Effects of topical application of Calendula officinalis gel on collagen and hydroxyproline content of skin in rats

Abstract

In this investigation, the effects of different concentrations of Calendula officinalis gel on collagen and hydroxyproline content of the skin are studied. Sixty-five mature male rats were randomly divided into
three groups (control, placebo, and treatment group). Under sterile conditions, a 2 × 2-cm piece of cervical skin was excised
in each animal. Treatment group received a daily topical application of 5%, 7%, and 10% C. officinalis gel, the placebo group received a daily topical application of the base gel, and the control group received no treatment
during this experimental study. Fourteen, 21, and 45 days later, the rats were euthanized and biopsies were taken from the
site of the initial incisions and samples were collected for biochemical investigation. Collagen production in the group treated
with 7% gel was significantly more than the placebo and control group. Upper and lower doses seem to be less effective, although
the reasons for this remain unclear.

Full text

ORIGINAL ARTICLE
Effects of topical application of Calendula officinalis gel
on collagen and hydroxyproline content of skin in rats
A. Tabatabai Naeini & R. Miri & N. Shafiei &
M. R. Tabandeh & A. Oryan & S. Nazifi
Received: 17 June 2010 / Accepted: 25 August 2010 / Published online: 10 September 2010
#
Springer-Verlag London Limited 2010
Abstract In this investigation, the effects of different con-
centrations of Calendula officinalis gel on collagen and
hydroxyproline content of the skin are studied. Sixty-five
mature male rats were randomly divided into three groups
(control, placebo, a nd trea tment group). Under sterile
conditions, a 2×2-cm piece of cervical skin was excised in
each animal. Treatment group received a daily topical
application of 5%, 7%, and 10% C. officinalis gel, the
placebo group received a daily topical application of the base
gel, and the control group received no treatment during this
experimental study. Fourteen, 21, and 45 days later, the rats
were euthanized and biopsies were taken from the site of the
initial incisions and samples were collected for biochemical
investigation. Collagen production in the group treated with
7% gel was significantly more than the placebo and control
group. Upper and lower doses seem to be less effective,
although the reasons for this remain unclear.
Keywords Calendula officinalis gel
.
Collagen
.
Hydroxyproline
.
Skin
.
Rat
Introduction
Many biological dressings and indigenous medicines have
been reported to possess wound healing properties. However,
none of these has been completely effective and free of side
effects. Many drugs of plant origin can produce wound
healing. Calendula officinalis of the family Asteraceae is one
of the most reliable cool season annuals. Flowers are single
or double and usually yellow or orange. Pot Marigold will
grow 1 to 2 ft tall and require full to partial sun (Edward
et al. 1999).Thisflowerhasbeenwidelyusedin
homeopathic medicine for the treatment of many diseases
(Zitterl-Eglseer et al. 1997).Ithasbeenreportedtopossess
many pharmacological activities, which include antioxidant
(Preethi et al. 2006), anti-inflammatory (Della logia et al.
1994),antibacterial(Dumeniletal.1980), antifungal (Kasiram
et al. 2000), antiviral (Barbour et al. 2004), anti-edematous,
immunomodulatory activity, and antimicrobial effects
(Danielski et al. 2007; Hamburger et al. 2003; Gazim et al.
2008). Marigold also possesses cytotoxic as well as tumor
reducing potential (Boucaud-Maitre et al. 1988; Chandran
and Ramadasan 2008). These effects are related to the
components of this flower such as sesquiterpenes, alcohol,
saponins, triterpenes, flavonoids, hydroxycoumarin, carote-
noids, tannin, and volatile oils (0.1–0.2 % ; Ao 2007;
Radulescu et al. 2000;Crabasetal.2003). Because of these
A. T. Naeini
:
N. Shafiei (*)
:
S. Nazifi
Department of Clinical Studies,
School of Veterinary Medicine, Shiraz University,
P.O. Box 1731, 71345 Shiraz, Iran
e-mail: nazanin_vet78@yahoo.com
R. Miri
Department of Pharmacology and Toxicology,
Faculty of Pharmacy, Shiraz University of Medical Sciences,
Shiraz, Iran
M. R. Tabandeh
Department of Biochemistry,
School of Veterinary Medicine,
Shahid Chamran University,
Ahvaz, Iran
A. Oryan
Department of Pathobiology,
School of Veterinary Medicine, Shiraz University,
P.O. Box 1731, 71345 Shiraz, Iran
Comp Clin Pathol (2012) 21:253–257
DOI 10.1007/s00580-010-1087-1

