Comparative Efficacy of <i>Tagetes erecta</i> and <i>Centella asiatica</i> Extracts on Wound Healing in Albino Rats

doi:10.4236/cm.2011.24023

Paper Menu >>

Journal Menu >>

Chinese Medicine, 2011, 2, 138-142

doi:10.4236/cm.2011.24023 Published Online December 2011 (http://www.SciRP.org/journal/cm)

Comparative Efficacy of Tagetes erecta and Centella

asiatica Extracts on Wound Healing in Albino Rats

Sandipan Chatterjee1, Tigari Prakash1*, Dupadahalli Kotrsha2, Nadendla Rama Rao3, Divakar Goli1

1Department of Phar macology, Acharya & B M Reddy College of Pharmacy, Bangalore, India

2Department of Parasi t ology, Faculty of Medicine, University of Malaysia, Kuala Lumpur, Malaysia

3Department of Pharmaceutical Chemistry, Chalapathi Institute of Pharmaceutical Science,

Guntur, India

E-mail: [email protected]

Received July 20, 2011; revised August 22, 2011; accepted August 29, 2011

Abstract

The study was undertaken to evaluate the comparative efficacy of hydroalcoholic extracts of leaves of Ta-

getes erecta (T. erecta) and aerial parts of Centella asiatica (C. asiatica) on Excision, Incision and Dead

space wound models in albino rats. Extract of T. erecta and C. asiatica (P < 0.001) showed significant in-

crease in rate of wound contraction, epithelization and formation of scar faster in excision wound model

compare to control group. T. erecta extract (250 and 500 mg/kg) showed significantly increased the wound

breaking strength in incision wound model and wet and dry granulation tissue weights, breaking strength in a

dead space wound model compare to control and C. asiatica treated group (P < 0.001). In this investigation

the work is conclude that the comparison made between the above two plants, the T. erecta extract showed

potent wound healing activity then the reported C. asiatica in different wound parameters.

Keywords: Centella asiatica, Tagetes erecta, Excision, Incision, Dead Space Wound Model

1. Introduction

Wound healing or wound repair, is the body’s natural pr-

ocess. Have regenerating dermal and epidermal tissues.

When an individual is wounded, a set of events takes

place in a predictable fashion to repair the damages. Wo-

unds were probably the first medical problem faced by

human race. The field of pharmacologic modulation in

wound healing is still in its infancy. Indians were acqu-

ainted with a far larger number of medicinal plants than

the natives of other country. In fact ancient Indian sur-

geon sushruta has enumerated large number of herbal dr-

ug in the management of wounds singly or in combina-

tion. Some Ayurvedic medicinal plants, namely, Centella

asiatica, Curcuma longa, Aloe vera, and Tagetes erecta

Linn, were found to be effective on wound healing in ex-

perimental models.

Tagetes erecta (T. erecta) is commonly known as Ma-

rigold. The plant is widely distributed throughout India,

Asia, Central Europe, USA and Africa. The leaves are

reported to be effective against piles, kidney troubles,

muscular pain, ulcers, wounds and earache. The pounded

leaves are used as an external application to boils and

carbuncles. The steam distillation of fresh leaves offer

0.3% of essential oil with a strong, sweet lasting odor

and contains d-limonene, ocimene, l-linalyl acetate, l-li-

nalool tagetone, n-nonyl aldehyde, lutein [1].

Centella asiatica (C. asiatica) is commonly known as

Indian pennywort. The plant is widely distributed in Asia,

Africa, and North and South America [2]. This plant is

reportedly useful in wound healing and protecting ag-

ainst ulcer formation and is reported to have antimicro-

bial properties. Extracts of the plant have been shown to

accelerate wound healing through topical and systemic

routes [3]. The extracts of C. asiatica plant are reported

to have sedative, antidepressant, analgesic, and anticon-

vulsive effects [4]. It is also reputed within the Indian

system of medicine for its use in treating chronic and ob-

stinate eczema, psoriasis, syphilis and leprosy [5]. The

titrated extract of C. asiatica contains three principal

ingredients like asiaticoside, asiaticacid, and madecassic

acid. All these ingredients are known to be clinically

effective in the treatment of systemic scleroderma, ab-

normal scar formation [6] and wound-healing activity [7,

8]. The present study was planned to evaluate the com-

parative efficacy of the hydroalcoholic extract of aerial

139

S. CHATTERJEE ET AL.

parts of leaves of T. erecta and C. asiatica on different

wound models.

