The document presents the formulation and development of a thymol loaded gum acacia and carbopol crosslinked hydrogel wound dressing. It discusses the need for natural antimicrobial agents for resistant microorganisms and the objective to incorporate thymol in a novel self-adhesive wound dressing that retains wound healing properties. The plan of work includes literature review, selection of excipients, preformulation studies, formulation development, and evaluation of the antimicrobial activity, in vitro studies, in vivo studies and mechanical properties of the developed dressing. Various characterization techniques were used such as NMR, SEM, drug release studies and texture analysis to analyze the crosslinking of the polymers in the hydrogel and the properties of the final formulation.

1. Presented By :-
Mr. Nikhil Sanjay
Lohe
M.Pharm. Sem. IV
Dept of
Pharmaceutics
Under Guidance
:-
Prof. Vinita Patole
Assistant
Professor
Dept of
Pharmaceutics
JSPM’S Rajarshi Shahu College of Pharmacy &
Research,
Tathawade, Pune-33.
11
Formulation And Development of Thymol Loaded
Gum-Acacia and Carbopol Crosslinked Hydrogel
Wound Dressing

2. Content
2
1. Introduction
2. Literature survey
3. Need and objective
4. Plan of work
5. Drug profile
6. Excipient profile
7. Method
8. Evaluation parameters
9. Project outcome
10. Acknowledgement
11. References

3. INTRODUCTION
3
 A dressing is a sterile pad or compress applied to a wound to promote
healing and protect the wound from further harm.
 A dressing is designed to be in direct contact with the wound, as
distinguished from a bandage, which is most often used to hold
a dressing in place.
 Many modern dressings are self-adhesive
 Uses :-
1. Stem bleeding – help to seal the wound to expedite
the clotting process
2. Protection from infection – defend the wound against germs and
mechanical damage
3. Debride the wound – remove slough and foreign objects from the
wound to expedite healing

4. Types of dressing
4
 Historically, dressings were made of a piece of material, usually a cloth,
but the use of cobwebs, dung, leaves and honey have also been
described.
 However, modern dressings include dry or impregnated gauze, plastic
films, gels, foams, hydrocolloids, alginates, hydrogels, and
polysaccharide pastes, granules and beads.
 They all provide different physical environments suited to different
wounds
1. Gauze dressings
e.g..- Band-Aid
1. Biologics, skin substitutes, and scaffolds
2. Self-adaptive dressings

5. Hydrogel dressings
5
 Hydrogel dressings use a hydrogel pad in contact
with the wound.
 They are designed to keep the wound slightly moist,
releasing water or absorbing exudate.
 In the slightly moist environment, rodent skin heals
faster than in a dry one, from both cuts and partial-
thickness burns.

6. Why Hydrogel dressings ??
6
 Creates moist nature to dry wound.
 Absorbs exudates from the wound.
 Provides thermal insulation by maintaining 37-38 0c.
 Provides homeostasis
 Exchange of gases and provides external protection.

7. Wound
7
Definition of wound - A wound is a type of injury which
happens relatively quick in which skin is torn, cut, or punctured
(an open wound), or where blunt force trauma causes
a contusion (a closed wound).
 In pathology, it specifically refers to a sharp injury which damages
the dermis of the skin.
Classification of wounds – there are three major types of
wound
Open wound Burn Cut wound

8. Types of wound
8
o According to level of contamination
1. Clean wound – made under sterile conditions where there are no
organisms present, and the skin is likely to heal without
complications
2. Contaminated wound – usually resulting from accidental injury; there
are pathogenic organisms and foreign bodies in the wound.
3. Infected wound – the wound has pathogenic organisms present and
multiplying, exhibiting clinical signs of infection (yellow appearance,
soreness, redness, oozing pus).
4. Colonized wound – chronic situation, containing pathogenic
organisms, difficult to heal (i.e. bedsore).
5. Burn - A burn is a type of injury to skin, or other tissues, caused
by heat, cold, electricity, chemicals, friction, or radiation. Most burns
are due to heat from hot liquids, solids, or fire.

