20150514_Santhosh Aruna et al IJAPR.pdf

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FORMULATION AND EVALUATION OF A WOUND HEALING GEL

CONTAINING SYZYGIUM CUMI NI LEAF EXTRACT

Santhosh Aruna Mamidi*, N.Santhi Priya, Sravani Avula, Bhavani B,

Gopi chand U, Fathima SK, Rama Rao.Nadendla.

Department of Pharmaceutics, Chalapathi Institute of Pharmaceutical Science, Lam, Guntur

Received on 09 – 01 - 2015 Revised on 14 – 01- 2015 Accepted on 25 – 02 – 2015

ABSTRACT Wounds are defined as the disruption of anatomical and functional integrity of living tissue. Wound healing is an

intricate and continual cascade of events, with various cellular and biochemical processes, ultimately resulting in thereconstruction and regeneration of damaged tissue. Plants and their extracts have immense potential for the

management and treatment of wounds. The phyto-medicines for wound healing are not only cheap and affordable

but are also safe as hyper sensitive reactions are rarely encountered with the use of these agents. These natural

agents induce healing and regeneration of the lost tissue by multiple mechanisms. Various plant products have been

used in treatment of wounds over the years. Wound healing herbal extracts promote blood clotting, fight infection,and accelerate the healing of wounds. The aim of present study was to prepare and evaluate the wound healing

activity of herbal gel. Our present study reveals that the new polyherbal formulations posses potent wound healing

activity, which could be a good choice of remedy for wound healing.

KEY WORDS: Wound healing, phyto-medicines, regeneration, polyherbal formulations

INTRODUCTIONPlants have bear the basis of many traditional

medicines throughout the world for thousands of

years and continue to provide new remedies to

mankind1.Correct knowledge of such crude drugs is

very important aspect in preparation, safety and

efficacy of the herbal products. Pharmacognosy is a

simple and reliable tool, by which complete

informations of the crude drugs can be obtained2.

Syzygium cumini Linn (family Myrtaceae),commonly known as Jamun (Hindi), is a medicinal

plant and utilizable species. Common names are Java plum, Black plum, Jambul and Indian Blackberry 3.

Corresponding authorM.Santhosh Aruna

Assistant Professor

Department of pharmaceutics

Chalapathi Institute of Pharmaceutical Sciences,

Lam, Guntur

Andhra Pradesh, India- 522034.

E mail: [email protected]

Mobile no: 91-8143275848

The original home of jamun is India, distributed

throughout India, in forest up to 1800m usually along

the bank and moist localities .The sprouts are

refrigerant, carminative &astringent to bowels.

Powdered seeds are used as remedy in diabetes and

in metrorrhagia4. As per Unani system of medicine,

they act as liver tonic, enrich blood, strengthen teeth

and gums and form good lotion for removing

ringworm infection of the head. Leaves have beenused in traditional medicine as a remedy for diabetes

mellitus in many countries. The leaves are also usedto strengthen the teeth and gums, to treat leucorrhoea,

stomachalgia, fever, gastropathy strangury,

Dermopathy, constipation and to inhibit in the

faeces5. The major phyto constituents are reported to

contain vitamin C, gallicacid, tannins, anthocyanins,

includes cyanidin, petunidin, malvidin, glycoside and

other components 6, 7.

Wound is defined as the disruption of the

cellular and anatomic continuity of a tissue. Wound

may be produced by physical, chemical, thermal,

microbial or immunological insult to the tissues

Research Paper

ISSN: 2230 – 7583

Santhosh Aruna, et al. / I nternational Journal of Advances in Pharmaceuti cal Research

IJAPRAvailable Online through

www.ijapronline.org

IJAPR /Apri l. 2015/ Vol. 6/I ssue.04/ 82 – 87 82

mailto:[email protected]






Wounds are physical injuries that results in an

opening and break of the skin that cause disturbance

in the normal skin anatomy and function. They result

in the loss of continuity of epithelium with or without

the loss of underlying connective tissue8. Wound may

be produced by physical, chemical, thermal,microbial or immunological insult to the tissues.

Topical gel drug administration is a

localized drug delivery system anywhere in the body

through ophthalmic, rectal, vaginal and skin as

topical routes. Skin is one of the most extensive and

readily accessible organs on human body for topical

administration and is main route of topical drug

delivery system. Topical application of drugs offers

potential advantages of delivering the drug directly to

the site of action and acting for an extended period of

time. It can penetrate deeper into skin and hence give

better absorption1. They are deemed more effectiveless toxic than conventional formulations due to the

bilayer composition and structure.

