Evaluation of In vivo Wound Healing Activity of dried leaf Ethanolic extract of Indigofera tinctoria on Albino Wistar Rat

 

P. Manimekalai1*, M. Gurumoorthy1, R. Dhanalakshmi2

1Swamy Vivekanandha College of Pharmacy, Tiruchengode, Namakkal (DT)

2Edayathangudy G.S Pillay College of Pharmacy, Nagapattinam-600117.

*Corresponding Author E-mail: mekalaivel@gmail.com

 

ABSTRACT:

Aim of the present study was to evaluate the wound healing properties of  ethanolic extract of Indigofera tinctoria  on  Albino wistar rat. The preliminary Phytochemical evaluation was carried out by standard methods. Burn was induced in wistar rat divided in to five groups as follow: Normal control Group-I received petroleum jelly. Group II Received Silver sulphadiazine-1%w/w,Group III received  5% extract of Indigofera tinctoria ointment, Group IV received  10% w/w Indigofera tinctoria  ointment and GroupV received 20% w/w Indigofera tinctoria  ointment topically twice daily for 16 days. The topical application of 20% w/w Indigofera tinctoria ointment, shows significant (P<0.001) reduction in the wound breaking strength when compared to III received 5% extract of Indigofera tinctoria ointment but not comparable with Silver sulphadiazine-1%w/w treated group.

 

KEYWORDS: Wound healing activity, Indigofera tinctoria, Hydroxyproline, Epithelialization.

 

 


INTRODUCTION:

Wound is an injury caused external or internal loss of breaking of cellular and anatomic or functional continuity of living tissue [1] Wound healing is a complex phenomenon involving a number of steps, including induction of an acute inflammatory process, regeneration of parenchymal and connective tissue cells, angiogenesis, collagen deposition, granulation tissue formation, epithelialization and wound contraction, Synthesis of extra cellular matrix (ECM) proteins and acquisition of wound strength [2-3] Wounds are generally classified as, wounds without tissue loss (e.g. in surgery), and wounds with tissue loss, such as burn wounds, wounds caused as a result of trauma, abrasions or as secondary events in chronic ailments e.g.: venous stasis, diabetic ulcers or pressure sores and Iatrogenic wounds such as skin graft donor sites and derma abrasions. [4].

 

 

During the formation of new tissue, endothelial cells proliferate and form new blood vessels. (S. Reddy et.al 2002). Several nutrional factors required for wound repair may improve healing time and wound outcome. Vitamin A, vitamin c, vitamin E [5], thiamine (vitamine B1), pantothenic acid (vitamin B5), glucosamine sulfate, chondrotin sulfate, trace elements such as zinc, copper, manganese, silicon is required for cross linking of connective tissue, epithelial, collagen and bone formation, immune function and as a tissue antioxidant for proper wound healing.

 

Wound healing management is complicated also expensive. Currently available morden medicines are showing promising effect with limitation. To overcome the limitation, research needed on alternative medicine. Medicinal plants have been reported to be very beneficial in wound care, promote the rate of wound healing with minimal scar. In Indian traditional system the Indigofera tinctoria leaf extract was used for numerous diseases. The isolated compounds of Indirubin, shows marked inhibition of Lewis lung cancer and walker carcinoma there by suggesting that the plant possess significant anti neoplastic activity [6]

 

The whole plant of Indigofera tinctoria is used in constipation, liver disease, heart palpitation and gout. The juice expressed from the leaves is useful in the treatment of hydrophobia. An extract of the plant is good for epilepsy and neuropathy. The plant possesses anti-toxic property.Amarnath V Bangar et al., (2011) were postulated that investigate anti-diabetic and nephroprotective activity of Indigofera tinctoria leaves, using STZ induced diabetic rats as model for clinical type-1 and type-2 diabetes. Pramodm K.Tyagi et al., (2010) elucidate the anti-inflammatory activity of Ethanolic extract of Indigofera tinctoria leaves (500 & 1000mg/kg). Gunasekaran balamurugan et al., (2009) investigated the anthelmintic activity of Indigofera tinctoria Linn (whole plant) against Pheretima posthuma. Anju Puri et al., (2007) investigated the antidyslipidemic activity of the alcoholic extract from Indigofera tinctori. [7] investigated the Gastro Protective effect of leaf extract of Indigofera tinctoriao on albino wistar rat.

