Original Article

診療と新薬 2017; 54: 593-602

Skin Enhancement Eﬃcacy of Supplements Containing
Four Amino Acids in Healthy Japanese Women:

診療と新薬 WebA Randomized Double-blind Placebo-controlled Study

Masatomo NAJIMA 1) ／ Akinobu MIYATA 2）／ Katsumi KURITA 3）／ Tsugio ODA 4）● Abstract
Objective: The objective of this research was to investigate the effectiveness of daily ingestion of a
supplement containing RHIZOPUS/RICE FERMENT FD POWDER, which features Glutamine acid, Aspartic
acid, Proline, and Arginine, on the condition of the human skin.
Methods: A randomized, placebo-controlled, double-blind study was conducted to verify the skin condition. In
this study we measured wrinkles, the amount of surface spots (visible spots and hidden ones), the amount ofmelanin, the whiteness of the complexion, the stratum corneum water content and the trans-epidermal water
loss (TEWL), three kinds of elasticity and also carried out the subjective reporting in form of questionnaire.
To evaluate the safety of the test foods, adverse events were collected by means of a written questionnaireduring the study.
Results: From all of 155 applicants, 108 were eliminated according to not meeting inclusion criteria. The 47
subjects were randomly assigned to intervention groups and made a start with ingestion. Among 47 subjects, 2
were withdrawn due to personal reason. Data obtained with 45 subjects (Test; 22, Placebo; 23) was used forthe analysis of eﬃcacy. After the 12-week ingestion the test group showed a signiﬁcant diﬀerence in Wrinkle
grade , The whiteness of the complexion , The stratum corneum water content , Melanin , andElasticityR2 compared to the Placebo. On the other hand, the tendency was not seen in Spot or The
trans-epidermal water loss . As for the subjective evaluation, we observed a signiﬁcant diﬀerence in 7 out of
20 items. No adverse eﬀects were observed after the ingestion of the test product.
Conclusion: We found out that the 12-week ingestion of the test product containing RHIZOPUS/RICEFERMENT FD POWDER, which has a plenty of amino acids such as glutamine acid, aspartic acid, proline, andarginine, contributed to the improvement of the skin condition such as elasticity, tightness or moisture. In
addition, no safety-related matter was observed during the 12-week test period.
Key Words: RHIZOPUS/RICE FERMENT FD POWDER, amino acid, skin condition, elasticity, wrinkle grade

1. INTRODUCTION
In modern Japanese society, the words skin care is
attracting much interest from people, irrespective of age
and sex. Society is flooded with many products orinformation about skin care, and the category of these
items or information varies from cosmetics tosupplements, massage technique and dietary treatment.
Recently, many people are inflicted with emotional or
physical harm caused by the lack of exercise, bad eating
habits or stress, and as a result they suffer from skinproblems such as rough skin, pale skin or lack of tension.
In addition, substantially more people start to have theskin problems by the external factors such as photo-aging
caused by the increase of ultraviolet 1) or dry air triggeredby the heavy usage of air-conditioning 2) . The skin1) JACTA (Japan Clinical Trial Association)2) Nihonbashi Mʼs Clinic
3) RISOU Co., Ltd
4) C-TECJAPAN Co., Ltd

condition is greatly influenced by the type of nutrition
consumed. Although there exists some individual
variation, people with an unbalanced diet tend to haverough skin. The recent dietary habit in Japan is shifting
to European-style, which is said to have problems such as
excessive intake of fat or lack of minerals 3). In terms of
the relationship between the skin condition and the foodconsumed, it is reported that the speciﬁc food ingredientssuch as collagen peptide 4), elastin 5) and placenta 6) exercise
a positive eﬀect on the skin condition.
In Japan, the fermentation technique with the use of
microorganisms (such as the production of Miso or soy
sauce) has been cultivated since ancient times.
Fermentation applies to the changing process of theoriginal food ingredients to another type with the help of
the development of microorganisms, and, at the sametime, a process which is beneﬁcial to humans. Therefore
the fermentation often produces ingredients of higher
nutrition than the original. Rice has been used forfermentation technique for many years since it has been
consumed from ancient times in Japan, and many

62 （594）

診療と新薬・第 54 巻

fermented foods that derived from rice have been
invented and developed so far. The representative
example of the rice-derived fermented foods is sake (ricewine). It is known that sake has more nutrition such asamino acids than the rice used as raw material 7). Rice
ferment extract is produced from rice (which has a wealthof carbohydrate), and by applying a high fermentation
technique to it. Therefore it is well known that the rice
ferment extract contains plenty of vitamins, amino acids
and carbohydrate, and it helps to keep skin enriched,moisturized high in tension. Also, the amino acids
contained in the rice ferment extract are reported to havea high function of moisture by the skin absorption 8).
RHIZOPUS/RICE FERMENT FD POWDER is a powder
produced by a filtration of extract which has a seed of
Oryza sativa as its substrate and undergoes a process offermentation via Rhizopus.
Although rice ferment extract is used in many cosmetics
and is renowned for its function, there are few studies
which focus on the eﬃcacy of rice ferment extract to theskin condition when it is ingested orally. Therefore in thisstudy, we conducted a randomized, placebo-controlled,
double-blind study to verify the efficacy of the foodcontainingRHIZOPUS/RICE FERMENT FD POWDER,
which is rich in amino acids, for the skin condition.