2. Materials and Methods

2.1. Plant Materials

Leaves of T. erecta and arial part of C. asiatica was col-

lected during the month of November from the local ar-

eas of Burdwan District, West Bengal, India and both the

plants were authenticated by Professor Udayan Sarkar,

HOD, Dept of Botany, Sonamukhi College, Bankura, In-

dia. A voucher specimen (Reference no-SC341/06) of

plants has been deposited in the department.

2.2. Preparation of Hydro Alcoholic Extracts

The plants material was cleaned in water and air dried,

powder was made using an electric mixer. The powder

(75 g) was packed into a Soxhlet apparatus and was ex-

tracted with 500 ml of hydro alcoholic solvent (350 ml

alcohol-95% and 150 ml water) at 65˚C to 70˚C for 24 h.

The extracts were dried under vacuum and weights of the

dried mass were recorded (Yield: C. asiatica-10% and T.

erecta-15%).

2.3. Animals

Healthy Wistar albino rats of either sex weighing be-

tween 180 - 250 g were used for the wound healing study

and albino mice weighing between 25 - 30 g were used

for the oral acute toxicity studies. The animals were

housed individually in Poly propylene cages and main-

tained at 24˚C ± 2˚C, a 12 h light/dark cycle fed with

standard pellet rat chow (Pranav agro industries Ltd.

Sangli, India) and water ad libitum. The experimental

protocol and procedures used in this study were ap-

proved by the Institutional Animal Ethics Committee

(Protocol no: IAEC /PP/06/07).

2.4. Toxicity Studies

The female albino mice weighing 20 - 25 g were used for

the study. The acute oral toxicity studies of hydro alco-

holic extracts of T. erecta and C. asiatica was carried out

by using fixed-dose method according to OECD [9]

guideline No. 401. Animals were kept fasting overnight

prior to the experimentation.

2.5. Surgical Procedures and Treatment

2.5.1. Excision Wound Model

In the present experimental model two different types of

creams were formulated of each extract (5% and 10%

w/w of T. erecta and the other having 5% and 10% w/w

of C. asia tica) and the wounds were medicated with one

local application (5 - 10 mg) of each cream for every day

until the day of epithelization and their excision wound

healing effect on rats was observed. The animals were

randomly allocated into five groups of six animals each.

Group 1: Wounds untreated served as control; Group 2:

Wounds treated with cream having T. erecta (5% w/w);

Group 3: Wounds treated with cream having C.asiatica

(5% w/w); Group 4: Wounds treated with cream having

T. erecta (10% w/w); and Group 5: Wounds treated with

cream having C. asiati ca (10% w/w).

This surgical procedure was performed according to

Panchatcharam. The wounding procedures were carried

out under anesthetized condition by thiopentone sodium

(40 mg/kg, ip), the backs of animals were shaved and

sterilized with 70% ethyl alcohol. Excision wounds sized

2.5 cm diameter in average were made after leaving at

least 5 mm space from the ears and treated topically

every day until the day of epithelization (Figure 1). The

physical attributes of wound healing viz wound closure

Excision wound Incision wound Dead space wound

Figure 1. Different surgical w ound models on r ats.

S. CHATTERJEE ET AL.

140

(contraction), epithelization and scar features were recor-

ded. The wound contraction was studied by traced 1 mm2

graph paper on the day of wounding and subsequently on

the alternate days, until healing was complete and calculated

as percentage reduction of initial wound area [10].

2.5.2. I n cision Wound Model

The animals were randomly allocated into five groups of

six animals each. The extracts were dissolved in water

and administered orally, daily for 10 days. Group 1: was

assigned as vehicle control group and received no treat-

ment; Group 2: received extract of T. erecta 250 mg/kg;

Group 3: received extract of C. asiatica 250 mg/kg;

Group 4: received extract of T. erecta 500 mg/kg; Group

5: received extract of C. asiatica 500 mg/kg, orally.

Under light ether anesthesia, the animals were secured

to operation table in its natural position. Two Para verte-

bral straight incision of 6 cm each was made through the

entire thickness of the skin, on either side of the vertebral

column of the rat with help of sharp blade [11]. Wounds

were closed with interrupted sutures, 1 cm apart and

showed in Figure 1. The sutures were removed on the

8th day. The tensile strength of wound was determined

on the 10th post wounding day by continuous constant

water flow technique [12].

2.5.3. Dead Spac e Wound Model

In the case of dead space wound model the animals were

randomly divided into five groups of six animals each.

