formulation and evaluation of herbal gel from different parts of cyamposis … · 2019-12-10 ·...

www.wjpps.com Vol 5, Issue 3, 2016.

740

Singh et al. World Journal of Pharmacy and Pharmaceutical Sciences

FORMULATION AND EVALUATION OF HERBAL GEL FROM

DIFFERENT PARTS OF CYAMPOSIS TETRAGONOLOBA (L.) TAUB.

FOR WOUND HEALING

Sumitra Singh* and Bhagwati Devi Rohilla

Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and

Technology, Hisar-125001 Haryana India.

ABSTRACT

The hydrogels prepared from ethanol extracts of leaves and fruits of

Cyamposis tetragonoloba (L.) Taub. were investigated for their healing

effect on excision and incision wound surface on swiss albino mice.

Different formulations were topically applied on the wound surface as

a single dose. Percentage of wound contraction, period of complete

epithilization of excision and tensile strength of incision wound and

histological studies of granulation tissue were used to evaluate effect of

wound healing. The activity was compared with that of the control and

metrogel (1% w/w) as standard drug. Both the formulation showed a

significantly higher rate of contraction and shorten epithilization period

in both the test models. In excision model healing was 99% in gel

containing 5% w/w fruit ethanol extract and 90% in gel containing 5%

w/w leaf ethanol extract (p<0.001) on 12th

day compared to 80% and 60% of healing with

standard metrogel (1% w/w) and control, respectively. In incision wound model there was

significantly increase in tensile strength (p<0.001). The result revealed that gel formulation

has got potential wound healing activity.

KEYWORDS: Excision wound model, Incision wound model, Cyamposis tetragonoloba

(L.) Taub.

INTRODUCTION

Cyamposis tetragonoloba (L.) Taub. (family- Fabaceae) are hardy and drought resistant

herb.[1]

It is cultivated in hotter parts of India particularly in Haryana, Panjab, Rajasthan and

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 6.041

Volume 5, Issue 3, 740-752. Research Article ISSN 2278 – 4357

Article Received on

24 Dec 2015,

Revised on 13 Jan 2016,

Accepted on 03 Feb 2016

*Correspondence for

Author

Dr. Sumitra Singh

Department of

Pharmaceutical Sciences,

Guru Jambheshwar

University of Science and

Technology, Hisar-

125001 Haryana India.

http://www.wjpps.com/


741


Gujarat for vegetable, fodder, green manure and seed gum. Oral administration of ethanol

extract of powdered pods has shown significant anti-ulcer, anti-secretory and cytoprotective

effects.[2]

Wounds are physical injuries that result in an opening or break of the skin. Proper

healing of wounds is essential for the restoration of disrupted anatomical continuity and

disturbed functional status of the skin.[3]

The wound-healing process consists of four highly

integrated and overlapping phases: hemostasis, inflammation, proliferation and tissue

remodeling or resolution.[4]

For a wound to heal successfully, all four phases must occur in

the proper sequence and time frame. Many factors can interfere with one or more phases of

this process, thus causing improper or impaired wound healing.[5]

The population is aging,

and advanced age is commonly identified as a risk factor for delayed wound healing.[6]

Many

Ayurvedic herbal plants have a very important role in the process of wound healing. Plants

are more potent healers because they promote the repair mechanisms in the natural way. The

healing process can be physically monitored by assessing the rate of contraction of the

wound.[7]

A survey of literature revealed that no systematic approach has been made to study

wound healing activity of this plant. To validate the ethnotherapeutic claim of the plant in

skin diseases, wound healing activity was studied. In this communication we report the

formulation and study of topical gels of the various extracts and their wound healing activity.

MATERIAL AND METHODS

Plant Material

The plant material of Cyamposis tetragonoloba (L.) Taub. was collected from cultivated field

at Charkhi Dadri (Haryana, India) in the month of August 2012. The plant were

taxonomically identified and authenticated by Dr. Roshni Nayar, Head, Raw Materials,

Herbarium and Museum Division, NISCAIR, New Delhi, vide reference no,

NHCP/NBPGR/2012-34, date 20/10/12. A voucher specimen of the same has been retained

in the Department for the future reference. The leaves and fruits part were used for the

experimental work.

