Wound healing activity of the aqueous alcoholic extract of jasminum grandiflorum Linn leaves

Abstract

The influence of leave extract of Jasminum grandiflorum was studied for its wound healing activity at a dose of
250 mg/kg body weight, using excision and dead space wound models in rats. The animals were divided into
three groups in excision wound model, the controls (n=6) were treated with 0.25% CM cellulose, reference
standard (n=6) were treated with sulfathiazole ointment and the experimental (n=6) were treated with extract of
J. grandiflorum leave till complete epithelialization. The animals in dead space wound models were divided
into two groups, controls were given plain drinking water and the experimental animals were administered with
extract orally for 10 days. The extract treated wounds were found to epithelize faster as compared to controls.
Extract treated rats exhibited 65% reduction in the wound area when compared to controls (54%). The wet and
dry granulation tissue weight, and hydroxyproline content in a dead space wound model increased significantly
(P<0.001) when compared to controls. Histological studies of the tissue obtained on day 10 from the extracttreated
group showed increased well organized bands of collagen, more fibroblasts and few inflammatory cells
when compared to controls which showed inflammatory cells, scanty collagen fibres and fibroblasts. The
demonstration of increased rate of wound contraction together with the biochemical and histological findings suggest the use of J. grandiflorum leave extract in the management of wound healing.

Full text

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WOUND HEALING ACTIVITY OF THE AQUEOUS ALCOHOLIC EXTRACT
OF JASMINUM GRANDIFLORUM LINN LEAVES.
Shanti Bhushan Mishra*1, Alok Mukerjee1, M.Vijayakumar2
1United Institute of Pharmacy, UCER, Naini, Allahabad, (UP) India
2Ethnopharmacology Division, National Botanical Research Institute, Lucknow (UP), India
Summary
The influence of leave extract of Jasminum grandiflorum was studied for its wound healing activity at a dose of
250 mg/kg body weight, using excision and dead space wound models in rats. The animals were divided into
three groups in excision wound model, the controls (n=6) were treated with 0.25% CM cellulose, reference
standard (n=6) were treated with sulfathiazole ointment and the experimental (n=6) were treated with extract of
J. grandiflorum leave till complete epithelialization. The animals in dead space wound models were divided
into two groups, controls were given plain drinking water and the experimental animals were administered with
extract orally for 10 days. The extract treated wounds were found to epithelize faster as compared to controls.
Extract treated rats exhibited 65% reduction in the wound area when compared to controls (54%). The wet and
dry granulation tissue weight, and hydroxyproline content in a dead space wound model increased significantly
(P<0.001) when compared to controls. Histological studies of the tissue obtained on day 10 from the extract-
treated group showed increased well organized bands of collagen, more fibroblasts and few inflammatory cells
when compared to controls which showed inflammatory cells, scanty collagen fibres and fibroblasts. The
demonstration of increased rate of wound contraction together with the biochemical and histological findings
suggest the use of J. grandiflorum leave extract in the management of wound healing.
Key words: Jasminum grandiflorum, wound healing, excision wound, incision wound,
Corresponding Author:
*Shanti Bhushan Mishra
Lecturer
United Institute of Pharmacy
UCER, Naini, Allahabad-211001
Uttar Pradesh, India
Email: shantipharma15@gmail.com
Ph: +918004279457

