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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: ashajyothivadlapudi@gmail.com 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).
V. Asha Jyothi* et al. International Journal Of Pharmacy & Technology
IJPT | Nov-2013 | Vol. 5 | Issue No.3 | 5879-5891 Page 5880
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
V. Asha Jyothi* et al. International Journal Of Pharmacy & Technology
IJPT | Nov-2013 | Vol. 5 | Issue No.3 | 5879-5891 Page 5881
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.
V. Asha Jyothi* et al. International Journal Of Pharmacy & Technology
IJPT | Nov-2013 | Vol. 5 | Issue No.3 | 5879-5891 Page 5882
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.
V. Asha Jyothi* et al. International Journal Of Pharmacy & Technology
IJPT | Nov-2013 | Vol. 5 | Issue No.3 | 5879-5891 Page 5883
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.
V. Asha Jyothi* et al. International Journal Of Pharmacy & Technology
IJPT | Nov-2013 | Vol. 5 | Issue No.3 | 5879-5891 Page 5884
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 ++
V. Asha Jyothi* et al. International Journal Of Pharmacy & Technology
IJPT | Nov-2013 | Vol. 5 | Issue No.3 | 5879-5891 Page 5885
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
V. Asha Jyothi* et al. International Journal Of Pharmacy & Technology
IJPT | Nov-2013 | Vol. 5 | Issue No.3 | 5879-5891 Page 5886
HEALING PATTERNS:
CONTROL STANDARD TEST
DAY 1
DAY 2
DAY 3
DAY 4
DAY 5
DAY 6
V. Asha Jyothi* et al. International Journal Of Pharmacy & Technology
IJPT | Nov-2013 | Vol. 5 | Issue No.3 | 5879-5891 Page 5887
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
V. Asha Jyothi* et al. International Journal Of Pharmacy & Technology
IJPT | Nov-2013 | Vol. 5 | Issue No.3 | 5879-5891 Page 5888
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)
V. Asha Jyothi* et al. International Journal Of Pharmacy & Technology
IJPT | Nov-2013 | Vol. 5 | Issue No.3 | 5879-5891 Page 5889
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
V. Asha Jyothi* et al. International Journal Of Pharmacy & Technology
IJPT | Nov-2013 | Vol. 5 | Issue No.3 | 5879-5891 Page 5890
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: ashajyothivadlapudi@gmail.com
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