The document describes the development and evaluation of dermal patches containing Calendula Officinalis for antimicrobial activity. Five formulations of dermal patches with varying drug content were prepared using HPMC and SCMC as polymers and polyethylene glycol as a plasticizer. The patches were evaluated for thickness, weight uniformity, folding endurance and moisture content. Scanning electron microscopy showed the patches had an irregular, rough surface which could aid in drug release. The patches were also tested for antimicrobial activity against Staphylococcus aureus using the disc diffusion method.

1. Under the guidance
Mr. Lalatendu Mohanty
Asst. Professor
Presented by-
Suraj Mohan
M.Pharm. 4th Sem
Department Of Pharmaceutical science, H.N.B.G.U. Srinagar Garhwal

2. Content
Introduction.
Objective of study
Protocol of work
Literature review
Plant description
Excipient profile
Methodology and evaluation
Result and discussion
Conclusion
Reference.

3. Introduction
 Dermal drug delivery system1
Dermal delivery should only be used to define
targeting to the pathological sites within the
skin, which involves ensuring minimal
systemic absorption, drug localization of this
type is important in the treatment of
dermatological conditions such as microbial
activity, skin cancer psoriasis, & eczema,
where the seat of disease located in the skin.

4. Dermal delivery system provides the following
advantages over conventional therapy.
 Gastrointestinal absorption problems are avoided.
 First pass effect is avoided, thus minimizing drug input.
 This approach is useful when oral administration is to
avoided or its contraindicated.
 Drug with narrow therapeutic range can be delivered.
 Patient compliance can be increased.
 Drugs with short half life are utilized.
 Therapy can be quickly terminated by simple removal
of the system from skin surface.

5. What is dermal patch?
• A dermal patch or skin patch is
a medicated adhesive patch that is placed on
the skin to deliver a medication into the skin.
• Patch accelerates hydration of stratum
corneum and consequently increases the
delivery of active ingredient to skin. It provide
the constant and prolonged release of the drug.

6. Merits
Avoid first pass metabolism.
Site specific action of drug as it directly
applied to affected area.
Convenient to use.
More stable.
Suitable dosage for bitter drug.
Controlled release for prolonged period.

7. Demerits
May cause staining.
Bulky to handle.
Application with finger may cause
contamination.
May cause irritation.
Some person may allergic to patches.

8. Components of dermal patch 1,2
The basic components of dermal patch consists
of
Polymer matrix / Drug reservoir.
Active ingredient (drug).
Backing laminates,&
Other excipients like plasticizers and solvents.

9. Objective of the study
• The objective of the study aims at the
preparation and evaluation of dermal patch
containing active ingredient of Calendula
Officinalis a natural herb for antimicrobial
activity.

10. Protocol of work
Literature survey.
Procurement.
Preparation of formulation.
Characterization of formulation.
Compilation of results and discussion.

11. Literature review
• S.Munch et al 2017, has practically studied on the dermal and
transdermal delivery system & demonstrated that there are few
benefits of allowing the dermal & transdermal delivery of
pharmaceutically relevant macromolecules to the skin & micro needle
have disadvantages concerning loading of macromolecules & creating
holes in for less than 24hrs.
• kush kumar Nayak et al 2017, has prepared dermal patches from the
blend of keratin with three different microbial exo-polysachharides.
The dermal patches were evaluated on the basis of therapeutic
properties like antimicrobial, antioxidant as well as cell debridement
and other physicochemical properties like tensile strength, porosity,
biodegradability & water swelling. The work justifies the suitability of
all three blends of keratin/ agar keratin/ alginate & keratin/ gellan as
therapeutic dressing of wound healing.

12. • Sara Bagherifard et al 2016, investigated dermal patch which
was prepared in a manner that utilizes thermo responsive drug
micro carrier encapsulated with in a hydro gel layer attached to
a flexible heater with integrated electronic heater control
circulatory for the topical administration of drug. The system
was used to release two different active molecules with
molecular weights similar to drug and growth factors for
treating skin disorders such as chronic wounds and burns.
• Mounyr Balouiri et al 2015, has given a review article on the
technique used in the antimicrobial assessment of antimicrobial
agents. In that review article, an exhaustive list of in-vitro
antimicrobial susceptibility testing method and detailed
information on their advantages and limitation are reported.

