Many plants have been traditionally utilized for curing male pattern baldness.
However, in most instances the efficacy was not determined (Takahashi
et al., 1998). E. cottonii is an edible species of Malaysian
seaweeds obtained from Sabah waters (Semporna) which have been exposed as a
novel sources for varieties of compounds such as dietary fibers, vitamin C,
α-tocopherol, minerals, fatty acid and protein (Matanjun
et al., 2008).
Seaweeds also are considered to be rich source of antioxidants. The potential
antioxidant compounds were identified as some pigments (fucoxanthin, astaxanthin,
carotenoid) and polyphenols (phenolic acid, flavonoid, tannins) (Chew
et al., 2008; Chandini et al., 2008).
The biological activity of antioxidants has been documented as anticoagulant,
anti-tumour, anti-inflammatory and antidiabetics (Hyun et
al., 2006). Takahashi et al. (1998) also
showed proanthocyanidins, a species of condensed tannin, extracted from grape
seeds is an active element in treatment of androgenetic alopecia. The objective
of this study was to evaluate the potential wound healing and hair growing properties
of ethanolic and aqueous extracts of Eucheuma cottonii.
MATERIALS AND METHODS
E. cottonii extract preparation: E. cottonii was collected
from the coastal areas of Semporna (Sabah, Malaysia). In the laboratory, the
fresh seaweeds were cleaned, washed with distilled water and their holdfasts
and epiphytes were removed. The seaweed was then dried at 40°C in dark room
for 3 days and then was grounded to fine powder using a Warring miller to be
able to pass through a 0.5 mm screen. Subsequently, it was stored in air-tight
containers covered by aluminum foil at -20°C. Plant extract was prepared
from the fine powder within a month. Ethanol and water extracts were prepared
based on method of Ponce et al. (2003) with modification.
The ethanolic extract was prepared from milled seaweed (200 g) using 1000 mL
of 80% (v/v) ethanol under mechanical stirring at room temperature for 24 h
and filtered. The residue was then dissolved in 3000 mL distilled water, stirred
at room temperature (25±2°C) for 8 h and then filtered. Subsequently,
the ethanolic and aqueous extracts were concentrated under negative pressure
at 40 and 70°C for 1 h, respectively. Both extracts were then oven dried
at 40°C overnight which produced powdered extracts. The powdered extracts
were then stored in air-tight containers at -20°C until application.
Animal and design: Forty eight 10-12 week old healthy male Sprague-Dawley rats weighing 300-350 g were used for wound healing and hair growth experiment. They were allotted randomly into four groups with twelve animals. The rats were acclimatized for 1 week prior to the study. Animals were housed in individual cages and kept in a ventilated room with temperature regulated at 23±2°C, with a 12 h light and 12 h dark cycle. They were fed with commercial rat pellets and water was supplied ad libitum. Experimental procedures and animal care had been approved by the Institutional Animal Care and Use Committee (IACUC), Faculty of Veterinary Medicine, Universiti Putra Malaysia (UPM/FPV/PS/184.108.40.2061/AUP-R-17).
Wound formation and treatment: Treatment groups were comprised of one positive and one negative control groups and 2 groups of animals treated with powdered extraction dissolved in distilled water to the desired concentrations. Four treatment groups in this study were Group A; animals treated by PO administration of ethanolic extract of seaweed, Group B; animals in this group received oral treatment of aqueous extract of seaweed Group C; positive control group that was treated by PO administration of honey and Group D; negative Control group which treated orally with distilled water. The rats were anaesthetized with ketamine HCL (120 mg kg-1 body weight) prior to wound induction. Wound site at dorsal interscapular area was prepared and shaved preceding to surgery. A full thickness circular wound with an area of 300 mm2 was incised. Immediately after wound induction, it was washed with normal saline and covered by a film dressing. Oral treatments commenced soon after wound creation. Rats were fed 100 mg kg-1, SID with extracts of E. cottonii using gastric tube.
