TYPE OF MANUSCRIPT: ORIGINAL RESEARCH ARTICLE
FORMULATION AND EVALUATION OF A HERBAL WOUND
HEALING GEL CONTAINING SYZYGIUM CUMINI LEAF EXTRACT
Santhosh Aruna M*, Sravani A, Bhavani B, Gopi chand U, Fathima SK, N.Santhi Priya,
Rama Rao.N
Department of Pharmaceutics
Chalapathi Institute of Pharmaceutical Science, Lam, Guntur
Corresponding author
M.Santhosh Aruna
Assistant Professor
Department of pharmaceutics
Chalapathi Institute of Pharmaceutical Sciences,
Lam, Guntur
Andhra Pradesh, India
522034.
E mail: santhosharuna.kathi@gmail.com
Mobile no: 91-8143275848
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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 the reconstruction 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, gel
formulations
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INTRODUCTION:
Plants 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. 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 metrorrhagia 4. 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 been used in traditional medicine as a remedy for diabetes
mellitus in many countries. The leaves are also used to 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 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.
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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
effective less 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
either small 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 tremendous development
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.
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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 featuresEpidermis: 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; the macerates were filtered through muslin cloth & concentrated using
rotary evaporator. The ethanolic extracts were tested for the presence of various phyto
constituents 9.
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
3
Benzene soluble extractive
1.8
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Table 2: Phytochemical Screening
Chemical constituents
Test
Result
Molisch’s Reagent
(+)ve
Benedict’s Reagent
(+)ve
Flavanoids
Shinoda Test
(-)ve
Phytosterols
Salkowski’s
(-)ve
Glycosides
Legal test
(+)ve
Alkaloids
Dragonodroff’s reagent
(-)ve
Tannin & phenolics
Ferric chloride solution
(+)ve
Lead acetate solution
(+)ve
Carbohydrates
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 in desiccator 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).
Table 3: Preparation of medicated formulations
INGREDIENTS
FI
F II
F III
CONTROL
Plant extract
1g
0.2ml
Sesame oil
-
0.2 ml
0.2ml
-
Carbopol 934
0.1 g
0.1 g
0.1g
0.1 g
-
(0.1%)
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Formulations of Gels:
Carbopol 934P NF 0.1gm was measured and was dispersed in 10ml of distilled water and
mixed by stirring 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 was
formed. 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 a mixture of sesame oil and
leaf extract and F III contains sesame oil F IV is control.
Evaluation of Gel:
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 4
2. Subjective Properties: Subjective properties such as consistency, texture and Irritation are
observed and shown 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 reading was 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 to which 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
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slip off from gel which is placed in between the slides under the direction of certain load.
Lesser the time taken for the separation of 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 stability testing 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 at 40C, 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 7
7. 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 organisms 11,
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 8 groups of six animals each. The back of the each animal 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.
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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 as color, 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 the color 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.6. The subjective
properties such as consistency were good and 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 room temperature are change in pH
is observed and shown in Table No.5. 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 and attained 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 gel formulations 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.
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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 antibacterial activity and this will help keep the wound area sterile, thus promoting wound
healing. This fact supports a faster wound healing in the treated groups compared with the
control group.
Wound contraction studies: The 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 wound contraction compared with the marketed formulations
Table 4: Physical Evaluation of Gels
Parameter
FI
F II
F III
Color
Dark green
Yellow
White
Appearance
Translucent
Translucent
Transcluent
Feel on application
Smooth
Smooth
Smooth
Time in days
FI
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
6.40
22
6.39
6.47
6.39
30
6.39
6.48
6.38
Table 5: pH of the Gels:
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Table 6: Subjective properties of Gels
Parameter
FI
F II
F III
Consistency
Good
Good
Good
Texture
Smooth
Smooth
Smooth
Irritation
_
_
_
Table 7: Spreadability and viscosity
Formulation
Spreadability (mm)
Viscosity (cp )
Formulation I
55
4500
Formulation II
48
4700
Formulation III
45
4500
Marketed
50
4600
Table 8: Stability testing
Formulation
Initial colour
Formulation I
Dark green
Formulation II
Formulation III
II month
III month
IV month
+
+
+
+
Yellow
+
+
+
+
White
+
+
+
+
+
+
+
+
Marketed
White
I month
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
Marketed
25
10
11
Table 10: wound contraction studies
Area of wound during different days of observation (%)
Treatment in days
Control
Formulation I
Formulation II
Formulation III
Marketed
4
8
12
16
21
06.60
15.60
36.94
53.44
66.38
±0.7160
±0.7065
±0.9410
±0.7819
±0.5671
1.88**
25.38**
47.71**
67.60**
79.05**
±0.5347
±0.5043
±0.5809
±0.5994
±0.5802
07.72*
18.16*
37.10*
54.27*
67.94*
±0.7276
±0.5369
±0.7287
±0.5468
±0.5477
8.27**
19.38**
38.88**
56.22**
72.27**
±0.7821
±0.6963
±0.5567
±1.060
±0.5802
1 3.88
29.38
53.94
73.27
88.33
±0.7487
±0.7272
±0.6522
±0.6408
±0.5671
Values are expressed as mean ± SEM, N= 6, *p<0.05, **p<0.01 Vs Group 6
One way ANOVA followed by Dunkeertannets test
CONCLUSION:
Various 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 antiacne activity on human volunteers.
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ACKNOWLEDGEMENTS
We 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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REFERENCES:
1. Patel SS. Morphology and Pharmacology of Passiflora edulis. Journal of Herbal
medicine and Toxicology. 2009; 3(1):1-6.
2. KR and Basu BD. Indian Medicinal Plants. Vol. II. 1975:1052-53.
3. Nadkarni KM. Indian Materia medica. Vol. I, Bombay, India, 1954; 516-18.
4. Sharma P and Mehta PM. In Dravyagunavignyan. (The Chowkhamba Vidyabhawan),
Part II & III, Varanasi. 1969; 586.
5. Gowri Shyamala S and Vasantha K. Phytochemical Screening and Antibacterial
Activity of Syzygium cumini (L.) (Myrtaceae)Leaves Extracts. International Journal
of Pharm Tech Research. 2010 ;( 2):1569-1573.
6. Martinez SB and Del Valle MJ. Storage stability and sensory quality of duhat
Syzygium Cumini Linn. Anthocyanins as food colorant. UP Home Economic Journal.
1981 ;( 9): 1.
7. Wealth of India. Raw materials, New Delhi: CSIR, Vol X. 1976; 100–104.
8. Charde M.S., Fulzele S.V.: Wound Healing and Anti-inflammatory potential of
Madhu Ghrita, Indian Journal of pharmaceutical science, 2006; 68(1); 26-31.
9. Khandwal KR. Practical Pharmacognosy.10th edition, Nirali Prakashan, Pune.
2003:158.
10. Harding K.G., Morris H.L.; healing chronic wounds. Science, medicine, and the
future .2002; 324,160-163.
11. Stadelmann WK, Digenis AG, Tobin GR. Physiology and healing dynamics of
chronic cutaneous wounds. Am. J. Surg. 1998; 176: 26-38.
12. Madden JW, Peacock J. Studies on the biology of collagen during wound healing. I.
Rate of collagen synthesis and deposition in cutaneous wounds of the rat. Surgery
1968; 64:288-294.
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