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Fruit Juice Supplementation Alters Human Skin …Fruit Juice Supplementation Alters Human Skin...
Transcript of Fruit Juice Supplementation Alters Human Skin …Fruit Juice Supplementation Alters Human Skin...
Biotechnology and Bioprocess Engineering 23: 116-121 (2018)
DOI 10.1007/s12257-017-0442-3pISSN 1226-8372
eISSN 1976-3816
Fruit Juice Supplementation Alters Human Skin Antioxidant Levels
In Vivo: Case Study of Korean Adults by Resonance Raman
Spectroscopy
Moon-Hee Choi, Han-Gyo Jo, Min-Ju Kim, Min-Jung Kang, and Hyun-Jae Shin
Received: 1 November 2017 / Revised: 27 December 2017 / Accepted: 4 January 2018
© The Korean Society for Biotechnology and Bioengineering and Springer 2018
Abstract Carotenoids, found in many fruits and vegetables,
are antioxidants that protect human skin from UV radiation.
In humans, fruit and vegetable intake increases carotenoid
contents in skin, which are conventionally assessed by
invasive blood tests. In this study, 47 healthy Korean subjects
(volunteers) consumed fruit juice containing tomato, apple,
strawberry, or grape three times per week for 6 weeks. Skin
antioxidant levels were measured by non-invasive resonance
Raman spectroscopy. The correlation between skin carotenoid
(SC) score with demographic data (age, height, weight) and
juice supplementation and changes in SC scores among
groups were analyzed. Variations in skin antioxidant levels
increased with juice supplementation (p < 0.05). Fruit juice
intake was significantly correlated with SC score, indicating
increased skin antioxidant levels. Grape and tomato increased
skin antioxidant levels and showed higher antioxidant
activity than other fruits. Fruit juices containing high levels
of carotenoids and antioxidants may provide modest benefits
to human health.
Keywords: skin carotenoid, antioxidant activity, Raman
spectroscopy, fruit and vegetable juice, Korean case study
1. Introduction
Carotenoids are the dominant pigments in autumn leaf
coloration in approximately 15 ~ 30% of tree species, but
many plant colors, particularly reds and purples, are
generated by other classes of chemicals [1]. Carotenoids
are found in numerous types of fruits and vegetables such
as tomato, carrot, spinach, and grape [2]. The food sources
and well-known structures of carotenoids are summarized
in Table S1. The two predominant carotenoids in human
tissues, β-carotene and lycopene, represent approximately
70% of the carotenoids in the skin and are found in the
liver, adrenal glands, fat, pancreas, lungs, testes, and kidneys
[3]. Carotenoids containing unsubstituted β-ionone rings,
include β-carotene, α-carotene, β-cryptoxanthin, and γ-
carotene, exhibit vitamin A activity [4]. These compounds
are involved in protection against light and contribute to
the prevention of external damage in humans, such as those
by UV-irradiation, smoke exposure, and contact with the
environment [5]. Numerous studies have examined the role
of antioxidants in the skin, particularly studies related to
anti-aging effects [6]. Among the many carotenoids, β-
carotene is an endogenous photoprotector whose efficacy
in preventing UV-induced damage has been widely
demonstrated [7,8]. Epidermal and dermal accumulation of
β-carotene may protect human skin from light-induced
disease [9]. In addition, lycopene is the major carotenoid in
tomatoes and provides photo-protection and antioxidation
to human skin [10,11]. Apples contain a carotenoid
biosynthesis pathway [12]. Given the increasing importance
of carotenoids in health and disease, methods for detecting
carotenoids in living human tissue are needed. Resonance
Raman spectroscopy (RRS) is a novel approach for nonin-
Moon-Hee Choi, Hyun-Jae Shin*
Department of Biochemical and Polymer Engineering, Chosun University,Gwangju 61452, KoreaTel: +82-62-230-7518; Fax: +82-62-230-7226E-mail: [email protected]
Han-Gyo Jo, Hyun-Jae ShinDepartment of Chemical Engineering, Graduate School of ChosunUniversity, Gwangju 61452, Korea
Min-Ju Kim, Min-Jung KangBio-Food Research Center, Hurom Co. Ltd., Gimhae 50969, Korea
RESEARCH PAPER
Fruit Juice Supplementation Alters Human Skin Antioxidant Levels In Vivo: Case Study of Korean Adults by … 117
vasive, laser optical carotenoid detection. The RRS technique
is precise, specific, sensitive, and well suited to clinical and
field studies [13-15]. Most studies on the nutritional effect
of fruits and vegetables have been performed using Western
people. To analyze the effects of food intake in different
ethnic groups, information regarding the effect of fruit or
vegetable juice and/or extract intake in Korean adults is
necessary. In this study, RRS has been conducted to
determine the changes in antioxidant levels in the skin of
Korean adults following supplementation and consumption
of fruit juices containing high carotenoid levels.
