INTRODUCTION:

Wounds are physical injuries that result in an opening or break of the skin. After injury, the objective of wound healing is to restore structure and function to an injured tissue in order to approximate pre wound characteristics. Healing is a complex and intricate process initiated in response to an injury that restores the function and integrity of damaged tissues (Evans 1983). Healing process can be broadly categorized into three stages; Inflammatory (consisting the establishment of homeostasis and inflammation), Proliferative phase (consisting of granulation, Contraction and epithelization) and the Remodeling phase which ultimately determines the strength and appearance of the healed tissue (Kerstein 1997)^.Several natural plant products (Suguna et al., 1999), which are composed of active principles, like triterpenes (Scoritchini and Pia Rossi 1991) and alkaloids and flavanoids and biomolecules have been reported to promote the process of wound healing. Collagen is a protein found in the skin; collagen fibers are woven together in a frame in which new cells grow.

Asoka tree (Saraca indica) (family-Fabaceae) grows throughout India. Bark of the Asoka tree contains tannin, catechin and organic compound of calcium, hemotoxylin and a ketosterol. (Saraca indica) bark most used in Ayurveda. Custard Apple (Annona squamosa) (family-Annonaceae) is a small, semi deciduous and also grows throughout India. This can treat burning sensation; it helps to heal the wound.

The present work includes the experimental induction of excision wounds and estimating the physical parameters like epitheilization period and wound breaking strength (Lee 1968). Further the estimation of biochemical parameters like hydroxyproline (Newman and Logan 1972), hexosamine, uronic acid, and Lowry’s levels.

MATERIALS AND METHODS:

Animals – Wistar strain of female albino rats 6 were taken from departmental house maintained in clean polypropylene cages and fed with standard rodent pellet diet and water ad libitum. Approval from the Institutional Animal Ethical Committee was taken prior to the experimental work.

Plant material- Riped, Custard Apple fruit (Annona squamosa) which has been collected during December from local areas of Chennai, Tamil nadu, India, Asoka tree (Saraca indica) bark has been collected in CLRI campus, Chennai, Tamil nadu, India.

Preparation of Extraction-Custard Apple (Annona squamosa) pulp has been separated from the seed, that pulp was smashed by use of mortar and pestle make it into a paste without adding any distilled water, fresh pulp (crude extract were used) was used in this experiment and Asoka tree bark was collected from the middle of the adult tree which has been air dried at room temperature and powdered coarsely and stored in vacuum disc.

Treatment protocol- The wound healing study was undertaken in excision wound healing model. The healing effect of graded doses of (Annona squamosa) Custard Apple fruit and (Saraca indica) Asoka tree (100mg/kg) were initially studied on excision wound model of rat, when given alternative of the day till the complete epithelization of wound. The effects of (Saraca indica) Asoka tree and (Annona squamosa) Custard Apple fruit were seen on various (a) physical parameters like epithelization period, wound breaking strength, and (b) healing biochemical parameters like hydroxyproline, hexosamine, total protein and uronic acid in the dried granulation tissue.

Experimental procedure –The animals were divided into 3 groups of 6 animals each. Rats were anaesthetized with Di ethylene ether.

Group I: Control group with wound alone;

Group II: Test group with wound and treated with Asoka bark powder;

Group III: Test group with wound and treated with Custard Apple pulp.

Excision wound (Morton and Malone 1972): - A square piece of skin of full thickness (2×2 cm²) was cut off from a predetermined area on the back of the rat. Wounds were traced on 1mm²graph paper on the day of wounding and subsequently at a gap period of 4 days till day 12 and then on the alternate days, until healing was complete. Change in wound area was calculated, giving an indication of the rate of wound contraction. Number of days required for falling of eschar without any residual raw wound indicated the period of epithelization.

% wound contraction = Healed area / total wound area × 100

(Healed area= original wound area- present wound area).

20mg of dried granulation tissue were used for the estimation of hydroxyproline (Woessner 1961), hexosamine (Dische and Borenfreund 1950), total protein, uronic acid (Bitter and Muir 1953) following the standard procedures. And further 40mg of dried granulation tissue were under go for histopathology test. The rats were sacrificed removed fully healed tissue in tensile strength (Clark 1996)to known the strength of the collagen.

