Dendricream For Wound Healing

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
Rani et al. World Journal of Pharmacy and Pharmaceutical Sciences

SJIF Impact Factor 6.041
Volume 5, Issue 8, 1483-1497 Research Article ISSN 2278 – 4357
FORMULATION, OPTIMIZATION AND EVALUATION OF

DENDRICREAM FOR WOUND HEALING ACTIVITY OF ARTEMISIA
INDICA.
Shalu Rani*, Amanjot, Harjaskaran, Narinder Singh, Surya Prakash Gautam,

Sukhbir Kaur
Department of Pharmaceutics, CT Institute of Pharmaceutical Sciences, Shahpur,

Jalandhar.
ABSTRACT
Article Received on
20 June 2016, The present study was intended to examine comparative wound
Revised on 10 July 2016,
Accepted on 30 July 2016
healing potential of herbal extract and herbal dendricream with
DOI: 10.20959/wjpps20168-7450 marketed standard formulations of Povidone-iodine using excision
wound models in normal rats. The crude extract was administered
topically in different doses for evaluating the wound healing potential
*Corresponding Author
Shalu Rani in excision wound model for fourteen days. WHC [Formulation No. 6
Department of (F-6)] showed significantly better wound healing activity than standard
Pharmaceutics, CT cream. F-6 also demonstrated complete epithelization and good
Institute of
collagen deposition as compared to standard cream. The experimental
Pharmaceutical Sciences,
data of wound size area that, expressed healing in the cream treated
Shahpur, Jalandhar.
group of animal was significant as compared to control group of
animal. It was observed that the formulated dendricreams showed better wound healing
activity.
KEYWORDS: wound healing, artimisia indica, dendricream, invitro study, invivo study,
stability studies.
INTRODUCTION
A wound is a breakage in the tissue continuity, from violence and trauma.[1] It can be
produced by physical, thermal, chemical or immunological damage to the tissue.[2] They do
not only affect the physical and mental health of patients but also impose the significant cost
on them.[3] Wound generally termed as physical injury that cause opening and breaking of
www.wjpps.com Vol 5, Issue 8, 2016. 1483

skin.[4] A wound consist of physical damage (pressure ulcers), thermal damage (burns),
mechanical damage (cut, abrasion, lacerations) etc. Wound healing is a process of cell
contraction, movement, re-adhesion after injury. Wound healing involves platelet
aggregation, blood clotting, formation of fibrin, an provocative response to damage,
angiogenesis and re-epithelization.[5,6]
Normal wound healing process involves four phases i.e. Homeostasis, Inflammation,
Maturation and Remodeling. In pharmaceutical drug industry, the accessibility of drugs able
to stimulating the process of wound repair is still limited.[7] Only 1–3% of the medications
listed in Western pharmacopoeias are intended for use on wounds; on the other hand, at least
one-third of natural cures are connected as wound healing agents.[8] Wound healing process is
mainly promoted by the use of herbal remedies which are based upon plant sources.[9,10,11]
Medicinal plants are important sources of new chemical substances that have beneficial
therapeutic effect.[12] Dendrimers has immense potential in drug delivery because of
nanoscale size-derived chemical, physical and biological properties. Dendritic polymers,
including dendrigrafts, dendrimers and dendrons are providing new directions in
nanomaterial based drug delivery due to their nanostructure, monodispersity, adaptability,
definite molecular size, custom-made surface groups and chemical stability. Dendrimers
based products are contributing significantly and efficiently. Acetylation is one more
approach for capping the free amine groups of dendrimers.[13] Dendrimers can function as a
drug carriers either by encapsulating the drugs within the dendritic structure or by acting
with medications at their terminal functional groups by electrostatic or covalent bonds
(prodrug).[14,15]
MATERIALS and METHODS

Chemicals and Reagents
The chemicals used during the experiments were of methodical grade. Stearic acid
(Qualikems Lab., Vadodara, Gujarat, India), Potassium Hydroxide, Petroleum jelly, Methyl
paraben sodium, Propyl Paraben sodium(Nice Chemicals, Kerala, India), Liquid paraffin,
Glycerine (Avarice Lab, Ghaziabad, Uttar Pradesh, India), Cetosteryl alcohol, Methanol,
Methyl acrylate (Loba Chem, India), Ethylenediamine (Merck Specialities, Karnataka),
Povidone-iodine IP 5%w/w, (Win-Medicare, Delhi, India) were used.

