Pharmaceutical Resonance 2023 Vol.

V - Issue II

RESEARCH ARTICLE

A NOVEL FORMULATION AND EVALUATION OF WOUND

HEALING CREAM CONTAINING CALENDULA EXTRACT
COATED ON SILVER NANO PARTICLES

R. K. Nanda*, Harshada Asutkar

Dr. D.Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune, Maharashtra, India

ABSTRACT :The cur r ent study pr esents a unique gr een pr ocess for the synthesis of silver nano par ticles
(AgNPs) by simple treatment of silver nitrate with ethanolic extract of Calendula officinalis and it’s Formulation
Development as a Wound healing cream containing the herbal nano particles. The major goal of this study was to
develop a cream with some special characteristics which can withstand very low temperature conditions as well.
Hence, it can be used by mountaineers, soldiers and people working in high altitude. Numerous reviews and
research papers have been published on Calendula officinalis [20-32].
A method was developed for rapid biosynthesis of metallic silver nano particles using extracts of C. Officinalis
petals. Formation of AgNPs was observed by visual colour change from colourless to dark brown and confirmed
by UV-Visible characteristic peak in the range of 440-460nm. Extracts of Petals provide non-toxic, eco-friendly
and cost effective reducing and capping agent for bio-reduction of AgNO3.This plant is available in tropical
countries like India.
Photosynthesized AgNPs were characterized by various advanced analytical methods. To identify the active bio
molecules responsible for the reduction of Ag+ ion and the stabilization of AgNPs synthesized the functional
group available in C. Officinalis petals extract was studied by FT-IR spectroscopy. The morphology and
topography of the AgNPs were determined using field emission scanning electron microscopy (FESEM). The size
of the herbal nano particles differed depending upon the concentration of extract used. The morphology of the
herbal AgNPs was found to be cubical or square shaped with diameter range from 123nm to 135 nm.
The biosynthesized AgNPs was studied for it’s antibacterial activity against the clinically isolated human
pathogens. The synthesized AgNPs were found to show potential antimicrobial activity against multidrug resistant
Gram-negative (Escherichia coli), Gram-positive (Staphylococcus aureus) bacteria and anti-fungal activity against
(Candida albicans) in clinically isolated human pathogens. The present results clearly indicated that the Calendula
is a good nontoxic source for extracellular synthesis of AgNPs in eco-friendly way as compared to other methods.
Thus C. officinalis petal extracts provide a potential source for development of commercially important nano
medicines and cosmetics .
Keywords : Silver nano particle , Calendula petal extract, Wound healing silver nano-Anti-freezing cream
INTRODUCTION: herbal medicines, neutraceuticals, phytocosmetics and
Herbal Nano Preparations: personnel care products. The global demand for these
products is increasing due to reinsured interest of
Herbal nano products have become a subject of consumers in natural products as they are considered
increasing global research for their health benefits and safer and more cost effective than synthetic
economic considerations. Some major categories of counterparts[1,2,5,18].
plant derived products include phyto-pharmaceuticals,
Why nano drug delivery:
1) Nano particulate system delivers the herbal
Rabindra Kumar Nanda formulation directly to the site of action.
Dr. D. Y. Patil Unitech Society’s, Dr. D. Y. Patil 2) Increased efficacy and therapeutic index.
Institute of Pharmaceutics and Research, Pimpri,
Pune, Maharashtra, India - 411018. 3) Increased stability via encapsulation.
eMail : rabindrananda@rediffmail.com 4) Improved pharmacokinetic effect.
5) It can be produced with various particle sizes and
© Published by DYPIPSR, Pimpri, Pune - 411 018 ( MH ) INDIA 14
 Pharmaceutical Resonance 2023 Vol. V - Issue II