The U.S.P. defines gels as a semisolid

system consisting of dispersion made up of eithersmall inorganic particle or large organic molecule

enclosing and interpenetrated by liquid. Gels consist

of two phase system in which inorganic particles are

not dissolved but merely dispersed throughout the

continuous phase and large organic particles are

dissolved in the continuous phase, randomly coiled in

the flexible chains 5.Research on wound healing agents is one of

the developing areas in modern biomedical sciences.

Many of the synthetic drugs currently used for the

treatment of wounds are not only expensive but also pose problems such as allergy, drug resistance etc

and this situation has forced scientists to seek

alternative drugs . In spite of tremendousdevelopment in the field of synthetic drugs during

recent era, they are found to have some or other side

effects, whereas plants still hold their own unique

place, by the way of having no side effects.

MATERIALS AND METHODS

Plant Collection The leaves of the plant syzygium cumini were

collected from the medicinal plant garden of

Chalapathi institute of pharmaceutical sciences.Collected plant material was subjected for

identification based on herbarium specimens at

department of Pharmacognosy, Chalapathi institute

of pharmaceutical sciences, Guntur. The voucher

specimen no ANU/CIPS/Identi /Tech/2014/24.

Macroscopic Study The leaves measuring about 10 to 15 cm long and 4

to 6 cm wide. These are entire, ovate- oblong,

sometimes lanceolate and also acuminate, coraceous,

tough and smooth with shine above. The fragrant

flowers of Jamun are small, nearly 5 mm in diameter.

These are arranged in terminal trichotomous panicles

greenish white in color.

Microscopy: Transverse section of S. cumini leaves

showed following features-Epidermis: Two to three layered epidermis.

Mesophyll: It is composed of isodiametric thin

walled parenchymatous ground cells which are

packed with simple starch grains. In the mid-rib

region, the vascular bundles show xylem, towards

upper epidermis and phloem on the lower side. Starch

grains, oil globules, tannin cells and stone cells are

also visible.

Phytochemical Investigation: The leaves were

washed properly & cut into small pieces before being

subjected to cold maceration for seven days. The

solvent used was ethanol (95%), after 7 days; themacerates were filtered through muslin cloth &

concentrated using rotary evaporator. The ethanolic

extracts were tested for the presence of various phyto

constituents like tannins, alkaloids, carbohydrates,flavonoids, sterols, & glycosides etc9.

Extraction of syzygium cumini :The powdered leaves were used for extraction. The

powder is extracted in soxhalet apparatus with

ethanol. The extract obtained was dried in a vacuum

evaporator under reduced pressure and below50°C to

give a dried residue. The product is stored indesiccator for further studies.

Animal’s collection: Wister Males rats (150-200gm) were procured from

Animal House of Chalapathi institute of pharmaceutical sciences, lam, Guntur & were fed a

standard diet, water was provided & they were

acclimated 7 days before entry into subsequent study.The protocol was approved by Institutional Animal

Ethics Committee (IAEC).

Formulations of Gels: Carbopol 934P NF 0.1gm was measured and was

dispersed in 10ml of distilled water and mixed bystirring continuously in a magnetic stirrer at 800rpm

for 1 h. Glycerol 5ml was added to the mixture under

continuous stirring. The mixture was neutralized by

drop-wise addition of 50% triethanolamine (w/w).

Mixing was continued until a transparent gel wasformed. The extract of syzygium cumini was

incorporated into the gel base and mixed

continuously for uniformity. Three types of gels are

formulated of which F I containing syzygium cumini

leaf extract and F II is a poly herbal gel where amixture of sesame oil and leaf extract and F IIIcontains sesame oil F IV is control.

Evaluation of Gel

Santhosh Aruna, et al. / I nternational Jour nal of Advances in Pharmaceuti cal Research

IJAPR /Apri l. 2015/ Vol . 6/I ssue.04/ 82 – 87 83



1. Physical evaluation: The color, appearance and

the feel on application of the prepared herbal gel

formulations were noted and the results are shown in

Table 42. Subjective Properties: Subjective properties such

as consistency, texture and Irritation are observed andshown in Table 5

3. pH measurement: The pH of the gel was

determined by using a digital dissolved in 50 ml

water and pH was determined by dipping the glass

electrode completely into gel solution system so as to

cover the electrode. Then instrument reading in terms

of pH are tabulated in the Table 6. The pH was

studied for 30 days.