Experimental Animals

The healthy swiss albino mice of either sex, weighing 20-25 g, were housed under standard

environmental conditions of temperature and humidity (25±0.50ºC) and 12 hours light/dark

cycle) were utilized for the studies. The animals were fed with standard pellet diet and water

ad libitum. The Institutional animal ethical committee (Guru Jambheshwar University of

Science and Technology, Haryana, India) reference no. Ph/2013/224, dated 20/3/2013



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approved the experimental protocol and care of laboratory animals were taken as per the

guidelines of CPCSEA, Ministry of Forest and Environment, Government of India

(Registration number 0436). The animals were divided into four groups. Each groups

contained six animals.

Group I - was assigned as control received simple gel base I.P.

Group II - received the standard drug (1% w/w metrogel).

Group III - received gel containing 5% w/w ethanol extract of leaves.

Group IV - received gel containing 5% w/w ethanol extract of fruits.

Preparation of Herbal Extract

The air-dried crude samples of leaves and fruits were pulverized and extracted respectively

with (95% v/v) ethanol using soxhlet apparatus for 24 hours. The solvent were removed

under vacuum. Semi solid masses with a yield of 15% and 19% w/w respectively were

obtained. The extracts were further used for evaluation of biological activity.

Preparation of Topical Formulations

Gel base IP

The simple gel was prepared according to the formula reported in Indian Pharmacopiea (IP)

as carbopol 934 (0.8%), propylene glycol (1 ml), ethanol (2 ml), triethanolamine (q.s.),

purified water (q.s.) for 20 gm. Carbopol was added in small amounts in purified water with

vigorous mechanical stirring, until the cloudy lump free and waited for air bubble to separate,

triethanolamine was added drop by drop and stirring by spatula, then remaining water added,

stirred the mixture thoroughly until gel formed. Packed it an gel jar. Simple gel base was

used as a control for the test animals.[8,9]

Medicated gel

The medicated gel were prepared by adding the ethanol extracts (5% w/w of leaves and 5%

w/w fruits respectively) were dissolved in purified water. These contents were added to the

contents of simple gel (IP) in similar manner. Stirred the mixture thoroughly until

homogenous gel was obtained. Packed it an gel jar.



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EXPERIMENTAL WORK

Monographic analysis of formulation

1. Content Uniformity

Five hundred milligram of the each gel were taken and dissolved in a small amount of buffer

pH 7.4. The volume were made up with buffer. The absorbance were measured at 284 for

each gel.[10]

2. Physical Stability

The gels were kept in collapsible tubes and were placed in the stability chamber, which was

maintained at 40°C and 75% relative humidity for 90 days. The gels were then observed for

any change in the consistency and colour.[11]

3. Spreadability

Test formulations 0.5 gm each were placed with in a circle of 1 cm diameter pre marked on a

glass plate over which a second glass plate was placed. A weight of 5 gm was allowed to rest

on the upper glass plate for 5 min. the increase in the diameter due to spreadability of the

formulation was noted.[11]

4. Drug Content

One gm of the gel was weight into a 100 ml beaker and dissolved in methanol, it was diluted

appropriately and analysis at 304.5 nm by UV spectrometry.[12]

5. Solubility Studies

The solubility of gels in water was determined by taking excess quantity of gel and adding to

screw capped 50 ml glass vials filled with water. The vials were shaken for 24 hours on

mechanical shaken for 24 hours on mechanical shaken at room temperature. The solution was

filtered through Whatmann filter paper No. 1 and the drug concentration was determined

spectrophotometrically at 260 nm.[12]

6. Viscosity

Viscosity of the gel prepared with different extracts were determined using a Brook Field

Digital Viscometer Helipath (LVDV 2P230). The spindle was inserted into the test solution

and the viscometer was sheared at different speeds of 20, 50 and 100 rpm at room

temperature. Viscosity values were recorded for different speeds of rotation. Graphs of

viscosity versus speed of rotation (in rpm) were plotted. Distilled water was used to prepare

gel formulations.[13]



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7. Permeability - In vitro release studies through excised rat skin (Franz’s diffusion cell

method)

The rat skin soaked in buffer for 6-8 hours was clamped carefully to one end of the dialysis

cell (donor compartment). Fifty milliliter phosphate buffer saline (PBS) containing 20%

ethanol was taken in a beaker (receptor compartment). Gel, of 1 gm quantity, was spread

uniformly on the membrane. The donor compartment was kept in contact with the receptor

compartment. The temperature was maintained at 37 ± 0.1°C. At predetermined time

intervals, 1 ml of the solution was pipetted out from the receptor compartment and

immediately replaced with 1 ml of fresh PBS. Thedrug concentration of the acceptor fractions

was determined against appropriate blank.[14]