2. Materials and Methods
2.1. Selection of test subjects and survey of health status
Changes in antioxidant levels following supplementation
and consumption of fruit juices were investigated. Forty-
seven healthy subjects (from among 60 people) from
Chosun University in Gwangju, aged 22 ~ 54 years, were
recruited for this study. Subjects completed a questionnaire
that included demographical, dietary, and lifestyle variables.
The questionnaires were reviewed to determine whether
the responses were inadequate and identify individuals
taking vitamin supplements (Table S2). This study was
approved by the IRB Committee of Chosun University
(IRB approval number: 2-1041055-AB-N-01-2015-0028).
2.2. Intake of fruit juices
Subjects were prohibited from taking other supplements for
1 month before and during fruit juice intake. The subjects
consumed fruit juices 3 times per week for 6 weeks (Fig. 1)
in consideration of the work schedules of the subjects. Fruit
juices were consumed a total 18 times. Fruits used in this
study included fresh tomato, apple, strawberry, or grape,
and juices were prepared with a blender (HE-DBF04,
Hurom, Gyeongsangnam-do, Korea). Participants were
divided into five groups: tomato, tomato-apple, strawberry-
apple, grape, and control. The control group did not
consume juice. The amount of juice provided was 300 mL
and the weight of fruit in the 300 mL of juice were
calculated by juice yield (Table 1).
Fig. 1. Experimental design for fruit-juice intake.
Table 1. Juice yield of fruits and amount of fruits per serving used in this study
FruitsJuice yield(%, v/w)
Amount of fruit per serving(g/300 mL)
Carotenoids content References
Tomato 44.6 ± 0.8 673 g 44552 6,620 μg/100 g dry wt. [18]
Apple 36.4 ± 2.2 824 g (412 g in mixed juice) 19.4 μg β-carotene/100 g [19]
Strawberry 32.8 ± 0.3 915 g (457 g in mixed juice) 500 ~ 2,000 μg/100 g dry wt. [20]
Grape 41.3 ± 0.8 728 g 567 μg/100 g dry wt [21]
Data were Mean ± S.D.
118 Biotechnology and Bioprocess Engineering 23: 116-121 (2018)
2.3. Detection of skin carotenoid score (SC score) by
resonance raman spectroscopy (RRS)
Skin carotenoid score as a measure of human antioxidant
level was detected using a resonance Raman spectro-
photometer (Bio Photonic scanner S3 Scanner, Pharmanex,
Provo, UT, USA). The S3 scanner uses blue light at a
wavelength of 478 nm; the wavelength shifts to 518 nm
upon detection of carotenoids because of photon reflection.
Photon reflection is quantified as the SC score (Fig. 2). An
iPad (Apple, Cupertino, CA, USA) was connected to an S3
scanner via Bluetooth and an iPad S3 scanner application
was used to operate the S3 scanner (Fig. 2A). Zero-
adjustment was conducted by covering the light exit
channel. Carotenoid levels in the skin of subjects were
measured on the palm of the hand. The SC score appeared
on the screen of the scanner when the measurement was
complete (Figs. 2B and 2C). The range of SC scores was
10,000 ~ 89,000, which was displayed as colors from red
to gray (Fig. 2D). The SC score of subjects was measured
once per week (Fig. 1B).
2.4. Antioxidant activity assay of fruits
In vitro antioxidant levels of fruits were measured by
determining the DPPH and ABTS radical-scavenging
activities. Measurements of scavenging activity of fruit
extracts against DPPH radicals were conducted as described
by Blois [16] with some modifications. Stock solutions of
samples (1.0 mg/mL) were diluted to final concentrations of
500 ~ 25 µg/mL in double-distilled water. Next, 0.3 mL of
a 0.1 mM DPPH methanol solution was added to 0.3 mL
of sample solutions of different concentrations and reacted
at 25°C. After 30 min, absorbance values were measured at
517 nm and converted into the percentage antioxidant
activity (AA) using the following formula:
DPPH scavenging activity of the fruit extract (IC50) is
the concentration required to reduce AA% by 50%.
Experiments were repeated three times. The ABTS assay
was conducted as described with some modifications [17].
The AA% formula was the same as that used in the DPPH
assay.
2.5. Statistical analysis
Statistical analysis was conducted using SPSS version 23
software (SPSS Inc., Chicago, IL, USA). T-test was performed
to analyze the antioxidant activities of fruits. Age, height,
and weight of subjects in each group were analyzed by
one-way analysis of variance (ANOVA). For the post-hoc
test, Duncan’s multiple range test was performed to identify
correlated variables. The correlation of age, height, or weight
and SC score was analyzed by two-way ANOVA. To
observe changes in the SC score, all scores were divided by
the SC score before fruit juice intake (normalization). The
normalized scores of each group were analyzed by one-
way ANOVA. The effects of fruits and time course in each
group were analyzed with the 95% confidence level.