surface properties (Lab Hosp, Mumbai), Ultrasonicator (Equitron), Deep

Why silver used as a core: Freezer (Voltas), Digital vernier Calliper (Mitutoyo-
Digimatic calliper) and Probe Sonicator (PCI
The development of techniques for manufacturing Analytics)
silver nano particles was selected as Silver nano
particles have been shown to have a higher Preparation of Calendula extract:
antibacterial activity than silver ions alone. The Collection of Raw Materials:
emergence of a wide range of antibiotic resistant Calendula officinalis (Marigold) flowers were
bacteria has created fresh interest in the use of silver as collected from the local market in the month of
a bactericidal agent. It appears that simply contact with October and November were used in this investigation
the surface of the silver nano particle destroys cell and were identified in our pharmacognosy Department
walls of the bacterium with high effectiveness. where voucher specimen is maintained.
However, the bactericidal effect is dependent on size
and shape. Smaller silver nano particles as well as Description of Calendula officinalis flower petals:
different shapes such as octahedral and decahedral are
more effective[14]. Literature survey reveals that there Table 1. Macr oscopical char acter s of Calendula of
is no formulation available in the market with the ficinalis
combination of Calendula coated silver nano particles
for the wound healing action. To add novelty and Quantity Collected 1 kg
uniqueness to this product, the formulation was
developed to maintain its flow properties without Month of collection October
solidifying at low temperature conditions .Keeping this
Colour Dark yellowish orange
point in consideration this formulation was developed
for wound healing action and to overcome the Odour Aromatic
infection associated with it.
Size 12mm in length
Procurement of Chemicals And Excipients:-
The Chemicals and Equipments used for various Geographical Source Maharashtra
experiments are enlisted as follows:
Taste Bitter
The following chemicals were procured.
Shape Slightly curved
Cetyl alcohol, Stearic acid, Disodium EDTA, Tri
ethanol amine, Propylene Glycol, Methyl paraben, Extra features
Propyl paraben, Glycerin, Silver Nitrate, Citrate of
Sodium and Sodium lauryl Sulfate from Loba Inflorescence Raceme
chemicals, Thane, Mumbai and Nutrient Agar, Potato
Calyx Dark green and ovate
dextrose Agar and Mackonkey Agar from Himedia,
Mumbai. Amoxicillin Trihydrate was obtained from Dark yellowish orange,
H.A.Ltd and Povidone-Iodine cream was obtained Corolla
oblanceolate
from local chemist’s shop.Calendula Flowers collected
1. Extraction from the Plant Material :
from Pimpri local market.
500 grams of dried ground petals of Calendula
The following equipments were used for carrying
officinalis flower wer e taken in 1 liter size
out the experiments:
beaker. To it, 1000 ml of ethyl alcohol was
UV-Visible double beam spectrophotometer added (BP). Then it is allowed to macerate for 3
(Shimadzu 1700), USP tablet dissolution test apparatus hours by using mechanical stirrer. It is then
Type I (Veego Scientific DT 60), Digital analytical stirred for 4 hours. Contents were filtered with
balance (Shimadzu AX 200), FTIR spectrophotometer the muslin cloth and clear filtrate so obtained
with diffused reflectance spectroscopy (DRS) was kept in refrigerator for further studies.
attachment (Shimadzu 8400S), Ultracryostat Bath
Prepared extract was then subjected to
(Modern Industrial Corporation MIC-1500),
phytochemical estimation, chromatographic
Laboratory centrifuge (Remi C-24), Orbital Shaking
techniques and FT-IR of plain extract for its
Incubator (Remi Motor), Magnetic stirrer with hot
qualitative tests and further it is used for the
plate (Remi Motor MS205), Mechanical Stirrer (Remi
preparation of herbal nanoparticles as a reducing
Motor), Stability Chamber (Remi Motor CHM 12 S),
and stabilizing agent.
Particle size analysis (Horiba), Scanning electron
microscope (PHILIPS CM200), UV cabinet (dual) 2. Evaluation of Extract:

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 Pharmaceutical Resonance 2023 Vol. V - Issue II
Phytochemical estimation of the prepared extract : 3. Chromatographic Separation:
The prepared extract was subjected to different 3.1 Thin Layer Chromatography
chemical tests as given below[3].
TLC was performed on the 20 × 20 cm plates pre-
Test for carbohydrates : A small quantity of extr act coated with microcrystalline cellulose. A volume of 1
was dissolved separately in 5 ml of distilled water and μL of 1% ethanolic extract was spotted on the plates.
filtered. The filtrate was tested to detect the presence One-dimensional TLC analysis was performed with
of carbohydrates. ethyl acetate : formic acid : acetic acid : water in
Molisch’s test : To 2 ml of extr act, 2 ml of volume ratio 100 : 11 : 11 : 26 as mobile phase. Spots
Molisch’s reagent was added. Then, 2 ml of were observed under UV light at 366 nm before and
concentrated sulphuric acid was added along the sides after spraying with the 2-aminoethyl diphenyl
of the test tubes. Disappearance in color on the boronate or anisaldehyde sulfuric acid reagent[11].
addition of excess solution indicated the presence of Preparation of spraying reagent:
carbohydrates.
1. 1g ethanolamine diphenyl borate was dissolved
Benedict’s test : To 0.5 ml of extr act, 5 ml of in 100 ml methanol.
Benedict’s reagent was added. The mixture was then
boiled for 5 minutes. Presence of a bluish green 2. 5g Polyethylene glycol was dissolved in 100ml
precipitate indicated the presence of carbohydrates. ethanol.
Test for Proteins and Amino acids Plate was sprayed with solution 1 first, followed by
Ninhydrin test : A small quantity extr act solution solution 2 and visualized under uv chamber.
was boiled with 0.2% solution of Ninhydrin. Purple After development and derivatization, plate showed
color indicated the presence of free amino acids. distinct spots for the components present into the
Test for Phytosterols and Tri terpenoids: Salkowski extract. The results are tabulated in Table 3 below.
test : To 2 ml of the extr act, 1 ml of concentr ated
Sulphuric acid added. Chloroform was added along the
sides of the test tube. A red color produced in the
chloroform layer indicated the presence of
Phytosterols or if it is yellow in color at the lower
layer indicated the presence of tri terpenoids.
Zinc hydrochloride reduction test : The extr act was
treated with mixture of zinc dust and concentrated Fig. 1 : A developed TLC plate
hydrochloric acid. Red color indicated the presence of
flavanoids.
Table 3 : Results of Thin layer chr omatogr aphy
Test for Flavonoids : 1 ml of extr act was added to
10 ml of 95% ethanol and kept in boiling water bath
Sr. Colour of
for 15 minutes and after filtration Magnesium ribbon Rf Value Compound
No spot
were added along with 2-3 drops of HCl.
To 1ml of extract, 2ml of Sodium hydroxide was 1 0.96 Yellow Quercetin
added and observed for the colour change.
Test for Steroids : 1 ml of extr act was extr acted Blue to
2 0.79 Caffeic acid
with methanol for 15 minutes and then Libermann light blue
Burchard reagent was added drop wise.
Yellowish Flavonoid
Calculation of Extract Yield : 3 0.42
Violet glycoside
Accurately weighed 10 gram of the previously
prepared liquid extract was taken and it was allowed to 4 0.34 Yellow Rutin
dry in the air. Semisolid mass so obtained is again
weighed and extract yield was calculated by using the 3.2 High Performance Thin Layer
formula - Chromatography ( HPTLC ) :
10 gram liquid extract contains 526 mg of semisolid Sample preparation and development of suitable
mass.500 gram liquid extract will contain 26.3 gram of mobile phase of solvent system are the two important
semisolid mass. steps in developing the analytical procedure, which
becomes more significant for herbal drugs because of
26.3/500*100=5.26

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 Pharmaceutical Resonance 2023 Vol. V - Issue II

Table 2 : Results of the Phyto chemical estimation

Sr. Therapeutic
Test Observation Inference
No. Activity

Test for Carbohydrates

Acts as reducing
To 2ml of extract, 1ml of Molisch’s Presence of purple Carbohydrates
1 agent and
reagent and few drops of concentrated or reddish color. present.
stabilizer.
sulphuric acid were added.

No formation of
Test for Tannins Hampers protein
dark blue or
2 To 1 ml of extract, 2ml of 5% ferric Tannins absent. synthesis in
greenish black
chloride was added. bacteria.
precipitate.

Test for Saponins

Formation of
To 2ml of extract, 2ml of distilled water
3 about 1cm layer of Saponins present. Anti mutagenic
was added and shaken in a graduated
foam.
cylinder for 15min lengthwise.

Test for flavonoides

Formation of Flavonoides Antibacterial
4 To 2ml of extract, 1mlof 2N sodium
yellow color. present. (Acts on cell wall)
hydroxide was added.