4. Viscosity: The measurement of viscosity of the

prepared gel was done with a Brookfield Viscometer.

The gels were rotated at 0.3, 0.6 and 1.5 rotations per

minute. At each speed, the corresponding dial readingwas noted. The viscosity of the gel was obtained by

multiplication of the dial reading with factor given in

the Brookfield Viscometer catalogues 10

.

5. Spreadability: It indicates the extent of area towhich gel readily spreads on application to skin or

affected part. The therapeutic potency of a

formulation also depends upon its spreading value.

Spreadability is expressed in terms of time in seconds

taken by two slides to slip off from gel which is

placed in between the slides under the direction of

certain load. Lesser the time taken for the separationof two slides, better the spreadibility. It is calculated

by using the formula

S = M. L / T

where,M = wt. tied to upper slide

L= length of glass slides

T = time taken to separate the slides

6. Stability testing: Since the period of stability

testing can be as long as two year, it is time

consuming and expensive. Therefore it is essential to

device a method that will help rapid prediction of

long term stability of drug. The accelerated stabilitytesting is defined as the validated method by which

the product stability maybe predicted by storage of

the product under condition that accelerated the

change in defined and predictable manner. The

stability studies of formulated gels were carried out at40C, 250C, 450C and at a room temperature for the

period of one month. The effect of temperature,

humidity and time on the physical characterization of

the gels was evaluated for assessing the stability of

prepared formulation. The result was shown in Table

77. Antimicrobial activity: The anti-microbial

activity of each formulation was assessed by

measuring the zone of inhibition in nutrient agar

medium, employing pseudomonas aeruginosa and

Staphylococcus aureus as test organisms11,

8. Wound healing studies: An excision wound

model was used for studying wound healing activity.

Albino rats (Wistar strain) of both sexes weighing

between 150 – 200 g were randomly divided into 8groups of six animals each. The back of the eachanimal was shaved and prepared after washing with

spirit. An area of about 2 sq.cm was defined with a

marker on the shaven back of the animals. The

circular marked area was excised with its full

thickness using a surgical sterile blade and scissors

under phenobarbitone anesthesia.

RESULT AND DISCUSSION

Physical Evaluation of Gels:The results of various physical parameters for

evaluation of the prepared herbal gel Formulation are

reported below. The physical parameter such ascolor, appearance, and feel on application are

observed and shown in Table No 4. From the

physical evaluation the color of prepared herbal gels

was dark green, faint yellow and light yellow as thecolor of extracts was green and yellow. Appearance

of gel was translucent and it was smooth on

application. So it shows significant physical

evaluation parameters. The subjective properties

such as consistency, texture and irritation are

observed. and are shown in Table No.5. The

subjective properties such as consistency were goodand texture of prepared herbal gel was found to be

smooth. No skin irritation was thereon application of

gel to the skin surface. So it can be used safely. The

pH value of gel formulation were studied at roomtemperature are change in pH is observed and shown

in Table No.6. pH value of prepared herbal gel

incorporating the medicinal plant extract was studied by using digital pH meter Systronics. (pH meter type

335). The pH was studied for 30 days at room

temperature. All four formulations were in range of

6.35 - 6.52 pH at initial phase. As we go from

epidermis to dermis, pH of the skin increases andattained the neutral value i.e. 7. So gel formulation

having pH range 6.2 to 7.0 are desirable to skin since

they do not interfere with the physiology of skin.

The Stability testing of the different formulation was

shown in Table No.8. The prepared herbal gelformulations were subjected to accelerated stability

testing. The prepared herbal gel were store at 4oC,

250C,450C in refrigeration, room temperature and

oven for a period of 30 days to study effect of

temperature and at different humidity condition. The physical parameter were evaluated during study period the result of study indicates that preparation

are physically stable at 450C.

Anti-microbial activity of the formulations





The gel base without the herbal extracts did not

shown any zone of inhibition. The zone of inhibition

was found to increase on increasing the herbal drug

concentration. Hence the results of this study confirm

that the herbs possess anti-bacterial activity and this

will help keep the wound area sterile, thus promotingwound healing. This fact supports a faster woundhealing in the treated groups compared with the

control group.

