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ORIGINAL CONTRIBUTION
Cosmeceutical effect of ethyl acetate fraction of Kombucha
tea by intradermal administration in the skin of aged mice
Nafiseh Pakravan PhD
1
|
Elaheh Mahmoudi PhD
2
|
Seyed-Ali Hashemi PhD
3
|
Jamal Kamali BSc
4
|
Reza Hajiaghayi PhD
5
|
Mitra Rahimzadeh PhD
6
|
Vajiheh Mahmoodi MSc
7
1
Division of Immunology, Medical School,
Alborz University of Medical Sciences,
Karaj, Iran
2
Department of Parasitology and Mycology,
Medical School, Alborz University of
Medical Sciences, Karaj, Iran
3
Department of Pathology, Medical School,
Alborz University of Medical Sciences,
Karaj, Iran
4
Department of Operating Room Nursing,
Paramedical School, Alborz University of
Medical Sciences, Karaj, Iran
5
Herbal Medicine Research Center, Institute
of Medicinal Plant, ACECR, Karaj, Iran
6
Health, Safety, and Environment Research
Center, Alborz University of Medical
Sciences, Karaj, Iran
7
Department of Food Science and
Technology, Faculty of Agriculture, Islamic
Azad University, Shahrekord, Iran
Correspondence
Elahe Mahmoudi, Department of
Parasitology and Mycology, Medical School,
Alborz University of Medical Sciences, Karaj
Iran.
Email: e_m592000@yahoo.com
Funding information
Deputy of Research, Alborz University of
Medical Sciences
Summary
Background/purpose: Natural ingredients have been always an interesting approach
to prolong youthful appearance of skin. One of the natural compounds is Kombucha
tea (KT), which has been mainly used as an energy drink in Asian countries for a
long time. Previous reports indicated that it has pharmaceutical and favorable
wound repairing effects. The beneficial properties of KT are thought to be mainly
due to the presence of fermentation products such as flavonoids and other polyphe-
nols with inhibition of hydrolytic and oxidative enzymes and anti-inflammatory
effects. These properties prompted us to study the anti-aging potential of KT and
investigate its effective fraction in aged mice,
Methods: Kombucha tea was fractionated into chloroform, butanol, and ethyl acet-
ate, and flavonoid content was determined. Young and old mice were used as con-
trol. KT ethyl acetate fraction (KEAf), which had the highest flavonoid content, was
intradermally administered to old mice.
Results: Administration of KEAf significantly increased the collagen content, NAD
+
/
NADH level, and concomitantly improved skin connective tissue abnormalities in
the aged skin. No sensitivity or irritation was observed.
Conclusion: This finding suggested that KEAf can be a suitable candidate as a cos-
metic product to improve aging-related skin abnormalities and regeneration of aged
skin.
KEYWORDS
aging, collagen, ethyl acetate fraction, Kombucha tea, NAD
+
/NADH
1
|
INTRODUCTION
Skin aging is a biological process during which changes in the struc-
tural integrity and physiological function of skin are induced. Visible
sign of aging includes development of dyschromia, roughness, fine
wrinkles followed by persistent deeper folds.
1-3
These changes occur
following microscopic structural changes including epidermal thin-
ning, dermal atrophy, reduction in connective tissue, decreased num-
bers of keratinocytes, reduced levels of elastin and collagen
synthesis, increased level of collagenase/matrix metalloproteinase
(MMP), increased oxidants,
2-4
reduction in the number and size of
vascular vessels,
5
loss of subcutaneous fat and elasticity, and
increased melanogen.
6
Thinning and loss of collagen fibers, which
form 90% of wet weight of skin connective tissue, are other promi-
nent features of aging skin.
7,8
Anti-aging products are used to protect skin against aging pro-
cess. These agents are capable of protecting the skin matrix by inhi-
bition of enzymatic degradation or promotion of collagen synthesis
Accepted: 9 October 2017
DOI: 10.1111/jocd.12453
J Cosmet Dermatol. 2017;1–9. wileyonlinelibrary.com/journal/jocd ©2017 Wiley Periodicals, Inc.
|
1
in the skin.