Acute Dermal Toxicity Studies

The study was carried out to determine the therapeutic dose of the ethanol extracts of leaf and

fruit. The acute dermal toxicity testing of the ethanol extracts gels were done by applying the

gels of the highest concentrations of 10% (w/w) on the shaved back of the mice. The OECD

guidelines no. 402 were followed for the study.[15]

Excision Wound Model

The mice were anesthetized by administering ketamine (0.2 ml/kg b.w. i.p.). A full thickness

of the excision wound of circular area (approx. 300mm2) and 2 mm depth was made on the

shaved back of the mice 10 minutes later the administration of ketamine injection.[16]

The

wounding day was considered as day 0. The wounds were treated with topical application of

the gels, as described above till the wounds were completely healed. The wounds were

monitored and the area of wound was measured on 4, 8, 12, 16 postwounding days and the

mean percentage (%) wound closure was report. The period of epithilization was calculated

as the number of days required for falling of the dead tissue remnants without any residual

raw wound.[17]

Wound healing rate: (percentage of wound closure) = 100× (wound area on day 0- wound

area on n day)/Wound area on day 0, where n =number of days 4

th, 8

th, 12

th and 16

th day.

Incision Wound Model

In the incision model the mice were anesthetized by administering ketamine (0.2 ml/kg b. w.

i.p.) and longitudinal paravertebral incision of about 1.5 cm length were made through the

skin and cutaneous muscle at a distance about 1 cm from the midline on the deplicated back



745


of the mice 10 minutes later the administration of ketamine injection.[16]

After the incision,

the parted skin was sutured 1cm apart using a surgical thread (no.000) and a curved needle

no. 11 for stitching. The continuous thread on both wound edges were tightened for good

closure of the wounds. The wounds of animals in the different groups were treated with

topical application of the gels once a day, till the complete healing. The wounding day was

considered as day 0. When wounds were cured thoroughly, the sutures were removed on the

8th post-wounding day and the tensile strength of the skin that is the weight in grams required

to break open the wound/skin was measured in anesthetized mice by tensiometer on the 10th

day reported.[17]

The tensile strength of a wound represents the degree of wound healing.

Usually wound healing agents promote a gain in tensile strength. The sutures were removed

on the 9th day after wounding and the tensile strength was measured on the 10th

day.[18]

Tensile strength was calculated using the following formula: Tensile strength = breaking

strength (g)/cross-sectional area of skin (mm2).

[19]

Estimation of Biochemical Marker

Circular wound with approximate area of 300mm2 was created using the procedure described

in excision wound model. The wounds were treated with topical application of gels as

described above for 10 days. On the 11th day, the animals from each group were euthanized

using diethyl ether and used to determine hydroxyproline content.[17]

Histopathological Studies

For histological study, granulation tissue were fixed in 10% neutral formalin solution for 24

hours and dehydrated with a sequence of ethanol - xylene series of solution. The materials

were infiltered and embedded with paraffin (40-600C). Microtome sections were taken at 10µ

thickness. The section were processed in alcohol - xylene series and stained with hematoxylin

- eosin dye.[18]

RESULTS

Monographic analysis of formulation

1. Content Uniformity

Both the gel formulations of ethanol extracts of leaves and fruits showed 99% drug content

uniformity in their formulations.



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2. Physical Stability

Both gels of ethanol extracts of leaves and fruits containing 5% w/w of each drug were found

to be stable in the accelerated stability studies, as there was no change in the color and

consistency. Besides, air bubbles and crystals did not appear during the studies.

3. Spreadability

The spreadability of gel was found to be 6.4 cm.

4. Drug Content

Release of drug content increases by time involved.

5. Solubility Studies

Increase in solubility was time dependent, highly soluble.

6. Viscosity

Viscosity of gels prepared with ethanol extracts of leaves and fruits were measured

504±0.014, 563±0.043 cPs.

7. In vitro release studies of gel through rat skin

In vitro diffusion of extract from their gel formulations in phosphate buffer saline (pH 7.4)

containing 20% ethanol through rat skin was studied. The release of the drugs from the

formulations was progressive and steady. At the third hour, the rate of release of gel prepared

from ethanol extracts of leaves and fruits formulations were 71.30% and 87.85%

respectively.

