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RESEARCH ARTICLE
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Astaxanthin and Wound healing
AtiyaRungjang MD1, SaranyooPonnikorn PhD2,SaorayaLueangarun MD
MSc1, WerayutYingmema DVM3, JitladaMeephansan MD PhD1
1
Division of Dermatology, Chulabhorn international college of medicine,
Thammasat University, PathumThani, 12120, Thailand
2
Chulabhorn international college of medicine, Thammasat University,
PathumThani, 12120, Thailand
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Laboratory Animal Centers, Thammasat University, PathumThani, 12120,
Thailand
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Abstract
Wound healing is a complex reaction of the organism to injury. The
successful wound healing requires the execution of three major overlapping phases:
inflammation, proliferation, and remodeling. Reactive oxygen species (ROS) are
involved in all phases of wound healing. It is shown that large amount of ROS,
which is called oxidative stress, is harmful to these processes. The correct balance
between oxidative and antioxidative forces is needed for favorable wound healing.
Astaxanthin, a member of the xanthophyll group, is a red-orange carotenoid. It is
recognized as a most powerful antioxidant. In this study, we investigated the effect
of topical astaxanthin on cutaneous wound healing. Full-thickness dermal wound
was done in 36 healthy female mice then divided into two groups. Mice were
treated with topical astaxanthin 78.9 uM (5% extraction) or vehicle twice daily for
15 days. Wound areas were determined on serial photographs on post injury days 1,
3, 6, 9, 12, and 15. We found that astaxanthin significantly accelerated wound
closure in treatment mice compared to control group. Astaxanthin treatment
improves wound healing since contraction takes place earlier in astaxanthin-treated
in comparison to vehicle-treated mice by wound area assessment method. These
results suggest that astaxanthin may have an effect on improving cutaneous wound
healing.
Keywords:wound healing, astaxanthin, fibroblast
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Introduction
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All phases in skin wound healing are involve with reactive oxygen species (ROS).
ROS may promote wound angiogenesis by inducing VEGF expression in woundrelated cells such as keratinocytes and macrophages (6). However, excess
quantities of such compounds are dangerous due to their very high reactivity
because they may react with various cellular components such as proteins, lipids,
carbohydrates, and DNA. This situation may cause oxidative damage that are also
involved in many natural and pathological processes, including aging cancer,
diabetes mellitus, atherosclerosis, neurological degeneration, angiogenesis, and
metastasis(7). Overexposure to ROS deleterious to wound healing process(8) due
to the harmful effects on cells and tissues. To inhibit wound site injury by oxidative
stress, antioxidants have been applied to balance oxidative stress on the wound
sites (9). Kumin et al. Reported that the over expression of peroxiredoxin, which is
an antioxidant enzyme that quench free radicals resulted in the enhancement of
wound closure in aged mice.(10)
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Astaxanthin, a biological antioxidant, is a pigment in xanthophylls family, the
oxygenated derivatives of carotenoids whose synthesis in plants derives from
lycopene. Common sources of natural astaxanthin are the green algae
haematococcuspluvialis and the red yeast, phaffiarhodozyma, It was reported to
exhibit strong free radical scavenging activity and to protect cells against lipid
peroxidation and the oxidative damage(11). Lee et al. Demonstrated that
astaxanthin inhibited the expression of a number of pro-inflammatory mediators
(such as nitric oxide),(12). Also, health benefits such as cardiovascular disease
Wound healing is a complex reaction of the organism to physical injuries that result
in an opening or break of the skin. Various cell types including leucocytes,
keratinocyte, fibroblasts, and macrophages and cytokines are involved in this
dynamic process that results in the closure of the wound and restoration of a barrier
function.(1-3) Repair of injured tissues occurs as a sequence of events briefly
divided into three overlapping phases “inflammation, proliferation and
remodeling”. Coagulation and inflammatory phase: cutaneous injury affects
primarily to the epithelial and endothelial compartments result in coagulation
cascade forming blood clot and the release of proinflammatory mediators. Blood
clot within the vessel lumen provides hemostasis; the clot within the injury site acts
as a provisional matrix for cell migration, further formation of new extracellular
matrix (ECM), a reservoir for cytokines and growth factors. Inflammatory white
cells functions are debridement of necrotic material and bacteria and production of
certain critical cytokines. 24–48 hours after injury, monocytes replace neutrophils
and change to tissue macrophages that phagocytose and kill bacteria, scavenge
tissue debris and release several growth factors stimulating migration and
proliferation of fibroblasts, endothelial cells and keratinocytes and production and
modulation of extracellular matrix “reepithelialization and angiogenesis”(3) of
proliferation/migration phases take place. The remodeling phase begins 5–7 days
after injury to breakdown of excess macromolecules. Cells within the wound are
returned to a stable phenotype and extracellular matrix material is altered.(4, 5)
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prevention, immune system boosting, bioactivity against helicobacter pylori, and
cataract prevention (13-15)
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The objective of the present study was to evaluate the effect of topical astaxanthin
extract on full-thickness cutaneous wound healing in mice model. We assess the
change of wound area during the process of wound repair.
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Materials and Methods
Drugs
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The astaxanthin material, composed of 78.9µMol (5.0%w/w) of astaxanthin
extracted from Haematococcuspluvialis, was supplied by China Jiangsu
International Economic and Technical Cooperation. The vehicle was Chremophore
RH 40 Glycerin.
