Evaluation
of Wound Healing, Anti-Inflammatory and Antioxidant Activity of Rhizomes of Curcuma longa
Rita
M Charde1*, Hemant J Dhongade1, Manoj S Charde2
and Siddheshwar B Joshi3
1J.L.Chaturvedi
2Kamla
3Department of Pharmaceutical Sciences,
ABSTRACT:
The present study deals with evaluation of
antioxidant, wound healing and anti-inflammatory activity of ethanolic extract
of Curcuma longa Linn rhizomes. The
ethanolic extract prepared by maceration technique was subjected to screen for
antioxidant activity using DPPH radical scavenging method and wound healing
activity using incision, excision, histopathological and dead space wound model
and the study was supported with evaluation of granuloma tissue to estimate
hydroxyproline content and histopathological evaluation. The anti-inflammatory
study was carried out by using carageenan induced rat paw odema method. The
tested extract of different dilutions in range 200΅g/ml to 1000 ΅g/ml shows
activity in range of 9.34% to 18.55%. Significant increase in wound closure
rate, skin breaking strength, granuloma breaking strength was observed. The
hydroxyproline content was also increased with decrease in scar area. The
initial healing action might be due to increased collagen deposition and better
alignment, with the obtained results it can be concluded that Curcuma longa
extract has significant wound healing activity and initial healing may be due
to presence of curcuminoids and antimicrobial agents. The extract shows
prominent anti-inflammatory activity as compared to that of standard (Ibuprofen
gel). The extract shows good anti-inflammatory activity on carageenan induced
rat paw odema method.
KEY
WORDS: Antioxidant, Wound
healing, Anti-inflammatory, Curcuma longa
INTRODUCTION:
Curcuma longa Linn (Zingiberaceae) is
distributed in
Wounds are visible results of individual cell death or
damage and can be classified by site, size, depth and causation surgery,
accident or circulatory failure15. Wound healing is a process which
is fundamentally a connective tissue response. Initial stage of this process
involves an acute inflammatory phases followed by synthesis of collagen and
other extracellular macromolecules which are latter remoulded to form scars16,17.
Inflammation is the process which may be due to release of histamine, kinins,
serotonins and prostaglandin18. Anti-inflammatory agents are the
agents which normally inhibit the release of these inflammatory mediators.
The present study deals with the use of traditionally
claimed use of Curcuma longa. The in-vitro antioxidant activity was
screened by DPPH method. The wound healing study was evaluated by incision and
excision and dead space wound model followed by histopathological study.
Anti-inflammatory study was screened by carageenan induced paw odema method.
MATERIAL AND METHODS:
Plant material and Preparation
of Herbal Extract:
Curcuma longa rhizomes was purchased from the local market
and authenticated in Department of Botany, Rashtrasant Tukdoji Maharaj Nagpur
University Campus,
The crushed mass were weighed and then placed with
ethanolic solution in a cylinder. 200g of Curcuma
longa rhizomes in 1.0 liter of ethanolic solution, were macerated for 7
days. The mensturm was removed and concentrated by vaccum distillation. Again
the crude material was allowed to undergo maceration for 4 days followed by 2
days for complete extraction of Curcuma
longa rhizomes. The mensturm was collected and concentrated by vaccum
distillation and then air dried in an evaporating dish till constant weight was
obtained. The percent yield of Curcuma
longa rhizomes extract is 17.26%.
Animals:
In the present study male wistar rats (150-200g) were
used for the study. They were individually housed and maintained on normal
standard diet (Gold Muhor Brand, Lipton
Antioxidant activity:
The DPPH (1,1-diphenyl-2-picryl hydrazyl) method was used for the determination of in vitro
antioxidant activity for crude extract. DPPH is stable free radicals scavenge
by antioxidant present in test solution.
To 1.0 mL of 0.01mM Solution of DPPH, 1.0mL of
ethanolic solution and 0.95 mL of tris HCl buffer of 0.05M was added. To this
solution 50΅l of extract of specific strength was added. It was kept for 20 min
for reaction at room temperature and then absorbance was recorded at 517 nm.
Control solution was also carried out without extract. Percent scavenging of
DPPH radical was calculated by comparing absorbance between the test and diluted
control mixture.19
Animals were wounded under light ether anesthesia,
semi-aseptically. The animals were assigned in to three groups, each group
containing six animals. First group was untreated group which was taken as
control (Group1). In Second group (Group-2) wounds received topical application
of Curcuma longa extract, while
animals in Group- 3 (Reference Standard) received treatment of Framycetine
Sulphate Cream (FSC) in both excision and incision wound models. No other topical
or systemic therapy was given to animals during the course of this study. The
experimental protocols were approved by the Institute Animal Ethical Committee.