Instruments
Viscometer (Brookfield DV-E viscometer, USA), Digital pH meter (Hanna Instruments,
Italy), FTIR-spectrometer (Bruker Optics, Germany), UV spectrometer (Shimadzu
Corporation, Japan), Magnetic Stirrer (REMI Laboratory Instruments, Mumbai, India), Hot
Air Oven (Gupta Scientific Industries, Ambala, India) were used.
Synthesis of PAMAM Dendrimers

Methylacrylate (MA), methanol, Ethylenediamine(EDA) were used for the synthesis of
PAMAM (Polyamidoamine) dendrimers. 2G PAMAM dendrimers were synthesized by
divergent growth method in which dendrimers grow from inside (i.e. core molecule) to
outside. The schematic flow diagram of 2G PAMAM dendrimers is given in Fig.1.
Figure 1: Flow diagram of 2G PAMAM dendrimers
2g of 1.5 G dendrimers and 42.7 ml EDA (Ethylenediamine) were dissolved in appropriate

amount of methanol in amber colored round bottom flask which was corked tightly and then
kept for 144 hours. Finally residual solvent is evaporated on the water bath. Prepared
dendrimers are analyzed by Ultraviolet Spectroscopy (UV- 1800, Shimadzu Corporation,
Japan) and Fourier Transform Infrared Spectroscopy.
Evaluation of 2G PAMAM dendrimers

Color Test
PAMAM dendrimers were treated with aqueous solution of copper sulphate (1%w/v).[16]

Ultraviolet Spectroscopy
0.01% w/v concentration of PAMAM dendrimers was scanned in the range of 200 nm to 400
nm against distilled water. The changes in λmax values were noted.[17,18,19]
FT-IR Spectroscopy
The 2G PAMAM dendrimer were subjected to FT-IR spectroscopy analysis (Bruker Optics,
Germany).[20]
Preparation of herbal extract

Fresh leaves of the plant, A. indica were collected from the hills of Sarkaghat, Himachal
Pradesh, India. Extraction of extract from A. indica was done by simple Maceration process.
Plant materials were collected in bulk, washed, shade dried and pulverized in a mechanical
grinder to obtain aqueous extract. It was stored in a well closed container.[21,22]
Evaluation of herbal extract

Evaluation of plant was carried out by determining their organoleptic properties, UV
spectroscopy and FTIR spectroscopy. The standard solution of A. indica in Methanol
(10µg/ml) was scanned (200-400) nm by use of UV spectrophotometer. FTIR spectrum of A.
indica was performed using wave number ranges.
Preparation of cream base

Firstly, Stearic acid, cetosteryl alcohol, liquid paraffin, petroleum jelly were melted on a
steam bath at 75°C. After that, the remaining ingredients were dissolved in water and boiled
at 75°C. The aqueous solution was then added to the above oily phase with agitation.
Glycerin was added finally and mixed. The formulated creams were filled in suitable plastic
container.[23]
Preparation of cream base

Table no 1: Preparation of cream base
S.no. Ingredients Quantity (% w/w)
1. Stearic acid 16
2. Potassium Hydroxide 2
3. Cetosteryl alcohol 5
4. Liquid paraffin 3.5
5. Petroleum jelly 3.5
6. Methyl paraben sodium 0.10
7. Propyl Paraben sodium 0.05
8. Glycerine 7
9. Purified water q.s. 100

Stearic acid, Potassium hydroxide, Cetosteryl alcohol, Petroleum jelly, Liquid paraffin,
Methyl paraben sodium, Propyl paraben sodium, Glycerin. Seven different variants of WHCs
(F1 to F6) were prepared by using different concentrations of herbal extract.
Preparation of dendricream
The 1%, 2%, 3%, 4% and 5% of the extracts were mixed with the above prepared cream base
individually with uniform stirring by using a mechanical stirrer. After that 1% dendrimer was
added in it. Glycerin was added and mixed. At last, a pinch colouring agent (Tartrazine) was
added in the formulation that gave yellow colour appearance. The formulated creams were
filled in suitable plastic container.
Formulation design for dendricream