components, this flower should affect cutaneous wound
healing and also collagen production.
The present study was designed to test the effect of C.
officinalis gel on cutaneous collagen production and also to
compare the effect of different concentrations of this gel on
cutaneous collagen production.
Materials and methods
Preparation of the gel
Fresh calendula flower t ops were used for extraction.
Calendula flowers (263.7 g) were extracted with 1,200 cc
ethyl alcohol 70%. For this, the flowers were placed in an
Erlenmeyer flask and the alcohol was added. This flask was
stoppered and sealed and then placed in a dark room at room
temperature and shaken every day for 1 week. Then the dark
liquid was decanted. A rotary device was used for the next
step. Ten days later, we had the oily extract of calendula
flowers. To prepare the gel, at first the extract was dried with a
freeze dryer and then the gel base was made. For this, 1 g
carbopol was added to 95 cc distilled water (5% gel), 1 g to
93 cc distilled water (7% gel), and 1 g to 90 cc distilled water
(10% gel); after 4 or 5 h, all carbopol powder dissolved and
sodium hydroxide were added to make the gel base. Finally,
5 g (5% gel), 7 g (7% gel), and 10 g (10% gel) were added to
the gel base and used for all experiments.
Experimental animals
Sixty-five white Sprague-Dawley male rats weighing
between 180 and 220 g were divided into five groups,
and each one housed individually in a separate standard
cage and fed with norm al mouse chow and water ad
libitum. Their information was written on their cage.
Temperature (25°C) and the ratio of daylight hours to
non-daylight hours (12:12 h ratio of light to dark) were kept
constant.
Rats were divided into five groups as follows: group Ι—
control without any treatment (n=15), group ΙΙ—gel base
with placebo treatment (n=15), group ΙΙΙ—5% gel (n=10),
group ΙV—7% gel (n=15), and group V—10% gel (n=10).
The control, gel base, and 7% gel group were divided into
three equal subgroups of five rats which were followed for
14, 21, and 45 days. The 5% and 10% gel group were
divided into two equal subgroups of five rats and followed
for 14 and 21 days. Care was taken to avoid unnecessary
stress to the animals throughout the experimental period.
Surgical procedures
The rats of all groups were anesthetized by injection of
0.8 cc ketamine (5%) and 0.2 cc xylazine intramuscularly in
the hamstring muscles. Before making incisions, the dorsal
aspect of the cervical area was shaved and washed with a
scrub solution of povidone–iodine. Under sterile condi-
tions, a skin incision was made in a square shape 2×2 cm in
the cervical region and then the skin was removed. The
duration of anesthesia was about 10 min for each rat.
Treatment regimes
In the control group, after making the incision on the
cervical region, no treatment was applied on the incision
and the incisions remained intact. Fifteen rats in this group
were followed 14, 21 and 45 days later. Daily observa tion
was performed and any wound fluid or any evidence of
infection or other abnormalities were noted. The rats from
group ΙΙ received gel base in a thin, uniform layer on the
wound daily for 14 days. In the treatmen t groups, the rats
received 5%, 7%, and 10% calendula flower gel.
Sampling
At the end of days 14, 21, and 45 postoperation, the rats were
euthanized by IV injection of 30 mg/kg thiopental sodium
(nesdonal) via tail vein and sampling was done. Samples were
Control Gel base 5% 7% 10%
14 54.79±12.44 56.53±3.12 70.25±13.29 88.52±3.66 69.23±11.77
21 62.71±3.88 63.15±6.57 71.79±15.36 89.15±7.14 86.75±4.81
45 69.13±6.52 63.96±6.13 89.34±3.86
Table 1 Collagen
content (grams per 100 g dry wt.
tissue)
Significant difference between
groups (P≤ 0.05)
Control Gel base 5% 7% 10%
14 6.84±1.55 7.06±0.39 8.77±1.66 11.06±0.45 8.65±1.47
21 7.83±0.48 7.89±0.82 8.97±1.91 11.14±0.52 10.84 ±0.6
45 8.63±0.81 7.97±0.75 11.16±0.48
Table 2 Hydroxyproline
content (grams per 100 g dry wt.
tissue)
Significant difference between
groups (P≤ 0.05)
254 Comp Clin Pathol (2012) 21:253–257

