference to below mentioned examples:
Example 1
Dewaxed shellac 2.0 g, casein 0.5 g, polyvinyl alcohol 0.5 g and maleic anhydride 0.05 g mixed well to obtain uniform powder. The powder placed in an iron pan and slowly heated until the entire mass melts. The heating was continued for 10 minutes while adding it to 30 ml of ethanol (absolute alcohol). There after 5 ml of propylene glycol is added to obtain clear and transparent solution is obtained. The pH of the solution was maintained at 7.8 adding 30 % ammonia solutions (Part A).
Melia azadirachta leaves (neem) 3 g and Curcuma longa 3 g were mixed and grinded to from paste in hot water. The slurry was taken in a 11t stainless steel vessel and to this 150 ml of water and 25 ml of propylene glycol was added and heated to 85°C for 3 hours while stirring with a spatula. The digested material filtered using double fold muslin cloth. The filtrate forms the active ingredient of neem leaves and turmeric (Part B).
100 ml of solution of Part A and 100 ml Part B was placed in 2 liter three necked reaction flask. In one of the three necks of the flask was fitted with a

thermometer in order to monitor the temperature during encapsulation and other neck was connected to a nitrogen gas cylinder to provide an inert atmosphere. The third neck of the flask connected to a remotely controlled glass stirrer fitted with Teflon blade. 0.2 gram of sodium salt of ethylene diamino tetra acetic acid is dissolved in 20 ml of hot water and poured in to the flask. The blending was continued for 6 hours at 90°C and thereafter cooled to 30°C. The solution was emulsified using polysorbate such as polyoxyethylene sorbitan monolaurate (Tween 20) as emulsifier in a homogenizer at 1000 rpm at ambient temperature. The resultant antimicrobial solution I was stored in a glass bottle at ambient temperature is ready for use as wound covering shelf life or Stability is more than one year and no mould or fungal growth was found even after one year storage of antimicrobial solution I.
Example 2:
Dewaxed shellac 3.0 g, casein 0.7 g, polyvinyl alcohol 0.7 g and maleic anhydride 0.08 g mixed well to obtain uniform powder. The powder placed in an iron pan and slowly heated until the entire mass melts. The heating was continued for 10 minutes while adding it to 45 ml of ethanol (absolute alcohol). There after 8 ml of propylene glycol is added to obtain clear and transparent solution is obtained. The pH of the solution was maintained at 8.0 adding 30 % ammonia solution (Part A).
Curcuma longa 2 g and Melia azadirachta leaves 3.0 g were mixed and grinded form to paste in hot water. The slurry was taken in a 11t stainless steel vessel and to this 250 ml of water and 35 ml of propylene glycol was added and heated to 95°C for 2.5 hours while stirring with a spatula. The digested material filtered using double fold muslin cloth. The filtrate forms the active ingredient of neem and turmeric (Part B).

90 ml of solution of Part A and 110 ml Part B was placed in 2 liter three necked reaction flask. In one of the three necks of the flask was fitted with a thermometer in order to monitor the temperature during encapsulation and other neck was connected to a nitrogen gas cylinder to provide an inert atmosphere. The third neck of the flask connected to a remotely controlled glass stirrer fitted with Teflon blade. 0.15 g of sodium salt of ethylene diamino tetra acetic acid is dissolved in 20ml of hot water and poured in to the flask. The blending was continued for 6 hours at 90°C and thereafter cooled to 30°C. The solution was emulsified using Tween - 20 as emulsifier in a homogenizer at 1000 rpm at ambient temperature. The resultant antimicrobial solution II was stored in a glass bottle at ambient temperature is ready for use as wound covering. Its shelf life or Stability is more than one year and no mould or fungal growth was found even after one year storage of antimicrobial solution II.
Example 3:
De-waxed shellac 2.5 g, casein 0.6 g, polyvinyl alcohol 0.6 g and maleic anhydride 0.06 g mixed well to obtain uniform powder. The powder placed in an iron pan and slowly heated until the entire mass melts. The heating was continued for 10 minutes while adding it to 40 ml of ethanol (absolute alcohol). There after 6 ml of propylene glycol is added to obtain clear and transparent solution is obtained. The pH of the solution was maintained at 7.8 adding 30 % ammonia solution (Part A).
Melia azadirachta leaves 2 g and Curcuma longa 3 g were mixed and grinded to fprm paste in hot water. The slurry was taken in a 1 It stainless steel vessel and to this 200 ml of water and 30 ml of propylene glycol was added and heated to 90°C for 2 hours while stirring with a spatula. The digested material filtered using double fold muslin cloth. The filtrate forms the active ingredient of neem and turmeric (Part B).