Table 2: Formulation design for dendricream
Formulation code Dendrimer (%) Plant extract (%) cream base q.s. (g)
DC1 1 1 20
DC2 1 1 20
DC3 1 2 20
DC4 1 3 20
DC5 1 4 20
DC6 1 5 20
Pharmaceutical evaluation of cream[24]

The formulations were evaluated for different pharmaceutical parameters:
Physical Appearance- The formulated creams were observed for their visual appearance,
transparency, color, consistency.
pH - The pH of formulated creams were determined by using digital pH meter ((Hanna

Instruments, Italy) by dissolving 1 gm cream in 100 ml of water.[25,26]
Consistency-The consistency of formulated creams were determined by hand. Take a pinch

of cream and rubbed it with fingers.[27]
Spreadability-The spreadability of formulated creams were determined by placing pinch of

cream base on glass slide and then place another slide on it and then observed.[28]
Viscosity- Viscosity of formulated creams was determined by Brookfield viscometer with the
help of spindle no. 6 which was rotated at 10 rpm. The sample of cream was allowed to settle
over 30 min before measurements were taken.[29]

In Vitro Release of herbal cream

Franz Diffusion cell was used for the drug release studies. The dendricream (1gm) was
applied onto the surface of Egg membrane evenly. The Egg membrane was clamped among
the contributor and the receptor chamber of diffusion cell. The receptor chamber was filled
with freshly prepared Acetate Buffer Saline (pH 5.5) solution to solubilize the drug. The
receptor chamber was stirred by magnetic stirrer. The samples (1.0 ml) were collected at
appropriate time interval of 15min. Samples were analyzed for drug content by UV visible
spectrophotometer at 211 nm after appropriate dilutions. Cumulative corrections were made
to obtain the total quantity of medicine liberate at each time period. The cumulative amount
of drug released across the Egg membrane was determined as a function of time.
In vivo evaluation of herbal cream

Skin inflammation test
The cream was evaluated for primary skin inflammation test on experimental animals
(lacking hair back of the rats) to evaluate the safety of cream.[30, 31, 32]
Evaluation of wound healing activity

The healthy Wistar albino rats of either sex, weighing 150–200 gm were housed under
normal environmental conditions of temperature, humidity (25 ± 0.50˚C) and 12 hr light/dark
cycle. The animals were fed with standard pellet diet and water ad libitum. The experiment
was conducted in accordance to the protocol approved by Institution Animal Ethical
Committee (IAEC-CTIPS), CT Institute Of Pharmaceutical Sciences, Shahpur, P.O.
Udhopur, Partappura Road, Jalandhar-144020 (Punjab)/ (Reg. no. 1704/PO/a/13/CPCSEA).
The rats were divided into four groups i.e. control (base cream treated group/without extract),
standard (Povidone-Iodine treated group), test1 (herbal extract treated) and test 2 (herbal
formulated dendricream) group. Rats were anesthetized by administering ketamine (80 mg/kg
i.p.). The rats were anaesthetized with ketamine hydrochloride (0.5 ml/kg b.w., i.p.). Animals
were anaesthetized by using Ketamine hydrochloride of an appropriate dose 80mg/kg. The
fur of back side of animals was removed by using an electric razor. A full thickness of the
excision wound with circular area of 176 mm2 (width 1.5 cm) was made on the shaved back
of the rats. The wounds was treated with topical application of the cream once daily. The
wounds and area of wound size was measured on 3, 6, 9, 12 and 14th of post-wounding
day.[33,34,35]

2.10 Stability studies

Different cream formulations were kept in tightly closed glass vials. The sample were kept in
dark (in amber colors vials) and light (in colorless vials) at 0°C. Room temperature (25°-30°C)
and 45°C for a periods of seven weeks. The samples were analyzed after every week for up to
seven weeks for any precipitation, change in color and consistency. The data obtained was
used for the analysis of any physical or chemical degradation.
RESULTS AND DISCUSSION