Acute Dermal Toxicity Study

The treatment of normal mice with different concentration (5%, 10%, 12%) of ethanol

extracts of leaf and fruit in simple gel base for 14 days revealed the non toxic nature of test

extracts. There was no change in general behavior or appearance, loss in body weight etc.

Toxicity study showed no mortality up to the selected dose of 5%, 10% (w/w) body weight

till the end of experiment.

Biochemical Marker Estimation

Biochemical analysis estimated out increase in hydroxyproline content. Collegen is the most

abundant protein in human body. In normal tissues collagen provides strength, integrity and

structure. When tissues are disrupted due to injury, collagen is needed to repair the defect and

restore anatomic structure and function of tissue. Major component of extracellular matrix is

the collagen which provides strength and support.[20]

The collagen, composed of amino acid,

hydroxyproline, is the major component of the extra cellular tissue, which gives strength and



747


support, Breakdown of collagen liberates free hydroxyproline and its peptides, Measurement

of hydroxyproline could be used as an index for collagen turnover, So the increment in

hydroxyproline is used for determining the collagen amount in the injured tissue.[21]

The

hydroxyproline content was found more increased in wound treated gel containing 5% w/w

fruits ethanol extract (13.98±0.19 µg/100mg) than gel containing 5% w/w leaves ethanol

extract (12.88±0.56 µg/100mg) and treated with standard drug (Table 1).

Excision Wound Study

In excision wound model, the percentage of wound contraction was calculated on 4th

, 8th

, 12th

and 16th

post wounding days for control (simple gel base treated group), standard (metrogel

treated group) and the test groups were gel containing ethanol extracts of leaves and fruits

(5% w/w each). Gel prepared from 5% ethanol extract of fruits showed the most significant

wound contraction rate (p<0.001) on 4th

, 8th

, 12th

and 16th

day post wounding days. The gel

prepared with 5% ethanol extracts of leaves showed very less significant (p<0.05) wound

contraction on 4th

day and significant (p<0.01) wound contraction on 8th

, 12th

and 16th

day.

Gel with 5% w/w fruits ethanol extracts gel with 5 % w/w exhibited the most significant

(p<0.001) period of epithelization. The gel prepared from 5% w/w leaves ethanol extracts

exhibited significant (p<0.01) effect which was comparable with standard effect on 12 day,

but less than effect of gel prepared with fruits ethanol extracts. The epithelization period was

18 days in case of animals treated with control gel base (Table 1).

Incision Wound Study

Incision wound healing activity in animals (Group I to IV) was evaluated depending upon

tensile strength of their incision wound skin. The increase in tensile strength of wounded skin

indicates the increase in collagen fibres that firmly knitting the disrupted skin of wounded

area. The results were expressed as the mean weight in grams ± SEM, the weight required to

break open the sutured wounds. The group treated with 5% w/w ethanol extract gel of fruit

showed the most significant (p<0.001) results than the group treated with 5%w/w ethanol

extract gel of leaves (p<0.01) and treated with standard drug. The group treated with simple

gel base showed insignificant results when compared with other test groups. (Table 2).

Histological Studies

The histological studies of the granulation tissue of the control animals demonstrated a more

aggregation of macrophages with few collagen fibers than the treated groups In the animals

treated with the 5% w/w ethanol extract of leaves gel, a moderate collagen deposition with



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fewer macrophages and fibroblasts was noticed, where as 5% w/w fruit ethanol extract gel

treated group exhibited a significant increase in collagen deposition with fewer macrophages

and fibroblasts (Fig. 1-6).

Histopathological examination: Microscopic appearance of wound sections are shown in

Fig. 1-6. (1) Histological section of the granuloma tissue of normal skin viewing in

complete healing with epithelialization showing macrophages and collagen formation.

(2) Histological section of the granuloma tissue of 0th

day viewing incomplete healing

with less epithelialization showing macrophages and lesser collagen formation. (3)

Histological section of granulation tissue of mice with gel prepared with 5% w/w fruits

ethanol extracts gel showing increased collagenation, with few macrophages. (4)

Histological section of granulation tissue of the mice treated with gel prepared with 5%

w/w leaves ethanol extracts showing increased collagenation, lesser macrophages. (5)

Histological section of the granuloma tissue of standard mice viewing complete healing

with elevated epithelialization showing macrophages and foremost collagen formation.