8-10
Several efforts have been made to replace synthetic
anti-aging agents with natural alternatives. Of particular interest is
the use of traditional foods and medicines containing active ingredi-
ents.
11-14
One of these natural compounds is Kombucha tea (KT).
This so-called long-life mushroom tea has been known as popular
natural remedy for a long time and mainly used as an energy drink
in Asian countries. It is prepared from the fermentation of sugared
black tea (BT) with a symbiotic culture of acetic bacteria and Kom-
bucha fungi.
14,15
Previous reports indicated that Kombucha has valu-
able cosmetic activities when applied topically. Kombucha
specifically fights against glycation and the various gaps of skin
metabolism leading to improvement in the aging signs and cutaneous
microrelief.
16,17
The beneficial properties of KT are thought to be
due to the presence of vitamins, amino acids, and a variety of
micronutrients produced during fermentation. KT is especially a good
source of flavonoid and vitamin B3.
18-20
Flavonoids are important
anti-aging components that their amount in KT is much more than
BT.
21
They are a group of polyphenolic compounds with free radical
scavenging, inhibition of hydrolytic and oxidative enzymes, and anti-
inflammatory activities and capable of physically blocking UV pene-
tration, influencing DNA repair, and induction of cytoprotective
pathways in skin.
21-25
Vitamin B3 is a precursor of nicotinamide ade-
nine dinucleotide (NAD), which declines during aging leading to
reduction in collagen expression within cells.
21-23
Apart from cosmeceutical effects of KT, it has also been claimed to
have pharmaceutical effects and favorable wound repairing effects
when applied topically.
18-20
However, there are reports attributing
side effects to this beverage
26
and specifically, to date no report has
determined the effective fraction of KT. To reduce possible side
effects, determination of the effective anti-aging fraction of KT can be
beneficial. To do so, in this study KT was fractionated using organic
solvents and the effective anti-aging fraction was determined.
2
|
MATERIALS AND METHODS
2.1
|
BT and KT preparation
Black tea (Golestan, Tehran, Iran) was added to boiling water (1.2% w/
v), mixed, and left to brew for 5 minutes. The tea was then filtered
through a sterile sieve, and sucrose (10%) was dissolved in the tea. To
prepare KT, 200 mL of the cooled BT was inoculated with 3% w/v tea
fungus plus 10% v/v previously fermented KT liquid and left to fer-
ment by incubating the Kombucha culture at 28°C for 14 days. The
resultant fermented tea was centrifuged at 600 gfor 20 minutes.
19
2.2
|
Fractionation of KT
According to the method defined by Jayabalan,
19
3 fractions of KT
including KCf (Kombucha tea chloroform fraction), KBf (Kombucha tea
butanol fraction), and KEAf (Kombucha tea ethyl acetate fraction) (1/
2 v/v) were concentrated using a vacuum rotary evaporator (R-200
model of Buchi, Sigma-Aldrich, Taufkirchen, Germany). This process
led to preparation of a viscid mass, which was dissolved in distilled
water, filtered through 0.22-lMillipore membrane filter, lyophilized,
and stored at 20°C.
2.3
|
Content of total flavonoids in BT and KT
fractions
The aluminum chloride colorimetric method was used to calculate
the content of flavonoids.
27
Briefly, 1 mL (100 mg/mL) solution of
each of BT, KT, KEAf, KCf, and KBf was mixed with 3 mL methanol,
0.2 mL of 10% aluminum chloride, 0.2 mL potassium acetate (1 mol/L),
5.6 mL of distilled water and incubated for 30 minutes at room tem-
perature. Quercetin (Sigma-Aldrich, Taufkirchen, Germany) was pre-
pared in dilutions of methanol (250-1000 lg/mL) and used to
prepare standard curve using spectrophotometry at a wave length of
415 nm. Total content of flavonoid compound (lg of QE/mg sample)
was calculated by the formula: T=(C9V)/M,C=concentration of
quercetin (mg/mL), V=volume of solution (mL), M=weight of
methanolic extract (gr). KEAf had the highest flavonoid content and
was further used to test for its anti-aging effect.