Evaluation of dendrimers
Physical appearance- The 2G dendrimers were light reddish yellow in colour.
Physical state- These 2G dendrimers had very viscous oily physical state.
Ultraviolet Spectroscopy
Absorption maxima (λmax) were recorded for 2G dendrimers and surface modified
dendrimers. The λmax of 2G PAMAM dendrimers were at 277.5.
Figure 2: UV Spectra of 2G PAMAM dendrimers
FT-IR spectroscopy
The 2G dendrimers were analyzed by FT-IR spectroscopy. The FT-IR peaks provide the
proof of modification of free amino group into amide group. The FT-IR spectra of 2G
PAMAM dendrimers were in fig.3.

Figure 3: FT-IR Spectra of 2G PAMAM dendrimers
Table 3: FTIR Spectrum of 2G PAMAM Dendrimers showing different wave number

with Assignment
IR Absorption Band IR Absorption Band
Sr.No Functional Groups
cm-1) Experimental) (cm -1) (Literature)
1. 3358 3366 (O-H stretching),
2. 2857 2942 Aliphatic C-H stretching
3. 1598 1575 (Aromatic C=N stretching)
4. 1043 1031 C-O stretching
Identification of dendrimers
All generations of dendrimers were identified by copper sulphate test. 1gm of CuSo4 is was
dissolved in 10ml water and treated with PAMAM dendrimers which showed violet color.
This violet color indicated the presence of 2G PAMAM dendrimers.
Identification of dendrimers
Figure 4: Identification of dendrimers with CuSO4 test

Table 4: Evaluation and cumulative % release of dendrcreams

Formulation Viscosity at cumulative %
Appearance pH Spreadability
Code 10rpm release
F1 Light yellow 7.5 Easily Spreadable 8600 84.99±0.14
Figure 5: Percentage Cumulative Plant Release of formulated Batches
Skin inflammation test

This test was conducted to evaluate the inflammation caused by the prepared cream on the
skin of animals. The results showed that the formulation (F6) was devoid of any primary skin
inflammation or sensation even after 48 hrs of application on the rat skin. None of the animal
showed any skin inflammation.
Wound healing activity

The results of wound healing activity by excision wound model are presented in Table 1. The
wounds and wound size was measured at 0, 3, 6, 9, 12 and 14 days. The results indicate that
dendricream and test herbal cream both significantly reduces the wound area as compared to
the control group.

Table 5: In-vivo study

Days Test 1(Extract) Test 2(formulated cream) Standard Control
12
14
Table no.6 Effect of herbal cream on Wound size at different days interval
Groups Wound size area in mm2 (mean ± SEM)
0 Day 3 Days 6 Days 9 Days 12 Days 14 Days
176.62± 165.43± 109.00 ± 43.57± 22.44± 11.39±
Control group
0.73 0.63 0.65 0.64 0.89 0.36
Standard 172.43± 125.66± 65.43 ± 32.13± 3.12± 0.25±
group 0.63 0.65 0.69 0.43 0.36 0.07
Test 1 (herbal 173.22± 126.78± 69.33 ± 28.16± 3.18± 0.21±
extract) 0.83 1.02 0.85 0.62 0.32 0.06
Test 2 173.25± 124.75± 65.31 ± 19.14± 3.11± 0.14±
(Dendricream) 0.79 0.89 0.75 0.52 0.22 0.04
Wound size area (mm2) at differents days interval.

Figure 6: Wound size area (mm2) at differents days interval
We have selected four groups i.e. control (base cream treated group/without extract), standard
(Povidone-Iodine treated group), test1 (herbal extract treated) and test 2 (herbal formulated
dendricream) group. The wounds were treated with topical application of the cream once
daily. The mean percentage of wound closure area was calculated on 3 th, 6th, 9th, 12th and
finally 14 days. All the formulations showed significant promotions of wound healing activity
with all four groups of animals. The test 2 group (dendricream) showed better wound healing
activity as compare to standard group because dendrimers already have wound healing
activity. The dendricream decreased wound size at faster rate as compared to standard and
herbal extract.
Stability study
Figure 7: IN-VITRO release of drug from Artemisia Indica dendrimer based cream in
pH 5.5 ABS before and after stability studies