2. METHODS
2.1. Trial design
A randomized, placebo-controlled, double-blind study was
conducted with the aid of a fund from RISOU Co., Ltd(Tokyo) at JACTA (Tokyo). The study period was 12
weeks, from March 26th to June 19th, 2016. This study was
conducted in accordance with the ethical principles of thedeclaration of Helsinki. The study protocol was approvedby the Institutional Review Board of Pharmaceutical Law
Wisdoms (Tokyo). Written informed consent was
obtained from all subjects.
The allocation of the test product to the subjects was
carried out by the person in charge of allocation. The
allocation list was sealed and strictly controlled in a safe
deposit box of JACTA until the end of the study.
2.2. Subject
Healthy subjects participated in the present study. All of
the subjects in this study were public volunteers who had
enrolled in the monitor bank of CROee Inc. (Tokyo),recruited from February through March, 2016.
2.2.1. Inclusion criteria(1) Females aged between 35 and 59 years.
(2) Females with wrinkle problems (evaluated between
grades 1 and 3 upon the guideline for evaluation of antiwrinkle products by a task-force committee for evaluationof anti-aging function 9)(3) Non-smoker
2.2.2. Exclusion criteria(1) Individuals diagnosed with photosensitive dermatosis;(2) Individuals with skin lesions at the test site (atopic

第 5 号（2017 年 5 月）

Table 1 Nutritional content of the test samples per 4 capsules
Item

RS

Placebo

Energy7.74 kcal

5.91 kcal

Protein
Lipid
Carbohydrate
Salt equivalent

0.02 g0.06 g1.77 g0.00 g0.00 g0.02 g1.43 g0.00 gdermatitis, hives, inflammation, eczema, injury, acne
spots, knots, speckles, or their traces);(3) Individuals on medication, including herbal medicinesthat may affect the test (adrenal cortex hormone,collagen, hyaluronic acid, etc.);(4) Individuals with a pollen allergy or may be allergic to
the test materials (collagen peptide, gelatin, dextrin,
ﬂavor, etc.);(5) Individuals who are pregnant, nursing, or likely to
become pregnant during the trial;(6) Individuals judged to be unsuitable to participate in
the trial by the principle investigator.
2.3. Randomization
From all of 155 applicants, 108 were eliminated according
to the wrinkle grade upon the guideline for evaluation ofanti-wrinkle products. The inclusion criteria was judged
by the principle investigator. All subjects weresequentially allocated to Group A (n=24) and Group B(n=23) using a random number table. In the process ofsubject assignment, background factors such as age wastaken into consideration to avoid biased distribution.
Subjects in Group A ingested the placebo and subjects inGroup B ingested the test sample for 12 weeks.
2.4. Description of test foods and blinding
The active material of the test product is RHIZOPUS/
RICE FERMENT FD POWDER, which features special 4amino acids; Glutamine acid, Aspartic acid, Proline, andArginine. The test product Repair Supple ( RS ) was
prepared by RISOU Co., Ltd. The amount of daily intake
was 4 capsules (1 capsule weighs 495 mg, therefore 4
capsules weigh 1,980 mg). The Placebo does not includeRHIZOPUS/RICE FERMENT FD POWDER. Bothcapsules were indistinguishable in shape, color, or taste,
and were managed by an identification symbol. All
involved were blinded. Table 1 shows the nutritional
contents of the samples.
2.5. Experimental procedures
2.5.1. Experimental protocol
Subjects consumed 4 capsules of the supplement with hotor cold water every day for 12 weeks. Subjects were
instructed as follows: to take the assigned foods as
indicated; to maintain their usual lifestyles and habits; toavoid excessive amounts of food, drink, or alcohol; toavoid direct sunlight; to avoid excessive exercise; tomaintain a daily record of that included the intake of test
foods (or not), the status of facial skin and physical診療と新薬・第 54 巻

Table 2Term
Item

Screening

Informed consent
Selection and/or allocation
WrinklesSurface spots
MelaninWhiteness of the complexion
Stratum corneum water content
TEWL
Elasticity
Subjective reporting
Ingestion of test foods
Log

第 5 号（2017 年 5 月）

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Schedule for the study
Pretrial test