13. • Nawazish et al 2013, has given a review article on the type,
preparation & evaluation methodology of transdermal patch, in
which they also discuss about the skin permeation mechanism &
give a brief idea about the preparation methodology for
transdermal patch & their evaluation parameters
• Efstratios Efstratiou et al. 2012, Investigated the antimicrobial
activity of methanol and ethanol extracts of C.Officinalis by disc
diffusion method. Extracts were prepared by using the dried and
ground petals (80 mesh) of plant and then it was transferred to the
flask containing 150 ml of solvents (methanol and ethanol) and the
material were stirred at 350 rpm , 350 C for 24hr by the use of
orbital shaker. Methanol and Ethanol extracts of C.Officinalis
petals were individually tested against a panel of pathogenic micro-
organisms.
• Jurga Bernatoniene et al 2011, has performed examination of
hydrophilic cream of calendula extract & proved its significant
antioxidant property & reported the suitability of use of cream for
regular use.

14. • Lucia Helena et al 2009, has reported a case in which an 18
year old young boy was successfully cured by the topical
application of the C.Officinalis preparation in the
exfoliative cheilitis disease.

15. Plant description 14
 Calendula Officinalis belong to the family Asteraceae
 Chemical constituent- A number of phytochemical
studies have demonstrated the presence of presence of
several classes of chemical compounds. Which are as
follows.
1. Terpenoids.
2. Flavanoids.
3. Coumarins.
4. Quinones.
5. Volatile oil.

16. Pharmacological activities
• Antibacterial and anti fungal activities.
• Wound healing activity.
• Anti-inflammatory and antioedematous
activities.
• Anticancer and lymphocyte activation dual
activities.
• Anti-HIV activity.

17. Excipients profile 15,16,17
HPMC SCMC
synonyms Methylcellulose propylene glycol
ether, methocel
CMC sodium, Sodium cellulose
glycolate, SCMC, Akucell, Blanose
Chemical name Cellulose Hydroxypropyl methyl
ether
Cellulose, carboxymethyl ether, sodium
salt
Molecular weight 10,000-1,500,000. 90,000- 700,000
Structural
formula
Functional
category
Coating agent, Film forming,
Suspending agent, Stabilizing
agent
Coating agent, stabilizing agent,
suspending agent, tablet binder,
viscosity-increasing agent.
Application in
Pharmaceutical
formulation
In oral products, As Tablet
binder, matrix for use inextend
release tablet, suspending,
emulsifier & thickening agent in
topical formulation
Used in oral and topical formulations
for its viscosity-increasing properties.
Also used as tablet binder and
disintegrant , emulsion stabilizer

18. HPMC SCMC
Description Odorless and Tasteless. White or
creamy-white fibrous or granular
powder
White to almost white, odorless, granular
powder.
solubility Soluble in cold water, mixture of
ethanol & dichloromethane,
methanol & dichloromethane and
water & alcohol. Insoluble in
chloroform, ethanol & ether
Practically insoluble in acetone, ethanol
(95%), ether, and toluene. It is easily
dispersed in water at all temperatures,
forming clear, colloidal solutions.
Stability and
storage
condition
It is stable powder &hygroscopic
after drying. Solutions are stable
at pH 3-11. increasing
temperature reduces the viscosity
of solutions
it is stable though hygroscopic material.
Aqueous solutions are stable at pH 2-10.
The bulk material should be stored in
well-closed container in a cool, dry place.
pH 5.5-8.0% w/v sol. in CO2 free
water
6.0-8.0 (1% w/v solution).
Incompatibility Incompatible with some
oxidizing agents
Incompatible with strongly acidic
solutions and salt of iron and other metal
Safety Regarded as non toxic and non
irritant material. Excessive oral
consumption may have laxative
effect.
Regarded as a nontoxic and nonirritant
material oral consumption of large
amounts of SCMC can have a laxative
effect.

19. Methodology and Evaluation
Dermal patch of Calendula Officinalis were
prepared by solvent casting technique using film
forming polymer like hydroxypropyl
methylcellulose (HPMC) and carboxymethyl
cellulose sodium (SCMC). Polyethylene glycol was
used as a plasticizer.. Ethanol was used as a solvent.
Methodology for preparation of dermal patch
containing Calendula Officinalis is shown in the
flow chart.