110 ml of solution of Part A and 90 ml Part B was placed in 2 liter three necked reaction flask. In one of the three necks of the flask was fitted with a thermometer in order to monitor the temperature during encapsulation and other neck was connected to a nitrogen gas cylinder to provide an inert atmosphere. The third neck of the flask connected to a remotely controlled glass stirrer fitted with Teflon blade. 0.1 gram of sodium salt of ethylene di-amino tetra acetic acid is dissolved in 20ml of hot water and poured in to the flask. The blending was continued for 6 hours at 90°C and thereafter cooled to 30°C. The solution was emulsified using Tween - 20 as emulsifier in homogenizer at 1000 rpm at ambient temperature. The resultant antimicrobial solution III was stored in a glass bottle at ambient temperature is ready for use as wound covering. Its shelf life or Stability is more than one year and no mould or fungal growth was found even after one year storage of antimicrobial solution III.
The above preparations were tested in vitro and vivo to evaluate their efficacies.
IN VITRO STUDIES:
The pathogenic test organisms namely E. coli, Staphylococcus aureus, Bacillus cereus, Pseudomonas and Salmonella typhimurium was procured for IMTECH, Chandigarh, India. Well diffusion assay method was used to evaluate antimicrobial properties of the antimicrobial solutions I, II and III obtained from examples 1, 2 and 3. The test organisms were grown in nutrient broth at 37°C. The overnight culture was diluted with distilled water (100ml) and 100 microlitres of the diluted inoculum was uniformly spread on the poured nutrient agar medium. Circular two wells of 8 mm diameter were cut on the agar surface. Sterilized distilled water was used as control. The plates were incubated for 24 hours at 37°C. The plates were visually examined for inhibition zones around the wells and the diameter of the zone was measured using a caliper. The results obtained are given in TABLES -1,2 and 3.

TABLE - 1
Anti-Microbial Activity of Medicinal Plants and Antimicrobial Solution I by Disc Diffusion Array (10mm)
(Table Removed)
TABLE - 2
Anti-Microbial Activity of Medicinal Plants and
Antimicrobial Solution II by Disc Diffusion Array (10mm)
(Table Removed)
TABLE - 3
Anti-Microbial Activity of Medicinal Plants and
Antimicrobial Solution III by Disc Diffusion Array (10mm)

(Table Removed)
Three antimicrobial solutions I, II and III, which were obtained from Examples 1,2 and 3 by varying the proportion of Melia azadirachta and Curcuma longa and there after encapsulating in polymer matrix were evaluated for in vitro anti-microbial properties by above procedure. It was observed that encapsulated Melia azadirachta and Curcuma longa in the ratio of 1:1 by weight has maximum anti-microbial properties. The inhibition disc diameter, is proportional to anti-microbial properties, and is more when Melia azadirachta and Curcuma longa blend is used as compared to individual components. This establishes the synergic effect.
IN VIVO STUDIES:
In vivo, tests were carried out by using test animal 6 to 8 rats weighing at least ½ kg in each group, were reared at room temperature (25°C) with relative humidity of 55±10%. Ten ml of emulsion was applied using cotton to intact and abraded skin respectively. The skin condition of the rats was observed after 24, 72 and 96 hours; with and without antimicrobial solutions. Results are given in TABLE 4.
TABLE - 4
In vivo, studies on rats -Red blisters and puss formation


(Table Removed)
Table 4, gives the antimicrobial effect of antimicrobial solutions I, II and III in rats. In this vivo studies, The Table 4, red blister and puss formation in rats were observed at different interval of time. There is no red blister or puss formation in antimicrobial solution I, in which Melia azadirachta leaves (neem) and Curcuma longa encapsulated in polymeric liquid in 1:1 ratio.