●
●
●

●
●
●
●
●
●
●
●

Test period
4w

8w

12 w

●
●
●
●
●
●
●

●
●
●
●
●
●
●

●
●
●
●
●
●
●
●

● : Implementation↔ : Daily practice during the test period

condition during the test period; and to send the diary to
the study coordinator.
2.5.2. Outcome
According to the schedule shown in Table 2, we
measured parameters on efficacy and safety. These
assessments were conducted upon pre-intervention andpost-intervention.
Wrinkles, the amount of surface spots (visible spots andhidden ones), the amount of melanin, the whiteness ofthe complexion, the stratum corneum water content, thetrans-epidermal water loss (TEWL), and the elasticity ofthe skin was measured to evaluate the skin condition as
the primary outcome.
The subjects stayed in the environmental laboratory(temperature; 22±2 ℃ , humidity; 50±10%) for twenty
minutes before starting the trial.
The wrinkles found around the corner of the left andright eye were graded by the study conductor throughoptical observation of the skin with photos taken by aprofessional photographer, according to the guideline for
evaluation of anti-wrinkle products by a task-force
committee for the evaluation of anti-aging function 9). The
average grade of the two eyes was then calculated per
subject. The amount of two kinds of surface spots on theface was measured by VISIA Ⓡ Evolution (CANFIELD
Imaging Systems, Inc.) 10). The amount of melanin wasmeasured by MexameterⓇ MX18 (Courage & Khazaka
electronic GmbH) 11) 12), and for the whiteness of thecomplexion, lightness (L*) was measured by aspectrophotometer, NF555 (Nippon Denshoku Industries
Co., Ltd) 13). For the stratum corneum water content, theTEWL, and the skin elasticity, CorneometerⓇ CM825,
TewameterⓇ TM300, and CutometerⓇ MPA580 (Courage& Khazaka electronic GmbH, Germany) were used
respectively 14)-16). For the amount of surface spots, theamount of melanin, the whiteness of the complexion,
stratum corneum water content, TEWL, and the skinelasticity, both sides of the face were measured. The
average value between both sides was then calculated per
subject. Regarding the skin elasticity, 3 kinds of indexes
were measured; R2 (Recovery ratio of the skin length),
R5 (Ratio of elasticity part when elongated and during
constriction), and R7 (Ratio of elasticity part during
constriction) 17) 18).
Further, subjective reporting of the skin was observed
by a questionnaire as the primary outcome. The
questionnaire covered 20 items (Appendix 1). Responses
to each question were rated on an ordinal scale of 0 to 9,
with higher scores indicating a better result.
To evaluate the safety of the test foods, adverse eventswere collected by means of a written questionnaireduring the study.
2.6. Data analysis
A full analysis set was adopted in the present study andno sample size design was used. All statistics wereexpressed as mean ± standard deviation (SD).
For the wrinkleʼs grade, changes from baseline in thesame group were assessed using Wilcoxon signed-rank
test. Mann‒Whitney U test was used for intergroupcomparisons of changes from the baseline. With respect
to the amount of the two kinds of surface spots, theamount of melanin, the lightness (L*), stratum corneum
water content, TEWL, the three kinds of elasticity,
scores of subjective reporting, and blood biochemical andurine analysis, changes from baseline in the same groupwere assessed using the paired t-test. Studentʼs t-test
was used for intergroup comparisons of changes frombaseline. Studentʼs t-test was used to compare subject
backgrounds between groups.
Multiplicity according to the occasions was not adjusted.

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Enrollment

第 5 号（2017 年 5 月）

Assessed for eligibility (n＝155)

Excluded (n＝108)¨Did not meet inclusion criteria (n＝108)¨¨Wrinkle s grade (n＝108)¨Declined to participate (n＝0)

Randomized (n＝47)
Group AGroup B

Allocation
Allocated to RS (n＝23)¨Received allocated intervention (n＝23)¨Did not receive allocated intervention (n＝0)

Allocated to Placebo (n＝24)¨Received allocated intervention (n＝24)¨Did not receive allocated intervention (n＝0)

Follow-Up
Lost to follow-up (n＝0)
Discontinued intervention (n＝1)¨Being unwell (n＝1)

Lost to follow-up (n＝0)
Discontinued intervention (n＝1)¨Being unwell (n＝1)

AnalysisAnalysed (n＝22)¨Excluded from analysis (n＝0)

Analysed (n＝23)¨Excluded from analysis (n＝0)

Figure 1 Flow diagram of subject disposition

Table 3 Subject demographics
Item

Unit

RS

Placebo

Subjects
Age ＊numbersyears

22
45.5 ± 5.1

2345.4 ± 4.9

mean ± SD＊

No signiﬁcant diﬀerence

Any subjects with missing values were eliminated fromthe analysis. Statistical analyses were performed using
Statcel 4 (Yanai, 2015). The results were consideredsigniﬁcant at a <5% level in the two-sided test.

3. RESULTS
3.1. Participant demographics
The 47 subjects were randomly assigned to intervention
groups and made a start with ingestion. 2 werewithdrawn (unwell), and 45 subjects completed the study.
Thus, data obtained from 45 subjects (RS; 22, Placebo;23) were used for efficacy analysis (Figure 1). There
were no signiﬁcant diﬀerences in the age between groups(Table 3).
3.2. Wrinkle grade9)
The results of the wrinkle grade are shown in Table 4.
The average score for wrinkles at the corner of the rightand left were significantly lower in RS compared with