RESULTS:

Wound parameters:

Excision wound – Control rats showed a dependant increase in percent wound contraction from 18 to 84.78% from 4^th day to 16^thday while complete wound closure and epithelization was observed on 22^nd day of wound induction compared with day 0 which was taken as 0%(fig.1).Graded dose of both (Saraca indica) Asoka tree and(Annona Squamosa) Custard Apple fruit (100mg/kg)showed dose dependent increase in percent wound contraction up to day 16^th of treatment from 30% to 97.6% Asoka tree (Saraca indica) (fig.1) and 26.32% to 94.72% (Annona Squamosa) Custard Apple fruit (Table-1) respectively, while complete epithelization was observed at 16^th day onwards.

(A) 4^th day

Asoka bark Custard Apple pulp Control

(B) 8^th day

Asoka bark Custard Apple pulp Control

(C) 12^th day

Asoka bark Custard Apple pulp Control

(D) 16^th day

Asoka bark Custard Apple pulp Control

Fig 1 – Photographic representation of contraction rate showing % wound contraction area on Excision wound days of Asoka bark, Custard Apple pulp and Control treated rats: Day 4 (Control-16.4,Asoka bark- 36.3, Custrad Apple pulp- 24.1); Day 8 (Control-40.1, Asoka bark- 63.1, Custrad Apple pulp- 63.5); Day 12 (Control-60.4,Asoka bark-86.0, Custrad Apple pulp- 81.6); Day 16 (Control-77.2, Asoka bark-81.6, Custrad Apple pulp-95.2);

Biochemical parameters:

Dead space wound model - Both Asoka tree (Saraca indica) and (Annona squamosa) Custard Apple fruit caused and increased in dry weight of granulation tissue compared to control. Both the test drugs showed an increase in hydroxyproline, hexosamine, uronic acid, and total protein level per g dried granulation tissue compared to control group indicating better effects on wound healing (Table-2).

Table – 1 Effect of extracts of Asoka bark and Custard Apple pulp on the excision wound parameters.

(Values are mean ± SD of 6 rats)

Treatment	Wound contraction (%)				Ephithelization period (Days)
	4th days	8th days	12th days	16th days	24.2 ± 0.94
Control	16.47±1.47	40.13±3.04	60.48±16.73	77.24± 7.2	24.2 ± 0.94
Asoka bark (100mg/kg)	36.38±4.24^c	63.17±2.73^a	86.00±2.78^a	98.76±0.92^a	18.7 ± 0.53
Custard Apple pulp (100mg/kg)	24.15±2.52^a	63.5±2.88^a	81.65±6.49^b	95.25±1.62^b	21.7 ± 0.50

Statistical analysis was done by one way analysis of variance (ANOVA) followed by Dunnett’s test for multiple comparisions. P values: ^a <0.001, ^b< 0.01,^c< 0.05 are compared to control values of respective dates or values.

Table – 2 Effect of Asoka bark and Custard Apple pulp on dead space wound parameters

(Values are mean ± SD of 6 rats)

Tissue content

Control mg/100mg of tissue

Asoka bark mg/100mg of tissue

Custard Apple mg/100mg of tissue

4^th day

8^th day

12^th

day

16^th day

4^th day

8^th day

12^thday

16^th day

4^th day

8^th day

12^thday

16^th day

Hydroxyproline

105.2

±3.9

215.9

±8.5

279.1

±8.4

307.8

±4.4

253.5

±3.8^a

307.8

± 4.0^c

345.3

±4.9^a

393.09

±3.9^a

232.04±6.7^b

285.8

±11.6^b

330.

±4.0^c

382.7

±3.8^a

Hexosamine

0.14 ±0.005

0.29 ±

0.008

0.33

±0.1

0.38±

0.005

0.65

±0.05^a

0.98± 0.053^a

1.086±0.009^b

1.08

±0.04^a

0.45±

0.03^a

0.70

±0.037^a

0.80

±0.035^a

0.95

±0.03^a

Protein

19.3 ±

0.836

47.6 ±3.98

83.3

±5.35

99.4

±2.5

58.41

±5.56^a

80.9

±3.2^a

126.4

±4.2^b

140.7

±5.7^c

42.5±

10.03^b

67.5

±10.8^b

108.3

±6.4^b

122.4

±8.6^a

Uronic acid

0.09 ±0.01

0.45 ±0.02

0.91

±0.02

1.10

±0.07

0.26

±0.08^c

0.79

±0.03^b

1.17

±0.02^a

1.24±

0.01^a

0.19±

0.01^a

0.68

±0.05^c

0.94

±0.03^c

1.15

±0.05^c

P values: - ^a :< 0.001, ^b :< 0.01,^c :< 0.05 are compared to control values of respective dates or values.