2.4
|
Experimental groups and study design
A total of 54 female NMRI mice were purchased from Pasteur Institute
Experimental Animal Center (Tehran, Iran). All animals were kept under
standard conditions of temperature (23 2°C) and humidity
(50% 10%) with an alternating 12-h light/dark cycles at the conven-
tional animal house of Alborz University of Medical Sciences. Given
free access to food and water, mice were housed for 1 week before
experiments and maintained under standard environmental conditions.
All experiments were done according to Animal Care and Use Protocol
of Ethics Committee of Alborz University of Medical Sciences.
According to the age, 5 experimental groups (n =9) were consid-
ered including: Group 1 as control young group (aged 2 months),
Group 2 as old group (aged 15 months) treated with KEAf at a dose of
5 mg/mL, Group 3 as old group (aged 15 months) treated with KEAf
at a dose of 10 mg/mL, Group 4 as control old group (aged 15 months)
treated with saline, and Group 5 as control old group (aged 15 months)
without any treatment. Treatments were carried out for 14 days and
performed via the intradermal route after the induction of anesthesia
and shaving a 2 92 cm region behind the animal’s neck.
2.5
|
Evaluation of skin irritation
Each animal was examined visually for signs of skin reactions, such
as erythema and edema, approximately 24 and 72 hours after intra-
dermal injection.
28
Also, pathological damage such as inflammatory
cell infiltration and fibroplasias was evaluated in hematoxylin and
eosin (H&E)-stained sections.
2.6
|
Histological analysis
To perform histological evaluation of the skin, the animals were sac-
rificed under ether anesthesia 72 hours after the last injection. Skin
2
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PAKRAVAN ET AL.
tissue samples, including epidermis, dermis, were taken using a scal-
pel No. 15. The skin samples from the control and test groups were
fixed in 10% buffered formalin and were processed for paraffin sec-
tioning. Sections of about 3 lm thickness were taken and stained
with H&E. To further assess the amount of total collagen, skin sam-
ples were stained with Masson’s trichrome. The stained sections
were examined with an Olympus cX41 microscope, (Olympus, Tokyo,
Japan), and photographed using an Olympus D330 digital camera.
The photographs were analyzed via Scion Image Software to analyze
markers of skin tissue alteration such as fiber length and width, fiber
fragmentation, number of fibroblasts, and the space between fiber
bundles of dermal connective tissue.
2.7
|
Evaluation of total collagen content
The content of hydroxyproline was measured according to the
method previously described by Kivirikko et al,
29
with some modifi-
cations. The skin tissue samples (0.3 g) were homogenized and
hydrolyzed in 6 mol/L HCl, at 105°C for 16 hours. Free hydroxypro-
line was oxidized using chloramine T. Then, Ehrlich`s reagent was
added and incubated at 60°C leading to formation of a chro-
mophore. Hydroxyproline product was further purified through
extraction of interfering chromophores with toluene and then acid
phase. The absorbance of the supernatant containing hydroxyproline
was measured by spectrophotometry at the wavelength of 543 nm.
Hydroxyproline content was calculated from the standard curve,
which was linear at 10-160 lg/mL.
2.8
|
Immunohistochemical evaluation of collagen
type I and III
Tissue sections of the skin samples were stained using antibody speci-
fic for collagen type I (1:100; Col 1A SC-59772, Santa Cruz, CA, USA)
and anti-collagen III antibody (Sigma-Aldrich, Taufkirchen, Germany)
as DAKO kit (Denmark). Anti-rabbit IgG antibody was used as sec-
ondary antibody, and the images were captured using a Zeiss LSM 5,
(Carl Zeiss, Tokyo, Japan) fluorescent microscope. Paraffin sections
obtained from each group were dewaxed and hydrated. Sections of
4lm were prepared from each sample, washed with PBS, incubated
in 3% H
2
O
2
for 20 minutes, washed once more with PBS, and then
incubated in a protein block solution for 10 minutes. Then, primary
antibody (diluted 1:100 in 0.01 mol/L PBS) was added to sections and
incubated for 1 hour, washed 3 times, and incubated with envision
solution (complex of secondary antibody diluted 1:200 in PBS, avidin,
and horseradish peroxidase). After 30-min incubation time, the
enzyme activity was visualized using 3, 30-diaminobenzidine (DAB). A
brown staining was regarded as a positive reaction. Data are pre-
sented as the mean of 3 randomly selected fields of microscopic view.