9. Wound Healing
9
 Wound healing is the complex cascade of cellular
and biochemical actions leading to the restoration of
structural and functional integrity with regain of
strength of injured tissues.
Stages of wound healing
1. Inflammatory stage
2. Fibroblastic stage
3. Maturation stage

10. Literature survey
10
Name of author ,
journal and year of
publication
Work done in the
research paper
Conclusion
Baljit Singh and
Abhishek Dhiman
American Chemical
Society publication
Aug. 2016
Prepared and evaluated
the Design of Acacia
Gum−Carbopol−Cross-
Linked-
Polyvinylimidazole
Hydrogel Wound
Dressings
The hydrogel wound healing dressing
was prepared and evaluated and
reported the synergic effects of the
mucoadhesive, antimicrobial, and
antioxidant nature of hydrogel
dressings
Mollarafie P and Khadiv
Parsi P
Journal of Medicinal
Plants
Feb. 2015
Antibacterial and
Wound Healing
Properties of Thymol
(Thymus
vulgaris Oil) and its
Application in a Novel
Wound Dressing
Thymol gave antibacterial and
wound healing properties for
a wound dressing.

11. Literature survey
11
Name of author ,
journal and year of
publication
Work done in the
research paper
Conclusion
Stoja Milovanovic
The Journal of Supercritical
Fluids
Oct. 2013
Solubility of thymol in
supercritical carbon dioxide
and its impregnation on
cotton gauze
Thymol was incorporated in
cotton gauze and shows
wound healing property
Stoja Milovanovic and
Marko Stamenic
The Journal of Supercritical
Fluids
Nov. 2015
Supercritical impregnation of
cellulose acetate with thymol
Strong antimicrobial effect of
impregnated samples against
Escherichia coli,
Staphylococcus aureus and
Candida albicans strains with
microbial reduction up to
99.9% was observed

12. Need and objective
12
 Need
 Most of the microorganism are resistant towards the synthetic drug.
 Need to search for natural antimicrobial agents.
 Need to explore Thymol as a wound healing agent
 To search for novel dressing which is self adhesive as well as having
antimicrobial property of its own.
 Objective
 To examine thymol for existence of its antimicrobial and wound healing
property
 To incorporate thymol in novel wound dressing and characterize the
resulting product ensuring that it retains wound healing property and
assuring the effect of its incorporation
 To prepare the novel dressing and evaluate it.

13. Plan of Work
13
Literature
review
Selection of
excipients
Preformulation
study
Formulation
development
Antimicrobial
activity study
In vitro study
In vivo study outcome

14. Drug profile
14
 Drug name: Thymol
 Category: Anti-microbial
 Solubility: Insoluble in water, soluble in
ethanol and methanol
Observed
peaks(cm-1)
Interpretation of
chemical group
3867.30 -O-H group
3745.61 -C-H group
3677.67 -C-H group
1699.70 C=C stretching
1517.86 C-O stretching
D:FTIR DATATHYMOL.5 THYMOL ATR eco ZnSe 13/11/2017
3867.30
3745.61
3677.67
1699.70
1517.86
100015002000250030003500
Wavenumber cm-1
96.597.598.599.5100.5
Transmittance[%]
Page 1/1

15. Drug profile
15
Parameters Properties
Melting point (°C) 51.5 °C
Boiling point (°C) 232.5 °C
Solubility Insoluble in water
Soluble in organic solvent
logP 3.30
pKa 10.6 (at 20 °C)
Categories • Anti-Infective Agents
• Anti-Infective Agents, Local
• Antifungal Agents
• Benzene Derivatives
• Monoterpenes

