2014 IEEE Conference on Biomedical Engineering and Sciences, 8 - 10 December 2014, Miri, Sarawak, Malaysia
Evaluation of Hibiscus rosa-sinensis Leaves Extracts as Wound Healing
Promoter on Rats
A. A. Ali, N. H. Jusoh, N. Saridin, M. S. A. Wahab and R. M. Zohdi
Abstract— The present study was designed to evaluate the
potential of aqueous and ethanol extracts of Hibiscus rosa-
sinensis leaves as wound healing promoter. The rats were
randomly divided into 5 groups of 6 rats each. Rats were
anesthetized using ketamine/xylazine cocktail and full thickness
incision wounds were created on the dorsal part of the rats. The
animals were topically treated with water and ethanol extracts
at a dose of 0.01g/ml once a day for 15 consecutive days. A
positive control group was treated with cetrimide 2% solution
and one group served as untreated controls. In another group,
wounds were left open without suture and further treatment.
The tensile strength and wound healing process was evaluated
macroscopically and histologically after 2 weeks. Wounds
treated with both Hibiscus rosa-sinensis leaves water and
ethanol extracts showed better healing with slightly visible fine-
line scar. Histological evaluation showed that wound treated
with both Hibiscus rosa-sinensis leaves extract was
reepithelialised, granulation tissues in the wound were nearly
replaced by fibrosis and hair follicles were almost healed. The
arrangement of collagen fibres in the skin treated with both
extracts was similar to conventional treatment. Moreover the
ethanol extracts of Hibiscus rosa-sinensis leaves-treated skin
had the highest tensile strength. In conclusion, both water and
ethanol extracts of Hibiscus rosa-sinensis are potentiate in
modulating the wound healing process and therefore could be a
possible alternative to conventional treatments.
INTRODUCTION
Wounds are one of the most widespread injuries in
accidents and remain a global public health issue [1].
Although many advances have been made in our
understanding and care of wound injuries, there are still
many wounds healed with scar formation, resulting in
significant aesthetic disfigurement and dysfunction [2].
Research supported by Research Management Institute (RMI), Universiti
Teknologi MARA (UiTM).
A.A. Ali is with the Department of Pharmacology & Pharmaceutical
Chemistry, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak
Alam, Selangor and Research on Affinity, Safety and Efficacy Studies,
Brain and Neuroscience Communities of Research, Universiti Teknologi
MARA, Shah Alam, Selangor, MALAYSIA (corresponding author phone:
603-32584784; fax: 603-32584602; e-mail:
aidaaz2790@puncakalam.uitm.edu.my).
N.H. Jusoh and N. Saridin were with Faculty of Pharmacy, Universiti
Teknologi MARA, Puncak Alam, Selangor. They are now with the
Ministry of Health, MALAYSIA.
M.S.A. Wahab is with the Department of Pharmacy Practice, Faculty of
Pharmacy, Universiti Teknologi MARA, Puncak Alam, Selangor,
MALAYSIA (e-mail: mohdsh2790@puncakalam.uitm.edu.my).
R.M. Zohdi is with the Department of Pharmaceutical Life Sciences,
Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam,
Selangor, MALAYSIA (e-mail: rozainizohdi@puncakalam.uitm.edu.my).
978-1-4799-4084-4/14/$31.00 ©2014 IEEE
Skin wound healing is a dynamic process in which
different cell types, such as fibroblasts, leukocytes, and
monocytes/tissue macrophages, as well as endothelial cells
and epidermal cells cooperate to restore the integrity of an
injured body surface [3]. The phases of normal wound
healing include homeostasis, inflammation, proliferation,
and remodelling [4]. After injury, blood vessels are
disrupted resulting in extravasations of blood components
such as proteins, coagulation factors and platelets. There is
still a need for effective and cost-effective therapies to
promote wound healing. Natural products offer an
alternative that needs to be developed [5.6].
Hibiscus rosa-sinens belongs to the family
Malvaceae. It is a glabrous shrub widely cultivated in the
tropics. It contain anthocyanins and flavonoids; cyanidin-3,
5-diglucoside, cyanidin-3- sophoroside-5 glucoside,
quercetin-3, 7-diglucoside, quercetin-3-diglucoside [7]. The
leaves are simple ovate or ovatelancolate, and are entire at
the base and coarsely toothed at the apex. Traditionally, this
plant is used for the control of dysfunctional uterine bleeding
and as an oral contraceptive [8]. Leaves extract of Hibiscus
rosa-sinens have been shown potentially to be effective in
maintaining hair growth in in-vivo and in-vitro methods [9].
Additionally, the aqueous-ethanolic extract of aerial parts of
Hibiscus rosa-sinensis was used in constipation and diarrhea
[10]. Cold aqueous extract of Hibiscus rosa-sinensis leaves
has also been reported by local traditional practitioners in
Western Nigeria to have aphrodisiac effect [11].
Anti-implantation of blastocyst activity of water
extract of leaves of Hibiscus rosa-sinensis was reported [7].
It has also been investigated that Hibiscus rosa-sinensis
extract exerts a protective effect against the tumour
promotion stage of skin cancer development [12]. Hibiscus
rosa-sinensis has been used for the treatment of a variety of
diseases as well as to promote wound healing. The wound-
healing activity of the ethanol extract of Hibiscus rosa-
sinensis flower was previously examined in rats, using
excision, incision, and dead space wound models [5]. This
study was carried out to investigate the healing properties of
Hibiscus rosa-sinensis Linn leaves aqueous and ethanol
extracts by histological and tensile strength studies.
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 2014 IEEE Conference on Biomedical Engineering and Sciences, 8 - 10 December 2014, Miri, Sarawak, Malaysia