2.9
|
NAD
+
and NADH content
The content of dinucleotides NAD
+
and NADH was determined in
skin tissue samples using NAD
+
/NADH assay kit (Abcam, London,
UK), according to the manufacturer’s instructions. Briefly, about
20 mg of tissue sample was washed with cold PBS and homogenized
with NAD/NADH extraction buffer. The supernatant was split into 2
aliquots. For NADH detection, the aliquot was incubated at 60°C for
30 minutes as NAD needs to be decomposed before the reaction.
To determine NADt (NAD and NADH), the other aliquot was kept
on ice to preserve NAD and NADH. The samples were transferred
into 96-well plates, and then a mixture of NAD cycling enzyme/buf-
fer was added to each sample. Finally, NADH developer was added
to each reaction. NADH standard was prepared according to the
manufacturer’s instructions. Total NAD (NADt) and NADH were esti-
mated directly, whereas the value of NAD
+
was estimated by sub-
tracting NADH from NADt and expressed as pmol/lg of skin tissue.
2.10
|
Statistical analysis
Statistical analysis was performed using GraphPad Prism 7.01. Data
are presented as meansSD. ANOVA was used to indicate any
significant difference among the groups. Value of Pwas considered
statistically significant when it was less than .05.
3
|
RESULTS
3.1
|
Flavonoid composition of BT, KT, and KT
fractions
Total flavonoid content of each of BT, KT, KEAf, KCf, and KBf is
shown in Figure 1. The flavonoid content increased in BT after fer-
mentation by Kombucha culture. The rate of flavonoid concentration
in KT and its derivatives was about 21%, 38.7%, 21%, and 25.4% in
KT, KEAf, KBf, and KCf, respectively. As Figure 1 shows, concentra-
tion of flavonoid in KEAf is about 22.3% more than KT. As KEAf had
the highest flavonoid content, it was further tested for anti-aging
effect.
BT
KT
KCf
KBf
KEAf
0
10
20
30
µg QE/mg Sample
*
**
**
FIGURE 1 Total flavonoid content of each sample (lg
QE/100mg); the flavonoid concentrations significantly increased in
Kombucha tea rather than unfermented black tea (BT). *P<.05 vs
BT, **P<.007 vs BT
PAKRAVAN ET AL.
|
3
3.2
|
Skin irritation assessment
Visual evaluation of skin reaction at 24 and 72 hours after ID injec-
tion did not show inflammatory reactions, such as edema and ery-
thema. The score of irritation in KEAf and PBS groups was 0%, and
the mean evaluation score was 0 (Table 1). Also, histological obser-
vation of the skin tissue sections did not show pathological damage
or accumulation of inflammatory cells in the treated animals (Fig-
ure 2) suggesting that KEAf is not cytotoxic and does not sensitize
the skin.
3.3
|
Histological observation
Histological view of H&E- and trichrome-stained skin tissue of mice
aged 2 and 15 months showed that age increment extends skin
tissue abnormalities represented as reduced fiber bundles width and
length, increased space between dermal connective fiber bundles
(P<.05, Figure 3A), and decreased numbers of fibroblasts (P<.05,
Figure 3B). Also, decreased number of the collagen bundles in the
upper layer of dermis, epidermal atrophy, and decreased interdigita-
tion between the epidermis and dermis were seen in old skin tissue
vs young samples (Figure 2). In the mice aged 2 months, the fibers
formed a fibrous network and were less regularly arranged compare
with in the older animals. Degrees of skin connective tissue abnor-
malities were significantly improved in samples treated with KEAf.