(6) Histological section of the granuloma tissue of control mice viewing incomplete

healing with less epithelialization showing macrophages and lesser collagen formation

indicated incomplete healing of the wound.

Table 1: Effect of topical application of gels containing ethanol extracts of Cyamposis

tetragonoloba (L.) Taub. leaves and fruits on wound contraction of excision wound

model.

Days Simple gel

base

Std (1%

metrogel)

Ethanol extract

leaves gel (5% w/w)

Ethanol extract

fruits gel (5% w/w)

0th

day 298±0.32

(0%)

297±0.42

(0%)

297.4±0.24

(0%)

298.2±0.02

(0%)



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4th

day 270±0.23

(10%)

240±0.24

(20%)

220.2±0.21

(23%)

205.3±0.26

(28%)

8th

day 210±0.14

(30%)

164.2±0.27

(45%)

150±0.26

(50%)

120±0.6*

(58%)

12th

day 120±0.25

(60%)

37.4±0.14**

(86%)

28.5±0.12**

(90%)

3.01±0.23***

(98.6%)

16th

day 30±0.26**

(90%)

2.01±0.23***

(99%)

2.5±0.32***

(99%)

------

Period of

epithelization 18 16 16 12

Cumulative amount

of hydroxyproline

(µg/100mg)

4.765±0.18

12.087±0.54

12.876±0.56

13.986±0.19

N = 6 animals in each group, Values are expressed as Mean ± SEM, in each group. Data was

analyzed by one way ANOVA followed by Dunnett’s t-test, *P < 0.05, ** P < 0.01, *** P <

0.001.

Table 2: Effect of topical application of gels containing ethanol extracts of Cyamposis

tetragonoloba (L.) Taub. leaves and fruits on breaking and tensile strength of the skin

having incision wound model.

S. N Group Breaking strength in

gm (mean ± SEM)

Tensile strength in

gm/mm2 (mean ± SEM)

1 Simple gel base 462.19 ± 0.76 15.42±0.32

2 Std (1% metrogel) 537.27 ± 0.16**

18.86±1.45**

3 Ethanol extract

leaves gel (5% w/w) 572.75 ± 0.06

** 22.91±2.31

**

4 Ethanol extract fruits

gel (5% w/w) 598.07 ± 0.96

*** 20.01±1.53

***

N = 6 animals in each group, Values are expressed as Mean ± SEM, in each group. Data was

analyzed by one way ANOVA followed by Dunnett’s t-test, *P < 0.05, ** P < 0.01, *** P <

0.001.

DISCUSSION

The study demonstrated that two different gel prepared with 5% w/w fruits ethanol extract

and 5% w/w leaves ethanol extract respectively were able to increase the rate of wound

healing for both incision and excision wound models. Ten days after inflicting the wound,

tensile strength in groups treated with 5% w/w fruit ethanol extract and 5% w/w leaf ethanol

extract respectively was significantly higher than control group. In incision wound, the

increase in tensile strength of treated wounds may be due to the increase in collagen

concentration and stabilization of the fibers.[23]

In excision wound, the ethanol extract of



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leaves and fruit showed faster healing with earlier wound contraction compared with control

groups may be due to increased cellular proliferation and collagen synthesis at the wound site

as evidenced by increase in total protein and total collagen contents reflected by

hydroxyproline content of granulation tissues.[24]

Several phytoconstituents like tannins[25]

,

phenolics, carbohydrates, proteins, saponins and flavonoids are also known to promote the

wound-healing process mainly due to their astringent and antioxidant activity, which seems

to be responsible for wound contraction and increased rate of epithelization.[26,27]

Presence of

Proteins, carbohydrates, arginine, glutamine, polyunsaturated fatty acids, vitamin A, vitamin

C, vitamin E, magnesium, copper, zinc and iron play a significant role in wound healing and

their deficiencies affect wound healing.[28,29]

These constituents are already reported on this

plant which supports the results.[2,29]

Thus present study revealed that the gel prepared with

5% w/w fruits ethanol extract and 5% w/w leaves ethanol extract respectively of Cyamposis

tetragonoloba (L.) Taub. possesses better wound healing potency, which was evident by the

increased rate of wound contraction, reduction in the period of epithelization, increase in

collagen deposition and increase in tensile strength in granulation tissue as compared to

control.

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