診療と新薬・第 54 巻

baseline data. Further, a signiﬁcant diﬀerence of changes
was observed between the two groups after 4, 8, and 12
weeks of ingestion.
3.3. Two kinds of surface spots 10)
The average amount of visible spots and hidden ones onboth sides of the face are shown in Table 4. In RS, valuesof both visible and hidden spots increased signiﬁcantly atweek 8 and 12.
3.4. Melanin 11) 12)
The average amount of melanin on both sides of the faceare shown in Table 4. Whilst in both groups, melaninincreased significantly at week4, 8, and 12, significant
differences between the two groups were found in⊿ 0-4w, ⊿ 0-8w, and ⊿ 0-12w.
3.5. The whiteness of the complexion (L*) 13)
For the whiteness of the complexion, Table 4 represents
the amount of L* on both sides of the face. At week 8, L*
decreased significantly in Placebo. Moreover ⊿ 0-8w,
and ⊿ 0-12w showed a signiﬁcant diﬀerence between thetwo groups.
3.6. The stratum corneum water content 14) 15)
Table 4 illustrates the average value of stratum corneum
water content on both sides of the face. Values of stratum
corneum water content was statistically signiﬁcant in RS
at week 8 and 12. Furthermore, ⊿ 0-8, and ⊿ 0-12w
show a signiﬁcant diﬀerence between the two groups.
3.7. The trans-epidermal water loss (TEWL) 16)
Table 4 shows the average value of TEWL on both sidesof the face. Both groups resulted in a signiﬁcant decreaseat week 4, 8, and 12 compared to the baseline, however
no significant difference was detected in the betweengroup comparison of changes from pretrial value.
3.8. The three kinds of elasticity 17) 18)
The results of the three kinds of elasticity are shown in
Table 4. At week 12 of R2, elasticity increased
significantly in RS. R2 also differed significantly in thecomparison of ⊿ 0-12w between the groups.
3.9. Subjective skin condition assessments
The results of subjective skin condition assessments areshown in Table 5. Moisture, Softness, Skin texture,
Foundation adhesion, Skin condition, Crowʼs feet, andEye bagʼs sagginess were signiﬁcantly diﬀerent after 12
weeks of between-group comparison of changes frompretrial value.
3.10. Safety
No adverse eﬀects associated with the test product wereobserved in the course of the reporting.

4. DISCUSSION
We conducted a randomized, placebo-controlled, doubleblind study in order to examine how the food containing
RHIZOPUS/RICE FERMENT FD POWDER affects theskin condition. As the primary outcome, the RS showed asigniﬁcant diﬀerence in Wrinkle grade , The whitenessof the complexion , The stratum corneum watercontent , Melanin , and Elasticity R2 after 12-week

第 5 号（2017 年 5 月）

65 （597）

ingestion, compared to the Placebo. Also, as for the
subjective reporting in form of a questionnaire, thebetween-group comparison of changes from pretrial value
showed a significant difference in the items ofMoisture , Softness , Skin texture , Foundation
adhesion , Skin condition , Crowʼs feet , and Eyebagʼs sagginess after ingestion of 12 weeks. On theother hand, the improvement tendency was not observedin Spot or The trans-epidermal water loss . In
addition, as the secondary outcome, no adverse effects
associated with the test product were observed in thecourse of the reporting, and the safety of ingesting thetest product was suggested.
Main Findings
This study showed the significant improvement in theitems related to moisture, wrinkle or elasticity, inboth test categories of the objective assessments(measurements) and the subjective assessments(questionnaire).
Skin consists of three layers: epidermis, dermis andsubcutaneous tissue. The epidermis has four layers ofstratum corneum, granular layer, stratum spinosum andbasal layer, while in the dermis, collagen ﬁber and elastic
fiber are complexly intertwined and the area between
these two fibers is filled with mucopolysaccharide andfibroblasts 19). The Skin aging is caused by two factors:
intrinsic aging and extrinsic aging 20). The increasing age
is categorized as the intrinsic aging, and because of theincreasing age, the whole epidermis becomes thinner andthe stratum spinosum adversely becomes thicker. Thesechanges cause the deterioration of moisture retention andthe acceleration of drying of the skin 21). They also degrade
the functions such as sebum secretion or sweat glands,
and the amount of the trans-epidermal water loss
increases 22). Furthermore, they cause the decrease of theamount of ceramide or amino acid in the dermis 23). The
extrinsic aging, on the other hand, is triggered by thedrying or oxidizing. Also, ultraviolet is a great enemy tothe skin; once the skin is exposed to ultraviolet, the agingof skin dramatically becomes advanced. UVB (290 〜
320 nm) mainly stimulates the epidermal keratinocyte andpromotes the production of cytokine. The cytokine
stimulates the pigment cells to help the melanin
production, or work on the fibroblasts of dermis andsevers collagen or elastic fiber. UVA (320 〜 400 nm)
directly stimulates the fibroblasts of dermis to promotewrinkle formation 21). In addition, the exposure of thehorny cell layer to ultraviolet leads to the decrease ofintercellular lipid or NMF (Natural Moisturizing Factor).
This decrease triggers the loss of moisture retention
f u n c t i o n o f t h e l a y e r, t h e i n c r e a s e o f m o i s t u r eevaporation and eventually the loss of moisture level ofthe layer 24). Based on the above aging phenomena, theskin is involved with several problems such as wrinkles,
spots, sagging, porous skin and/or dullness.
RHIZOPUS/RICE FERMENT FD POWDER contained66 （598）