20. Process for preparation of dermal patch
containing Calendula Officinalis
HPMC & SCMC were weighed accurately and
dispersed in ethanol.
Drug is dispersed in the polymeric solution and
polyethylene glycol was added as plasticizer.
The whole solution was poured into the glass petridish.
Inverted funnel was placed over the dish to avoid
sudden evaporation.
The mould containing polymeric solution of drug was
kept for 24hrs at room temperature for drying.
Prepared patches were separated and kept in aluminum
foil in self sealing plastic envelopes.
Mixed
with the
help of
magnetic
stirrer

21. Composition of Dermal patch of
Calendula Officinalis
Ingredients Formulations
F1 F2 F3 F4 F5
Drugs (ml) 1.5
(10%)
2.25
(15%)
3
(20%)
3.75
(25%)
4.5
(30%)
HPMC (mg) 500 500 500 500 500
HCMC (mg) 100 100 100 100 100
Polyethylene
glycol (ml)
.18 .18 .18 .18 .18
Ethanol (ml) 15 15 15 15 15

22. Evaluation parameters
Physico-chemical
evaluation
Physical appearance
Thickness
weight uniformity
Folding endurance
Moisture content/loss
Antimicrobial assay
Primary skin irritation
testing

23. 1. Physico-chemical evaluation
Physical appearance: All the prepared dermal
patches were evaluated for color, flexibility,
smoothness and clarity.
Thickness : The thickness of dermal patch was
measured by using vernier caliper at different points
of the patches.
Weight uniformity: The weight uniformity of each
patch was done by calculating the average weight
(weighing 3 randomly selected patches). The
individual weight should not vary significantly from
the average weight.

24. Folding endurance: Folding endurance of the patches
was determined by repeatedly folding one patch at the
same place until it breaks. The number of times the film
could be folded at the same place without breaking
gives the value of the folding endurance.
Moisture content/loss: The prepared patches were
weighed individually and kept in desiccators containing
calcium chloride at room temperature for 24hrs. The
patches were reweighed again and again after a
specified time interval until they show a constant
weight.
% Moisture content = [Initial weight – Final
weight /Final weight] × 100

25. 2. Antimicrobial assay3
Biological antimicrobial assays, or bioassays as they
are frequently known, most commonly used method
for bioassay is agar diffusion method. Agar diffusion
method can be done by the following two methods.
A. Disc diffusion method
B. Well diffusion method

26. A. Disc diffusion method
In this well-known procedure, agar plates are
inoculated with a standardized inoculum of the test
microorganism. Then, filter paper discs (about 6 mm
in diameter), containing the test compound at a
desired concentration, are placed on the agar
surface. The Petri dishes are incubated under
suitable conditions. Generally, antimicrobial agent
diffuses into the agar and inhibits germination and
growth of the test microorganism and then the
diameters of inhibition growth zones are measured.

27. B. Agar well diffusion method
Agar well diffusion method is widely used to evaluate
the antimicrobial activity of plants or microbial
extracts. Similarly to the procedure used in disk-
diffusion method, the agar plate surface is inoculated
by spreading a volume of the microbial inoculum over
the entire agar surface. Then, a hole with a diameter of
6 to 8 mm is punched aseptically with a sterile cork
borer or a tip, and a volume (20–100 µL) of the
antimicrobial agent or extract solution at desired
concentration is introduced into the well. Then, agar
plates are incubated under suitable conditions
depending upon the test microorganism. The
antimicrobial agent diffuses in the agar medium and
inhibits the growth of the microbial strain tested .

28. 3. Primary skin irritation test 7,8,9
Test for irritation was performed on human volunteers.
For each patch, three volunteers were selected and
patch in a circular shape (area=4.91cm2) was applied to
hand. The volunteers were observed for irritation.

29. Formulation 1 Formulation 2 Formulation 3
Images of Calendula loaded dermal patches.

30. Formulation 4 Formulation 5
Images of Calendula loaded dermal patches.

31. Result & Discussion
Formulations Thickness
(mm)
n=3
Folding
endurance
n=3
Weight
uniformity (mg)
n=3
Moisture
content
n=3
F1 0.15±0.01 109.33±8.14 83.66±1.52 1.190±0.707
F2
0.07±0.005
112.66±8.02 33.00±2.64 5.88±1.414
F3 0.12±0.005 107.00±12.1 32.33±2.51 12.90±2.828
F4 0.06±0.005 118.66±4.16 53.66±1.52 15.55±4.949
F5 0.08±0.001 108.66±6.65 42.33±1.52 12.82±3.535

32. • The thickness of patches range for 0.07±0.005mm to
0.15±0.01mm which shows the uniformity in the
thickness.
• Weight was seen in the range of 32.33±2.51 mg to
83.66±1.52 which indicate the variation in weight of
formulation is high.
• Folding endurance was calculated in the range of
107.00±12.1 to 118.66±4.16 which indicates that the
concentration of the Calendula Officinalis mother
tincture in different formulation doesn’t effect on the
strength of the patch.
• The low moisture content in the formulation F2
(5.88±1.414) is highly capable for protection of
bulkiness of patches and help them to remain stable
from being a completely dried.