16. CALIBRATION CURVE OF THYMOL
16
R² = 0.995
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
5 10 15 20 25 30
ABS
ABS
Linear (ABS)
CONC. (µg/ml)
CONC (µg/ml) ABS
5 0.113
10 0.243
15 0.359
20 0.483
25 0.667
30 0.783

17. ANTI MICROBIAL STUDY OF THYMOL
17
2 % THYMOL
ZOI= 20 MM
Pseudomonas
aeruginosa
PLAIN DMSO
ZOI = 0 MM
ZOI – Zone of
Inhibition
2% THYMOL
ZOI= 15 MM
Staphylococcu
s aureus

18. EXCIPIENT PROFILE
18
ACACIA
 It is the dried gummy exudation obtained from the stem and branches
of Acacia belonging to family Leguminosae.
 Gum arabic (Acacia gums) is carbohydrate but contain 2-3% protein as
an part of its structure.
 Advantages of natural gums
 Biodegradable
 Biocompatible and non-toxic
 Low cost
 Local availability
 Anti-microbial activity

19. EXCIPIENT PROFILE
19
CARBOPOL
 Carbopol polymers are polymers of acrylic acid cross-linked with
polyalkenyl ethers or divinyl glycol.
 Colour: White, light, acidic, hygroscopic powder.
 Particle size: Flocculated powder having a median diameter of 2 to 7
microns.
 Solubility / swelling properties: Insoluble due to their cross linked
nature and high molecular weight. Get swell in water and some polar
solvents.
 Uses: Gelling agent
thickening agent

20. EXCIPIENT PROFILE
20
N-N, METHYLENEBIS ACRYLAMIDE
 Chemical Names: N,N'-METHYLENEBISACRYLAMIDE;
 Molecular Formula: C7H10N2O2
 Molecular Weight: 154.169 g/mol
 IUPAC Name:N-[(prop-2-enoylamino)methyl]prop-2-enamide
 Uses: Crosslinking agent

21. EXCIPIENT PROFILE
21
Ammonium Persulfate
 Chemical Names: Ammonium persulfate;
 Molecular Formula: H8 N2 O8 S2/ (NH4)2S2 O8
 Molecular Weight: 228.19 g/mol
 Industry Uses:
1. Bleaching agents
2. Fuels and fuel additives
3. Intermediates
4. Ion exchange agents
5. Oxidizing/reducing agents

22. EXCIPIENT PROFILE
22
Tri Ethyl Citrate
 Triethyl citrate is an ester of citric acid
 Colorless, odorless liquid used as a food additive
 Also used as a plasticizer for pharmaceutical formulation
 IUPAC name: Triethyl 2-hydroxypropane-1,2,3-tricarboxylate

23. RATIONAL OF DRUG AND EXCIPENTS
SELECTION
23
 THYMOL – Thymol is potent anti microbial agent and also
natural extract T.VELGERIS
It also work as wound healing agent by initiating cell
multiplication
 ACACIA - Acacia is natural gum which shows anti microbial
action and biodegradable property.
 CARBOPOL - Carbopol is inert gum which has a sufficient
adhesive properties.

24. METHOD
CROSSLINKING OF ACACIA AND CARBOPOL
24
MIXTURE OF
POLYMERS
• ACACIA
• CARBOPOL
• Mix in 100 ml
water
ADDITION OF
CROSSLINKING
AGENTS
• N,N-Methylene
Bisacrylamide
• Ammonium
Persulphate
Formulation of
cross-linked
hydrogel
• Mix it for 24 hrs
with continuous
stirring
• Allow the
hydrogel to dry
at 50℃ for 24 hrs

25. Preliminary batches of hydrogel
25
2% Thymol was dissolved in sufficient ethanol which was dispersed in the each
batch of hydrogel formed with continuous stirring
Batches Acacia : Carbopol 934 Mucoaddhasive Tensile strength
F1 1:4 ---- 78 mm
F2 2:3 35 mj 50.07 mm
F3 2.5:2.5 27 mj 43.5 mm
F4 3:2 20 mj 38.6 mm
F5 4:1 ---- 30.01 mm