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Table 4Item
〈unit〉

第 5 号（2017 年 5 月）

Results of test analyses
Scores/ Values 1）
P-value 2）Time points
RS（n=22）

Placebo（n=23）

2.63 ± 0.51
2.14 ± 0.48
Baseline
2.22 ± 0.37 ＊＊2.15 ± 0.48
4-week
− 0.41 ± 0.26
⊿ 0-4 w0.01 ± 0.05
Wrinkles
8-week
2.18 ± 0.41 ＊＊2.15 ± 0.48
〈scores〉⊿ 0-8 w
− 0.44 ± 0.28
0.01 ± 0.05
2.15 ± 0.48
12-week
2.13 ± 0.38 ＊＊0.01 ± 0.05
⊿ 0-12 w− 0.50 ± 0.28
Baseline
102.5 ± 25.1
103.3 ± 30.4
4-week
100.4 ± 26.3
105.8 ± 31.9
⊿ 0-4 w
− 2.1 ± 13.8
2.4 ± 9.3
Visible spots
106.2 ± 31.4
8-week
108.1 ± 28.0 ＊〈numbers〉⊿ 0-8 w
5.5 ± 12.4
2.8 ± 10.4
107.5 ± 29.9 †12-week
107.9 ± 25.6 ＊4.2 ± 10.6
⊿ 0-12 w
5.4 ± 10.2
Baseline
302.3 ± 35.0
302.1 ± 39.6
4-week309.3 ± 29.8
306.8 ± 45.7
⊿ 0-4 w
7.1 ± 20.6
4.7 ± 20.8
Hidden spots
302.1 ± 56.3
8-week
312.8 ± 30.8 ＊〈numbers〉
0.0 ± 28.5
⊿ 0-8 w
10.6 ± 21.7
12-week
315.5 ± 35.8 ＊＊306.3 ± 51.3
⊿ 0-12 w
13.3 ± 20.1
4.2 ± 24.6
126.2 ± 32.0
123.2 ± 27.8
Baseline
152.5 ± 29.5 ＊＊173.0 ± 29.7 ＊＊4-week26.3 ± 18.3
⊿ 0-4 w
49.7 ± 15.7
Melanin
8-week161.6 ± 27.0 ＊＊176.5 ± 32.0 ＊＊〈index〉⊿ 0-8 w
35.4 ± 17.7
53.2 ± 17.2
190.8 ± 42.3 ＊＊12-week
165.3 ± 25.1 ＊＊67.6 ± 29.0
⊿ 0-12 w
39.1 ± 20.7
Baseline
62.80 ± 3.04
62.32 ± 2.85
4-week
61.34 ± 6.59
61.77 ± 2.36
⊿ 0-4 w
− 1.45 ± 6.43
− 0.55 ± 3.04＊
L
8-week
62.59 ± 2.00
60.06 ± 2.35 ＊＊〈index〉⊿ 0-8 w
− 0.21 ± 2.16
− 2.26 ± 3.65
12-week
63.48 ± 2.23
61.31 ± 1.78
⊿ 0-12 w
0.68 ± 2.21
− 1.01 ± 2.86
Baseline
56.14 ± 15.78
44.76 ± 12.09
4-week50.26 ± 13.35 †34.45 ± 15.55 ＊⊿ 0-4 w
− 5.89 ± 14.80− 10.32 ± 19.12
Stratum corneum
water content
39.32 ± 12.11
8-week
64.89 ± 12.22 ＊＊〈index〉− 5.44 ± 15.58⊿ 0-8 w
8.75 ± 13.11
12-week
80.65 ± 11.44 ＊＊39.38 ± 15.38⊿ 0-12 w
24.50 ± 12.60− 5.38 ± 21.97
18.21 ± 7.09
Baseline
17.13 ± 4.63
14.97 ± 6.58 ＊＊4-week
14.48 ± 4.04 ＊＊− 3.24 ± 3.76
⊿ 0-4 w
− 2.65 ± 2.95
TEWL
8-week
13.57 ± 4.92 ＊14.37 ± 6.04 ＊＊〈g/hm2〉⊿ 0-8 w
− 3.57 ± 6.05
− 3.85 ± 4.47
13.20 ± 8.22 ＊＊12-week
11.90 ± 6.13 ＊＊− 5.01 ± 7.63
⊿ 0-12 w− 5.23 ± 7.21
0.575 ± 0.046
Baseline
0.683 ± 0.083
0.551 ± 0.052 ＊4-week
0.634 ± 0.074 ＊＊− 0.023 ± 0.045⊿ 0-4 w
− 0.049 ± 0.052
Elasticity（R2）8-week
0.645 ± 0.070
0.537 ± 0.053 ＊＊〈ratio〉⊿ 0-8 w
− 0.038 ± 0.106− 0.038 ± 0.049
0.569 ± 0.056
12-week
0.745 ± 0.085 ＊− 0.006 ± 0.051⊿ 0-12 w
0.062 ± 0.111
0.352 ± 0.045
Baseline
0.450 ± 0.062
0.305 ± 0.048 ＊＊4-week
0.402 ± 0.052 ＊＊− 0.047 ± 0.039⊿ 0-4 w
− 0.048 ± 0.039
Elasticity（R5）8-week
0.398 ± 0.069 ＊＊0.319 ± 0.054 ＊＊〈ratio〉⊿ 0-8 w
− 0.051 ± 0.066− 0.033 ± 0.053
12-week
0.457 ± 0.083
0.334 ± 0.058⊿ 0-12 w
0.007 ± 0.065− 0.017 ± 0.055
Baseline
0.312 ± 0.052
0.247 ± 0.034
0.227 ± 0.036 ＊＊4-week
0.297 ± 0.039 †⊿ 0-4 w
− 0.015 ± 0.037− 0.020 ± 0.026
Elasticity（R7）8-week
0.293 ± 0.054 †0.231 ± 0.038 ＊〈ratio〉⊿ 0-8 w
− 0.019 ± 0.044− 0.016 ± 0.029
0.242 ± 0.041
12-week
0.332 ± 0.064 †− 0.005 ± 0.037⊿ 0-12 w
0.020 ± 0.052
Scores and Values are expressed as the mean ± SD.
1）† p ＜0.1, ＊ p ＜0.05, ＊＊ p ＜0.01 against baseline.
2）‡ p ＜0.1, # p ＜0.05, ## p ＜0.01 between-group diﬀerence in change from baseline.

