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V. Asha Jyothi* et al. International Journal Of Pharmacy & Technology

IJPT | Nov-2013 | Vol. 5 | Issue No.3 | 5879-5891 Page 5879

ISSN: 0975-766X

CODEN: IJPTFI Available Online through Research Article

www.ijptonline.com PHYTOCHEMICAL EVALUATION & PHARMACOLOGICAL SCREENING OF WOUND

HEALING & ANTIOXIDANT ACTIVITY OF PLUMBAGO ZEYLANICA

V. Asha Jyothi*& Butool Fathima

Shadan Womens College of Pharmacy, Khiratabad, Hyderabad & Department of Pharmacology

E-mail: [email protected] Received on 18-11-2013 Accepted on 29-11-2013

Abstract:

Traditional medicine in India with thousands of flora in it has not yet been explored. Many such medicines need

scientific evaluation and evidence based research to be conducted on those plants for them to become medicines of great

potential, one of such plants is Plumbago zeylanica. A wound is defined as a break in the epithelial integrity of skin.

Wound is defined simply as the disruption of the cellular and anatomic continuity of a tissue that may be produced by

physical, chemical, thermal, microbial or immunological insult to the tissue. In the present study the wound healing

activity of the plumbago zeylanica is studied in the incision woung model on Sprague dwaley rats. The tensile strength

of the wound was measured by the tensiometer. The results in comparison of untreated sham operated group with the

standard and the test the asymptomatic significance is displayed and the significance level is p<0.05.

Key words: Wound healing, Skin, Plumbago zeylanica.

Introduction:

A wound is defined as a break in the epithelial integrity of skin. Wound is defined simply as the disruption of the cellular

and anatomic continuity of a tissue that may be produced by physical, chemical, thermal, microbial or immunological

insult to the tissue (Robson et al., 2001; Raina et al., 2008).Wound healing process begins with the restoration of a

damaged tissue as closely as possible to its natural state and wound contraction is the course of shrinkage in wounded

area. The healing primarily depends on the repairing ability of the tissue in addition to the type and degree of damage and

general health status of the tissue (Robbins and Cotran, Pathologic basis of disease, 8th edition, 2009).



Materials:

Plant: Plumbago zeylanica plant was collected commercially from cultivator. The plant was chopped and dried in shade.

It was the subjected to size reduction by mechanical grinder. The powdered material was subjected to soxhlet extraction

with ethanol; the extract obtained was dried and used for preclinical studies.

Chemicals: All the chemicals: Ethanol (China-Changshu Yangyuan Chemical), Ketamine (Sigma Aldrich), Xylazine

(Sigma Aldrich), Atropine sulphate (Sigma Aldrich), Neosporin (Glaxo Smith Kline) , Clobeta GM cream- Laborate

(Clobetasol propionate, Gentamicin, Miconazole nitrate & Zinc oxide with borax cream) used for the study were of

analytical grade.

Animals: Twenty four (24) albino Male Sprague Dawley rats weighing between 120-200 gms were used for the study.

They were kept in polypropylene cages and allowed to acclimatize to the environment for two weeks before the

commencement of the experiment. The animals were fed with standard animal pellet grower mash and allowed access to

water ad libitum.

Methodology:

Incision Wound Model:

Adult male Wistar or Sprague-Dawley strain weighing 120-200g were used. They were divided into four groups of six

each. After fasting overnight, rats were anaesthesized with Ketamine (70mg/kg, IP) or Xylazine (5mg/kg, IP) and

Atropine sulphate (0.2 ml, SC). Under anaesthesia the dorsal skin was shaved using electronic clipper. A 2cm long

incision wound was made through the skin and cutaneous muscles in the dorso-lumbar region with the help of sharp

scalpel (Ehrlich and Hunt, 1969). After the incision was made, the parted skin was kept together and stitched with black

silk at 0.5 cm intervals; surgical threads (No.2) and a curved needle (No.11) were used for stitching. The wounds were

left undressed (Lee K.H., 1968). Extracts were topically applied to the wound once a day. The animals were placed in

individual cages for recovery with free access to food and water. The experimental room was maintained between 25°C

and 28°C with natural light and dark cycles. When wounds were cured thoroughly the sutures were removed on the day

before the tensile strength was tested i.e. on 8th day and formulations were continued to be applied regularly. For the

measurement of wound tensile strength, the rats were anaesthetised on days 9th after surgery. Regular observation of



healing patterns are to be done from day 1-10 and parameters to measure wound healing are tensile strength, percentage

wound contraction and period of epithelialisation.