26. Formulation of Film
26
 The formulation of the film was done by the solution casting
method
 Pour the hydrogel in the petri plate and allow to dry for 24 hrs.
Fig- Formulated patch in proper packaging

27. Evaluation of Dressing
27
1. Cross linking of acacia and carbopol
2. Biodegradability of dressing
3. Water vapour transmission study
4. Drug content
5. Drug release study- diffusion cell apparatus
6. Antimicrobial properties
7. Animal study
8. Mechanical properties
I. Tensile strength
II. Folding endurance
III. Surface pH
IV. Total area of patch

28. Cross linking of acacia and carbopol
28
 NMR STUDY OF ACACIA, THYMOL AND
CROSSLINKED HYDROGEL
C13 NMR OF ACACIA AND HYDROGEL
ACACIA THYMOL HYDROGEL

29. 29
Observed peaks Interpretation of chemical group
Thymol Gum-Acacia Hydrogel
209 178 C=O aromatic ring
205 174 COOH aromatic ring
152 173 C=C aromatic ring
136 171 C=C aromatic ring
126 129 C=C aromatic ring
116 116 C=C aromatic ring
81 C=C starching non aromatic
76 C-C stretching
73 73 C-C stretching
72 63 C-C stretching
69 62 C-OH stretching
65 57 C=O non aromatic
61 43 C-CH2-CH3
40-10 40-10 -CH3

30. Cross linking of acacia and carbopol
30
SEM OF ACACIA
SEM OF HYDROGEL

31. Biodegradability of dressing
31
DAY 1 DAY 5

32. Water vapour transmission study
32
BEFORE HEATING AFTER HEATING
i. After test patch retains 0.5 gm of water
ii. Water transmitted across the patch is 0.1 gm
iii. % increase in weight of patch = 62.23 %
iv. % of transmitted water = 12.5%

33. Texture Profile Analysis Of Dressing
33
FORMULATION TENSILE STRENGTH
F1 74.8 mm
F2 50.07mm
F3 43.5 mm
F4 38.65 mm
F5 30.01 mm
Hardness : 1920g
Hardness work : 5.7 mJ
Adhesive force : 107g
Adhesiveness : 61.8 mJ

34. Surface pH Folding Endurance
 Film of F2 = 7.02
 Plain hydrogel = 7.5
 Plain acacia carbopol
film = 78 folds (without
plasticizer)
 Thymol loaded hydrogel
film = 88 folds (without
plasticizer)
 Thymol loaded hydrogel
film = 124 folds (with
plasticizer)
34
Mechanical properties

35. DRUG RELEASE OF ALL
BATCHES IN 8 HRS
DRUG RELEASE GRAPH
Batch Drug release %
F1 80 ±0.2
F2 94.72 ± 0.5
F3 89.08 ± 0.6
F4 87.91± 0.4
F5 83.008± 0.15
35
In- Vitro Drug release study

36. Drug content of final batch
36
 Drug content = ABS – Intercept
× dilution factor
Slope
•100 ml of hydrogel contains 2000 mg of thymol
•25 ml of hydrogel contains 500 mg of thymol
•So the film of 72.8 cm2 contains 500 mg of thymol

37. Anti microbial test of HYDROGEL
37
 Zone of inhabitation
0
5
10
15
20
25
30
35
2%
Thymol
Loaded
Hydrogel
2%
Thymol
0 %
thymol
loaded
hydrogel
dmso
control
ZOI (mm)
zoi (MM)

38. MICROBIAL PENETRATION TEST
38
DAY 1 DAY 2

39. Animal Study
39
 The animal study of the patch was done by the giving the external
cut wound method and observed for the 15 days
 The whole animal study was clarified by the histopath study of
the skin tissue.
 The study was carried out by the observing the 6 groups of
animals as stated in the following table.
Group of animals No. of animals
Thymol loaded hydrogel dressing 3
Plain hydrogel dressing 3
Plane acacia 3
Marketed formulation 3
Plain thymol 3
Control 3