Test for Alkaloides Antibacterial

To 2ml of extract, 2ml of concentrated Presence of green Alkaloides (Metallic
5
HCl was added, then few drops of color. present. byproducts)
Mayer’s reagent were added.

Test for Glycosides

Formation of Glycosides Wound healing
6 To 2ml of extract, 3ml of chloroform
reddish pink color. present. activity
and 10% ammonia solution was added.

Test for Terpenoides

Formation of red
To 0.5 ml extract, 2ml of chloroform Terpenoides Anti-
7 brown colour at
was added and concentrated sulphuric present. inflammatory
the interphase.
acid was added carefully.

Test for Phenols

To 1ml of the extract, 2mlof distilled Formation of
8 Phenols present. -
water followed by few drops of 10% bluish green color.
ferric chloride was added.

Test for Anthraquinones No appearance of

Anthraquinones
9 To 1ml of plant extract, few drops of pink colored -
absent.
10% ammonia solution was added. precipitate.

Test for Carotenoides

To 2ml of plant extract add 2ml of Colourless Carotenoides Supress tumour
10
sulphuric acid and 1ml of Sodium appearance. present. growth.
nitrite.
© Published by DYPIPSR, Pimpri, Pune - 411 018 ( MH ) INDIA 17

the complexity of chemical compounds and their
affinity towards various solvents[12].
Chromatographic condition
Instrument: A Camag HPTLC system equipped
with a sample applicator Linomat IV, twin through
plate development, chamber, TLC Scanner III and
Integration software CATS 4.05.
Absorbent: TLC Aluminium plate pr ecoated with
silica gel 60 F 254 (E.Merck)
Solvent system: Chlor ofor m : Glacial acetic acid :
Methanol: water(6.4: 3.2: 1.2: 0.8).
Solvent run up to: 80 mm.
Scanning wavelengths: 254 nm, 280nm, 335nm,
510nm, 765nm.
Sample preparation and Development of
chromatogram
Application of 2 and 5 μl of ethanol extract as bands
on silica gel 60 F 254 pre-coated aluminium plates
(Merck) using Linomat IV sample applicator. The
speed of applicator was maintained at 10μl /sec. The
width of the band was kept at 6 mm. The
chromatogram was developed up to 80 mm under
chamber saturation condition. The plate was dried and
scanned at 254 nm, 280nm, 335nm, 510nm, 765nm in
absorbance mode. Different components of the
extracts were determined qualitatively depending upon
their Rf vlues.
The desired resolution of different components with
symmetrical and reproducible peaks was achieved by
using chloroform: glacial acetic acid: methanol: water
(6.4: 3.2: 1.2: 0.8) using the proposed HPTLC method.
From the Rf values, presence of following components
were confirmed from the literatures available.
After the development when plate was scanned under
different wavelengths, characteristic picks were
obtained and the components are analysed depending
upon their Rf values. Components and their observed
Rf values along with the reported values are given in
Table 4.
Prepared formulation was evaluated for various
evaluation parameters as that of conventional
Fig. 2 : A developed HPTLC plate seen befor e
derivatisation (A) & (B), and after derivatisation (C)
© Published by DYPIPSR, Pimpri, Pune - 411 018 ( MH ) INDIA
Pharmaceutical Resonance 2023 Vol. V - Issue II
formulations. The additional data were generated by
studying stability and all the evaluation parameters at
low temperatures.
4. Preparation of Herbal Nano particle:
Aqueous solution (1 mM) of silver nitrate (AgNO3)
was prepared and used for the synthesis of silver
nanoparticles. Dropwise addition of previously
prepared calendula extract to the Silver nitrate solution
was carried out. The synthesis of silver nano particles
was carried out at room temperature (25 C ± 2C) till
the end point is observed. On adding light yellow color
flower extract to color less silver nitrate solution,
formation of silver nano particles(AgNPs) occurred
and they exhibited a color change to surface plasmon
resonance. The intensity of color was directly
proportional to the formation of AgNPs. The color
change was very rapid and as soon as the two solutions
were mixed, the solution turned brown and within 10
min, it turned to dark brown and after 2 hours, the
solution turned black. The color change in the reaction
mixture (i e. Silver metal solution + flower extract)
was recorded through the visual observations. The
bioreduction process of silver ions in aqueous solution
was monitored by periodic sampling of aliquots (1
mL) and subsequently measuring UV-Visible spectra
of the solution[7,10,16].
01 02 03 04
01) Before 02) End point of reaction