Figures 2 and 3 together show that treatment with KEAf on average
decreased skin connective tissue abnormalities approximately 48.2%
in aged skin sample (15 month at age) vs vehicle-treated old skin
(P<.007). There was no significant difference between samples
treated with 5 or 10 mg/mL KEAf.
TABLE 1 Evaluation of skin irritation in Kombucha tea ethyl acetate fraction (KEAf)- and PBS-treated animals; total score/number of mice
(n =9) =Ms
Groups Vehicle L (PBS) KEAf
Hours 24 72 24 72
Skin reaction Erythema Edema Erythema Edema Erythema Edema Erythema Edema
Mean score (Ms) 0 0.1 0 0 0 0 0 0
FIGURE 2 Formalin-fixed skin sections were stained with H&E and trichrome. (A) control 2-months old, (B) focus on (A), (C) control 15-
months old, (D) focus on (C), (D) 15-months old mice treated with 5mg/ml KEAf, (E) focus on (D), (F) 15-months old mice treated with 5mg/ml
KEAf, (E) focus on (F), (a) Trichrom staining of 2-months old, and (b) trichrom staining of 15-months old control mice skin. Mice aged 15
months showed significant increase in abnormalities in connective tissue structure: thin, disorganized fiber bundles present throughout the
dermis, and reduced numbers of fibroblasts. Also, patches of ground substance deposition increased in areas resembling degraded fiber
bundles. Skin connective tissue alterations due to age increment are partially improved by KEAf treatment. Data is presented as the mean of
three randomly selected fields of microscopic view. (Scale bar 50 lm).
4
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PAKRAVAN ET AL.
3.4
|
KEAf stimulated type I and III collagen
synthesis in the skin
Based on hydroxyproline estimation, the total collagen content was
different in the skin tissue of the 2 different age groups. The aged
skin tissue samples showed a mean reduction of 35.5% in hydrox-
yproline content comparing to the young skin tissue samples
(P<.05, Table 2). Treatment with KEAf led to a 31.8% increase at
the hydroxyproline content of the skin connective tissue samples
of the old animals compared with those treated with PBS
(P<.007, Table 2). Immunohistochemical staining of the skin from
old and young animals with monoclonal antibody against collagen
type I revealed that type I collagen content decreased in the aged
skin compared to the skin tissue from young mice (Figure 4A).
Analysis of the images by Image Scan software showed that there
is about 29.3% lower collagen type I in the skin tissue samples
from old animals than the young animals (P<.05, Figure 4B). How-
ever, immunohistochemical staining of collagen type III showed no
prominent change in the skin connective tissue in mice aged
15 months (Figure 5). Based on hydroxyproline content and
immunohistochemical analysis of collagen type I, KEAf treatment
restored abnormalities (Figure 2), and increased total collagen in
the skin of aged mice (Table 2). Intradermal treatment of skin of
aged mice with KEAf revealed a significantly higher content of col-
lagen type I and III compared with that of the vehicle group (Fig-
ures 4 and 5).
3.5
|
NAD
+
/NADH content
In the final set of experiments, level of NAD
+
/NADH production
upon administration of KEAf was investigated. The findings showed
notable reduction in NAD
+
/NADH content in the aged skin vs skin
of the young animals. Parallel to this, the NAD
+
/NADH level in the
skin of old animals treated with KEAf was significantly higher than
the skin tissue samples of their age-matched treated with PBS. There
was no difference in NAD
+
/NADH level between 5 and 10 mg/mL
KEAf (P<.01, Figure 6).
4
|
DISCUSSION
The health benefits of KT have made some researchers consider it
as a probiotic product.
18
During the fermentation process, effective
components are formed and cause the beneficial properties of this
drink. Antioxidant and free radical scavenging properties of KT,
which are mainly attributed to flavonoid and polyphenols, are
formed during fermentation.