Sampling, wound healing and hair growth evaluation: Wounds were traced
on 1 mm2 graph paper on the day of induction and planimetrically,
every 3 days for 2 weeks. Wound contraction was calculated as percent reduction
in wound area (Nayak et al., 2005). Half rats
in each group were sacrificed on day 15 post operation with ketamine overdose
and the rest maintained to be check for period of epithelization. Collected
samples were rinsed with 0.2 M phosphate buffered saline (PBS, pH 7.4) and fixed
in 4% paraformaldehyde. They were then embedded in paraplast wax after further
processing and sectioned into 5 μm thick ribbons. The tissue samples were
then mounted stained with hematoxylin and eosin (H and E) and observed under
light microscope. Samples were evaluated for extent of wound contraction and
Statistical analysis: All results were revealed as means±Standard Deviation (SD) and were analyzed by one-way Analysis of Variance (ANOVA) followed by Duncan multiple range tests. All procedures were performed at 95% confidence level using the Minitab 14.
RESULTS AND DISCUSSION
Wound contraction: Wound contraction is the factor indicating rate of
reduction of unhealed area during the period of treatment. Wound contraction
occurs as the myofibroblasts contract. Increased wound contraction in treated
rats might be a result of enhanced activity of fibroblasts in the treated rats.
Wound contraction is mediated by specialized myofibroblasts found in the granulated
tissue (Moulin et al., 2000). Table
1 shows the rate of wound contraction expressed in terms of the percentage
of healed wound area. On 15th day there was a significantly difference in percentage
of wound closure (p<0.05) between the control and the test groups. It is
manifested that group. A rats showed 100% healing, whereas group B rats showed
83.44% and group C showed 93.76% when compared to the controls (52.66%). The
least percentage of wound healing was seen in negative control group which had
not received any treatment.
Period of epithelization: Some of the phases of healing such as wound
contraction and epithelialization run concurrently and independently (Kaushal
et al., 2007). Epithelization is the process in which epithelial
cells around the margin of the wound or in residual skin appendages (hair follicles
and sebaceous glands) begin to migrate into the wound by the process known as
epiboly (Stenn and Cotsarelis, 2005).
||The wound healing (%) of different groups of rats fed with
E. cottonii extracts over a period of 15 days
|Value are mean±SE of 6 animals in each group; means
with different superscripts within a column were significantly different
at p<0.05; (A, ethanolic extract group; B, aqueous extract group; C,
treated group with honey; D, no treated group)
Re-epithelization had taken 9 days in ethanolic extract treated groups in
contrast to 20 days in the control group. On the 15 day post treatment, all
groups completely covered with fibrin except for the negative control group.
It can be concluded that effect of E. cottonii extract oral treatment
on skin lesions could accelerate the reepithelization and remodelling phases
Percentage of hair growth: In this research, hair growth on shaved skin
near the wound site was monitored. As reported by Geoffrey
et al. (2008), dermal structures such as hair follicles, sweat glands
and sebaceous glands that are lost during injuries would not be regenerated.
Group which was treated with ethanolic extract of E. cottonii (Group
A) showed the best result for hair growth. In this group 95-100% of the shaven
area showed hair re-growth. Growth of hair in rats treated with aqueous extract
of the seaweed (Group B) was less than that of the rats treated with honey (the
percentages of covering were 45-50% and 75-80%, respectively).
||Period of epithelialization (day) of rats fed with E. cottonii
|Values are mean±SD (p<0.05); (A: Ethanolic extract
group; B: Aqueous extract group; C: Treated group with honey; D: No treated
Control group showed the least hair growth (25-30%). Figure 1
shows the faster wound healing and hair growth in ethanolic extract of the seaweed.
There was a direct relation between hair follicle growth and dermis repair approach.