AA % 100Abssample Absblank–( )
Abscontrol----------------------------------------------- 100×–=
NormalizationSC score after fruit juice intake
SC score before fruit juice intake-------------------------------------------------------------------------------=
Fig. 2. Resonance Raman apparatus used in this study. (A) Operation method, (B) function keys and monitors of S3 scanner, (C)instructions of the scanner, and (D) skin carotenoid score (SC score) range and its color variation.
Fruit Juice Supplementation Alters Human Skin Antioxidant Levels In Vivo: Case Study of Korean Adults by … 119
3. Results and Discussion
The carotenoid content in the fruit juices were measured
(Table 1). The antioxidant activity was highest in the grape
and tomato groups. The IC50 values for the DPPH
antioxidant activities of grape and tomato were 164 and
172 μg/mL, respectively (Fig. 3). The IC50 values of the
ABTS antioxidant activities of grape and tomato were 123
and 209 μg/mL, respectively. No significant differences in
demographic data were found, including age (F(47) = 0.136,
p > 0.05), height (F(47) = 1.385, p > 0.05), and weight
(F(47) = 0.955, p > 0.05), among the groups (Table 2).
There must be other flavonoids and phenolic compounds in
the juices. Thus, the antioxidant levels are thought to be not
only due to carotenoids. The correlation of age, height, and
weight with SC score were evaluated. The SC scores of
subjects are shown in Table 3. Age and height had no
correlation with SC score. However, significant SC score
changes in the tomato-apple group and control group were
found to be affected by weight [tomato-apple group F(47)
= 6.859, p < 0.01; control group F(47) = 3.708, p < 0.05].
After fruit juice intake, significant changes in the SC score
were observed starting after 3 weeks (3 weeks F(47) =
3.793, p < 0.05; 4 weeks F(47) = 2.748, p < 0.05; 5 weeks
F(47) = 2.756, p < 0.05; 6 weeks F(47) = 4.312, p < 0.01)
(Tables 2 and 3). Duncan’s multiple range test (p < 0.05)
Fig. 3. Antioxidant activity of fruits used in this study by (A) DPPH assay and (B) ABTS assay. Symbols: gallic acid (black circles),grape (black triangles), tomato (white inverted triangles), tomato and apple (black rectangles), and strawberry and apple (whitediamonds). Error bars indicate the variability of replicates within a single representative experiment (mean ± SD).
Table 2. Characteristics of subjects classified by dietary group in this study
Variables Age (years) Height (cm) Weight (kg)
Tomato (N = 11) 32.5 ± 8.3a 169.6 ± 6.2a 63.7 ± 13.3a
Tomato-apple (N = 9) 31.8 ± 9.1a 167.8 ± 8.8a 62.8 ± 12.6a
Strawberry-apple (N = 8) 31.8 ± 7.1a 164.4 ± 4.4a 59.1 ± 9.5a
Grape (N = 9) 30.9 ± 7.2a 164.2 ± 6.6a 62.8 ± 14.1a
Control (N = 10) 30.3 ± 3.3a 169.8 ± 8.3a 69.8 ± 9.5a
Mean ± S.D.avalues in the same column not sharing a common superscript were significantly different by Duncan’s multiple range test (p < 0.05).
Table 3. Normalized average of skin carotenoids score
Groups Before intake 1st week 2nd week 3rd week 4th week 5th week 6th week
Tomato (N = 11) 1.00 ± 0.00 1.02 ± 0.10a 0.99 ± 0.09ab 0.97 ± 0.11ab 0.97 ± 0.11ab 0.99 ± 0.11ab 0.97 ± 0.10b
Tomato-apple (N = 9) 1.00 ± 0.00 1.00 ± 0.08a 1.05 ± 0.13b 1.01 ± 0.02b 1.06 ± 0.17b 1.05 ± 0.18b 1.03 ± 0.03b
Strawberry-apple (N = 8) 1.00 ± 0.00 1.02 ± 0.07a 0.97 ± 0.04ab 0.95 ± 0.06ab 0.92 ± 0.10a 0.98 ± 0.12ab 0.98 ± 0.14b
Grape (N = 9) 1.00 ± 0.00 1.00 ± 0.06a 0.96 ± 0.07a 1.03 ± 0.12b 1.01 ± 0.10ab 1.04 ± 0.14b 1.06 ± 0.15b
Control (N = 10) 1.00 ± 0.00 0.98 ± 0.03a 0.95 ± 0.06a 0.90 ± 0.06a 0.91 ± 0.06a 0.88 ± 0.07a 0.87 ± 0.08a
Data were Mean ± S.D.a–b values in the same column not sharing a common superscript were significantly different by Duncan’s multiple range test (p < 0.05).