22,23
The result of the present study
indicated an increase in the flavonoid content of KT and especially
KEAf in BT by about 21% and 38.7%, respectively, after fermenta-
tion of BT by Kombucha culture. The increase in the flavonoid con-
tent of KT and especially KEAf might be due to the secretion of
some enzymes by the microbial biofilm that degrades the polyphe-
nols to flavonoids.
18
Anti-inflammatory effects of flavonoids
23,30
are
consistent with the lack of skin sensitization upon KEAf administra-
tion. Also, a large body of literature attributed anti-aging activities of
flavonoids to their potent antioxidant properties.
31,32
Histological comparison of the dermal sections between mice
aged 15 months and 2 months revealed that increasing of age is
associated by thinner fiber bundles, more open space within and
between bundles in the dermis, fragmentation of collagen fibers, epi-
dermal atrophy, and decreased numbers of fibroblasts. Previous
FIGURE 3 (A) Average of skin connective tissue abnormalities was measured with an image analysis (image J: imagej.nih.gov/ij). ¤space
between dermal connective fiber bundles was scored using a scale of 1-10. (B) Number of fibroblast, per unit area: about 0.5 mm2 per skin
tissue sample. Correlation analysis between the numbers of fibroblasts in the dermis and age revealed a significant effect of age increment on
the decrease of fibroblasts in the dermis. The amount of abnormalities decreased significantly with KEAf treatment. Values are means of 9
measurements. *P < 0.05 vs. 2 month-old mice, **P < 0.007 vs. vehicle-treated skin.
TABLE 2 Total collagen content evaluated by hydroxyproline
Mice skin tissue Old mice skin tissue
Young Old Vehicle KEAf
Hydroxyproline (lg/0.1 g sample)
294.19 189.5* 173.14 253.93**
*P˂.05 vs. young skin connective tissue.
**P˂.007 vs. vehicle.
PAKRAVAN ET AL.
|
5
study showed that decreased numbers of keratinocytes in aged skin
are largely responsible for the epidermal thinning.
33
Also, thinning of
dermis in aged skin is due to reduction in the amount and
organization of connective tissue comprising febrile collagen bundles
and elastic fibers along with a complex array of proteoglycan and
other extra cellular matrix molecules.
4
The rate of such abnormalities
FIGURE 4 (A) Immunostaining of the skin connective tissue from old and young animals with monoclonal antibody collagen type I revealed
that type I collagen content decreased in aged skin, compared with younger mice skin. Intradermal treatment of skin of aged mice with KEAf
revealed a significantly increased content of type I collagen (secondary antibody conjugate to FITC, magnification 2009). (B) Content of
collagen type I was quantified for each image using the ImageJ analysis (imagej.nih.gov/ij(in 4 nonconsecutive tissue sections (*P<.05 vs. 2-
mo-old mice, **P<.007 vs. vehicle)
FIGURE 5 Immunohistological evaluation of type III collagen in old mice skin aged 15 mo revealed prominent staining vs. 2 mo old
(collagen fibers stained brown). This dermal staining was significantly increased in the skin of aged mice when treated with KEAf (n =9, Scale
bars =100 lm)
6
|
PAKRAVAN ET AL.
was significantly improved in aged mice skin treated with KEAf com-
pared to the control groups. It suggests that these alterations in the
dermal connective tissue are largely responsible for the thin, fragile,
and finely wrinkled quality of naturally aged skin.
4,8
Immunohistochemical evaluation of collagen type I in the skin
of old subjects revealed a decrease in collagen type I by 29.3%
compared with the young mice skin. Many studies have also
demonstrated that collagen type I content decreases with age.
However, there are differences in the collagen type I content of
naturally aged skin,
8
sun protected skin,
34
and photo-aged skin.
35
As for type III collagen, there was no significant difference
between skin of the old and young mice. This is consistent with
previous studies indicating that type III collagen content does not
change in covered skin with increasing age.
36,37
Nevertheless,
other studies have shown that type III collagen decreases with age
increment, although it is noteworthy that these researchers studied
skin of a sun exposed site.
35,38
In addition, it should be considered
that type III collagen constitutes a small portion of the total colla-
gen in the skin,
36
and type I collagen is the main constituent of
skin connective tissue by approximately 80% of skin wet weight.