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Animals
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Young female Balb/c mice (8-week-old) were procured fromNational Laboratory
Animal Center. A total of 36 studied mice were assigned to treatment group (N=18)
and control group (N=18). During the experiments, the animals were housed under
Strict hygienic conventional standard, maintained under controlled environmental
conditions (12-hour light/darkcycle, temperature approximately 23°C ), and
provided with standard laboratory food and water ad libitum. Study protocol was
approved by Thammasat University’s Animal Ethical Committee and conducted
according to Ethical Principals and Guidelines for the Use of Animals for Scientific
Purpose
Full-thickness wounds were created on the back of mice under sterile conditions.
Mice were anesthetized with inhaled Isofurane before the procedure. After shaving
and cleaning with70% ethanol, the dorsal skin was picked up and punched through
two layers of skin with a sterile disposable biopsy punch (4 mm in diameter) to
generate two wounds on the dorsal skin. Each wound site was digitally
photographed until complete wound closure was observed as shown in Figure1.
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The wounds were treated topically twice daily with 5% astaxanthin in treatment
group and vehicle in control group (0.025mL/wound). A digital image of each
wound with a scale was daily recorded, For the wound contraction study, A
template containing a10mm diameter circular window was used to standardize the
size of each wound and wound areas were determined on photographs using Adobe
Illustrator CS3. Changes in wound areas overtime were expressed as the percentage
of the decrease wound areas. The mice were humanely euthanized with 5-8%
Isofurane at experimental end-point.
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Statistical analysis was done using SPSS statistic program. The data were
expressed as mean ± standard deviation. Mann–Whitney U-test was conducted for
comparisons. A p value of < 0.05was considered a statistical significance.
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A
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a.
B
Figure1. Wound measurement and analysis. A,On day 0, two circular excisional
wounds (4 mm in diameter) were created in the dorsal skin of female Balb/c mice.
B. Each wound site was digitally photographed at the indicated time intervals, and
wound areas were determined on photographs using Adobe Illustrator software.
Changes in wound areas over time were expressed as the percentage of the wound
reduction.
b.DAY
Figure 2.Accelerate wound healing in Astaxanthin treated mice.
a. Representative photographs from astaxathin treated and control mice showing
the macroscopic wound closure on different days postinjury. The size of wounds
was determined from photographs. b. At the time points indicated, the wound area
was determined using image analysis and expressed as mean wound area of
astaxanthin-treated mice(A) in comparison with the control group(C).
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Results
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Wound healing process is regulated by numerous factors, including cells, growth
factors, cytokines, and hormones.(1, 16) During inflammation, tissue formation,
and tissue remodeling ROS are involved in all of these phases.(17)In addition to
these, several studies have demonstrated that the appropriate balance of ROS is an
important factor in the wound healing(18, 19), as they provide defense against
invading microorganisms and assist in cellular signaling. When the generation of
free radicals exceeds the capacity of the defenses, these highly active radicals may
produce structural changes that may contribute to reversible or irreversible cell
injury which is called oxidative stress. For example, it has been shown that excess
amount of H2O2 impairs wound closure, whereas low doses fairly facilitated
closure.(10, 20)This suggested that the favorable wound healing requires a delicate
balance between oxidative and anti-oxidative forces. Many studies have reported
using astaxanthin as an antioxidant.(21-23) Our study results support that
astaxanthin, a powerful quencher of reactive oxygen and nitrogen species shorten
the period of wound healing by clinical assessment. Thus, further study, for
example, histology and molecular evidence may influence astaxanthin as a novel
redox-based strategies to treat wounds. In addition, Mizuta and colleagues have
demonstrated that the expression of procollagen type1 increased significantly in the
astaxanthin-treated group.(19) Collagen is essential as a scaffold for wound
healing. Therefore, astaxanthin may promote healing through inducing expression
of procollagen type 1.
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Astaxanthin accelerated healing of full-thickness dermal wounds. Astaxanthin 5%
extract applied topically on the wounds showed significant acceleration of wound
closure that was observed at the 5th and 7th day of the experiment (figure.2a).
We evaluated wound changes in female Balb/c mice. Two full-thickness skin
circular excision wounds were created on the backs of mice (figure. 1A). Wound
areas were analyzed throughout the healing process (figure. 2). Wound closure was
accelerated in astaxanthin treatment group. On day 9 after wounding, the
astaxanthin-treated wounds had already lost their eschars and appeared completely
epithelialized, whereas the wounds of control mice demonstrated only partial
epithelialization and still carried scab. A complete wound closure of control group
was observed by day 12 after the injury.
Statistical analysis indicated that wound closure in astaxanthin-treated mice was
significantly accelerated as compared with control mice at p value <0.05.A
significant difference of wound area between 2 groups was observed on day of the
study.
Discussion
Conclusion
In the present study, Astaxanthin accelerates healing of full-thickness dermal
wounds. Considering the obtained results, we believe that the astaxanthin extracted
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from Haematococcuspluvialis, might become a useful alternative for the cutaneous
wound healing.
Acknowledgements
The authors are very thankful to the Laboratory Animal Center, Thammsat
university and its staff members for providing facilities and encouragement given
to carry out this work.
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