Original Article J. Clin. Biochem. Nutr.

, 43, 58–64, September 2008

Journal
JCBN
0912-0009
the
Kyoto,
Original
10.3164/jcbn.2008043
jcbn2008043
1880-5086
Society
Effect Japan
ofArticle
Clinical
offorCalendula
FreeBiochemistry
Radical Research
and Nutrition
Japan
officinalis Flower Extract on Acute Phase
Proteins, Antioxidant Defense Mechanism and Granuloma
Formation During Thermal Burns

Preethi K. Chandran, and Ramadasan Kuttan*

Amala Cancer Research Centre, Amala Nagar Thrissur-680555, Kerala, India

98
1
5
2
43
Received
;64
008
accepted1.2.2008
3.2.2008

Received 1 February, 2008; Accepted 3 February, 2008

Copyright © 2008 Effect

Summary JCBN of Calendula officinalis flower extract was investigated against experi-
mentally induced thermal burns in rats. Burn injury was made on the shaven back of the rats
under anesthesia and the animals were treated orally with different doses of the flower extract
(20 mg, 100 mg and 200 mg/kg body weight). The animals treated with the extract showed
significant improvement in healing when compared with the control untreated animals. The
indicators of the wound healing such as collagen-hydroxyproline and hexosamine contents were
significantly increased in the treated group indicating accelerated wound healing in the treated
animals. The acute phase proteins—haptoglobin and orosomucoid which were increased due
to burn injury were found to be decreased significantly in 200 mg/kg body weight extract treated
animals. The antioxidant defense mechanism, which was decreased in the liver during burn
injury, was found to be enhanced in treated animals. The lipid peroxidation was significantly
lowered in the treated group when compared to control animals. Tissue damage marker
enzymes- alkaline phosphatase, alanine and aspartate transaminases were significantly lowered
in the treated groups in a dose dependant manner. The histopathological analyses of skin tissue
also give the evidence of the increased healing potential of the extract after burn injury.

Key Words: acute phase proteins, antioxidant enzymes, Calendula officinalis, granuloma tissue,
thermal injury

leukins [3]. Acute phase responses are initiated to restore

Introduction homeostasis after trauma. Clinical studies have shown that a
sustained or increased acute phase response can contribute
Thermal injury produces profound systemic changes such to multiorgan failure, hyper metabolism, complications, and
as oligemic shock, anemia, renal failure and metabolic death [4]. There are only very few drugs, which are effective
disturbances. It causes direct tissue damage as well as in the treatment and management of thermal burns.
inflammatory reactions. Infection is another major compli- Many drugs of plant origin can produce wound healing
cation of thermal injury [1]. It also leads to increased oxidant and are used in burn injury. Calendula officinalis. Linn of
stress in the cells as seen by decreased endogenous non- Family Asteraceae has been widely used in homeopathic
enzymatic and enzymatic antioxidant activity [2]. The medicine for the treatment of many diseases [5]. It has been
mediators of burn shock include histamine, serotonin, kinins, reported to possess many pharmacological activities, which
oxygen free radicals, prostaglandins, thrombaxene and inter- include antioxidant [6] anti-inflammatory [7] antibacterial
[8], antifungal [9] and antiviral [10]. It also possess cyto-
toxic as well as tumor reducing potential [11].
*To whom correspondence should be addressed.
Tel: +91-487-230-4190 Fax: +91-487-230-7698 In the present study we have evaluated the potential
E-mail: [email protected], [email protected]. activity of Calendula officinalis flower extract against