Hairs were removed from dorsal thoracic central region
of anaesthetized rats. Full thickness from the demarketed area was excised to
produce wound measuring around 300 mm2 as shown in Fig-1. Wound was
cleaned with cotton swab soaked in alcohol. The test extract and FSC cream was
applied on the excised wound once daily for 24 days starting from the first day
of wounding. Wound contraction was measured as percent reduction in wound area21.
FIG. 1: Excision Wound Model
Animals were anaesthetized and paravertebral incisions
(2.5 -3.0 cm long) were made through the entire length of skin. After the
incision was made, the parted skin was kept together and stitched with nylon
thread at 0.5 cm apart with curved needle (No. 11).The Photographic
representation of incision wound model with the stitches on the incised skin
are shown in Fig 2. The sutures were removed on day 8 and healing (tensile)
strength was measured on day 1022.
FIG. 2: Incision Wound Model
Dead space wound model:
Under light ether anesthesia, subcutaneous dead space
wounds were inflicted in the region of axilla and groin, by making a pouch
through a small nick in the skin. Granuloma formation was induced by implanting
either sterile cotton pellets or grass piths. Two sterile cotton pellets
weighing 10 mg (sterilized by autoclaving) were implanted in axilla by
technique of DArcy et al as describe by Turner23, but the granuloma
were removed on 10th day. Thus one animal has had two cotton. The
sutures were mobbed with an alcoholic swab and animals were placed into their
individual cages after recovery from anesthesia.
Physical, mechanical and histopathological changes in
granuloma tissue were studied in this model. Excision of granuloma from the
surrounding tissues were performed on
the 10th post wounding day under light ether anesthesia cotton
pellet granuloma excised from dead space wound were dried overnight at 60°C so
as to obtained a constant dry weight, their weights were noted and expressed as mg/100gm body weight24.
The excised tissue was cut into two approximately equal halves. One half of the
granuloma tissue was used for determination of hydroxyproline content25.
The other part is kept in 10% formalin solution for histopathological studies
to evaluate the effect of extract on collagen formation.
Histopathological study:-
The histopathology study was done to evaluate the
healing promoters like keretenization, epithelization, collagenation, fibrosis
and neovascularisation, which were evidenced to promote wound healing in the
experimental animals. The section from 10 day old regenerated tissue of
incision wound was taken and washed with normal saline solution to study the
healing markers. After fixing the section in 10% formalin solution the tissues
were dehydrated with 90% ethanol, cut into thin sliced section (7mm thick),
stained with haemotoxyline-eosin dye and observed under light microscope for
keretinization, epithelization, fibrosis, collagen formation and
neovascularization26. The interpretation of the results were
numbered from 1 to 5 of which 5 stands for maximum similarity and 1 stands for
least similarity form normal tissue around the wound area in the Curcuma longa treated and untreated
wounds27.
Anti-inflammatory Study:
Statistical difference
between the groups were evaluated using one way analysis of variance
(ANOVA) followed by Turkeys Kramer Multiple comparison test (P < 0.001).
RESULT:
The present study deals with evaluation of antioxidant,
wound healing and anti-inflammatory activity of ethanolic extract of Curcuma longa rhizomes. The method used
to determine the peroxide value or scavenging activity is based on
decomposition or scavenging of DPPH which is a very stable free radical. The
comparative percent peroxide value of Curcuma
longa rhizomes extract is shown in Fig. 3. Percent peroxide value for Curcuma longa extract is in the range of
9.34% to 18.55% for the extract with 200΅g/ml to 1000 ΅g/ml strength extract.
The wound healing study was screened by four models incision, excision, dead
space wound model and histopathological study. The anti-inflammatory study was
performed by carageenan induced rat paw odema method. The acute toxicity study
of ethanolic extract of Curcuma longa
rhizomes do not show any signs of toxicity up to 3g/kg body weight. Since there
was no mortality at higher dose 1/10th of maximum dose of extract
tested for acute toxicity was screened for evaluation of wound healing activity
i.e., 300mg/kg.
FIG. 3: Percent
Peroxide Value for Curcuma longa
extract
In the excision wound study the wounds are treated with
FSC cream and Curcuma longa rhizomes
extract shows complete healing in day 24-25. The results of these groups were
compared with the healing activity of untreated group which took more than 30
days for wound closure and fall of eschar. The results are shown in Table 1.
In incision wound study the tensile strength was
measured on day 10 of regenerated tissues. The tensile strength of wounds
treated with FSC cream and Curcuma longa
rhizomes extract is 396.17±3.55 and 379.00±6.32 respectively was compared with
mean tensile strength ±SEM of untreated group i.e., 281.30±5.82. The
comparative tensile strength is shown in Fig. 4.