Hair follicle dermal cells produce wound healing fibroblasts and their participation
in wound healing reduces the probability of scarring. Fibroblasts are prominent
in granulation tissue and important for wound contraction (Naik-Mathuria
et al., 2008). Hair follicle epithelial stem cells and its contribution
to wound healing have attracted particular attention (Taylor
et al., 2000). Jahoda and Reynolds (2000)
have shown that labeled dermal sheath cells introduced into skin wounds make
up parts of healed dermis several weeks later and could also create a dermis
from dermal sheath cells in rodents which supports growth of a normal-looking
epidermis. Taylor et al. (2000) also suggested
that hair follicle dermal sheath cells act as specialized progenitor fibroblast
populations that are activated in response to wound induction. In another major
study, Naik-Mathuria et al. (2008) found that
these myofibroblasts are also prominent in granulation tissue and important
in wound contraction procedure.
Hair follicle counts: Table 3 shows the average number
of hair follicles, area size of hair bulb and length of hair follicle around
the wound site in animals treated with various extractions of E. cottonii
at day 15.
||Photograph showing the hair growth in the different groups
at the 15 day post treatment. Group A treated with ethanolic extract of
E. cottonii, Group B treated with aqueous extract of the seaweed
in Group C honey was used and Group D received distilled water. Shaved skin
site covered with hair completely only in Group A
||Number of hair follicle, area size of hair bulb and length
of hair follicle around the wound in rats treated with various extractions
of E. cottonii at day 15
|Means with different superscripts (a-d) within a row were
significantly different at p<0.05, (A, ethanolic extract group; B, aqueous
extract group; C, treated group with honey; D, no treated group)
||Micrographs of hair follicle after 15 days oral treatment
in different treated groups (H and E stain; x40). Group A treated with ethanolic
extract of E. cottonii, Group B treated with aqueous extract of the
seaweed in Group C honey was used and Group D received distilled water
Ethanolic extract of seaweed (Group A) showed a significant increase (p<0.05)
in hair follicle number at 15 day post treatment. All major structures of the
hair follicle were in mature phase with biggest bulb and maximum length. In
aqueous extract group (Group B) there were early signs of newly formed hair
follicles with an averages number of hair follicles. Hair follicle had a smaller
bulb and a narrower dermal papillaries. Hair follicle length was shorter in
comparison with the ethanolic extract group. In the positive control group there
was modest numbers of newly formed hair follicles whereas negative control group
revealed the least population of follicle (Fig. 2). The results
manifested earlier show the potentials of seaweed ethanolic extracts in boosting
hair growth. It contains flavonoids which are antioxidants and undetermined
substances which might cause proliferation of fibroblasts. Takahashi
et al. (1998) described the potent positive influence of flavonoids
on hair growth. They purified proanthocyanidins linked by catechin and epicatechin
which possessed higher proliferation and hair re-growth activities.
Oral application of E. cottonii extracts could enhance cutaneous healing
however it showed a faster trend in ethanolic extract which manifested a complete
healing in 15 days. Also this group showed a faster hair growth rate during
the time. There is a direct association between hair follicle growth and rate
of dermis repair. It is shown that skin containing large actively growing follicles,
heals more rapidly than skins with not well growing well follicles. Literature
scan showed that phenotype of dermal sheath cells is closely relevant to dermal
wound healing. Although, the controversies regarding nature and origin of wound
healing fibroblasts, it is widely believed that specialized wound healing cells
(myofibroblasts) are prominent in granulation tissue formation and wound contraction
(Naik-Mathuria et al., 2008).
This study reported effectiveness of E. cottonii extracts in management of wound healing, hair growth and androgen-induced alopecia in the shortest possible period of time. It gives the impression that E. cottonii extracts (especially ethanolic form) could be used as a proper treatment approach in healing procedures and hair re-growth instances successfully and effectively however, further studies with purified components are required to appreciate the complete mechanism of wound healing and hair growing activity of ethanolic extract of E. cottonii.
Researchers would like to thank Mr. Kufli Che Noor for his supports in the animal study. This study was supported by the Ministry of Higher Education Fundamental Research Grant Scheme (project no: 91030).