＜0.001 ##＜0.001 ##＜0.001 ##

0.205
0.428
0.709

0.702
0.169
0.182

＜0.001 ##0.001 ##
＜0.001 ##0.546
0.028 #
0.033 #

0.391
0.002 ##
＜0.001 ##

0.561
0.859
0.922

0.089 ‡
0.990
0.011 #

0.948
0.312
0.174

0.620
0.819
0.071 ‡

診療と新薬・第 54 巻

Table 5Item

第 5 号（2017 年 5 月）

67 （599）

Results of questionnaire analyses

Time points

Scores 1）
RS（n=22）

Placebo（n=19）

P-value 2）

4.5 ± 1.3
Baseline
3.7 ± 1.3
4.6 ± 1.1
12-week
5.9 ± 1.5 ＊＊0.1 ± 0.9
＜0.001 ##⊿ 0-12 w
2.2 ± 1.7
Baseline
4.3 ± 1.0
4.3 ± 1.2
2 Refreshing feeling
12-week
4.8 ± 1.3 ＊4.7 ± 1.5 †⊿ 0-12 w
0.5 ± 0.9
0.4 ± 1.2
0.840
Baseline
4.3 ± 1.4
4.6 ± 1.1
3 Greasiness
12-week
4.8 ± 1.3
5.2 ± 1.3 ＊⊿ 0-12 w
0.5 ± 1.3
0.6 ± 1.3
0.698
Baseline
4.0 ± 1.2
4.6 ± 1.2
4 Softness
12-week
5.7 ± 1.4 ＊＊4.5 ± 1.2
⊿ 0-12 w
1.8 ± 1.2
0.0 ± 0.7
＜0.001 ##Baseline
3.5 ± 1.3
3.4 ± 1.55 Skin elasticity
12-week
5.5 ± 1.3 ＊＊4.6 ± 1.4 ＊＊⊿ 0-12 w
2.0 ± 1.5
1.2 ± 1.2
0.072 ‡
3.1 ± 1.9
Baseline
3.4 ± 1.6
4.0 ± 1.9 ＊＊6 Skin texture
12-week
5.2 ± 1.6 ＊＊0.9 ± 1.4
0.041 #
⊿ 0-12 w
1.9 ± 1.6
Baseline
2.9 ± 1.4
2.6 ± 1.8
3.8 ± 1.7 ＊＊7 Sagginess
12-week
4.8 ± 1.4 ＊＊⊿ 0-12 w
1.9 ± 1.7
1.2 ± 1.2
0.111
Baseline
3.3 ± 1.7
2.9 ± 1.9
8 Pore distinctiveness12-week
4.7 ± 1.8 ＊＊3.7 ± 1.8 ＊＊⊿ 0-12 w
1.3 ± 1.5
0.8 ± 1.2
0.165
Baseline
3.0 ± 1.3
2.9 ± 1.5
9 Facial wrinkle
12-week
4.8 ± 1.8 ＊＊4.2 ± 1.6 ＊＊⊿ 0-12 w
1.8 ± 1.6
1.3 ± 1.5
0.291
Baseline
2.6 ± 1.5
2.7 ± 1.9
10 Surface spots
12-week
4.5 ± 1.7 ＊＊3.7 ± 1.7 ＊＊⊿ 0-12 w
1.9 ± 1.7
1.0 ± 1.4
0.054 ‡
Baseline
3.3 ± 1.2
3.8 ± 1.3
11 Foundation adhesion
12-week
5.3 ± 1.7 ＊＊4.0 ± 1.3 †⊿ 0-12 w
2.0 ± 1.4
0.2 ± 0.5
＜0.001 ##3.8 ± 1.3
Baseline
3.7 ± 1.3
4.8 ± 1.5 ＊＊12 Make up degradation
12-week
5.1 ± 1.7 ＊＊1.1 ± 1.5
0.481
⊿ 0-12 w
1.4 ± 1.3
3.6 ± 1.2
Baseline
3.7 ± 1.3
5.8 ± 1.8 ＊＊13 Skin condition
12-week
4.7 ± 1.5 ＊＊2.2 ± 1.7
⊿ 0-12 w
1.0 ± 1.5
0.022 #
Baseline
3.4 ± 1.3
2.8 ± 1.6
14 Dark circle
12-week
4.8 ± 1.3 ＊＊3.9 ± 1.9 ＊＊⊿ 0-12 w
1.3 ± 1.0
1.1 ± 1.6
0.606
Baseline
3.2 ± 1.4
2.7 ± 2.1
15 Dullness
12-week
4.9 ± 1.5 ＊＊4.1 ± 1.9 ＊＊⊿ 0-12 w
1.7 ± 1.5
1.4 ± 1.5
0.612
Baseline
3.5 ± 1.3
3.2 ± 2.0
16 Skin luster
12-week
4.8 ± 1.6 ＊＊4.8 ± 1.6 ＊＊⊿ 0-12 w
1.2 ± 1.2
1.6 ± 1.3
0.312
Baseline
4.4 ± 1.2
4.3 ± 2.0
17 Acne/ Pimple
12-week
5.5 ± 1.4 ＊＊5.1 ± 1.6 †⊿ 0-12 w
1.0 ± 1.3
0.8 ± 1.80.581
4.0 ± 1.7
Baseline
3.7 ± 1.1
4.5 ± 1.3
18 Redness
12-week
4.5 ± 1.1
0.419
0.5 ± 1.2
⊿ 0-12 w
0.7 ± 1.0
Baseline
3.2 ± 1.3
3.8 ± 1.6
19 Crow s feet
12-week
4.8 ± 1.9 ＊＊4.4 ± 1.4 ＊⊿ 0-12 w
1.6 ± 1.5
0.6 ± 1.0
0.012 #
3.5 ± 2.0
Baseline
3.5 ± 1.2
3.7 ± 1.8
20 Eye bag s sagginess
12-week
4.7 ± 1.7 ＊＊0.1 ± 0.6
＜0.001 ##⊿ 0-12 w
1.2 ± 1.2
Scores are expressed as the mean ± SD.
1）† p ＜0.1, ＊ p ＜0.05, ＊＊ p ＜0.01 against baseline.
2）‡ p ＜0.1, # p ＜0.05, ## p ＜0.01 between-group diﬀerence in change from baseline.
1