FIG. 4: Effect of Curcuma longa on Tensile Strength
In histopathological study some healing markers were
evaluated like keretinization, epithelization, fibrosis, Collagenation and
neovascularization. After the histopathological evaluation of slides the Curcuma longa treated wound shows
prominent promotion of keretinization, epithelization, fibrosis and collagen
formation, the results are compared with untreated group and wounds treated wit
FSC cream was consider as reference standard.
The comparative promotion of these markers are shown with the help of
photomicrographs in Fig. 5 (a), (b) and (c).The promotion of healing markers is
four times more than that of untreated wound. The results are shown in Table 2.
FIG. 5: Histopathology of 10
D Old Regenerated Tissue
Photomicrographs of histology of regenerated tissues
collected from a) Untreated control, b) Curcuma
longa treated wounds, and c) FSC (1% w/w) treated after 10 d.
Maginification si 100 X. K - Keretinization, E - Epithelization, F- Fibrous tissue
and N- Neovascularization
Similarly in dead space wound model there was
significant increase in granuloma breaking strength, dry granuloma weight and
hydroxyproline content of granulation tissues as compared to controls. The
results are shown in Table 4.
The topical anti-inflammatory activity of Curcuma longa extract was carried out.
The percentage protection (inhibition) of odema for Curcuma longa extract and Ibuprofen gel was found to be 47.76
(1hr), 71.49 (2hr), 27.96 (3hr), 56.79 (4hr) and 36.54 (1hr), 64.85 (2hr),
63.83 (3hr), 67.62 (4hr) respectively. The tested extract showed significant
anti-inflammatory activity when the results are compared with untreated group
and decreased in inflammation by Ibuprofen gel was taken as reference standard.
The results are highly significant (p< 0.001) and are shown in Table 3.
DISCUSSION:
Herbal drug for the treatment of chronic diseases or as
raw material from which more or less complex chemical compounds with particular
biological activity are isolated, the deeply held beliefs in the efficiency of
natural products/herbals should be proven by scientific investigation bringing
the tradition and experience in the ancient knowledge with in the realm of
sciences29.
In excision wound healing study from the observed
values it were assumed that Curcuma longa
extract shows better and faster healing as compared to untreated group. During
the initiation of the study day 0 the wound closure with Curcuma longa extract was very slow as compared to untreated and
FSC treated wounds. As the study progress the wound healing efficiency
increases shows complete healing on day 18-21 which is same for group 3 (wounds
treated with FSC cream), But the untreated group took more time to complete
wound closure. The study was carried out till fall of eschar leaving no scar
behind. This shows healing potential of Curcuma
longa extract with better and faster wound closure.
The result obtained for tensile
strength shows higher tensile strength for Curcuma
longa extract treated wounds as compared to wounds of untreated group. The
increase in tensile strength may be due to promotion of collagen formation
which significantly contributing to effective and better wound healing. The Curcuma longa extract also promotes
healing markers as compared to that of control group. In addition to this
increase in granuloma breaking strength, dry granuloma weight and
hydroxyproline content strongly emphasize the positive wound healing potential
of ethanolic extract of Curcuma longa rhizomes. Accordingly hydroxyl proline (marker of
collagen) was significantly increased in treated group as compared to control
and further histopathological studies of granulation tissue, which recorded
increase in collagen content in treated group.
The determination of anti-inflammatory
activity is based on plethysmographic measurement of odema produce by sub
planer injection of carageenan in hind paw of rat. The increase odema in animal
treated standard (Ibuprofen gel) and Curcuma
longa extract were composed with increase in odema of untreated control
animals at constant interval of 1,2,3 and 4hrs. The percentage inhibition of
odema at known interval in treated animals was used for the purpose of
calculating the percent inhibition of odema of control. The present study revealed
that the Curcuma longa extract showed
significant inhibition of odema. The maximum activity showed during 1st
and 2nd hrs, the results are highly significant (p<0.001) as
compared to standard. The anti-inflammatory activity may be due to inhibition
of release of histamine, serotonin and kinins in first hour after injection of
carageenan and also retard the release of prostaglandin and like substances30,
shows anti-inflammatory activity of Curcuma
longa extract.