03) Agglomeration 04) Increased particle size
Fig. 3 : Progression of the reaction of nanoparticle
synthesis.
UV–Vis spectra of synthesized nanoparticles was
monitored as a function of time of reaction on a
spectrophotometer (Shimadzu UV-1601) in 400–600
nm range operated at a resolution of 10 nm. The UV
absorbance for silver nanoparticles was found in the
range of 440-460nm (Figure 4. ).The formulations
showed good homogeneity and extrudability. The pH
of all the formulations was in between 6.0-7.0 which
lie in the normal pH range of the skin. The
formulation did not produce any skin irritation. The
results for rheological behavour of the cream showed
increase in shear stress and decreased viscosity with
the increase in torque. The comparative study of
spreadability and viscosity showed that as viscosity
of formulation increases, spreadability decreases and
18
 Pharmaceutical Resonance 2023 Vol. V - Issue II
vice versa. From the stability studies, all the creams IR absorption spectrum of prepared extract was
showed no change in pH, viscosity, spreadability, recorded by specialized technique for sticky
extrudability, consistency and phase separation after semisolids in which chloroform extracts of the sticky
keeping them at freezing temperatures to -35º C for materials were placed in sample holder and infrared
almost a month. spectrum was recorded using FTIR. The FTIR (Fourier
The formulation prepared was studied for its wound transform infra-red spectroscopy) was recorded in the
healing activity and antimicrobial action against range of 400–4000 cm-1 (SHIMADZU 8400S).
different infections associated with it. Various modes of vibrations were identified and
assigned to determine the different functional groups
Prepared formulations showed significant promotion present in the Calendula officinalis.
of wound healing activity with statistically significant
results in all the groups of animals. The mean
percentage closure wound area was calculated on the
2nd, 4th, 8th, 11th, 15thand finally on 18th day. Post
wounding days are also shown in tables. The wound
healing activity was found comparable with that of the
reference standards (commercial product- povidone
iodine, calendula cream, silver nano gel) and control
base. The percentage closure of excision wound area
in animals treated with 20 and 40 microgram
concentration of the active nanoparticles was found to
be 100% and 98.49% respectively.
After the wound healing activity of the optimized
formulations was found significant, 20µg/g was Fig. 4: Absor ption spectr um of silver nanopar ticles.
selected as a final formulation. Both the formulations Inset of the figure shows end point of the reaction for
gave excellent wound healing activities but it was formation of silver nanoparticles by using calendula
decided to avoid the formulation which was having extract.
40µg/g in order to keep the metal toxicities under
effective control.
5. Results and Discussion:
The current study summarizes a unique green process
for the synthesis of silver nanoparticles (AgNPs) by
simple treatment of silver nitrate with ethanolic extract
of Calendula officinalis and Formulation Development
of Wound healing cream containing those herbal
nanoparticles. The major goal of this study was to
develop a cream with some special characteristics that Fig. 5: IR Spectr um of Calendula extr act
it can withstand the very low temperature conditions.
Hence it can be used by people who are staying in low
temperature climatic condition. Table 5: Char acter istic fr equencies in IR spectr um
of extracts.
The present study developed a method for rapid
biosynthesis of metallic silver nanoparticles from C.
Officinalis petals extracts. Formation of AgNPs was Sr. Wave number (cm-1) Functional Groups
observed by visual color change from colorless to dark
brown and confirmed by UV-Visible characteristic 1 3311.89 O-H stretching
peak in the range of 440-460nm.
Petals provide non-toxic, eco-friendly and cost 2 2856.67 C-H stretching
effective reducing and capping agent for bioreduction
of AgNO3 and that are readily available in tropical 3 2937.68 Diphenolic OH stretch
countries including India.
4 1730.21 C=O stretch
Photosynthesized AgNPs were characterized by
various advanced analytical methods. 5 1458.23 O-H bending
FT-IR of the Extract
C-O stretch in phenolic
6 1209.41
acid.
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 Pharmaceutical Resonance 2023 Vol. V - Issue II