 

The previous research of Indigofera tinctoria on various effects shows the plant leaves has medicinal value. So far no scientific evidence was found during literature survey for that activity. So, the present study was focused on wound healing activity of Indigofera tinctoria leaves ethanolic extract justify its traditional use. Wound healing activity of the leaves was evaluated in different wound models .using Wistar rats.

 

MATERIALS AND METHODS:

Plant collection and authentification:

Fresh leaves of Indigofera tinctoria Linn. was collected during the month of September-December nearby the area of Thanjavur, Tamilnadu, India. India. The plant was authenticated by Prof. P. Jayaraman, Ph.D, National Institute of Herbal Science, Chennai, India. Plant authentification number: PARC/ 2009/ 466.

 

Preparation of extract:

Leaves were dried under shade, pulverized by an electric blender and passed through 40 mesh sieve. It was stored in an air tight container, away from sunlight at room temperature. The coarse powdered leaves were extracted in Soxhlet apparatus for 24h with Ethanol then concentrated and dried under reduced pressure. The extract was weighed and the yield was 15% (w/w). The semisolid mass (black colour) was obtained.

 

Phytochemical analysis:

Preliminary phytochemical screening: The phytochemical examination of the methanoloic extract of Indigofera tinctoria was performed by the standard methods, Phytochemical methods, a guide to modern technique of plant analysis Alkaloids were tested by the method of Dragendroff's test, carbohydrates and glycosides was tested using Borntragers test. Salkowski test was used for the identification of steroids. Proteins and flavonoids are tested by the method of biuret and Shinoda test respectively. [7]

 

Pharmacological evaluation:

Acute oral toxicity studies:

Acute oral toxicity studies was carried out as per OECD -425 up and down method guideline. The animals were fasted overnight prior to the experiment and maintained under standard conditions. The extract was administrated orally in increasing doses and found safe up to a dose of 2000 mg/kg.

 

Preparation of ointment:

Ethanol-free extract was used for ointment preparation. A 5, 10, 20 % w/w Indigofera tinctoria ointment was prepared from the plant extract using a petroleum jelly (PJ) base. The 5, 10, 20 % w/w ointment was prepared by mixing 5, 10, 20 g of Indigofera tinctoria crude extract with 95, 90, 80 g of petroleum jelly [8] for a total of 100 g respectively.

 

Animals:

The animals were divided in to five groups of six rats each as follows: group 1 rats were treated with simple ointment base –Petroleum jelly base (control), group 2 treated with reference standard silver sulfazidine 1% w/w [9] group 3, 4, 5 rats treated with 5, 10 and 20 % w/w of extract ointments respectively.

 

Burn wound model:

Male albino rats (wistar strain) of weight ranging 100-200 g were used for the present study. Burn wounds were inflicted on overnight starved animals under Thiopental sodium (40 mg/ kg, i.p) anesthesia [10]. The shaved area was disinfected with 70% (v/v) ethanol. Burn wounds were created on dorsal part of shaved rats using a metal rod (1.5 cm diameter) heated to 80-85ºC and exposed for 20 s after 24 h, dead tissues were excised using sterile surgical blade.[11]

 

     Heal area

% Wound contraction = --------------- x 100

     Total area

 

Control rats were dressed with simple ointment base alone, while experimental rats were dressed with the 5, 10, 20 % ointment formulated. All the rats were given regular dressing changes at every alternative day. [12.] Wound area was measured by tracing the wound on a millimeter scale graph paper. The percentage of wound healing was calculated of original wound size (500 mm2) for each animal group on pre determined days i.e., 4, 8, 12, 16 days [13] post wounding for final analysis of results. Falling of scar leaving no raw wound behind was taken as end point of complete epithelization and the days required for this was taken as period of epithelization. [14]