CONCLUSION
This concludes that Formulation No.6 is statistically significant better wound healing agent as
compared to various wound healing creams. The prepared cream was pleasant, easily
spreadable and washable thus there is a possibility of increased the patient compliance.
Formulated cream considerably promotes wound healing than control group. The activity
may be mainly due to active constituents present in the herbal extract. Some of herbs reported
to act by promoting tissue regeneration. However, further depth and structured study, would
be beneficial to assess its usefulness and mechanisms more exactly. Formulation No.6 is
statistically significant better wound healing agent as compared to various wound healing
creams. This study can be helpful for upcoming researchers to select these herbs for the
formulation and evaluation of other cosmetic applications which can be claimed for their
efficacy with scientific data.
DISCUSSION
F6 formulation or cream was observed to be best as compared to other formulations. It had
light yellow appearance and gave a smooth feel on application which was maintained after
tested the stability study. The formulation didnot produce any irritation. Stability was
determined by exposing the formulation to various temperatures such as 4°C, 27°C & 37°C
for specified period. The pH of the formulation was found to be 6.1 which is good for skin
(pH=6.8). The creams also showed good spreadability when using slides. A comparative
study of viscosity showed that the viscosity of the optimized formulation increases. The
prepared cream was easily spreadable with small amount of shear. All the creams showed
slight difference in release profile at particular time period. From the stability study, cream
showed no changes in pH, consistency, spreadability and viscosity after keeping at different
temperatures for 90 days. All the formulations showed significant promotions of wound
healing activity with all four groups of animals in Table 6. The mean percentage of wound
closure area was calculated on 3th, 6th, 9th, 12th and finally 14 days. The test 2 group
(dendricream) and test 1 group (herbal extract) showed better wound healing activity as
compare to control group because of active constituents (borneol) present in Artemisia indica.
Further the rate of wound contraction for dendricream in applied rats was significant higher.

ACKNOWLEDGEMENT
Authors are grateful to Department of Pharmaceutics, CT Institute of Pharmaceutical
Sciences, Shahpur Campus, Jalandhar, Punjab for providing necessary facilities to complete
this research.
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WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

Rani et al. World Journal of Pharmacy and Pharmaceutical Sciences

Volume 5, Issue 8, 1483-1497 Research Article ISSN 2278 – 4357

FORMULATION, OPTIMIZATION AND EVALUATION OF

Shalu Rani*, Amanjot, Harjaskaran, Narinder Singh, Surya Prakash Gautam,

Department of Pharmaceutics, CT Institute of Pharmaceutical Sciences, Shahpur,

www.wjpps.com Vol 5, Issue 8, 2016. 1483

MATERIALS and METHODS

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Synthesis of PAMAM Dendrimers

Figure 1: Flow diagram of 2G PAMAM dendrimers

2g of 1.5 G dendrimers and 42.7 ml EDA (Ethylenediamine) were dissolved in appropriate

Evaluation of 2G PAMAM dendrimers

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Preparation of herbal extract

Evaluation of herbal extract

Preparation of cream base

Preparation of cream base

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Formulation design for dendricream

Pharmaceutical evaluation of cream[24]

pH - The pH of formulated creams were determined by using digital pH meter ((Hanna

Consistency-The consistency of formulated creams were determined by hand. Take a pinch

Spreadability-The spreadability of formulated creams were determined by placing pinch of

www.wjpps.com Vol 5, Issue 8, 2016. 1487

In Vitro Release of herbal cream

In vivo evaluation of herbal cream

Evaluation of wound healing activity

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2.10 Stability studies

RESULTS AND DISCUSSION

Figure 2: UV Spectra of 2G PAMAM dendrimers

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Figure 3: FT-IR Spectra of 2G PAMAM dendrimers

Table 3: FTIR Spectrum of 2G PAMAM Dendrimers showing different wave number

Figure 4: Identification of dendrimers with CuSO4 test

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Table 4: Evaluation and cumulative % release of dendrcreams

Figure 5: Percentage Cumulative Plant Release of formulated Batches

Skin inflammation test

Wound healing activity

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Table 5: In-vivo study

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Figure 6: Wound size area (mm2) at differents days interval

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