Moisture68 （600）

診療と新薬・第 54 巻

in the RS has a wealth of amino acids. In particular, it
contains plenty of glutamine acids, aspartic acid, proline
and arginine. Amino acids constitute the protein that
formulates the human organs such as skin, internalorgans or blood vessels. Twenty types of amino acids
formulating protein become the ingredients for producing
collagen, the chief element of the skin. NMF inside thestratum corneum is also composed of amino acids. There
are reports that the introduction of amino acids (i.e.
iontophoresis) encourages the production of collagen
which is damaged as a result of aging 25), or the intake ofamino acids improves the barrier functions of skin andallow it heal quickly in case of a wound 26). The otherreport illustrates that the ingestion of natural
moisturizing amino acids improves dry skin 27). Proline andaspartic acid, which are found in large quantities in
RHIZOPUS/RICE FERMENT FD POWDER, exist in thedermic layer of skin. They are the major component ofcollagen which dictates the skin elasticity, and they arealso the constituent amino acid of NMF 28). Therefore, it isconsidered that the ingestion of RHIZOPUS/RICEFERMENT FD POWDER promoted the production ofcollagen and/or NMF. Also, since there is a report thatthe arginine contained in RHIZOPUS/RICE FERMENTFD POWDER stimulates the production of NO andcontributes to blood circulation improvement 29), thefunctions such as increasing overall blood flow and/or
alleviating the skin dullness can be expected by theingestion of the test product. Based upon the discussion
above, we can speculate that the ingestion of
RHIZOPUS/RICE FERMENT FD POWDER containing
amino acids contributed to the promotion of the collagenproduction on the skin, the promotion of the productionof NMF or the blood circulation eﬃcacy, all of which leadto the improvement of skin elasticity or tightness.
Although at the randomization phase prior to the study,
we allocated the test subject as equally as possible, thebaseline at the beginning of the test showed a signiﬁcantdiﬀerence between the RS and Placebo. However, it did
not inﬂuence the assessment of the test result since theassessment was carried out based on the amount ofchange. Therefore we can determine that wrinkle
grade improved significantly compared to the Placebo.
R2 (of Elasticity ) also showed a significant
improvement.
For the assessment of elasticity, we used R2 (Recovery
ratio of the skin length), R5 (Ratio of elasticity part whenelongated and during constriction), and R7 (Ratio ofelasticity part during constriction), and among them R2
and R7 in particular are an important elasticity index of
the cutometer. It is reported that R2 and R7 decrease
with aging over 30); especially, R7 dramatically decreases
with advancing of age. As for the test result of R7, thetest group showed an improvement tendency comparedto the Placebo, and this result indicates tendency that theingestion of the RS improved the skin elasticity. On the