Table – 3 Effect of Asoka bark and Custard Apple pulp on Tensile strength.

SAMPLE	Displacement at Max.Load (mm)	Load at maximum Load(N)	Stress at max.Load (MPa)	% Strain at Max.Load (%)
Control	7.875	7.874	1.516	39.373
Group-1(Asoka bark)	8.739	7.600	1.365	43.695
Group-2(Custard Apple)	8.799	7.198	1.242	43.997

Safety profile – (Saraca indica) Asoka tree and (Annona squamosa) Custard Apple fruit (100mg/kg) did not show any change in spontaneous and voluntary motor activity up to 24 hour; seen at interval of 30 min, 1 hour, 2 hour,4 hour and 24 hour and spontaneous activity till 7days after treatment. No changes in the colour of skin, fur, eyes and mucous membrane and other CNS (behavior, gait, convulsion lethargy, and coma) and ANS (diarrhoea and salivation) parameters were found when observed daily till 21 days after treatment.

The result of tensile strength wound model (tensile strength measurement) is shown in the (Table-3). (Saraca indica) Asoka tree applied rat shows higher tensile strength than (Annona squamosa) Custard Apple fruit and control. Histological observations of the granulation tissue in extract treated group (fig.2) revealed a well developed matrix. Collagen was well organized and formed bundles between the cells. There was better neovascularization in the extract treated group as compared to all other groups. Due to the better formation of ground substance the cells appeared to be spread apart in extract treated group when compared to control group (fig.2).

(A) 4^th day

(i) Control (ii) Asoka bark (iii) Custard Apple pulp

(B) 8^th day

(i) Control (ii) Asoka bark (iii) Custard Apple pulp

(C) 12^th day

(i) Control (ii) Asoka bark (iii) Custard Apple pulp

(D) 16^th day

(i) Control (ii) Asoka bark (iii) Custard Apple pulp

Fig 2 (A-D): Histopathology of old granulation tissue. (i) – Control -Maximum number of fibroblasts, Collagen bundles are indistinguishable. (ii) – Asoka bark treated – well developed blood vessels with well organized collagen bundles. (iii) – Custard Apple pulp – Moderate cell population with some matrix formation. (H and E, 10 X magnification).

DISCUSSION:

Wound represents a major problem; wound healing is a fundamental response to tissue injury that results in restoration of tissue integrity. It mainly depends on the repairing ability of the tissue, type and extent of damage and general state of the health of the tissue (Biswas and Mukherjee 2003). Plant products are potential agents for wound healing and largely preferred because of their widespread availability, non-toxicity, absence of unwanted side effects and their effectiveness as a crude preparations (Ehrlich and Hunt 1969). Collagen is a major protein of the extracellular matrix and is the major component that ultimately contributes to wound strength (Pradhan 2009). Tannins promote the wound healing through several cellular mechanisms¹⁴; chelating of the free radicals and reactive species of oxygen, promoting contraction of the wound and increasing the formation of capillary vessels and fibroblasts (Deters et al., 2001). (Annona squamosa) Custard Apple fruit having astringent property but (Saraca indica) Asoka tree having tannin property. Tannin heals the wound faster than astringent. Similar findings have been reported with the extracts of the plants containing tannins by earlier workers (Rane and Mengi 2003). The result of this study also revealed that tannins are one of the important phytoconstituents responsible for wound healing.

CONCLUSION:

Thus, the results of present study it was concluded that the wounds treated with (Saraca indica) Asoka tree bark powder and (Annona squamosa) Custard Apple fruit pulp exhibited better healing capacity than the control rats. Enhanced wound contraction, increased biochemical parameters and histological findings were observed in the (Saraca indica) Asoka tree bark powder treated Albino rats than (Annona squamosa) Custard Apple fruit pulp. This confirms the use of (Saraca indica) Asoka tree bark powder in management of wound healing.

ACKNOWLEDGEMENT:

The authors are thankful to Dr. Asit Baran Mandal, Director of CLRI to give this opportunity in CLRI Chennai, Tamilnadu, India.

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Received on 10.02.2011 Modified on 16.03.2011

Research J. Pharm. and Tech. 4(6): June 2011; Page 928-931