TABLE- 1: Effect of Topical
Application of Curcuma longa extract
on Excision Wound
|
Post wounding days |
Wounding area (mm2) |
F values |
||
|
Group 1 |
Group 2 |
Group 3 |
||
|
0 |
311.86±3.03 (0) |
311.31±2.96 (0) |
369.50±4.38 (0) |
F(2,15) = 90.16 |
|
3 |
272.51±5.06 (12.62) |
253.11±1.74 (18.29) |
308.61±1.68 (16.47) |
F(2,15) = 75.51 |
|
6 |
212.29±4.86 (27.76) |
261.26±1.80 (31.48) |
245.89±2.04 (33.45) |
F(2,15) = 27.70 |
|
9 |
184.11±4.28 (41.01) |
171.21±2.30 (44.73) |
217.23±4.62 (41.20) |
F(2,15) = 37.52 |
|
12 |
160.72±4.49 (46.54) |
116.14±2.36 (62.51) |
157.87±3.94 (57.27) |
F(2,15) = 45.23 |
|
15 |
135.81±3.76 (56.45) |
62.94±1.579 (79.68) |
129.09±3.79 (65.06) |
F(2,15) = 156.55 |
|
18 |
126.46±3.04 (59.45) |
7.09±0.66 (97.71) |
92.63±2.86 (74.93) |
F(2,15) = 634.28 |
|
21 |
114.44±5.71 (63.30) |
00 (100) |
44.56±3.24 (87.94) |
F(2,15) = 231.44 |
|
24 |
97.26±3.58 (68.81) |
0.00 (100) |
13.85±1.32 (96.25) |
F(2,15) = 570.40 |
|
27 |
66.69±2.57 (78.61) |
0.00 (100) |
0.00 (100) |
F(2,15) = 671.29 |
Values are mean ± SEM of animals (n=6) in each group.
Number in parenthesis indicates percentage of wound contraction. All the values
are significant at p<0.001 as compared to Group 1
TABLE- 2: Histopathological
Examination of Wounds Treated with Curcuma longa extract at the End of 10 Days
|
Parameters |
Group 1 |
Group 2 |
Group 3 |
F values |
|
Keretinization |
0.48±0.03 |
4.1±0.03 |
3.66±1.22 |
F(2,15) = 672.89 |
|
Epithelization |
1.23±0.55 |
4.33±0.10 |
4.16±0.04 |
F(2,15) = 526.22 |
|
Fibrosis |
2.01±0.07 |
3.60±0.20 |
4.20±0.05 |
F(2,15) = 75.917 |
|
Collagenation |
1.85±0.42 |
4.3±0.05 |
4.43±0.05 |
F(2,15) = 766.74 |
|
Neovascularization |
0.50±0.06 |
4.4±0.09 |
4.42±0.12 |
F(2,15) = 520.89 |
Values are reading of mean ± SEM of six animals, Value
5 refers to maximum similarity and 0 refers to least similarity of wound from
normal tissues. All the values are significant at p<0.001. Group 1
Untreated group, Group 2 Treated with Curcuma
longa extract, Group 3 Treated with framycetine sulphate cream
TABLE 3: Topical
Anti-Inflammatory Activity of Curcuma
longa extract
|
Group |
Dose |
Percent inhibition of paw volume at different time
intervals |
|||
|
1hr |
2hr |
3hr |
4hr |
||
|
Standard (Ibuprofen gel) |
300 |
36.54 |
64.85 |
63.83 |
67.62 |
|
Test (Curcuma longa extract) |
300 |
47.76 |
71.49 |
27.96 |
56.74 |
TABLE 4: Effect of Curcuma
longa extract on Dead Space Wound Models
|
Dead Space Wound Models |
|||
|
Parameter Studied |
Granuloma Breaking
Strength (g) |
Dry Granuloma weight (mg %
of B.W.) |
Hydroxyproline content |
|
Control |
278.89±2.603 |
43.08±2.381 |
6.89±0.4604 |
|
Ethanolic extract |
379.85±1.748 |
79.98±1.827 |
10.10±0.692 |
|
F values |
1.925 |
1.096 |
2.082 |
It has been reported that terpenoids posses an ability
to increase the collagen content, which is one of the factor for promotion of
wound healing31. As the title plant is rich in curcuminoids, it may
be responsible for wound healing activity.
The plant extracts also shows antioxidant activity
i.e., free radical scavenging activity which might reduces lipid peroxidation,
may not only prevent or slows down the onset of necrosis but also improve
vascularity32. The experimental plant also shows antimicrobial
activity as reported earlier these are the factors count for wound healing
activity of Curcuma longa rhizomes.
Thus it may be concluded that the rhizomes of Curcuma longa shows significant
antioxidant, wound healing and anti-inflammatory activity. Further studies are
in progress to isolate the bioactive component of plant extract.
ACKNOWLEDGEMENTS:
The authors are thankful to Head, Department of
Pharmaceutical Sciences, Rashtrasant Tukdoji Maharaj,
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Received on 06.10.2009
Accepted on 10.12.2009
© A&V Publication all right reserved
Research J. Pharmacology and
Pharmacodynamics 2(1): Jan. Feb. 2010: 42-47