第 5 号（2017 年 5 月）

other hand, it can also be said that the 12-week test
period was not suﬃcient for R7 to show a deﬁnite sign ofsignificant difference due to its characteristic ofdramatically decreasing with advancing age. As for thetest result of R5, it did not even show the improvement
tendency. It is unclear that this result was triggered byan insuﬃcient test period (same as that of R7), or it was
because the RS is not effective for the improvement of
the condition speciﬁed in R5. Therefore further study will
be needed to clarify the matter. To sum up, it isconsidered that all test outcomes discussed above were
yielded by the increase of collagen (which is forced todecrease by the skin aging) after the ingestion of the RS.
In addition, this study showed an improvement tendency
in stratum corneum water content, an index of skinmoisture 31). This result was possibly produced as a resultof the increase of NMF by the ingestion of the RSG.
Furthermore, L* index score which represents thebrightness of skin showed a significant improvementcompared to Placebo. As for the trans- epidermal water
loss (TEWL) which is regarded as an index of skin barrier
function 32), we could observe signiﬁcant improvement in
the intra-group comparison, whereas the improvement
was not seen in the inter-group comparison. Although wecannot determine these results were achieved byingesting the RS since the same improvement tendency
was seen in Placebo, it can be said that the ingestion of
the RS somewhat contributed to the improvement of theskin condition by providing some effects such as the
improvement of blood circulation and/or skin moisture,
and also the improvement of the balance between waterand oil on the surface of skin. As for spots or melanin, on
the other hand, the study did not show an improvement.
Among the amino-acids, cysteine or glycine are reported
to have the function of melanin inhibition 33) 34). However,the RS contains only a small quantity of cysteine, and thisfact might aﬀect the test result. The results of subjective
reporting (questionnaire) also showed a significant
improvement in the items related to moisture or the skincondition. It is considered that these results were basedon the objective test outcome discussed above. To sum
up, it is speculated that the ingestion of RS contributed tothe improvement of the skin condition such as skinelasticity or skin moisture.
Secondary Findings
In this study, adverse events were collected by means ofa written questionnaire during the study, and noabnormal change caused by RS was observed during theingesting period. During the test period two test subjects
discontinued the test. The reasons of discontinuancewere personal ones such as being unwell, and had nothing
to do with the ingestion of RS. These results indicatedthe safety of the ingesting the test product for the 12week test period
General Information
Skin is a part which primarily catches anotherʼs attention,

診療と新薬・第 54 巻

and therefore aﬀects oneʼs ﬁrst impression. Also, it is thelargest organ in the body overlying the whole body, andplays an important role in the internal metabolism while
protecting the vital activity from outward threats.
RHIZOPUS/RICE FERMENT FD POWDER is an extract
which contains plenty of nutrition such as amino acids.
Since the protein is bio-degraded to the form of aminoacid (which is easily utilized in the body), the metabolism
speed of the extract is fast when ingested. Also, as aminoacid is a component material of living organisms, it is safe
for ingestion. To sum up, it is contemplated that if we can
improve the skin condition by ingesting food which iseasily-prepared and safe, it may contribute to not only
improving the QOL of women from the standpoint ofbeauty, but also maintaining the health of the body, suchas the prevention or improvement of skin disorders.
Limitations
In this study we used the food containing RHIZOPUS/
RICE FERMENT FD POWDER. The manufacturer of thefood confirmed that the category and amount of aminoacids contained in RHIZOPUS/RICE FERMENT FDPOWDER are stable and without variation from productto product. On the other hand, since it contains quite a
lot of amino acids, the outcome of this study was possibly
yielded by the complex interactions of the various (notsingle) amino acids or the admixture contained in theextract. However, the above discussion is merely aspeculation since we could not explain rationale for theseinteractions in this study. Therefore, it is expected thatthese interactions are studied by way of in vitro or in vivo
analysis.

5. CONCLUSION
In conclusion, we found out that the 12-week ingestion of
RS containing RHIZOPUS/ RICE FERMENT FDPOWDER, which has plenty of amino acids such as
glutamine acid, aspartic acid, proline, and arginine,
contributed to the improvement of the skin condition
such as elasticity, tightness or moisture. In addition, no
safety-related matter was observed during the 12-weektest period.
CONFLICT OF INTEREST
All parts of this study were funded by RISOU Co., Ltd. Katsumi Kurita is
the principal. All authors state that the study was conducted in theabsence of any other relationships that could be interpreted as a conﬂict ofinterest.

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第 5 号（2017 年 5 月）
Appendix 1 . Questionnaire of skin condition
Scale of 0 to 9, with higher scores indicating a better result.
Question #1: Moisture
Question #2: Refreshing feeling
Question #3: Tightness
Question #4: Softness
Question #5: Skin elasticity
Question #6: Skin texture
Question #7: Sagginess
Question #8: Pore distinctiveness
Question #9: Facial wrinkle
Question #10: Surface spots
Question #11: Foundation adhesion
Question #12: Make up degradation
Question #13: Skin condition
Question #14: Dark circle
Question #15: Dullness
Question #16: Skin luster
Question #17: Acne/ Pimple
Question #18: Redness
Question #19: Crowʼs feet
Question #20: Eye bagʼs sagginess