Determination of the hydroxyproline content:

On the 16th day, the animals from each group were euthanized using thiopental sodium (40 mg/kg) and used to determine hydroxyproline content. [15] The wound tissue was excised and its weight recorded. The tissue was dried in oven at 60°C for 12 h and the dry weight was again noted. They were hydrolyzed in 6N HCl for 24 h at 110°C in sealed glass tubes. The hydrolysate was neutralized to pH 7.0. The samples were mixed with 1ml of 0.01M CuSO4 followed by the addition of 1ml of 2.5 N NaOH and then 1ml of 6% H2O2. The solution was mixed and shaken occasionally for 5 min. All the tubes were incubated at 80°C for 5min with frequent vigorous shaking. Upon Cooling, 4ml of 3N H2SO4 was added with agitation. Finally, 2ml of 5% p-dimethyl amino benzaldehyde was added. The samples were incubated at 70°C for 16 min, cooled by placing the tubes in water at 20°C, and the absorbance was measured at 500 nm using a digital photo colorimeter (EI Products, India). The amount of hydroxyproline in the samples was calculated using a standard curve prepared with pure L-hydroxyproline at the same time [16]

 

STATISTICAL ANALYSIS:

Results are expressed as mean ± S.D. The difference between experimental groups were compared by one-way Analysis of Variance (ANOVA) followed by Bonferroni’s test. The results were considered statistically significant when P<0.05. statistical analysis done by graph pad prism.

 

RESULT AND DISCUSSION:

Preliminary Phytochemical Screening

Table. 1 Phytochemical investigation of Ethanolic extract of Indigofera tinctoria:

S.no

Phyto constituents

Ethanolic extract

1

Alkaloids

+

2

Carbohydrates

+

3

Flavanoids

+

4

Glycosides

+

5

Proteins

+

6

Saponin

+

7

Steroids

8

Tannin

+

+ Indicates presence - Indicates Absence

 

 

Phytochemical investigation of Ethanolic extract of Indigofera tinctoria showed the presence of alkaloids, flavonoids, tannins, proteins, saponin, carbohydrate and glycosides. These compounds scavenge the free radical and play an important role in the prevention and therapy of diseases. The details of qualitative chemical tests and phyto constituents present in the extracts are shown in Table-I.

 

Acute toxicity study:

Before the study of wound healing activity, preliminary toxicity studies of the tested extracts were carried out. The tested extracts did not cause any mortality when administered up to a dose of 2000 mg/kg body weight orally.

 

 

Figure 1 Effect of Ethanolic extract of Indigofera tinctoria on wound healing site

 

The rate of contraction of wounds was determined by tracing the wound surface on to a transparent graph paper and measuring the surface area in each 4 days interval and healed are calculated by subtracting from the original wound area. On day 4, the wound contraction of standard and extract ointment treated groups was found to be significant (p<0.05) in comparison to simple ointment base treated group on day 16, standard ointment treated wound was completely healed while extract ointment treated group was also almost at complete healing stage.

 


 

Table 2: Effect of ethonolic extract of Indigofera tinctoria leaf on burn wound.

Days

Group I (Petrolum jelly)

Group-II (sulfazidine 1 %w/w)

Group III (5%Indigofera tinctoria ointment)

Group- IV ((10%Indigofera tinctoria ointment))

Group V (20% Indigofera tinctoria ointment))