The extracts were dissolved in water and administered

orally, daily for 10 days. Group 1: was assigned as con-

trol group and received no treatment; Group 2: received

extract of T. erecta 250 mg/kg; Group 3: received extract

of C. asia tica 250 mg/kg; Group 4: received extract of T.

erecta 500 mg/kg; Group 5: received extract of C. asiat-

ica 500 mg/kg, orally.

Physical and mechanical changes in the granulation

changes in the granuloma tissues were studies in this

model. These wounds were created by implanting two

polypropylene tubes (0.5 × 2.5 cm each), one on either

side in the lumber region on the dorsal surface of each rat

and it showed in Figure 1. The wounds were sutured and

mopped with cotton swabs in 70% alcohol. Animals were

placed independently in cages after recovery from anes-

thesia. On the 10th post wounding day, the granulation

tissue formed on the implanted tubes was carefully dis-

sected out under anesthetic condition. The wet weight of

granulation tissue was noted. The breaking strength of

granulation tissue was measured by the method of Lee.

Later, these granulation tissues were collected, dried at

60˚C for 24 h. The weight of the dried granulation tissue

was noted and expressed as mg/100g body weight.

2.6. Statistical Analysis

The data are represented as mean ± S.E.M, and statistical

significance between treated and control groups was

analyzed by using one way ANOVA followed by Tukey

multiple comparison test using Graph Pad Instate soft-

ware (GPIS) (Version 1.13). P < 0.05 was considered

statistically significant.

3. Results

The acute toxicity studies of the hydroalcoholic extract

of leaves of T. erecta and aerial part of C. asiatica were

found to be non-lethal up to dose of 2 g/kg body weight.

Hence 1/8th and 1/4th of 2 g/kg dose (i.e. 250 and 500

mg/kg, orally) was selected for incision and dead space

wound model. In the excision wound model, the T.

erecta extract treated animals showed a significant in-

creased percentage of wound contraction, decreased in

the epithelization period and formation of scar, compare

to C. asiatica extract treated animals. Data are presented

in Table 1 and Figure 2. In the incision wound model, a

significant difference (P < 0.001) was observed with the

treatment of C. asiatica and T. erecta in wound breaking

strength group has compared to control group. Results

are represented in Table 2. The effect of C. asiatica (500

mg/k g ) and T. erecta extract (250 & 500 mg/kg) signifi-

cantly increased the granulation tissue weight (wet and

dry) and granulation breaking strength (P < 0.001) in the

dead space wound model. By comparison, a significant

decrease was observed in the T. erecta 250 mg/kg (P <

0.05) and non significant in C. asiatica 250 mg/kg

treated group, data are presented in Table 2.

4. Discussion

The objective in wound management is to heal the wound in

the shortest time possible, with minimal pain, discomfort,

100

120

812 16

Da y s

Control C. asiatica 5%C . a sia tic a 10

T. erecta 5% T . erecta 10%

Figure 2. Effect of hydroalcoholic extracts of Centella asiat-

ica and Tagetes erecta for percentage of wound contraction

(in days), in excision wound model.

141

S. CHATTERJEE ET AL.

Table 1. Effect of hydroalcoholic extracts of Centella asiatica and Tagetes erecta for percentage of wound contract ion (in days),

epithelization (in days) and formation of scar in exci sion wound model.

% Wound contraction in days

Treatment

4 8 12 16

Period of

epithelization

( in days)

Mean size

of scar area

(in mm2)

Control 36.29 ± 1.52 54.59 ± 1.90 83.94 ± 1.40 90.41 ± 0.80 21.80 ± 0.65 55.50 ± 1.78

C. asiatica

5% (w/w) 47.95 ± 2.06** 67.80 ± 1.40** 88.28 ± 0.70* 96.77 ± 0.64*** 19.33 ± 0.76 48.33 ± 2.20

T. erecta

5% (w/w) 49.74 ± 1.49** 71.45 ± 2.30** 92.79 ± 0.74*** 98.82 ± 0.39*** 18.00 ± 0.51* 44.66 ± 2.50*

C. asiatica

10% (w/w) 55.11 ± 2.30*** 76.50 ± 2.22*** 94.12 ± 1.16*** 99.48 ± 0.21*** 17.66 ± 0.71* 43.33 ± 2.54*

T. erecta

10% (w/w) 59.59 ± 3.08*** 83.73 ± 4.01*** 99.72 ± 0.12*** 100.00 ± 0.0*** 14.16 ± 0.75*** 29.00 ± 2.80***

The value are expressed as mean ± SEM, (n = 6). If *P < 0.05, **P < 0.01, ***P < 0.001 vs. control.