I. MATERIALS AND METHODS E. Measurement of tensile strength

The tensile strength was measured TA XT-plus Texture
A. Plant collection
Analyser machine. The distance between the two grips was
Hibiscus rosa-sinensis (HRS) plant materials for this
30 mm and the crosshead speed was 100 mm/min.
study was collected around the university campus following
confirmation of the taxonomic identity of the plants by the
horticulturalist.

B. Preparation of the extracts II. RESULTS

Two types of extracts were prepared in this experiment
which was aqueous and ethanol extracts. For aqueous A. Histological examination
extract, 500 g of the powdered leaf was macerated in 1.5 L
of distilled water. The extracts are then were filtered several Figure 1 : Photomicrograph at 2 weeks post-wounding. (A) Control group,
times through a sieve. Extract was evaporated to dryness (B) Opened wound group, (C) Cetrimide group, (D) Aqueous extract and
using freeze dry and were weighed. For ethanol extract, 200 (E) Ethanol extract group. (H&E staining with manification 20x)
g of the powdered leaf is soaked with ethanol as a solvent
and leaved for about one day. The extracts are then filtered
and evaporated to dryness by using rotary evaporator. The
dose for treatment was 0.01 g/ml for both extracts.

C. Experimental animals and wound creation

30 healthy, female, Sprague–Dawley rats weighing180–
250 g were used and maintained in standard laboratory (A) (B)
conditions at animal house of Faculty of Pharmacy of
University of Teknologi MARA (UiTM). All surgical and
experimental animals were treated in accordance with the
criteria outlined in UiTM Animal Care guidelines, and the
protocols were approved by the UiTM Animal Research and
Ethics Committee (Reference No 18/2012).
(C) (D) (E)
Rats were anesthetized using an intraperitoneal injection
of ketamine (90mg/kg) and xylazine (10 mg/kg). The dorsal
skin was prepared by removing the hair with clippers and the
surgical area was disinfected with 70% ethanol. After the Figure 2 : Photomicrograph at 2 weeks post-wounding. (A) Control group,
dorsum of thoracic region of each rat was shaved, a 30 mm (B) Opened wound group, (C) Cetrimide group, (D) Water extract and (E)
midline incision was made through the full skin thickness Ethanol extract group (Masson’s trichrome staining with manification 20x)
with a sharp surgical scalpel. Each incised wound except for
the open wound model was closed with silk sutures to secure
the edges of the incision wound.

After wound creation, experimental animals were

randomly divided into the following groups (n = 6):
Group I: wounds left untreated (controlled group),
Group II: wounds treated with 2% cetrimide, (A) (B)
Group III: wounds treated with HRS aqueous extract,
Group IV: wounds treated with HRS ethanol extract
Group V: wounds left open without suture and further
treatment.