Fig. 6: IR spectr um of Her bal nanopar ticles

Table 6: Char acter istic fr equencies in IR spectr um

Table 7: Par ticle char acter ization data
of herbal nanoparticles

Sr. Wave number (cm-1) Functional Groups

Polydi -
Particle Zeta
1 711.76 Aromatic C-H bending Particulars spersibility
size(nm) potential
Index
2 1527.67 C=C stretch
Silver
3 1051.24 C-O single bond stretch 101.1 -19.2 0.488
nanoparticles
4 3082.35 Diphenolic OH stretch
Herbal
135.2 -6.93 0.185
5 2721.65 C-H stretch nanoparticles

To identify the active biomolecules responsible for the

reduction of Ag+ ion and the stabilization of AgNPs
synthesized the functional group available in C.
Officinalis petals extract was studied by Fourier
transmission infrared (FT-IR) spectroscopy.
The morphology and topography of the AgNPs were
determined using field emission scanning electron
microscopy (FESEM). The size of the herbal
nanoparticles differed depending upon the
concentration of extract used. The morphology of the Fig. 7: FESEM image of her bal nanopar ticles.
herbal AgNPs was found to be cubical or square
shaped with diameter ranging from 123nm to 135 nm. (Candida albicans) on clinically isolated human
pathogens.
The biological synthesis of AgNPs was demonstrated
using AgNO3 and also studied their antibacterial The present results clearly indicated that the Calendula
activity against the clinically isolated human is a good nontoxic source for extracellular synthesis of
pathogens. The synthesized AgNPs were found to AgNPs in eco-friendly way as compared to other
show potential antimicrobial activity against Gram- methods. Thus C. officinalis petal extracts provide a
negative (Escherichia coli), Gram-positive potential source for development of commercially
(Staphylococcus aureus) and anti-fungal activity important industrial products including nanomedicines

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 Pharmaceutical Resonance 2023 Vol. V - Issue II

Table 8: Zone of Inhibition showing antibacter ial and anti-fungal activity.

Microbial Species S. aureus E. coli C. albicans

I 12±2 14.66±2.08 13±2.64
I
II 12.33±2.08 10.33±1.15 14.66±3.46
Extract
III 14.33±3.05 14±0.57 13.66±3.60
I 13.33±2.51 16.33±2.88 12.66±2
II
II 13.66±1.52 14.33±3.05 12.66±3.78
Plain AgNps
III 14.33±3.60 13.66±2.08 13±3.60
I 21±1.73 21±3.05 19.33±0.57
III
II 21±2.51 20.66±2.51 21±1.73
Herbal AgNps
III 22±2.88 22±2.64 26.33±3.60
I 16.33±4.72 17±1 18.33±3.60
Standard
Amoxycillin II 18 15±2 15.66±6.11
Trihydrate
III 17±1.52 16.66±3.21 14±3.60
healing action. The formulation also withstood free
Table 9: For mulation of the Cr eam.
flow characteristics at low temperature conditions.
Ingredients Quantity (%) Prepared formulation was evaluated for various
evaluation parameters as that of conventional
formulations. The additional parameter was studying
Cetyl Alcohol 2.5% stability and all the evaluation parameters at low
temperatures.
Stearic acid 2.5%
The formulations showed good homogeneity and
Disodium EDTA 0.002% extrudability. The pH of all the formulations was in
between 6.0-7.0 which lie in the normal pH range of
the skin. The formulation did not produce any skin
Triethanolamine 1% irritation. The results for rheological behavior of the
cream showed increase in shear stress and decreased
Water 35% viscosity with the increase in torque. The comparative
study of spreadability and viscosity showed that as
Propylene Glycol 3% viscosity of formulation increases, spreadability
decreases and vice versa. From the stability studies, all
Methyl paraben 0.025% the creams showed no change in pH, viscosity,
spreadability, extrudability, consistence, phase
Propyl paraben 0.125% separation after keeping them at freezing temperatures
to -35º C for almost a month.
Glycerin q. s. 7. Wound healing activity and antimicrobial
action:
and cosmetics by tagging nanoparticles with Quercetin
in the future. The formulation prepared was studied for its wound
healing activity and antimicrobial action against
6. Formulation different infections associated with it[3,4,8,19].
Literature survey reveals that there is no formulation Fig. 8: Image r epr esentation of wound healing
available in the market with the combination of process in control, Calendula standard, Silver standard,
Calendula coated silver nano particles for the wound Povidone iodine, 20µg Herbal nano and 40µg herbal