Table 2. Effect of hydroalcoholic ext ract of Centella asiatica and Tagetes erecta in incision and dead space wound models.

Incision wound Dead space wound

Treatment Breaking Strength, (g) Wet tissue weight mg/100 g ratDry tissue weight mg/100 g rat Braking Strength, (g)

Vehicle control 259.20 ± 7.45 152.30 ± 4.20 14.20 ± 1.04 256.10 ± 9.14

C. asiatica 250 mg/kg 281.20 ± 9.20* 175.60 ± 5.00 21.20 ± 0.99 280.60 ± 9.50

T. erecta 250 mg/kg 300.70 ± 13.60** 185.30 ± 4.20 22.28 ± 2.20* 297.10 ± 12.90

C. asiatica 500 mg/kg 324.50 ± 11.30*** 193.50 ± 5.30** 25.27 ± 2.24** 331.70 ± 14.70***

T. erecta 500 mg/kg 351.40 ± 11.07*** 214.50 ± 8.30*** 27.21 ± 2.30*** 353.60 ± 5.90***

The value are expressed as mean ± SEM, (n = 6). If *P < 0.05, **P < 0.01, ***P < 0.001 vs. Vehicle control.

and scarring to the patient. At the site of wound closure a

flexible and fine scar with high tensile strength is desired

[13,14]. The healing process begins with tissue reposi-

tion by cell proliferation presented in connective tissue.

The main cells that trigger the wound healing process are

the macrophage cells that remove foreign bodies and

direct granular tissue development. Sequentially fibro-

blast and endothelial cells migrate towards the injured

area increasing tissue permeability and collagen fiber

production and it decreases vascularization by a contrac-

tion process by the scar process [15,16].

The ability of the wound is to heal is a biological

process, which follows a definite pattern of cellular and

molecular events, ultimately leading to complete repair

of the injured tissues. High order animal’s posses very

limited regenerative capabilities except for those of some

organs e.g. liver, parenchyma and outer surface of the

skin. The healing rate differs among species and tissues

and even between sites of the same tissue. However, in

general the pattern of wound healing in domestic rumi-

nants appears to be similar. The initial events of wound

healing mechanism occurred smoothly under favorable

condition. The normal process of wound healing may be

disrupted. During this condition before using drugs,

many herbal preparations are extensively used in the

rural area [17].

The result suggest that treatment with hydroalcoholic

extract of C. asiatica and T. erecta have beneficial in-

fluence various phase of wound healing fibroplasias, col-

lagen synthesis and wound contraction result in faster

healing.

In the present study the significant increase in the

breaking strength was observed in the animals treated

with extract of T. erecta when compared to extract of C.

asiatica on 10th post wound healing day.

In addition to excision and incision wound model,

dead space wound model also revealed a significant in-

crease in the dry granulation tissue weight indicating hi-

gher protein content and significant in breaking strength

of granulation tissue both resutured incision wound was

observed in the hydroalcoholic extract of T. erecta

treated group compare to C. asiatica treated group.

The observation and result obtained in this study indi-

cated that crude extract of T. erecta significantly stimu-

lated wound contraction, breaking strength of the inci-

sion wound and increased in the dry granulation weight

in the treated group compared with extract of C. asiatica.

In this study one more attempt has been made to inclu-

S. CHATTERJEE ET AL.

142

sion T. erecta plant in the management of wound healing

in folk medicine is justifiable.

5. Conclusions

In the present work it can be concluded that the com-

parison made between the above plants of hydroalcoholic

extract of T. erecta showed promising wound healing

activity then the reported C. asiatica in different wound

parameters.

6. Acknowledgements

The authors are grateful to Premnath Reddy, Chairman

Acharya Institute, and Principal, Acharya & B. M. Re-

ddy College of Pharmacy, Bangalore, India, for the con-

stant support and facilities to this study.

7. References

[1] T. Ghosh, A. Bose, G. K. Dash and T. K. Maity, “Wound

Healing Activity of Tagetes erecta Linn Leaves,” 2004.

URL http://www. Pharmainfo.net/exclusive/reviews

[2] B. Brinkhaus, “Centella asiatica in Traditional and Mod-

ern Phytomedicine―a Pharmacological and Clinical Pro-

file, Part I: Botany Chemistry Preparations,” Perfusion,

Vol. 11, 1998, pp. 466-474.

[3] B. Brinkhaus, “Centella asiatica in Traditional and Mod-

ern Phytomedicine―a Pharmacological and Clinical Pro-

file, Part II: Botany Chemistry Preparations,” Perfusion,

Vol. 11, 1998, pp. 508-520.