33. Scanning electron microscopy
• The formulations were further subjected for SEM analysis.
The SEM for all formulations is shown in following figures .
SEM image of F1

34. SEM image of F2

35. SEM image of F3

36. SEM image of F4

37. SEM image of F5

38. • Scanning Electron Microscopy images of all
formulation showed that the prepared dermal
patch were of irregular shapes. The surface of
all formulation seems to be non uniform and
rough. On the basis of SEM analysis it was
concluded that due to irregular or rough
surface of patches the release of drug from the
patches was easy as it provides easy absorption
of moisture of skin into the patches, and
release drug quickly from the patches. If drug
release will be more than more drug will
release which gives better antimicrobial effect.

39. Antimicrobial assessment
• All the formulation along with povidone iodine was
screened against one pathogenic bacterial strain. The
tested organism was Staphylococcus Aureus. For
testing the antibacterial activity, the disk diffusion
method was used. Nutrient agar plates were made by
pouring 15-20 ml of sterilized nutrient agar medium
and allowed to solidify for a few minutes in aseptic
condition. A volume of 60µl prepared inoculums
was poured into each plate. The poured inoculums
were spread on the whole surface of the agar
uniformly by sterilized cotton swab.

40. Pf
Pl C Cl
Cf
• Cf For formulation
• Pf For povidone iodine formulation
• Cl For calendula mother tincture
• Pl For pure povidone iodine solution
• C For blank formulation containing only polymer patch
Schematic diagram for formulation placed in the culture media
where,

41. zone of inhibition shown
by F1
zone of inhibition shown
by F2
zone of inhibition shown
by F3
zone of inhibition shown
by F4
zone of inhibition shown
by F5

42. comparative zone of inhibition shown by two skin patch brand (A & B) and
calendula tincture by well diffusion(C) and disc diffusion(D)

43. Formulations Zone of inhibition (mm2)
Calendula (Fc) Povidone (Fp)
F1 7.8 11.6
F2 6.2 5.3
F3 4.7 2.9
F4 11.6 13.7
F5 11.6 16
zone of inhibition shown by the formulation containing
calendula and povidone iodine
Zone of inhibition shown by the pure povidone liquid is 23.7mm2
Zone of inhibition shown by Skin patch containing benzalkonium chloride is 2.1 mm2
Zone of inhibition shown by Skin patch containing nitrofurazone is 28.4 mm2
After the antimicrobial assay it is seen that the highest zone of inhibition is shown
by F4 & F5 (11.6 mm2) among the dermal patch containing calendula mother
tincture. And F5(16mm2) among the dermal patch containing povidone iodine

44. Formulation code Volunteer (1) Volunteer (2) Volunteer(3)
F1 0 0 0
F2 0 0 0
F3 0 0 0
F4 0 0 0
F5 0 0 0
Skin irritation test performed on volunteers
3. Primary skin irritation test
Any skin irritation were not found to be produced by the dermal
patch.

45. Conclusion
 The thickness of patches range for all formulations as found
between 0.06±0.005 to 0.15±0.01 which indicates that they
are uniform in thickness.
 Weight was found to be in the range between 32.33±2.51mg
to 83.66±1.52mg which indicates that different patches were
having slightly difference in weight.
 Folding endurance was found to be in the range between
107.00±12.1 to 118.66±4.16. Highest folding endurance was
found for F4 (118.66±4.16) and the lowest for F3
(107.00±12.1). From the results it was concluded that the
formulation F4 have the concentration of excipients in the
optimized manner which provide good mechanical strength to
patch and it would not break and maintain their integrity when
applied

46.  The moisture content for all formulation was found to
be in range 1.190±0.707 to 15.55±4.949. The lowest
moisture content was found in F1 (1.190±0.707) is
highly capability of for contamination and bulkiness of
patches and help them to remain stable from being a
completely dried patches.
 Scanning electron microscopy images of all
formulations showed that the prepared dermal patches
were of irregular shapes. The surface of the all
formulations seems to be non uniform and rough. On
the basis of SEM analysis it was concluded that due to
irregular or rough surface of dermal patches release of
the drug from the patches will be very easy as it
provides easy absorption of the body fluid into the
dermal patches, thus releasing the drug in
bioavailability.