© Published by DYPIPSR, Pimpri, Pune - 411 018 ( MH ) INDIA 21

 Pharmaceutical Resonance 2023 Vol. V - Issue II

Table 10: Evaluation of Pr epar ed For mulation

Evaluation
I II III IV V VI VII VIII IX
parameter

Colour Cream White

10 rpm 368 234 538 760 450 121 25000 740 14245
Viscosity
(cp)
50 rpm 744 748 760 13900 912 745 26250 450 14630

Spreadability 11 sec 14 sec 12 sec 11 sec 13 sec 16 sec 14 sec 22 sec 26 sec

pH 6.43 6.82 6.15 6.77 6.49 6.42 6.66 6.84 6.75

Stability Not stable Stable

Microbial contents No growth

Fig. 8: Image r epr esentation of wound healing pr ocess

© Published by DYPIPSR, Pimpri, Pune - 411 018 ( MH ) INDIA 22


nano containing creams at 2, 4, 8, 11,15 and 18 days of
post wounding Prepared formulations showed
significant promotion of wound healing activity with
statistically significant results in all the groups of
animals. The mean percentage closure wound area was
calculated on the 2nd, 4th, 8th, 11th, 15thand finally
on 18th day. Post wounding days are also shown in
tables. The wound healing activity was found
comparable with that of the reference standards
(commercial product- povidone iodine, calendula
cream, silver nano gel) and control base[3,4,8,19]. The
percentage closure of excision wound area in animals
treated with 20 and 40 microgram concentration of the
active nanoparticles was found to be 100% and
98.49% respectively.
After the wound healing activity of the optimized
formulations was found significant, 20µg/g was
selected as a final formulation. Both the formulations
gave excellent wound healing activities. The decision
to reject the formulation which is having 40µg/g
concentration of AgNps is based on keeping the metal
toxicities under safer limit. The selected formulation
was then studied for its antimicrobial activities against
the same two bacterial and one fungal strain.
Fig. 9: Antimicr obial activity shown by cr eam
containing herbal nanoparticles. (a) C. albicans, (b) S.
aureus, (c) E. coli
After carrying out the microbial study, it was found
that combination of calendula coated silver
nanoparticle cream was a superior antimicrobial
formulation than the plain calendula extract as well as
plain silver nano suspension[14,15].
Antibacterial results were supported by the existing
literature which suggests that silver in nanocrystalline
forms not only reduces wound infection but also
promotes wound healing. It further helps in reducing
the frequency of dressing changes which would
© Published by DYPIPSR, Pimpri, Pune - 411 018 ( MH ) INDIA
Pharmaceutical Resonance 2023 Vol. V - Issue II
Fig 10: Differ ent batches of cr eam
subsequently reduce associated pain and can be
manufactured at low cost. It can also withstand
freezing conditions without solidifying where all
marketed formulations solidify.
8. Conclusion:
Herbal nano particles are better than the plain silver
nano particle as it reduces the skin irritation and has
better wound healing property. Avoiding other toxic
chemical reducing agents helps to eliminate toxicity
and gives better drug delivery system. Herbal nano
particle containing cream has better antimicrobial
activity compared to extract and plain nano containing
creams. As a result, they have a better wound healing
property compared to marketed formulations available.
Formulation is novel and no such marketed
preparations are available currently which can
withstand freezing conditions without solidifying.
Formulation characteristic meets all the criteria
regarding viscosity, pH, spreadability, skin irritation,
etc. Hence, it has potential for commercialization.\
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[1] Rasheed A, Reddy S, Roja C. A Review on
Standerdization of Herbals; International
Journal of Phytotherapy 2011. Vol 2 (2). 74-88
[2] Traditional Medicine, World Health
Organization, Geneva 1978.
[3] Pulok K. Mukherjee, Kakali Mukherjee, M.
Rajesh Kumar, M. Pal1 and B. P. Saha.
Evaluation of Wound Healing Activity of Some
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