[4] R. P. Lubadie, “An Ehnopharmacognostic Approach to

the Search for Immunomodulators of Plant Origin,”

Planta Medica, Vol. 55, 1989, pp. 339-348.

doi:10.1055/s-2006-962024

[5] S. Chaudhary, S. Poddar, S. Sarkar and P. K. Das, “New

Multidrug Regimen with Indigenous Drugs and Dapsone

in the Treatment of Lepromatous Leprosy,” Indian Jour-

nal of Dermatology, Vol. 32, No. 3, 1987, pp. 63-67.

[6] S. S. Hong, J. K. Kim, H. Li and C. K. Shim, “Advanced

Formulation and Pharmacological Activity of Hydrogel

of the Titrated Extract of Centella asiatica,” Archives of

Pharmacal Research, Vol. 28, No. 4, 2005, pp. 502-508.

doi:10.1007/BF02977683

[7] A. Shukla, A. M. Rasik, G. K. Jain, R. Shankar, D. K.

Kulashrestha and B. N. Dhawan, “In vitro Wound Heal-

ing Activity of Asiaticoside Isolated from Centella asiat-

ica,” Journal of Ethnopharmacology, Vol. 65, No. 1,

1999, pp. 1-11. doi:10.1016/S0378-8741(98)00141-X

[8] S. Somashekar, S. L. Udupa and A. L. Udupa, “Effect of

Centella asiatica Linn (Umbelliferae) on Normal and

Dexmetasone Suppressed Wound Healing in Wistar Al-

bino Rats,” The International Journal of Lower Extremity

Wounds, Vol. 5, 2006, p. 136.

[9] Organization for Economic cooperation and development

(OECD), “OECD Guidelines for Testing of Chemicals

Acute Oral Toxicity,” OECD, Paris, 1993. p. 401.

[10] J. J. P. Morton and M. H. Malone, “Evaluation of Vul-

nary Activity by an Open Wound Procedure in Rats,”

Archives Internationales de Pharmacodynamie, Vol. 196,

1997, pp. 117-126.

[11] H. P. Ehrlich and T. K. Hunt, “Effect of Cortisone and

Anabolic Steroids on the Tensilestrength of Healing

Wounds,” British Journal of Surgery, Vol. 170, 1969, pp.

203-206.

[12] K. H. Lee, “Studies on the Mechanism of Action of Sali-

cylates II. Retardation of Wound Healing by Aspirin,”

Journal of Pharmaceutical Sciences, Vol. 57, No. 6, 1968,

pp. 1042-1043. doi:10.1002/jps.2600570633

[13] B. S. Reddy, R. K. K. Reddy, V. G. M. Naidu, K. Mad-

husudhana, S. B. Agwane, S. Ramakrishna and P. V. Di-

wan, “Evaluation of Antimicrobial, Antioxidant and

Wound Healing Potentials of Holoptelea integrifolia,”

Journal of Ethnopharmacology, Vol. 115, No. 2, 2008,

pp. 249-256. doi:10.1016/j.jep.2007.09.031

[14] I. Süntar, U. Koca, H. Keleş and E. K. Akko, “Wound

Healing Activity of Rubus sanctus Schreber (Rosaceae):

Preclinical Study in Animal Models,” Evidence-Based

Complementary and Alternative Medicine, Vol. 2011,

Article ID: 816156.

[15] P. J. Michelle, M. Cristiana, T. G. R. Ana Lu´ cia, T. F.

Alik, A. F. R. Rodney, M. O. S. Ilza, A. F. Mary and E. C.

Jo˜ao, “Evaluation of Wound Healing Properties of Ar-

rabidaea chica Verlot Extract,” Journal of Ethnophar-

macology, Vol. 118, No. 3, 2008, pp. 361-366.

doi:10.1016/j.jep.2008.04.024

[16] S. K. Prasad, R. Kumar, D. K. Patel and S. Hemalatha,

“Wound Healing Activity of Withania Coagulans in Str-

eptozotocin-Induced Diabetic Rats,” Pharmaceutical Bi-

ology, Vol. 48, No, 12, 2010, pp. 1397-1404.

doi:10.3109/13880209.2010.486837

[17] M. M. Alam, S. A. Islam, Y. Mohammed, N. S. Juyena

and M. A. Hashim, “Comparative Efficacy of Two Me-

dicinal Plant Extracts and an Antibiotic on Wound Heal-

ing,” Pakistan Journal of Biological Sciences, Vol. 8, No.

5, 2005, pp. 740-743. doi:10.3923/pjbs.2005.740.743