0

181±0.54 (0%) ns

182±0.663 (0%) ns

182.±1.020 (0%) ns

180±0.583 (0%) ns

181±0.800 (0%) ns

4

175±5.01 (4%) ***

120.±1.600 (34%) ***

128.±1.568 (70%) ***

135±1.356 (25%) ***

137.±5.609 (25%) ***

8

131±3.3(27%) ***

82±2.518 (54%) ***

93±1.949 (49%) ***

92±2.135 (49%) ***

82±1.806 (55%) ***

12

98.±4.41 (46%) ***

55±5.941 (70%) ***

83±3.826 (55%) **

61±2.804 (67%) ***

46±3.878 (75%) ***

16

86.400±6.266 (47%) ***

24.±2.159 (87%) ***

61±4.202 (67%) ***

42±1.249 (77)***

30.±3.992 (84%) ***

The results are expressed as mean ± SEM. For five animals in each group and statistical significance was calculated by one way ANOVA; ns = p > 0.05; *** P< 0.001;** P < 0.01 vs. control; in parenthesis values showing percentage closure of burn wound area.

 

Table 3: Effect of ethonolic extract of Indigofera tinctoria leaf on Hydroxyproline content in burn wound.

Days

Group I (Petrolum jelly)

Group-II (sulfazidine 1%w/w)

Group III (5%Indigofera tinctoria ointment)

Group- IV ((10%Indigofera tinctoria ointment))

Group V (20% Indigofera tinctoria ointment))

16

21.130±0.927

38.986±1.245***

29.090±1.057***

31.508±1.325***

36.828±0.813***

Values are expressed in mean ± S.E.M from five rats. ***p< 0.0001 statistically difference in comparison with control group.

 


Table 2 and figure 1 indicates the studies on the burn wound, which reveals all the five groups showed the decreased wound area day by day. However, on the 16 th post wounding day, control group showed 47 % of healing which may be due to self immunity of the animal, whereas standard (Silver Sulphadiazine) treated animals showed 87 % healing. For the Ethanolic extracts of Indigofera tinctoria of the low medium and high dose exhibited 67 %, 77 % and 84 % wound healing respectively. When compared with the controls, the activity of the extract was found to be highly significant.

 

Determination of the hydroxyproline content in burn wound model:

The Measurement of Hydroxyproline can be used as an index for collagen turnover (Nayak and Pinto Pereira, 2006). Increase in Hydroxyproline content indicates increased collagen synthesis which in turn leads to enhanced wound healing. In our study, the Hydroxyproline content was found to be significantly increased in Test-III (p < 0.0001) as compared with the control group. The content was found to be increased in the Test- III as well, but due to larger variation among animals, the difference was not significant.

 

Table: 4 Effect of ethonolic extract of Indigofera tinctoria leaf on Epithelialization time

S. No

Group

Epithelialization days

1

Group I ( Petrolum jelly)

33.333 ± 0.803

2

Group-II

(sulfazidine 1 %w/w)

16.833 ± 0.601 ***

3

Group III (5% Indigofera tinctoria ointment)

27.167 ± 0.543 ***

4

Group- IV ((10% Indigofera tinctoria ointment))

23.833 ± 0.477 ***

5

Group V (20% Indigofera tinctoria ointment))

16.000 ± 0.477 ***

 

 

The epithelialization time was found to be significantly reduced in test-II, IV, V, (p < 0.001) as depicted in Table 3.The treatments with Extracts incorporated in simple ointment as well as conventional burn-treatment cream were superior to untreated and ointment-treated groups. Finally, the epithelialization time was also found to be shorter in animals treated with simple ointment base containing extracts (Table 1). Moreover, the role of simple ointment base as a suitable vehicle for delivery of Indigofera tinctoria extracts cannot be neglected owing to its adhesive properties. It may therefore be concluded that dried Indigofera tinctoria extract possesses burn-healing properties as depicted by the increment in the Hydroxyproline content, faster wound contraction and shortening of the epithelialization time. Hence, the results support the Ethonolic extracts of indigofera tinctoria to treat skin disorders including burns. Indigofera tinctoria have also been reported that it contain flavonoids which may involve in the mechanisms contributing to enhanced wound healing.

 

CONFLICT OF INTEREST:

None.

 

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Received on 15.10.2018            Modified on 10.11.2018

Accepted on 30.11.2018           © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(2):827-830.

DOI: 10.5958/0974-360X.2019.00143.4