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Introduction
Wound healing and tissue repair are complex processes that involve a dynamic series of events including
clotting, inflammation, granulation tissue formation, epithelization, collagen synthesis and tissue remodeling1,2.
These phases run either concurrently or intimately inter-linked through some chemical, biochemical and cellular
pathways. A treatment could influence the healing of wounds by intervening in one or many phases of wound
healing. No treatment, either systemic or local, could be considered inert on the healing process unless it is
proved experimentally. Wounds are defined as breach in the continuity of living tissues. Thus, humans cannot
escape from an event of injury in their lifetime. Depending upon the causation, site of injury, condition of the
patient, extent of trauma etc., the wounds could be minor or major. Wound care and maintenance involve a
number of measures including dressing and administration of painkillers, use of anti-inflammatory agents,
topical and systemic antimicrobial agents and healing promoting drugs. Jasminum grandiflorum Linn.
(Oleaceae) is commonly knows as Jasmine. It is a wellknown glabrous twining shrub widely grown in gardens
throughout India. Its leaves are mostly ternate or pinnate; the flowers, usually white with a tubular, five- or
eightcleft calyx, a cylindrical corolla-tube, with a spreading limb and two stamens enclosed in the corolla-tube.
The flower is acrid, bitter with a sharp taste. It is useful in treating diseases of the mouth and teeth, especially
for toothache3. The J. grandiflorum flowers and leaves are largely used in folk medicine to prevent and treat
breast cancer. Flowers of J. grandiflorum are useful to women when brewed as a tonic as it aids in preventing
breast cancer and stopping uterine bleeding4. It is widely used in the Aurveda, as an antiulcerative, antileprotic,
skin diseases and wound healing. The extensive studies on this species for its wound healing potential are yet to
be ascertained. A large number of materials have been reported to affect the healing differentially. However, the
intensive research in wound healing has not yielded, until today, a safe, economic and efficacious pro-healing
agent that could obviate the long hospitalization of patients following surgeries, wounds etc. Some rural
communities still apply a paste made from the dried or wet leaves and flowers of several plants including
Jasminum. There is however, a need to study and provide evidence for the efficacy of Jasminum extract in the
treatment of wounds.
Materials and Methods
Plant material and extract preparation
The leaves of J. grandiflorum was collected from Botanical Garden of N.B.R.I. (National Botanical Research
Institute), Lucknow, India in month of July 2008. The plant materials were authenticated by taxonomist. The
freshly collected plant materials (4 kg) of J. grandiflorum were washed with distilled water and air-dried at 30 ±
20C. Then dried it in tray drier under the control conditions and powdered. The powdered plant materials
(1000g) was macerated with petroleum ether to remove fatty substances, the marc was further exhaustively
extracted with of 50% ethanol for 3 days (3 X 3L) and centrifugation at 10,000 rev/min. The extract was
separated by filtration and concentrated on rotavapour (Buchi, USA) and then dried in lyophilizer (Labconco,
USA) under reduced pressure obtain 95.0 g of solid residue (yield 9.5 % w/w). The extract obtained was further
subjected to toxicological and pharmacological investigations.
Animals
Healthy outbred male albino rats of dr strain weighing 150–200 g were used for the study. They were
individually housed and maintained on normal food and water ad libitum. Animals were periodically weighed
before and after experiments. The rats were anaesthetized prior to and during infliction of the experimental
wounds. The surgical interventions were carried out under sterile conditions using ketamine hydrochloride as

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anesthesia (120 mg/kg) body weight. Animals were closely observed for any infection and those which showed
signs of infection were separated and excluded from the study. An acute toxicity study was conducted for the
extract by the stair-case method5.
Wound healing activity
Excision and dead space wound model was used to evaluate the wound-healing activity of J. grandiflorum.
Excision wound model
The rats were inflicted with the excision wounds6. The rats were anaesthetized prior to creation of the wounds,
with 1 ml of intravenous ketamine hydrochloride (120 mg/ kg body weight). The dorsal fur of the animal was
shaved with an electric clipper, and the area of the wound to be created was outlined on the back of the animal
with methylene blue using a circular stainless steel stencil. A full thickness of the excision wound of circular
area of about 300 mm2 and 2 mm depth was created along the markings using
toothed forceps, a surgical blade and pointed scissors. The animals were distributed into three groups of six
each: group 1-control treated with 0.25% CM cellulose, group 2– reference standard-treated with sulfathiazole
ointment, group 3-experimental-treated with extract of J. grandiflorum leaves (250 mg/ kg/ day) till complete
epithelialization. The parameters studied were wound closure and epithelialization time. The measurements of
the wound areas of the wound were taken on day 1, 5 and 11 post-wounding using transparent paper and a
permanent marker. The recorded wound areas were measured with AutoCAD RL 14 computer analysis since it
was more accurate, reliable and less time consuming.
Dead space wound model
Dead space wounds were inflicted by implanting sterile cotton pellets (10 mg each), one on either side of the
groin and axilla on the ventral surface of each rat by the technique of D’Arcy et al as described by Turner7. The
animals were distributed into two groups of 6 each. The test group rats were given leave extract orally in their
drinking water at a dose of 250 mg/ kg daily for 10 days. An average, rat consumes 110 ml of water/kg/day, we
dissolved 250 mg of leave extract in 100 ml of drinking water. The control group animals were administered
with plain drinking water. On the 10th post wounding day, the granulation tissue formed on the implanted
cotton pellets was carefully removed under anaesthesia. The wet weight of the granulation tissue was noted.
These granulation tissues were dried at 60°C for 12 hours, weighed and recorded the dry weight. To the dried
tissue 5 ml of 6N HC1 was added and kept at 110°C for 24 hours. The neutralized acid hydrolysate of the dry
tissue was used for the determination of Hydroxyproline8. Additional piece of wet granulation tissue was
preserved in 10% formalin for histological studies.
Estimation of Hydroxyproline
Dry granulation tissue from both control and treated group was used for the estimation of hydroxyproline.
Hydroxyproline present in the neutralized acid hydrolysate were oxidized by sodium peroxide in presence of
copper sulfate and subsequently they were complexed with para-dimethylaminobezaldehyde to develop a pink
color which was measured at 540 nm by spectrophotometery.