taken from only the wound or only the scar of the wound.
Then they were collected for biochemical investigations.
Biochemical studies
After shaving, the skin containing the incision area was
excised with a biopsy 3 mm punch. The samples were frozen
(−70°C) promptly after sampling before being tested. (Oloumi
et al. 2007). After thawing, the punched skin pieces were
dried in a hot-air oven at 60–70°C until a consistent weight
was achieved. The samples were then hydrolyzed with 6 N
HCl for 2 h at 120°C. The hydrolyzed samples were adjusted
to pH 7 and subjected to chloramines T oxidation, and
finally, the colored adduct formed with the aldehyde–
perchloric acid reagent at 60°C was read at 550 nm after
cooling for 5 min. Actually, we used a modified assay to
determine a hydroxyproline in a tissue hydrolizate (Edwards
and Obrien 1980).
Statistical analysis
One-way analysis of variance and Duncan’s multiple range
tests were used to compare the means. A value of P≤ 0.05
was considered as significant.
Results
Collagen and hydroxyproline contents (grams per 100 g dry
wt. tissue) were presented in Tables 1 and 2. Hydroxyproline
and collagen content in the treatment group from day 21 was
significantly higher in the 7% and 10% gel groups than the
others, while on days 14 and 45, the 7% gel group was
significantly higher than the other groups (Figs. 1, 2,and3).
Discussion
Wound healing is a dynamic, interactive process involving
soluble mediators, blood cells, extracellular matrix, and
parenchymal cells (Adam and Richard 1999). Though the
healing process takes place by itself and does not require
much help, various risk factors such as infection and delay
in healing have been mentioned to promote this process
(Shanmuga Priya et al. 2002). C. officinalis flowers have an
anti-inflammatory effect (Della Logia et al. 1994), @@@so
this can promote wound healing and also several compo-
nents that affect the healing of burn wounds (Chandran and
Ramadasan 2008), promote anti-tumor (Ukiya et al. 2006)
and anti-edematous activity (Zitterl-Eglseer et al. 1997) and
result in the treatment of venous leg ulcers (Duran et al.
2005) are isolated from this flower. But there is no
academic or classified report about the effect of this flower
on collagen deposition and the difference between different
ccc 45
7.001.00.00
Mean of B
100
90
80
70
60
Fig. 3 Collagen content on day 45
col21
10.007.005.001.00.00
Mean of B
100
90
80
70
60
Fig. 2 Collagen content on day 21
col14
10.007.005.001.00.00
Mean of B
100
90
80
70
60
50
Fig. 1 Collagen content on day 14
Comp Clin Pathol (2012) 21:253–257 255

concentrations of it on this process. There is almost
unanimous agreement that collagen performs a major role
in restoring strength and remodeling scar tissue (Madden
et al. 1971). Biochemical analysis showed increased
hydroxyproline content, which is a reflection of increased
collagen synth esis (Nayak et al. 1999). Collagen is one of
the most dominant extracellular matrix proteins in the
granulation tissue and appears to be significantly high by
the fifth day of wounding, and after day 7, collagen
production is further advanced (Grillo 1964). In this
research, different concentrations of flower gel and three
different days are noted, but numerous studies about this
flower were done in 1 day and one concentration, so the
comparison of different concentrations on different days is
incomplete. As seen in the tables, topical application of C.
officinalis flower gel on day 14 is more efficient than on the
other days. This means that this gel effect is better on this
day, and also, the 7% concentration of this product can act
much better than the other concentrations, causing signif-
icant wound healing activity because of the collagen
synthesis; however, the 10% gel is less effective than the
7% gel. Perez-Car reon et al. (2002) and Barajas-Farias et al.
(2006) s ho wed tha t some conce nt ratio ns of cale ndu la
flower extract can be toxic (1 0 and 20 mg/kg doses
produced an increment in the number of carcinogenic
hepatic cells 25 days later, but there is no application about
this effect on the other days), so decreased collage n
deposition in the 10% gel to the 7% gel may be due to
this effect, and also each group contained only five rats and
this can increase the error risk. To summarize the results of
this study, C. officinalis gel can affect collagen deposition
and wound healing, but not all of its concentrations because
it has both a dual and an opposite effect in different
concentrations of this gel. Low concentrations have no
effects and high concentrations have cytotoxic effects.
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