58
 Effect of Calendula Extract on Thermal Burns 59

thermal burns in animals. The enzyme markers of tissue Burn wound model
damage, the level of antioxidant enzymes and the acute Burn wounds were created on the shaved dorsal part of
phase proteins were evaluated and compared with the rats after anaesthetizing them with ketamine hydrochloride
untreated thermally injured animals. (100 mg/kg body weight). The animals were immersed in
water bath of 90°C for 12 s to produce thermal burns [13].
Materials and Methods To achieve uniform area of burn the animals were kept on a
box with a hole so that the body exposed through the hole
Chemicals will only be burned. The method was standardized and
Nitro blue tetrazolium (NBT), 5-5'dithiobis (2-nitro confirmed through histopathological analysis of burned skin.
benzoic acid) (DTNB) and riboflavin were purchased from
Sisco Research Laboratories Pvt. Ltd., (Mumbai, India), Experimental protocol
Thiobarbaturic acid was purchased from Himedia laborato- Fifty four animals were used for the study. Animals were
ries, (Mumbai, India). Para dimethyl amino benzaldehyde treated with the extract orally 24 h prior to burning. The
was purchased from E Merck, (Mumbai, India). Ketamine animals were grouped as follows: Group I: Normal without
hydrochloride was purchased from Neon laboratories Ltd., any treatment or burning (n = 6), Group II: Burned animals
(Mumbai, India). Span Diagnostics Ltd., (Surat, India) sup- without any treatment (n = 12), Group III: Burned animals
plied the biochemical kits for determining alkaline phos- treated with 20 mg/Kg body weight Calendula extract
phatase, aspartate transaminase, alanine transaminase activ- (n = 12), Group IV: Burned animals treated with 100 mg/Kg
ities and bilirubin content. The kits for haptoglobin and body weight Calendula extract (n = 12), Group V: Burned
orosomucoid were supplied by Orion Diagnostica, (Espoo, animals treated with 200 mg/Kg body weight Calendula
Finland). All other chemicals and reagents used were of extract (n = 12). Treatment with the drug was continued
analytical grade. upto 10 consecutive days. Six animals from each group
were sacrificed on 5th day and the rest of the animals were
Preparation of the extract sacrificed on 10th day. Blood was collected; serum was
Fresh Calendula flower tops were used for extraction of separated for analyzing alkaline phosphatase, glutamate-
the active components. They were collected from Govern- pyruvate transaminase, glutamate-oxaloacetate transaminase
ment Botanical Gardens, Ooty, Nilgiris during January 2006 activities, and bilirubin levels using commercially available
and were authenticated by Dr. S. Rajan, Field Botanist, kits. Haptoglobin and alpha-1 acid glycoprotein (orosomu-
Central Council for Research in Homeopathy, Ooty, India coid) were estimated in serum by turbidometric method.
and the voucher specimen was deposited at Amala Granuloma tissue of the burned skin was carefully excised
Ayurvedic Research Centre (Voucher No: Co05). Extraction and washed in cold saline. A small portion of the skin was
was done as per Pharmacopoeia [12]. Calendula flowers fixed in 10% formalin and sectioned for histopathological
(700 g) were extracted with 450 ml ethyl alcohol by analysis. The rest of the tissue was lyophilized. These tissues
masturation. For this, the material was placed in a wide were then hydrolyzed with 6N hydrochloric acid and
mouth bottle and the alcohol was added. The jar was estimated for the content of hexosamine by the method of
stoppered and sealed to prevent evaporation. It was placed in Elson & Morgan [14] and collagen hydroxyproline by
a dark room at room temperature and shaken everyday for method of Newman & Logan [15].
two weeks. Then the clear liquid was decanted and the Liver was excised out and washed in ice-cold saline and a
residue was pressed out through clean linen, which was 25% homogenate was prepared in Tris-HCl buffer (pH 7.0)
added to the decanted liquid. Volume was made upto 1 L and centrifuged at 10,000 rpm for 30 min at 4°C. The
with alcohol. 100 ml of this tincture was evaporated to supernatant was collected and analyzed for glutathione
dryness in a shaker water bath at 42°C. The yield was found content [16], protein content [17], lipid peroxidation [18],
to be 1.1 g. Dried extract (1 g) was resuspended in a known superoxide dismutase activity [19] and catalase activity [20].
amount of distilled water and used for all experiments.
Statistical analysis
Experimental animals The results are expressed as mean ± SD Statistical
Female Wistar rats (150–200 g) were obtained from Small evaluation of the data was done by one-way ANOVA
Animal Breeding Station, (Mannuthy, Thrissur, Kerala). followed by Dunnet’s test (post-hoc) using In Stat 3 software
They were housed in well-ventilated cages and fed with package. The control animals were compared with the
normal mouse chow (Sai Durga Feeds and Food, Bangalore, normal whereas the drug treated with that of the control
India) and water ad libitum. All the animal experiments were values.
done after approval from the Institutional Animal Ethical
Committee.

Vol. 43, No. 2, 2008

60 P.K. Chandran et al.

Table 1. Effect of Calendula officinalis on hexosamine and collagen hydroxy proline content in the skin granuloma tissue of thermally
burned animals
Hexosamine (mg/100 mg dry weight tissue) Hydroxy proline (mg/gm dry weight tissue)
5th day 10th day 5th day 10th day
Group I
6.6 ± 1.4 — 9.70 ± 1.85 —
(Normal)
Group II
1.72 ± 0.14*** 2.96 ± 0.27*** 7.56 ± 2.15 6.99 ± 1.50
(Burned untreated)
Group III
1.85 ± 0.17 3.08 ± 0.33 9.25 ± 0.93 11.05 ± 0.98**
(Burned + 20 mg/kg body weight drug)
Group IV
1.88 ± 0.07 3.52 ± 0.16** 9.54 ± 1.06 12.70 ± 2.1**
(Burned + 100 mg/kg body weight drug)
Group V
2.20 ± 0.28** 3.72 ± 0.17** 12.95 ± 0.87** 13.26 ± 1.84**
(Burned + 250 mg/kg body weight drug)
**: p<0.01, ***: p<0.001