40. % WOUND HEALING
40
POST WOUNDED
DAYS
GROUP 1 GROUP 2 GROUP 3 GROUP 4 GROUP 5 GROUP 6
1 0 0 0 0 0 0
3 43.57 18.94 22.25 19.09 52.49 16
7 98.88 63.08 75.48 62.21 98.79 35.2
10 100 84.96 85.18 80.54 100 49.5
15 100 94.73 98.48 89.7 100 61.75
• Group I- Thymol Loaded Hydrogel film
• Group II- Acacia Carbopol crosslinked film
• Group III- Plain Thymol
• Group IV- Plain Acacia
• Group V- Marketed
• Group VI- Control

41. 41
0
20
40
60
80
100
120
0 2 4 6 8 10 12 14 16
% wound healing
GROUP 1
GROUP 2
GROUP 3
GROUP 4
GROUP 5
GROUP 6
%woundhealing
post wounded days
%woundhealing
post wounded days
• Group 1- Thymol Loaded
Hydrogel film
• Group 2- Acacia Carbopol
crosslinked film
• Group 3- Plain Thymol
• Group 4- Plain Acacia
• Group 5- Marketed
• Group 6- Control

42. 42
(A) (B)
Fig- A- skin of control rat with wounded structure
B- Skin of Thymol loaded hydrogel wound dressing treated rat

43. PROJECT OUTCOME
43
 The 2% thymol loaded wound dressing shows
property, self adhesiveness and antimicrobial
property of its.
 The 2% thymol loaded novel biodegradable bandage
shows the anti microbial activity and wound healing
property .

44. Poster on project
44
1. NIPICON-2018 at
Nirma University
2. DPU conference-
2018 at DYPU
Pimpri

45. Acknowledgement
45
 I will thank to my guide and colleagues for helping me out in
my project
 I am also thankful to my parents for there support and love.
 Very special thaks to Dr. Pandit mam, Dr. Atul Sayare and Dr.
Pranshant Ghode for there valuable Guidance.
 Thanks to G K Bioscience for providing me excellent
Excipients for my work
 Thanks to Faculty of Instrumentation SPPU for evaluation
studies….
 Also thankfull to PRADO Research institute for animal study..

46. REFERANCES
46
1. Baljit Singh and Abhishek Dhiman “Design of Acacia Gum−Carbopol−Cross-
Linked-Polyvinylimidazole Hydrogel Wound Dressings for
Antibiotic/Anesthetic Drug Delivery” Industrial & Engineering Chemistry
Research, Aug 2016.
2. Mollarafie P and Khadiv Parsi P “Antibacterial and Wound Healing Properties
of Thymol (Thymus vulgaris Oil) and its Application in a Novel Wound
Dressing” Journal of Medicinal Plants, Feb. 2015.
3. Stoja Milovanovic “Solubility of thymol in supercritical carbon dioxide and its
impregnation on cotton gauze” , The Journal of Supercritical Fluids, Oct. 2013.
4. Stoja Milovanovic and Marko Stamenic , “Supercritical impregnation of
cellulose acetate with thymol” , The Journal of Supercritical Fluids Nov. 2015

47. References
47
5. Khalil Ettayebi and Jamal El Yamani, “Synergistic e¡ects of nisin and thymol
on antimicrobial activities in Listeria monocytogenes and Bacillus subtilis” ,
FEMS Microbiology Letters, Dec. 1999.
6. C.C. Liolios, “Liposomal incorporation of carvacrol and thymol isolated from
the essential oil of Origanum dictamnus L. and in vitro antimicrobial activity”
Food Chemistry, May. 2008.

48. 48