The treatment groups received topical application of 0.5 ml

of cetrimide, HRS aqueous and ethanol extracts respectively (C) (D) (E)
once a day till for 15 days.

D. Histological analysis
Tissue samples were fixed in 10% formalin, then embedded
in paraffin following dehydration and stained by
hematoxylin-eosin or Masson’s trichrome reagents.

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 2014 IEEE Conference on Biomedical Engineering and Sciences, 8 - 10 December 2014, Miri, Sarawak, Malaysia
Table 1 : Tensile strength of the wounded skins treated with different
treatments.
Experimental Groups Tensile strength(g/mm2)
Control 48.039 ± 8.58
Cetrimide 10% solution 62.433 ± 7.33 *
Water extract of hibiscus 56.322 ± 10.17
Ethanol extract of hibiscus 62.855 ±12.41 *
Open wound 39.492 ± 13.45
* (p<0.05) compare with the control group
III. DISCUSSION
Wound healing is an orderly process of repair that
follows injury to the skin and other soft tissues. Wound
healing occurs in 4 stages: homeostasis, inflammation,
proliferation, and remodelling. This study was focused on
the proliferative phase that usually occurs after 2 weeks.
Proliferative phase is dominated by the formation of
granulation tissue and epithelialization. Chemotactic and
growth factors released from platelets and macrophages
stimulate the migration and activation of wound fibroblasts
that produce a variety of substances essential to wound
repair [4].
No significant difference was observed between
Hibiscus rosa-sinensis leaves water and ethanol extract
demonstrating that both extracts have the same effectiveness
in enhancement of the wound healing process. To the best of
our knowledge, this is the first study reporting on the
efficacy of Hibiscus rosa-sinensis leaves extracts on incision
wound healing. Therefore, this study can be the one in
proving the effectiveness of the water extract in incision
wound healing. Meanwhile, a study by [5] has demonstrated
that an ethanol extract of Hibiscus rosa-sinensis flower has
properties that render it capable of promoting accelerated
wound healing activity. The incised wound of the both
extracts treated groups showed a complete reepithelialised
and granulation tissues were nearly replaced by fibrosis and
hair follicles were almost healed. Meanwhile for control and
open wounded groups, the incised wound was partially
reepithelialised and necrotic tissues were not completely
replaced by granulation tissues. Besides that, the collagen
fibres were showed a fine thickness in the wound treated
with both extracts and the arrangement was almost similar to
that of cetrimide (positive control) group as compared to the
control and open wounded group which shown abnormal
thickness and disordered fibres in the wound.
Stimulation of wound strength is one of the
important factors in the healing process of the wound.
Tensile strength indicates how much the repaired tissue
resists from breaking under tension. It may also indicate the
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quality of the repaired tissue. Collagen is a major protein of
the extracellular matrix and is the component that ultimately
contributes to wound strength [13]. Besides that, collagen is
also responsible for the mechanical properties of the skin.
The amount of wound collagen deposition will depend and is
a reflection of collagen synthesis minus collagen breakdown.
[14]. The changes in the collagen fibrils and the diameter
will also affect the strength of skin. Evidently thick collagen
fibrils will resist greater tensile strength as compared to thin
collagen fibrils [15]. Once the skin is injured, the scar
collagen will replace the normal collagen and the original
highly organized and the strength will not regain. Thus, the
healing skin is weaker and result in weaker tensile strength
as compared to the normal skin [14].
In the present study, there are significant
differences in tensile strength between the groups. The
tensile strength of the cetrimide, water extracts and ethanol
extract groups were higher compared to the control group.
The study done by Nayak et al., [5] has also proven the
wound healing ability of the ethanol extract. Because of only
small differences in the tensile strength of the cetrimide
compared to the ethanol extract, it shows that, they exhibit
the same role in wound healing. In conclusion, both water
and ethanol extracts of Hibiscus rosa-sinensis are potentiate
in modulating the wound healing process and therefore
could be a possible alternative to conventional treatments.
ACKNOWLEDGMENT
We would like to acknowledge Faculty of Pharmacy,
UiTM Puncak Alam and Research Management Institute
(RMI) UiTM, Shah Alam. This research is supported by
Ministry of Education (MOE) under the Research
Acculturation Grant Scheme (RAGS) fund with project
code: 600-RMI / RAGS 5/3 (107/2012).
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