Table 2. Effect of Calendula officinalis on level of acute phase proteins in the serum of thermally burned animals
Haptoglobin (g/l) Serum orosomucoid (g/l)
5th day 10th day 5th day 10th day
Group I
5.01 ± 1.33 — 0.49 ± 0.23 —
(Normal)
Group II
9.74 ± 4.05** 8.97 ±1.66*** 2.18 ± 0.94*** 2.85 ± 0.88***
(Burned untreated)
Group III
4.76 ± 1.5* 5.03 ± 0.91*** 0.97 ± 0.31* 0.65 ± 0.18***
(Burned + 200 mg/kg body weight drug)
*: p<0.05, ***: p<0.001

Results scattered lymphocytes. Histopathological analysis supported

the healing potential of Calendula extract (Fig. 1).
Effect of Calendula officinalis extract on granuloma
formation during thermal injury Effect of Calendula officinalis extract on antioxidant defense
The granuloma tissue excised from the burned skin was system and lipid peroxidation in the liver after thermal
estimated for hydroxy proline and hexosamine contents, injury
which are the indicators of extracellular matrix. The result Glutathione was found to be increased in the liver of
obtained is shown in Table 1. There was a significant higher animals with thermal burns possibly to scavenge the free
amount of hydroxyproline in the granuloma tissue of drug radicals generated during burning. This was found to be
treated animals compared to controls on both the time further increased by treatment with the extract. On 5th day
periods. The hexosamine content was found to be decreased for extract treated animals the glutathione content was
during thermal burn but was significantly more in drug significantly higher than that of untreated animals. On 10th
treated groups on both 5th as well as 10th day of burning. day the level reached almost normal level in all treated
There was also a significant increase in the level of acute groups whereas it was significantly low in the untreated
phase proteins, haptoglobin as well as orosomucoid in the control group (Table 3).
serum of burned animals on both 5th and 10th day of thermal Lipid peroxidation was significantly high in the liver of
burn (Table 2). The increase in the level was found to be control animals. Lipid peroxidation was lowered in the drug
significantly lowered by treatment with the extract. treated group (200 mg/kg body weight) on 5th day. On 10th
Histopathology section of the burned skin of control day lipid peroxidation attained the normal level in treated
animals on 5th day showed denuded epidermis, diffuse infil- animals whereas the control animals had significant high
tration of plasma cells, lymphocytes and polymorphs. The lipid peroxidation (Table 3).
stroma also showed edema and congestion. But in treated Superoxide dismutase activity was found to be increased
animals only the sub epidermal region and dermis showed in burned animals on 5th day possibly to reduce the oxidative

J. Clin. Biochem. Nutr.

 Effect of Calendula Extract on Thermal Burns 61

Fig. 1. Histopathological section of skin on 5th day of burning. (i) Normal skin, (ii) Skin of thermally burned animal without treatment,
(iii) Skin of thermally burned animal treated with 200 mg/kg body weight Calendula extract

Table 3. Effect of Calendula officinalis on antioxidant parameters in liver of thermally burned animals
LPO GSH SOD CAT
(n moles of MDA formed/mg protein) (n moles/mg protein) (Units/ mg protein) (Units/mg protein)
5th day 10th day 5th day 10th day 5th day 10th day 5th day 10th day

Group I
0.83 ± 0.03 — 14.1 ± 2.5 — 0.63 ± 0.13 — 8.50 ± 1.50 —
(Normal)
Group II
(Burned 3.70 ± 0.88*** 1.18 ± 0.15 19.3 ± 2.9** 9.2 ± 0.89** 0.82 ± 0.10* 0.54 ± 0.06 7.43 ±1.24 7.78 ± 0.89
untreated)
Group III
(Burned +
3.70 ± 0.57 0.90 ± 0.19** 23.5 ± 2.7* 12.0 ± 0.29** 0.85 ± 0.12 0.65 ± 0.10 8.05 ± 0.98 9.24 ± 2.09
20 mg/kg
body weight drug)
Group IV
(Burned +
3.41 ± 0.90 0.84 ± 0.01** 23.6 ± 2.7* 13.5 ± 0.59** 1.09 ± 0.22* 0.91 ± 0.06** 8.35 ± 1.59 9.27 ± 1.88
100 mg/kg
body weight drug)
Group V
(Burned +
2.68 ± 0.20 0.80 ± 0.15** 23.6 ± 2.9* 12.1 ± 1.88** 1.11 ± 0.18* 1.10 ± 0.22** 10.91 ± 2.55** 10.03 ± 2.24*
250 mg/kg
body weight drug)

*: p<0.05, **: p<0.01, ***: p<0.001

stress. The level was further increased in drug treated groups Effect of Calendula officinalis extract on hepatic function
(p<0.05). On 10th day when control animals possessed much markers after thermal injury
low level of SOD activity 100 mg and 200 mg/Kg body There was a significant change in the biochemical para-
weight extract treated animals had significantly high SOD meters of serum in the burned animals. Serum ALP activity
activity (p<0.01). Similarly catalase was also found to be was significantly increased in burned animals and was found
enhanced in the extract (200 mg/kg body weight) treated to be significantly lowered by Calendula treatment
animals (p<0.01) (Table 3). (Table 4). Serum GPT activity was significantly increased in

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62 P.K. Chandran et al.