120 Biotechnology and Bioprocess Engineering 23: 116-121 (2018)
was performed to evaluate the results from 3 to 6 weeks. At
3 and 5 weeks, the tomato-apple and grape groups showed
significant differences from the control group. At 4 weeks,
only the tomato-apple group showed a statistically significant
difference from the control group. All groups were
statistically different from the control. Next, analysis of SC
score changes by time course during fruit juice intake was
conducted in each group (Table 3). In the control group,
there was a significant difference in SC score (F(70) = 7.516,
p < 0.001) after 3 weeks. In contrast, no fruit juice-intake
groups showed significant differences in SC score (tomato
group F(77) = 0.346, p > 0.05; tomato-apple group F(63) =
0.516, p > 0.05; strawberry-apple group F(56) = 1.054,
p > 0.05; grape group F(63) = 0.871, p > 0.05).
Carotenoids are fat-soluble pigments obtained by fruit
and vegetable ingestion and are transported into the blood
as lipoproteins to accumulate in the lipid layer of the
human epidermis. Carotenoid levels can be assessed by
measuring their concentration in the blood or skin. As
reported previously, 2.75 ~ 11 mg carotenoid-rich juice
(2.75 mg/30 mL) significantly increased skin carotenoid
status over an 8-week period among children aged 5 ~ 17
years [22]. Similarly, a study of children who received rice
supplemented with fruits (3.5 mg carotenoids) or β-carotene
powder (5.0 mg carotenoids) compared to a control diet for
30 days showed significantly higher plasma β-carotene
concentrations [23]. However, no significant differences
were detected in skin carotenoid status between participants
consuming a carotenoid-rich high-dose and carotenoid-rich
low-dose juice during the study [24]. These results are
similar to those of following feeding of a high (11 mg
carotenoids), low (4.4 mg carotenoids), and no-carotenoid
muffin to 10 children [25]. The reason for this large variation
in serum carotenoid is unclear, but has also been observed
in other high-carotenoid foods and supplements [26,27].
Previous studies revealed strong correlations (r2 = 0.81 and
r2 = 0.79, respectively; both p < 0.001) between the amounts
of carotenoids measured in the blood by high-performance
liquid chromatography and those measured in the skin by
resonance Raman spectroscopy [15,28].
This study chose five fruits as sources of fruit juice
according to the productivity and consumption of fruits in
Korea. For SC score data, fruit juice intake positively
influenced SC scores. A correlation was observed between
SC score and fruit juice supplementation. The tomato-apple
group showed a larger change in SC score than the tomato
group. Two factors may explain the SC score changes in the
two groups. First, apples contain high levels of flavonoids
and a strong carotenoids biosynthesis pathway [12,29].
Thus, skin carotenoid levels may be increased when
consumed with mixed juices containing tomato and apple.
Second, these results may be related to differences in the
absorption of carotenoids or differences in the ability to
cleave beta-carotene to vitamin A. Significant correlations
between skin carotenoids and fruit and vegetable intake,
particularly high-carotenoid vegetable intake, were previously
observed based on the questionnaires like Table S2 and
24 h recall [15,28,30]. Intake of orange-colored fruits
increased β-carotene concentration in the serum of Indonesian
children compared to intake of green-colored vegetables
[31].
A high body weight is significantly correlated with a
decreased SC score. In previous studies reported that
decreases in SC score occurred with weight loss in all
intervention groups [32]. This result differs, who found a
positive association between skin carotenoid score and fruit
and vegetable intake, whereas, not between skin carotenoid
score and age, height, weight, or body mass index [33].
Although results suggest variations in the effects of
high-carotenoid fruits and vegetables on serum carotenoid
levels, most previous studies examined children. In this
study, participants who consumed fruit juices generally had
higher SC scores than those who did not consume fruits
juices (control group). These findings were significant, and
thus skin carotenoid status may be used as a biomarker to
determine changes in carotenoid intake in human skin.
This is the first study to measure antioxidant levels in the
skin of Korean adults using non-invasive resonance Raman
spectroscopy.
4. Conclusion
The information provide in this paper is rather preliminary
but this study is of great significance to measure the effect
of fruit juice intake on Korean adults. In conclusion,
supplementation with fruit juice containing high levels of
carotenoids and antioxidants may provide benefits to
human health, particularly antioxidant-related effects. For
practical application, this simple method can be applied as
a juice-quality indicator to determine antioxidant levels.
Electronic Supplementary Material (ESM) The online
version of this article (doi: 10.1007/s12257-017-0442-3)
contains supplementary material, which is available to
authorized users.
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