Measurement of Tensile strength:

Two stands were arranged facing opposite to each other. Thread was attached to both the stands. Stand L was kept

stationery. Artery forceps was attached to stand L. Stand R was allowed to move and artery forceps was attached to it

with the help of thread. On the other side of the thread a cup was hanged in which weights were placed accordingly. The

arrangement was setup as shown in Fig

Incision Wound Model

Adult male Sprague-Dawley strain rats weighing 120–200 gm. They are divided into four groups of six each. After

fasting overnight. Rats are anaesthesized with ketamine (70mg/kg, IP) or xylazine(5mg/kg, IM) and atropine (0.2 ml,

SC).

Under anesthesia the dorsal skin is shaved using electronic clipper. A 2cm long incision wound is made through the skin

and cutaneous muscles in the dorso-lumbar region with the help of sharp scalpel. After the incision was made, the parted

skin was kept together and stitched with black silk at 0.5 cm intervals; surgical threads (No.2) and a curved needle

(No.11) were used for stitching. The wounds are treated according to specific group. Then rats are treated with

formulations beginning on the day of surgery (day 0) till day 10. during this time the healing pattern of the wounds is

noticed.

The groups were divided into:

Fig-1: Incision wound model for the wound healing activity.



a. Group 1: Blank the animals with no wound and no vehicle.

Fig-2: Healthy animal

b. Group 2: Control group the animals with incision wound and liquid paraffin.

Fig-3: Untreated animal

c. Group 3: Standard group the animals with incision wound and standard drug (Neosporin in Clobeta

GM cream).

Fig-4: Treated animal

d. Group 4: Test group animals with incision wound and ethanolic extract of Plumbago zeylanica.


IJPT | Nov-2013 | Vol. 5 | Issue No.3 | 5879-5891 883

Fig 5: Extract treated animal

The animals are carefully observed from day 0. They are placed in individual cages for recovery and should have free

access to food and water. The experimental room is maintained between 25°C and 28°C with natural light and dark

cycles. When wound were cured thoroughly the sutures were removed on the day before the tensile strength is tested i.e.

on 8th day and formulations were continued to be applied regularly. For the measurement of wound tensile strength, the

rats are anesthetised on days 9 after surgery. Regular observation of healing patterns was done from day 1-10 and

parameters to measure wound healing are tensile strength, percentage wound contraction and period of epithelialisation.

Measurement of Tensile Strength:

Both the artery forceps were clipped to the either sides of the incision wound. Weights were added in the cup until wound

dehiscence takes place. These weights determine the wound breaking strength.

Fig 6: Dehiscence of wound during the tensile strength measurement using tension meter.



Results:

Yield Calculation

Calculate the % dry weight as follows:

%�� ℎ� =��.��

��.�� ×100

% yield of the extract was found to be 96.4%

Phytochemical Evaluation:

The preliminary phytochemical investigation of extract of Plumbago zeylanica shows presence of Carbohydrate, Tannin,

Alkaloid, Saponins and Flavonoids in ethanolic extract. The presence of Carbohydrates was confirmed by Molisch’s test,

Fehling’s test & Barfoed’s test .While, Alkaloids by Mayer’s test, Wagner’s test, & Dragendroff’s test .The presence of

Flavonoid was confirmed by Shinoda test & Lead acetate test. While of Glycoside by Borntrager’s test; Phenolics &

Tannins by Bromine water test; Saponins by Foam test (reported by Khandelwal). As, ethanolic extract shows presence

of most of these compounds, its extract were selected for these study.

Table-1: Phytochemical Investigation of Extract of Plumbago zeylanica.

+ = slightly present; ++ = moderately present; +++ =highly present; - = absent

S.NO

.

NAME OF

COMPOUNDS

ETHANOLIC EXTRACT

OF PLUMBAGO

ZEYLANICA

1. Alkaloids ++

2. Carbohydrates +++

3. Glycosides +

4. Saponins +

5. Fat and Oils +

6. Flavonoids +++

7. Tannins +++

8. Steroids ++



Antioxidant Activity

Table-2: Standardisation and % Inhibition of Ascorbic Acid.

S.NO CONC (µg) MEAN ± S.D %INHIBITION

1 1 1.226 ±0.173 42.87

2 2 1.247 ±0.178 41.89

3 4 1.638 ±0.101 23.67

4 6 1.259 ±0.196 41.33

5 8 1.191 ±0.251 44.5

Table-3: Measurement of anti-oxidant activity.

S.

No

.

Volu

me

(µl)

Conce

ntrati

on

(mg)

Abs 1 Abs 2 Abs 3 Abs 4 Abs 5 Abs 6 Mean Std.

Dev

Std

Err

%

Abs

%

Inhibi

tion

1 1 0.1 1.405 1.496 1.354 1.518 1.496 1.578 1.474 0.08

9

.03 68.68 31.31

2 2 0.2 1.504 1.599 1.359 1.497 1.555 1.899 1.568 0.18 .07 73.06 26.93

3 4 0.4 1.251 1.334 1.352 1.512 1.394 1.058 1.316 0.15 .06 61.32 38.67

4 6 0.6 1.37 1.963 1.358 1.331 1.36 1.361 1.457 0.24 .10 67.89 32.1

5 8 0.8 1.33 1.334 1.375 1.198 1.371 1.321 1.321 0.06 .02 61.55 38.44

6 10 1 1.211 1.188 1.132 1.199 1.124 1.022 1.146 0.06 .02 53.4 46.59

7 20 2 1.041 1.017 0.979 0.978 0.944 0.938 0.982 0.03 .01 45.75 54.24

8 40 4 0.916 0.922 0.916 0.897 0.899 0.888 0.906 0.01 .005 42.21 57.78

9 60 6 0.954 0.938 0.954 0.937 0.945 0.927 0.942 0.01 .004 43.89 56.1

10 80 8 0.974 0.96 0.964 0.954 0.95 0.955 0.959 .008 .003 44.68 55.31

11 100 10 0.979 1.023 0.988 0.995 0.969 0.99 0.99 0.01 .007 46.13 53.86

IC 50 Value of The Standard Ascorbic Acid is 1.2µg/ml.

Graph 1: % Inhibition of Ascorbic acid

0

10

20

30

40

50

1.226

±0.173

1.247

±0.178

1.638

±0.101

1.259

±0.196

1.191

±0.251

1 2 4 6 8

%INHIBITION

%INHIBITION



HEALING PATTERNS:

CONTROL STANDARD TEST

DAY 1

DAY 2

DAY 3

DAY 4

DAY 5

DAY 6



DAY 7

DAY 8

DAY 9

DAY 10

Fig 7: Healing patterns of Untreated, Standard & Test groups from Day 1-10.

Table 4: Measurement of Tensile Strength.

S.NO UNTREATED

(gm)

STANDARD

(gm)

TEST

(gm)

1. 180 190 350

2. 170 230 350

3. 170 210 350

4. 175 200 350

5. 170 220 350

6. 180 215 350



Graph 2: Comparison of Tensile Strength of Test, Standard & Untreated groups.

Table 5: SIGNIFICANT DIFFERENCES BETWEEN ALL THE GROUPS

GROUPS N MEAN STANDARD

DEVIATION

STANDARD

ERROR

UNTREATED 6 174.17 4.916 2.007

STANDARD 6 210.83 14.289 5.833

TEST 6 350.00 0 0

TOTAL 18 245.00 78.366 18.471

No tearing of wound was observed above 350gms. Independent samples of distribution of tensile strength across the

group using Kruskal-Wallis test was found to be 0.000 and rejected the Null hypothesis. Asymptomatic significance is

displayed. The significance level is 0.05.