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Statistical analysis
The means of wound area measurements between groups at different time intervals was compared using a one-
way ANOVA, followed by Dennett’s comparison test.. One-way ANOVA was used to examine the mean
differences in wound healing between the groups in incision and dead space wound models.
Results and Discussion
Wound healing is an extreme complex phenomenon involving a number of well-orchestrated processes,
including regeneration of parenchymal cells, migration and proliferation of both parenchymal and connective
tissue cells, synthesis of extracellular matrix protein, remodeling of connective tissue parenchymal components,
collagenization and acquisition of wound strength. The LD50 of J. grandiflorum leave extract was found to be
2500 mg/kg, b.w. In the excision wound model (Table I, Fig. 1), extract treated rats showed 65% reduction in
the wound area when compared to controls which was 54%. The extract treated wounds were found to
epithelize faster as compared to controls. This was comparable to the study done by Popova et al in which they
reported the physiological regeneration and epithelialization using fractions isolated from Calendula
officinalis9. The wet and dry granulation tissue weight, and hydroxyproline content in a dead space wound
model increased significantly when compared to controls (P<0.001). (Table 2, Fig. 2) Upadhya and others
noticed the similar wound healing effects with the leaf extract of J.grandjflorum10.The estimated increase in
hydroxyproline content of the granulation tissue indicated rapid collagen turnover thus, leading to rapid healing
of wounds11. The above mentioned prohealing actions of the extract may be due to the constituents present in it.
Many researchers reported the similar type of prohealing actions of constituents present in Aloe Vera,
Peperomia galioides, Anredera diffusa and Jatropha curcas12,13. Histological studies of the tissue obtained on
day 10 from the extract-treated animals showed increased well organized bands of collagen, more fibroblasts
and few inflammatory cells when compared to controls which showed inflammatory cells, scanty collagen
fibres and fibroblasts. The J. grandiflorum is known to contain methyl anthranilate, indol, benzyl alcohol,
benzyl acetate, and the terpenes linalol and linalyl acetate. The present study has demonstrated that an ethanol
extract (50%) of J. grandiflorum leave has properties of promoting wound healing activity compared with
controls. Wound contraction and increased hydroxyproline content support the J. grandiflorum in the topical
treatment and management of wounds.
Table -1: Wound healing activity of J. grandiflorum leaves in the excision wound model.
Parameter
(Wound area in mm2) Control Standard Experimental
Day 1 260.40± 0.160 260.60± 0.22 260.60± 0.22
Day 5 176.44±0.17 (32%) 171.40±0.15 (35%) 163.44± 0.17 (37%)
Day 11 80.91± 9.50 (54%) 59.36± 11.16 (65%) 57.00±0.51** (65%)
Epithelialization time (days) 16.60± 0.30 14.2± 0.13 12.30± 0.13**
n=6 *P.<0.05, **P<0.001 vs. control. Values are mean ±SE.

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Table -2: Wound healing activity of J. grandiflorum leaves in the dead space wound model.
Parameter Control Experimental
Wet granulation tissue
weight (mg/100 g rat) 128.2 ± 4.20 395.9 ± 3.4**
Dry granulation tissue
weight (mg/100 g rat) 30.3 ± 0.68 60.0 ± 1.2**
Hydroxyproline (mg/g
tissue) 48.3 ± 2.29 95.1 ± 1.4**
n=6*P.<0.05, **P<0.001 vs. control. Values are mean ±SE.
Fig.1
Fig.2

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Conclusion
The repair of wounds involves different phases including contraction, the formation of epithelialization and
fibrosis. The biological response regulating the body’s own cellular defense mechanisms contributes to the
wound and its repair.14 Thus from this study it is concluded that the J. grandiflorum leaf extract has a
reproducible wound healing potential and thereby justifies its use in folklore medicine in India.
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