47.  Antimicrobial activity of all formulations has showed different
zone of inhibition. F3 (4.7) showed the minimum zone of
inhibition and F4 &F5 (11.63) showed the maximum zone of
inhibition. Which clearly indicates the concentration of the
excipients doesn’t have any effect on the therapeutic effect of
patch on F4 & F5 with respect to the concentration of
calendula tincture. On the other hand dermal patch prepared by
using povidone iodine as active ingredient, has shown less
activity then pure povidone liquid which shows that the drug
may show less activity when they are used as formulation.
 Skin patch which contains benzalkonium chloride has shown
less activity and another skin patch which have nitrofurazone
as active ingredient has shown high antimicrobial
susceptibility then the dermal patch containing calendula
officinalis tincture. This shows that the dermal patch
containing calendula is much better then skin patch containing
benzalkonium chloride with respect to activity and its natural
origin

48. • Primary skin irritation shows that it is safe to use that it
doesn’t shows any irritation or redness to the skin.
• After evaluating all the five formulation F4 & F5 were
selected for further comparison due to their highest
zone of inhibition among all the remaining
formulations which is 11.6mm2. In the comparison
study between both F4 & F5 it is concluded that F4 is
the best fit formulation because of its high folding
endurance then F5 which is 118.66±4.16 which shows
the high physical strength and ensure the stability of
patch during its use. On the other evaluation parameter
there is not any large margin of variation is see so on
this basis of evaluation it is concluded that F4 could be
better substitution for the present marketed skin patch
containing synthetic drugs and F4 is to be selected for
the further study in the future.

49. Reference
1. Alam I, Alam N, Singh V, Alam S, Ali S, Anwer T, Sajhi M. Types, Preparation and
Evaluation of Transdermal Patch: Review, World Journal of Pharmaceutical Sciences,
(2013); 02: 2199-2233.
2. Sahoo BK, Mishra AK, Formulation and Evaluation of Transdermal patch of Diclofenac,
World Journal of Pharmaceutical Sciences, (2013), 2(6): 4965-4971.
3. Balouri M, Sadiki M, Ibnsouda SK, Method for in-vitro Evaluating Antimicrobial Activity:
Review, Journal of Pharmaceutical Analysis, (2016); 6: 71-79.
4. E. Efstratiou,Antimicrobial activity of Calendula officinalis petal extracts against fungi, as
well as Gram-negative and Gram-positive clinical pathogens, Complementary Therapies in
Clinical Practice, 18 (2012) 173-176.
5. Leach, Matthew John ‘Calendula officinalis and wound healing: a systematic review.'
Wounds: a compendium of clinical research and practice, vol. 20(2008),236-243
6. Lúcia Helena DenardiRoveroni-Favaretto, Topical Calendula officinalisL. successfully
treated exfoliativecheilitis: a case report,(2009),

50. 7. Chandan T, Parahar B, Arora S, Design and Evaluation of Diclofenac Sodium Gel,
International Journal of Pharmaceutics and Chemical Sciences, (2013): 72-81.
8. Shivhare VD, Jain KB, Roy AA, Formulation, Development and Evaluation of
Diclofenac Sodium sing water soluble Polyacrylmide Polymer, Digest Journal of
Nanoparticles and Biostructures (2009); 4(2): 285-290.
9. Niyogi P, Raju NJ, Formulation and Evaluation of Anti-inflammatory Activity of
Solanum Bubescens wild extracts gel on Albino Wistar Rats, International Journal
of Pharmacy, (2012); 2(3): 484-490.
10. Monica Butnariu and Cristina ZepaCoradini, Evaluation of Biologically Active
Compounds from Calendula officinalis Flowers using Spectrophotometry,
Butnariu and Coradini Chemistry Central Journal 2012.
11. BP Muley, SS Khadabadi and NB Banarase, Phytochemical Constituents and
Pharmacological Activities of Calendula officinalisLinn (Asteraceae): A
Review,Tropical Journal of Pharmaceutical Research, October 2009,455-465.
12. https://www.sigmaaldrich.com/life-science/nutrition-research/learning-
center/plant-profiler/calendula-officinalis.html
13. Kush Kumar Nayak, Pratima Gupta, study of the keratin-based therapeutic dermal
patches for the delivey of bio active molecules for wound treatment, materials
science and engineering C,( 2017) 1088-1097.

51. 14.http://bioinfo.bisr.res.in/project/domap/plant_details.php?plantid=0013&bname=Calen
dula%20officinalis
15. Rowe RC, Sheskey PJ, Owen SC. Handbook of Pharmaceutical Excipients, 5th edition,
published by the pharmaceutical press (2006); 346-348, 120-122, 554-559.
16. Lewis RJ, ed. Sax’s Dangerous Properties of Industrial Materials, 11th edition, New York
Wiley (2004): 2054.
17. Fletcher J. The benefits if using hydrocolloids, Nurs Times (2003); 99(21):57.