Table 4. Effect of Calendula officinalis on hepatic function of thermally burned animals

Serum ALP KA units SGPT IU/L SGOT IU/L Bilirubin mg/dL
5th day 10th day 5th day 10th day 5th day 10th day 10th day
Group I
14.8 ± 2.2 — 19.5 ± 3.4 — 24.5 ± 3.8 — 0.48 ± 0.15
(Normal)
Group II
(Burned 38.9 ± 8.7*** 56.7 ± 6.8*** 18.2 ± 0.8 27.0 ± 5.1** 46.2 ± 7.2** 108.3 ± 13.6*** 0.53 ± 0.13
untreated
Group III
(Burned +
30.6 ± 3.7 44.5 ± 4.7** 17.2 ± 1.2 25.6 ± 1.9 45.8 ± 3.8 87.5 ± 5.2** 0.58 ± 0.09
20 mg/kg
body weight drug)
Group IV
(Burned +
24.5 ± 5.0** 42.9 ± 2.5** 16.5 ± 0.5* 19.3 ± 5.7* 45.3 ± 10.0 85.0 ± 5.4** 0.53 ± 0.11
100 mg/kg
body weight drug)
Group V
(Burned +
19.3 ± 4.7** 37.3 ± 5.3** 16.3 ± 1.0** 12.6 ± 4.1** 35.7 ± 3.2* 80.8 ± 7.3** 0.34 ± 0.16**
250 mg/kg
body weight drug)
*: p<0.05,**: p<0.01, ***: p<0.001

control animals on 10th day. In the extract treated group it cation of foreign compounds, hydrogen peroxide and free
was significantly (p<0.01) decreased and was almost normal. radicals [22]. There was a significant increase in the
Serum GOT activity was also significantly increased on glutathione content in all the burned animals, which may be
5th day in control group of animals whereas significant through the triggering of the antioxidant system. This level
reduction was found in 200 mg/Kg body weight extract was further increased by the treatment with the Calendula
treated group (p<0.01). On 10th day there was further extract. Similar results were also found with SOD and
increase in serum GOT activity in all the groups. However catalase, which were increased by treatment with the extract.
GOT was significantly low in treated animals (p<0.01). Results indicate the effectiveness of Calendula officinalis
Bilirubin was found to be increased on 10th day of burning extract on enhancing the antioxidant defense mechanism
but reduced significantly in treated group, especially in thereby decreasing the burn injury.
200 mg/kg body weight (p<0.01). The significant increase of both hexosamine and hydroxy-
proline content in the granuloma tissue shows the effective-
Discussion ness of the extract in enhancing the collagen content in
burned tissue. This could be either due to increased synthesis
Healing of burned tissue is a complex process, which or decreased catabolism of collagen due to presence of
involves re-epithelisation, granulation tissue formation and flavonoids in the extract which can produce artificial cross
remodeling of extracellular matrix. There is also experi- linkage between collagen molecules.
mental evidence that indicates the involvement of super- The systemic inflammatory response after trauma leads to
oxide radical in the pathogenesis of burn wound [21]. Earlier protein degradation, catabolism, and hyper metabolism. To
studies have shown that there is a close relationship between restore systemic homeostasis, the liver reprioritizes its
a lipid peroxidative reaction and secondary pathological synthesis from constitutive hepatic proteins toward acute-
changes following thermal injury [13]. It has been known phase proteins. A prolonged increase in the acute-phase
that severe burning not only affects skin but also internal response, however, has been shown to increase morbidity
organs. An attack of biomolecules by ROS can result in an and mortality [4]. The increased level of the acute phase
alteration of the structure of biological membranes of proteins in thermally injured animals were found to be
tissues. A local burn injury produces oxidant-induced organ decreased in Calendula extract treated animals. The
changes as evidenced by increased lipid peroxidation in administration of Calendula officinalis extract significantly
remote organs. The body’s innate mechanism to protect decreased the serum level of marker enzymes of tissue
itself from the deleterious effect of free radicals is anti- damage like ALP, GOT and GPT indicating that Calendula
oxidants. Glutathione plays an important role in the detoxifi- extract reduce the injury of the internal organs during

J. Clin. Biochem. Nutr.

 Effect of Calendula Extract on Thermal Burns 63

thermal burning. antibacterial properties of Calendula officinalis flowers and

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