7
Presumably, reduction in collagen content and declining trend in
collagen fiber number and width with increasing age are conse-
quences of destruction and impairment in the replacement of col-
lagen type I. Treatment of aged mice skin with KEAf led to a
measurable restoration of the dermal connective tissue abnormali-
ties as a consequence of the natural aging process. KEAf could
also significantly increase total collagen content of the skin and
increase in the type I and III collagen content. This could be
mainly attributed to flavonoid and polyphenols formed during fer-
mentation causing antioxidant and free radical scavenging proper-
ties of KEAf.
22,23,27
In addition, Jayaban et al
18
reported that ethyl
acetate fraction of KT contains dimethyl malonate and vitexin.
These compounds show strong radical scavenging activity with
anti-aging properties.
39,40
Alternatively, the present study demonstrated that ethyl acetate
fraction of KT can stimulate energy production. Accordingly, the
treatment of aged mice skin with KEAf significantly increased
NAD
+
/NADH content whose level normally decreased in old mice.
In parallel with these findings, a previous report demonstrated a cor-
relation between NADPH levels and type I collagen expression in
adult human skin fibroblast cell in culture. The type I collagen
expression was decreased in parallel with decrease in NAD
+
/NADH
level and increased with treatment with a complex of niacinamide.
41
Gomes et al. directly measured that during aging, decline in NAD
+
content inducing a pseudohypoxic state led to distribution of
nuclear-mitochondrial communication and finally decline in mito-
chondrial function with age. They demonstrated that this phe-
nomenon can be apparently reversible with adding NAD
+
derivatives
to cell.
42
When there is a sufficient level of NAD
+
/NADH on fibrob-
lasts, production of collagen is high.
43
Based on the findings of other
studies and the finding presented in this study, it is hypothesized
that the reduction in collagen content in aged animals occurs as a
consequence of reduced NAD
+
/NADH level. Hence, it is hypothe-
sized presumably that the beneficial effects of KEAf are due to fla-
vonoids and energy-producing compounds such as great source of
Vit B3, which can stimulate collagen production and repair abnormal-
ities of connective tissue in naturally aged skin. Finally, the results of
the current study suggest that KEAf treatment is useful to improve
thickness and flexibility of aged skins. Accordingly, KEAf can be
applied as a potential candidate for natural cosmeceuticals/functional
cosmetics either used in this study or as a topically supplement in an
attempt to prolong youthful skin appearance. However, more
research is required to elucidate the mechanisms underlying this
phenomenon and conduct an investigation on human skin.
Altogether, our results suggest that KEAf treatment is useful to
improve thickness and flexibility of aged skin. The beneficial effects
of KEAf are presumed to be due to flavonoids and energy-producing
compound capable of repairing connective tissue in naturally aged
skin. This study suggests KEAf as a potential candidate for natural
cosmeceuticals/functional cosmetics and long-term use in aged pop-
ulations. However, more research is required to elucidate the mecha-
nisms beneath this phenomenon.
ACKNOWLEDGMENTS
This work was financially supported by the Deputy of Research,
Alborz University of Medical Sciences under reference No. 2427656.
CONFLICT OF INTEREST
The authors have no conflict of interest.
ORCID
Nafiseh Pakravan http://orcid.org/0000-0002-9355-1738
FIGURE 6 NAD
+
/NADH ratio is measured by spectrophotometric
assay. All KEAf-treated samples had significantly greater NAD
+
/NADH
ratio compared with controls (N = 9, *P< .05 vs. 2-mo-old mice,
**P< 0.01 vs. vehicle)
PAKRAVAN ET AL.
|
7
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How to cite this article: Pakravan N, Mahmoudi E, Hashemi S-A,
et al. Cosmeceutical effect of ethyl acetate fraction of Kombucha
tea by intradermal administration in the skin of aged mice. J
Cosmet Dermatol. 2017;00:1–9.
https://doi.org/10.1111/jocd.12453
PAKRAVAN ET AL.
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