We Claim
1. An antimicrobial solution for wound covering comprising encapsulated active ingredient with film forming adhesive polymeric solution in ratio 1:1-1.2 in presence of 0.05-0.1% sodium salt of ethylenediaminotetra acetic acid and neutral emulsifier.
2. An antimicrobial solution for wound covering as claimed in claim 1 wherein the active ingredient is natural phytochemical/s such as Melia azadirachta leaves, Curcuma longa either alone or in combination.
3. An antimicrobial solution for wound covering as claimed in claim 1 wherein neutral emulsifier is polysorbate.
4. A process for preparation of antimicrobial solution for wound covering comprising
encapsulating aqueous propylene glycol extract of active ingredient with film forming adhesive polymeric solution in ratio 1:1-1.2 by adding 0.05-0.1% sodium salt of ethylene diamino tetra acetic acid and heating at 85-95°C; and
emulsifying with neutral emulsifier to obtain clear antimicrobial solution for wound covering.
5. A process for preparation of antimicrobial solution as claimed in claim 4,
wherein film forming adhesive polymeric solution is obtained by the
steps:
heating mixture of 60-80% dewaxed shellac, 10-20% casein, 10-20% polyvinyl alcohol and 1 -2% maleic anhydride to melt, adding 10-20 times absolute alcohol slowly while heating, adding 1-3 times aliphatic glycol to obtain clear solution,

adjusting pH to 7.8-8.5 by adding 30% ammonia solution to obtain film forming adhesive polymeric solution.
6. A process for preparation of antimicrobial solution as claimed in claim 4,
wherein the aqueous propyleneglycol is a mixture of water and
propylene glycol in ratio 3-6:1.
7. A process for preparation of antimicrobial solution as claimed in claim 5, wherein the alcohol is selected from the C1-C4 alcohols.
8. A process for preparation of antimicrobial solution as claimed in claim 5, wherein aliphatic glycol is selected from the C2-C4 glycol.
9. An antimicrobial solution for wound covering substantially such as herein
described and exemplified.
10. A process for preparation of antimicrobial solution for wound covering
substantially such as herein described and exemplified.

Documents:

85-DEL-2007-Abstract-(26-08-2010).pdf

85-del-2007-abstract.pdf

85-DEL-2007-Claims-(26-08-2010).pdf

85-del-2007-claims.pdf

85-del-2007-Correspondence-Others-(20-04-2011).pdf

85-DEL-2007-Correspondence-Others-(26-08-2010).pdf

85-del-2007-correspondence-others-1.pdf

85-DEL-2007-Correspondence-Others.pdf

85-DEL-2007-Description (Complete)-(26-08-2010).pdf

85-del-2007-description (complete).pdf

85-DEL-2007-Form-1-(26-08-2010).pdf

85-del-2007-form-1.pdf

85-del-2007-form-18.pdf

85-DEL-2007-Form-2-(26-08-2010).pdf

85-DEL-2007-Form-2.pdf

85-del-2007-Form-3-(20-04-2011).pdf

85-DEL-2007-Form-3-(26-08-2010).pdf

85-del-2007-form-3.pdf

85-DEL-2007-Form-5.pdf

85-DEL-2007-GPA.pdf


Patent Number 248504
Indian Patent Application Number 85/DEL/2007
PG Journal Number 29/2011
Publication Date 22-Jul-2011
Grant Date 20-Jul-2011
Date of Filing 12-Jan-2007
Name of Patentee DIRECTOR GENERAL, DEFENCE RESEARCH & DEVELOPMENT ORGANISATION
Applicant Address MINISTRY OF DEFENCE, GOVT OF INDIA, ROOM NO 348, B-WING, DRDO BHAWAN, RAJAJI MARG, NEW DELHI 110 011
Inventors:
# Inventor's Name Inventor's Address
1 JAMBUR HIRIYANNAIAH JAGANNATH DEFENCE BIOENGINEERING & ELECTROMEDICAL LABORATORY (DEBEL) BANGALORE-560093
2 RADHIKA MADAN URS DEFENCE BIOENGINEERING & ELECTROMEDICAL LABORATORY (DEBEL) BANGALORE-560093
3 VINOD CHIDAMBAR PADAKI DEFENCE BIOENGINEERING & ELECTROMEDICAL LABORATORY (DEBEL) BANGALORE-560093
4 AGRAM SRINIVASAMURTHY KRISHNA PRASAD DEFENCE BIOENGINEERING & ELECTROMEDICAL LABORATORY (DEBEL) BANGALORE-560093
PCT International Classification Number A61K 35/78
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA