Evaluation of Wound Healing Potential of Ethanolic Extracts of Various Parts of Phyllanthus simplex Retz.
Arasan Elayaraja1*, M Vijayalakshmi2 and Devalarao Garikapati1
1KVSR
2Department of
Microbiology,
ABSTRACT
Wound healing is a
complex dynamic process that results in the restoration of anatomic continuity
and function. The present study has proved that the wound healing potential of ethanolic extract obtained from roots of Phyllanthus simplex Retz showed a significant
effect than the same extract obtained from other parts of the plant. That
extract showed a facilitatory epithelisation
and wound contraction than other parts extracts. The root extract animal groups
showed a high collagen content than other extracts and the control groups. Hexosamine content and ascorbic acid levels showed an
increased level of the extract. Ascorbic acid is reported to have scavenging
activity and inhibition of lipid peroxidation, which
helps to show a better healing effect for the test groups.
KEYWORDS: Wound
healing, Collagen, Hydroxy proline,
Hexose amine, Epithelization.
INTRODUCTION:
Phyllanthus simplex Retz. is
commonly called as “ Kaya-an”, “Bhuiamali” and “Kayut-bulang”. (Fam:
Euphorbiaceae). It is a glabrous twining perennial
herb, which has a taproot and its branches are compressed. Its flowers are
solitary and its leaves are distichous. The siddha and ayurvedhic form of
medications text showed that the Hindus used equal parts of the fresh leaves,
flowers, fruit and cumin seeds with sugar, made into an electuary for the
treatment of gonorrhea by taking a teaspoonful for twice a day. The fresh
leaves, bruised and mixed with buttermilk, make a wash to cure itches in
children. The root is used in Chota Nagpur as an
external application for abscesses1-3.
Many
Indian indigenous plants have a very important role in the process of wound
healing. Those plants are more potent healers because they promote the skin
repair mechanisms in natural way. Wounds cause the activation of a cytokine
cascade that result in the formation of oxygen free radicals to lipid per
oxidation. Any drug that shows inhibition of lipid peroxidation
is believed to increase the viability of cells by improving circulation,
preventing cell damage, promoting DNA synthesis, increase in tensile strength
of collagen, increases lysyl oxidase
formation, contraction of the wound and showing null effect of necrosis have
excellent wound healing activity 4-7.
The present study is
undertaken to investigate the wound healing activity of ethanolic
extracts obtained from various parts of Phyllanthus
simplex Retz.
Fresh
whole plant materials of Phyllanthus simplex
Retz had been collected from Chota-Nagpur of
Jharkhand and was identified and authenticated by Dr. S. M. Khasim,
Asst. Professor, Department of Botany,
One of
the plant specimen had been planted in KVSR Siddhartha College of
Pharmaceutical Sciences Vijayawada and a voucher (No: PS/PCRL/No: 0041/BN) had
been deposited after planted in the herbal garden.
Preparation
of Ethanolic extract:
The
various parts of the plant material such as leaves including flowering tops,
stem part closed with barks and roots had been separated and shade-dried. About
1kg of the milled powder of powdered parts was extracted successively with 95%
ethanol (60-70°C) by Soxhlet apparatus and was concentrated under reduced
pressure to semisolid consistency. Their yield and amount used for evaluation
are given in table-1. A preliminary phytoscreening
was also done to identify the presence of various phytoconstituents
by using standard tests.
Preparation
of Ointment Base:
About 35% of Bees wax was melted in a china dish using a
water bath at a temperature of 60-80°C. Hot paraffin oil (5%) was also boiled in a water bath
at the same temperature. Then transferred into the china dish
with constant stirring using a glass rod. Finally 60% of petroleum gel was
added and boiled for 15 minutes with constant stirring until the ointment base
was cooled and semisolid in nature.
Experimental work:
The
experiment protocol was duly approved by Institutional Ethical Committee. Adult wistar rats
of either sex (200-250gms) were procured from King’s
Group I
Wound control (WC)
Group II
Wound + ointment prepared from Flowering tops
and leaves extract (WFTLE)
Group III
Wound + ointment prepared from Stem bark extract (WSBE)
Group IV
Wound + ointment prepared from Root extract (WRE)
Group V
Wound + ointment prepared from Standard drug –Soframycin
(WSDS)
A steel rod of diameter 2.5cm was heated to
80-85°C and burn wounds were created on the dorsal
side of the rat. After 24h, dead tissues were excised using surgical blade and
the wound was sterilized with surgical spirit. Control rats were dressed with
paraffin and gauze. The tested groups were treated with formulation. 1gm of
each formulation was used to treat the wounds. A similar quantity of soframycin was used to treat the Group-V animals. All the
rats were kept under an observation period of 20 days. The rate of wound
contractions was also monitored by measuring the wound areas for every 5days
until the healing of the wound. The contraction was studied by tracing the raw
wound area on a tracing sheet and measured. Its diameter was recorded.
Granulation
tissues were collected from both control and drug treated rats and washed with
cold saline (0.9%w/v NaCl) to remove blood tissues
and stored for analyzing various parameters such as collagen and hexosamine after lyophilized.
The blood samples were collected by
decapitation in the cervical region using a sterile syringe rinsed with EDTA.
Then the plasma was separated from blood by centrifugation for 15 minutes at
1500rpm. The plasma was subjected for estimation of malondialdehyde
and ascorbic acid.
1. Hydroxy Proline: 8
Samples of varying concentrations were taken
for analysis. Hydroxy proline
was oxidized by adding 1ml of chloramine T to each
tube. The contents were mixed thoroughly by shaking and allowed to stand for
20minutes at room temperature. Then 1ml of 70% perchloric
acid was added to each tube for destruction of chloramines T. The contents were
mixed and allowed to stand for 5minutes. Finally 1ml of PDAB (
2. Hexose amine:9
Samples of various extracts were taken for
analysis. The solutions were treated with 1ml of freshly prepared 2% acetylacetone in 0.5M Na2CO3 in
capped tubes and kept in boiling water bath for 15 minutes. After cooling in
tap water, 5ml of 95% ethanol and 1ml of Ehrlich’s reagent were added and mixed
thoroughly. The purple red colour formed was read by UV spectrophotometer
(JASCO-Japan) at 530nm after 30 minutes.
3. Malondialdehyde:10
To 0.1ml of the supernatant liquid 0.9ml of
10% TCA (Trichloroacetic acid) and 2.0ml of 67% thiobarbituric acid reagent were added and kept in boiling
water bath for 20minutes. The tube was cooled after centrifugation and the
mixture was measured at 532nm by UV spectrophotometer.
4. Ascorbic acid:11
To 0.5ml of plasma, 0.5ml of ice cold 10% TCA
was added and mixed thoroughly and centrifuged for 20 minutes. About 0.5ml of
the supernatant was mixed with 0.1ml of DTC reagent, mixed well and incubated
at 37°C for 3hours. Then 0.75ml of ice-cold 65%
conc. H2SO4 was allowed to stand at a room temperature
for 30minutes. The yellow colour was read at 520nm by using UV
spectrophotometer. Ascorbic acid was used as a standard.
All the results were expressed as mean ±
SEM. P<0.05 was considered statistical significant.
|
S. No |
Parts Used |
Amount yield w/w
|
Quantity used |
|
1. |
Flowering tops and leaves extract |
0.425 |
40%extract |
|
2. |
Stem bark extract |
0.724 |
40%extract |
|
3. |
Root extract |
1.235 |
40%extract |
|
S. No |
Days |
Group
I |
Group
II |
Group
III |
Group
IV |
Group
V |
|
1 |
0 |
4.97±0.004 |
4.97±0.004 |
4.97±0.004 |
4.97±0.004 |
4.97±0.004 |
|
2 |
5 |
4.02±0.032 |
4.17±0.042 |
3.86±0.014* |
3.78±0.019* |
3.98±0.012 |
|
3 |
10 |
3.71±0.023 |
3.28±0.0042 |
2.97±0.088* |
2. 43±0.79* |
2.36±0.0092 |
|
4 |
15 |
2.98±0.076 |
2.75±0.032 |
2.14±0.043* |
1.75±0.043* |
1.84±0.015 |
|
5 |
20 |
1.35±0.014 |
2.14±0.069 |
1.74±0.014* |
0.96±0.065* |
1.23±0.069 |
Values are mean ±
S.E.M. (n = 4), *P<0.05 statistically significant
when compared with control group.
|
Biochemical
Parameters |
Group
I |
Group
II |
Group
III |
Group
IV |
Group
V |
|
L-Hydroxy proline |
0.126±
0.0048 |
0.156
± 0.0013 |
0.183
± 0.0045 |
0.207
± 0.0017* |
0.224
± 0.0021 |
|
Hexosamine |
0.0413
± 0.0017 |
0.0672
± 0.0021 |
0.0972
± 0.0031 |
0.1132
± 0.0078* |
0.1242±
0.008 |
|
Malondialdehyde |
6.429
± 0.0039 |
5.321
± 0.0014 |
4.45 ±
0.0021 |
3.321
± 0.0034* |
2.231
± 0.0056 |
|
Ascorbic
acid |
5.34 ±
0.013 |
4.2 ±
0.104 |
5.19 ±
0.104 |
8.3 ±
0.104* |
9.2 ±
0.054 |
Values are mean ±
S.E.M. (n = 4), *P<0.05 statistically significant
when compared with control group.
The
wounds treated with plant extracts showed a higher rate of wound contraction
and they are significant to soframycin. Also the
extracts obtained from roots are showing an increase in the wound contraction.
The rate of contraction of the wounds increased with treatment showed an
appreciable decrease in wound size as showed in table-2. The tensile strength
of the collagen fiber was found to increase in tensile strength and hence
showed an increase in biochemical parameters showed by the ointment prepared
from Root extract treated animals (Group IV) as showed in table-3.
Hexose amine content in
group IV animals indicate that the fibroblasts actively biosynthesized, ground
substances on which the collagen can be laid on12. The
cytokine cascade activated after a burn injury with stimulation of phagocytic cells that result in the formation of oxygen
free radicals and lipid peroxidation. The control
group showed an elevation in the lipid peroxidation
levels which indicates the decreased free radical scavenging capacity of the
wounded tissues. Lipid peroxidation is oxidative deterioration
of PUFA. It leads to cell injury leading to generation of peroxides and lipid
peroxides. Ascorbic acid was used as a standard drug for scavenging activity.
In the present study it was found that the ascorbic acid levels were higher in
the test group when compared to the control group and hence a decline in the
lipid peroxidation.
Finally
the present study showed that the wound healing potential of the root extract
showed the significant results with the standard drug soframycin.
Though many synthetic medications are available for the treatment of wounds
these natural sources would serve better in the treatment of wounds at a faster
rate.
REFERENCES:
1.
Kirtikar and Basu. In: Indian Medicinal Plants, International Book
Publishers and Distributors, 2nd edn,. Reprint 2007; 3: 2224-2225.
2.
Agarwal V.S. In:
Drug Plants of
3.
Rajkishor.S.N. and Thawra M.F. Phytochem 1988; 27
(9): 3027-3028.
4. Nadiga Shoba. Ind J Pharmacol 1999; 31: 1 and 77.
5.
Khan
M, Patil P A and Shoba J C.
J Nat Remedies 2004; 4:1.
6.
Udayan P S, Satheesh
George K V, Tushar and Indira
Balachandran. J of Nat Remedies 2005; 5: 1.
7.
Prashant S Hegde, Anitha B and T S Chandra.
8.
Woessner J F Jr. Biochem
J 1961; 83: 304-314.
9.
Elson
L A and Morgan W T J. Proc Soc Exp Biol 1933; 58:
97-100.
10.
Yagi K. In: Assay for blood plasma or serum
methods in Enzymology, 1984; 105: 328-331.
11.
Omayer et al, In:
selected methods for the determination of ascorbic acid in animal cell tissues
and fluids methods in Enzymology 1979; 62: 3-8.
12.
Karthikeyan J and Rani P.Ind J Exp Biol 2003; 41: 135-140.
Received on 08.10.2009
Accepted on 30.11.2009
© A&V Publication
all right reserved
Research Journal of Pharmacognosy and Phytochemistry. 2(1): Jan.-Feb. 2010, 34-36