Wound contraction studiesThe results of wound contraction studies indicate that

all the formulations enhance wound healing in open

wounds. The rate of wound contraction was found to

reach a maximum on the 12th

day in the treated

groups. The gel formulations produced better woundcontraction compared with the marketed formulations

Table 1: Physicochemical Parameters

Parameter Value (%)

Total Ash 12

Acid Insoluble Ash 1.5

Water soluble ash 3

Loss on drying 32

Methanol soluble extractive 4

Ethanol soluble extractive 4.2

Petroleum ether soluble extractive 3Benzene soluble extractive 1.8

Table 2: Phytochemical Screening

Chemical constituents Test Result

Carbohydrates Molisch’s Reagent

Benedict’s Reagent

(+)ve

(+)ve

Flavanoids Shinoda Test (-)ve

Phytosterols Salkowski’s (-)ve

Glycosides Legal test (+)ve

Alkaloids Dragonodroff’s reagent (-)ve

Tannin & phenolics Ferric chloride solution

Lead acetate solution

(+)ve

(+)ve

Table 3: Preparation of medicated formulations

INGREDIENTS F I F II F III CONTROL

Plant extract 1 g 0.2ml -

Sesame oil - 0.2 ml 0.2ml -

Carbopol 934 (0.1%) 0.1 g 0.1 g 0.1g 0.1 g

Table 4: Physical Evaluation of Gels

Parameter F I F II F III

Color Dark green Yellow White

Appearance Translucent Translucent Transcluent

Feel on application Smooth Smooth Smooth

Table 5: Subjective properties of Gels

Parameters F I F II F III

Consistency Good Good Good

Texture Smooth Smooth Smooth

Irritation _ _ _





Table 6: pH of the Gels:

Time in days F I F II F III

0 6.37 6.50 6.39

2 6.38 6.49 6.38

7 6.40 6.47 6.39

14 6.41 6.48 6.4022 6.39 6.47 6.39

30 6.39 6.48 6.38

Table 7: spread ability and viscosity

Formulation Spreadability (mm) Viscosity (cp )

Formulation I 55 4500

Formulation II 48 4700

Formulation III 45 4500

Table 8: Stability testing

Formulation Initial colour I month II month III month IV month

Formulation I Dark green + + + +Formulation II Yellow + + + +

Formulation III White + + + +

Table 9: Anti – microbial studies (Zone of inhibition shown by the formulations).

Formulation P. aeruginosa (mm*) S.aureus (mm*)

Formulation I 14 6

Formulation II 24 10

Formulation III 26 13

Table 10: wound contraction studies

Area of wound during different days of observation (%)

Treatment in days 4 8 12 16 21Control 06.60

±0.7160

15.60

±0.7065

36.94

±0.9410

53.44

±0.7819

66.38

±0.5671

Formulation I 1.88**

±0.5347

25.38**

±0.5043

47.71**

±0.5809

67.60**

±0.5994

79.05**

±0.5802

Formulation II 07.72*±0.7276

18.16*±0.5369

37.10*±0.7287

54.27*±0.5468

67.94*±0.5477

Formulation III 8.27**

±0.7821

19.38**

±0.6963

38.88**

±0.5567

56.22**

±1.060

72.27**

±0.5802

Marketed 1 3.88

±0.7487

29.38

±0.7272

53.94

±0.6522

73.27

±0.6408

88.33

±0.5671

Values are expressed as mean ± SEM, N= 6, *p<0.05, **p<0.01 Vs Group 6 One way ANOVA followed byDunkeertannets test

CONCLUSIONVarious topical application dosage forms like creams,

ointments, liniments, lotions, gels and jellies have

been in use for many decades. Gels and jellies are

although age old formulations, they have now gained

more and more importance and the extensive studies

on their release properties have revealed that the

active ingredients in gel based formulations are better

percutaneous absorbed than from creams and

ointment bases. Thus the present research work

suggests that herbal gel formulation holds a

tremendous potential against wound healing and can





prove to be a safe and efficacious remedy for treating

wounds. However an elaborate protocol for the

clinical trials is needed to be designed and

implemented to check the anti-acne activity on

human volunteers.

ACKNOWLEDGEMENTSWe would like to sincerely thank the management

and Principal of Chalapathi institute of

pharmaceutical sciences, Guntur, for letting us avail

the facilities of the College. We are also thanking to

department of biotechnology and department of

pharmacology, their co-operation for the Invivo

studies.

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