Discussion:

The ethanolic extracts of the roots of Plumbago zeylanica belonging to the family Plumbaginaceae, demonstrated the

highest wound healing potential in experimental rats. The plant was selected based upon less amount of work done to

substantiate its effects.

The phytochemical results reveal the presence of tannins, alkaloids, reducing sugars and steroids in the ethanolic root

extract. The constituents of the root extract, such as terpenoids and alkaloids, may play a major role in the wound healing

process observed in this study. Studies have shown that flavonoids promote the wound-healing process mainly due to

their astringent and antimicrobial properties which appear to be responsible for the wound healing. The wound healing

0

100

200

300

400

500

600

700

800

1 2 3 4 5 6

TEST (gm)

STANDARD (gm)

UNTREATED (gm)



effect of ethanolic root extracts of Plumbago zeylanica may be due to the presence of more than one active principle

mentioned above.

Recent studies provided strong evidence for a role of oxidative stress in the pathogenesis of non-healing ulcers. The

normal physiology of wound healing depends on low levels of reactive oxygen species and oxidative stress, an

overexposure to oxidative stress leads to impaired wound healing. Antioxidants are postulated to help control wound

oxidative stress and thereby accelerate wound healing. The mechanism of action of the extract may be attributed to the

excellent DPPH antioxidant activity which may be due to the presence of tannins, flavonoids in the extract.

In the present study, wound healing potential of this plant was evaluated by creating incision wounds in rats. This method

is advantageous as these wounds are less painful, heals faster with less noticeable scar. The animals are maintained in

individual cages to minimize the risk of infection.

The measurements of the progress of the wound healing induced by the Neosporin in Clobate GM cream, Ethanolic

extract and the control group were reported. Wound healing progression was observed for a period of 10 days during

which extract showed significant closure of wounds when compared with the standard drug (Neosporin in Clobetasol

GM cream) and control group.

It was observed that the wound contracting ability of the extract were significantly greater than that of the control, which

was comparable to that of the reference standard. A better healing pattern with complete wound closure was observed in

rat using test extract as shown.

There were significant differences between groups in the reduction of weight. The average weight of animal in all groups

increased after the treatment period. Although, hair growth was very significant in Plumbago zeylanica extract treated

group since day 4 and reference standard group when compared to control.

The neurological profile of all animals was observed. It was found that control group showed very strong aggressive

behavior throughout the experimentation period, whereas extract treated animals showed moderate aggressiveness when

compared to standard drug treated animals.

In the incision wound studies, there was very excellent increase in tensile strength of the 10 day old wound due to

treatment with Plumbago zeylanica extract when compared with the reference standard Neosporin in Clobate GM cream



when compared with the respective control. The above observation for tensile strength indicates the effect of Plumbago

zeylanica root extract in maturation of collagen fibres. This shows wound healing potential of tested extract.

Conclusion:

The aim of the present study was to investigate the wound healing potential of ethanolic extract of roots of Plumbago

zeylanica by using incision wound model in Sprague-dawley rats. The wound healing activity was conducted to evaluate

the unexplored herbal plant material to provide a scientific evidence of their claimed pharmacological properties. The

wound healing activity of the plant may be due to phytochemicals like Flavonoids, Tannins, Alkaloids, Carbohydrates

and Saponins. One of these compounds may be responsible for this activity. Incision wound model is one of the

simulatory modules of post – operative care which replicates the true clinical conditions. This method is highly reliable,

versatile and effective.

The ethanolic extract has shown a greatly significant result when compared to control and standard. Wound healing

properties were remarkable and highly reproducible. The tensile strength was nearly equal to healthy skin and also in

comparison with the standard and control. There was a significant increase in tensile strength, and decrease in

epithelization period in extract-treated group as compared to control and standard drug-treated groups. The results were

significant. The study is concluded to confirm their wound healing potential and create a proof for further study.

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Corresponding Author:

V. Asha Jyothi*,

Email: [email protected]

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