Application of Butterﬂy Pea Flower Extract in
Li Hsien Chen, I Chia Chen, Pei Yen Chen and Ping Hsin Huang *
Cardinal Tien Junior College of Healthcare and Management, New Taipei 23143, Taiwan;
firstname.lastname@example.org (L.H.C.); email@example.com (I.C.C.); firstname.lastname@example.org (P.Y.C.)
*Correspondence: email@example.com; Tel.: +886-2-2219-1131 (ext. 5114)
Received: 13 November 2018; Accepted: 5 December 2018; Published: 5 December 2018
(1) Background: Clitoria ternatea (butterﬂy pea), a plant species belonging to the Leguminosae
(Fabaceae) family, is useful for medical treatments and has been used in folk medicines and to cure
different diseases. The antioxidation ability of the total phenolic compounds of butterﬂy pea is useful
for preserving ﬂavor, and colour and for preventing vitamin destruction in processed foods. In this
study, a butterﬂy pea ﬂower fermentation solution was added to cosmetics as a whiting ingredient.
(2) Methods: After the phenolics, ﬂavonoids and ascorbic acid content of the butterﬂy pea ﬂower
extraction had been determined, lactic acid bacteria fermented the extraction. The whitening and
moisturizing effect was assayed by SSC3 and NF333 analyzers. (3) Results: This study demonstrated
that the butterﬂy pea ﬂower fermentation solution has free radical scavenging ability, a reducing
power in high concentrations, a moisturizing effect, and a whiting effect. (4) Conclusions: The results
showed that the butterﬂy pea ﬂower fermentation solution not only inhibits redness, itching, allergies,
and irritation to the skin, but also has antioxidation properties and promotes moisture retention and
whitening effects, and the results increase as the concentration increases. Therefore, butterﬂy bean
ﬂowers may be suitable as a raw material for natural beauty care products.
Keywords: butterﬂy pea ﬂower fermentation solution; moisture retention; whitening
The butterﬂy pea ﬂower (Clitoria ternatea) originates from subtropical regions and is widely
distributed in Africa, Asia, Australia, North America, South America, and the Northwest, Central
South, and Southwest Paciﬁc. Butterﬂy pea, like vine plants, is a perennial climbing plant or herbaceous
plant. It is self-pollinated and spreads by seeds. It prefers moist and neutral soil (pH 5.5 to 8.9). Butterﬂy
pea ﬂower is an axillary, solitary, or twin ﬂower. It is harvested in summer. The most apparent feature
of the butterﬂy pea ﬂower is its dark blue petals with a yellow mark. According to Zakaria et al. [
the ﬂavonoids found in the butterﬂy pea ﬂowers can reduce infections in the upper respiratory tract.
They have been proven to be anti-inﬂammatory in animal tests and have antioxidative power. Butterﬂy
pea ﬂowers contain about 0.9 mg ash, 8.94 mg soluble minerals, 41.27 mg crude protein, and 29.18 mg
soluble carbohydrate per 100 g of dry weight. Terahara et al. [
] used reversed-phase High Pressure
Liquid Chromatography (HPLC) to isolate ﬁve major structures of anthocyanins, which are delphinidin
derivatives that are highly acylated.
Tantituvanont et al. [
] pointed out that the anthocyanins in butterﬂy pea ﬂowers are mainly
delphinidin-glucosides. Anthocyanins are water-soluble macromolecular substances that give orange,
red, or blue-violet colour to fruits, vegetables, ﬂowers, leaves, or stems. Saptarini et al. [
that the anthocyanins in butterﬂy pea ﬂowers appear blue at pH 4, green at pH 9, and yellow at pH
12, making it an acid-base titration indicator. Anthocyanins are susceptible to environmental and
chemical inﬂuences, including pH variation, ambient temperature, light, oxidation, and enzymes,
Sci. Pharm. 2018,86, 53; doi:10.3390/scipharm86040053 www.mdpi.com/journal/scipharm
Sci. Pharm. 2018,86, 53 2 of 9
making their application in the food industry difﬁcult (Mohamed et al. [
]). Tantituvanont et al. [
Mohamed et al.
] found that the anthocyanins in butterﬂy pea ﬂower are more stable at low
temperatures, and the ﬂowers are purplish red in acidic environments and blue in an alkaline
environment. Compared with alkaline environments, the stability and antioxidative activity of the
anthocyanins are higher in weakly acidic environments. Rabeta et al. [
] pointed out that the blue
ﬂowers of butterﬂy peas have good free radical scavenging activity and have potential as antioxidants.
Rajamanickam et al. [
] suggested that the antioxidative power of the methanol extract of butterﬂy pea
ﬂowers is equivalent to that of L-ascorbic acid.
The butterﬂy pea ﬂower contains anthocyanins and thus, is a natural antioxidant that can delay
the aging of the skin and is good for the skin. Therefore, in this study, butterﬂy pea ﬂowers extracted
by cold water extraction and hot water extraction were analyzed for their total phenolics, ﬂavonoids,
and ascorbic acid content as well as their antioxidative power. Fermentation solution from lactic acid
bacteria contains lactic acid, peptides, and polysaccharides, which are excellent for skin whitening
effects. In this study, butterﬂy pea ﬂowers were fermented by the lactic acid bacteria in a safe and
pollution-free environment. The performance of the fermentation solution was evaluated for its
whitening and moisture retention effects in order to determine the value of using a butterﬂy pea ﬂower
fermentation solution in cosmetic applications.
2. Materials and Methods
2.1. Preparation of the Butterﬂy Pea Flower Fermentation Solution
The Butterﬂy Pea Flower was kindly provided by bluetopurple Corp. agency (Wujie Township,
Yilan County, Taiwan). Take 1 L of the culture medium listed in Table 1and sterilizer for 1 h at 121
at a concentration of 1.2 kg/cm
. Wait until room temperature is reached and then mix 50.0 g of
butterﬂy bean ﬂowers, 50.0 g of sugar-free soy milk, 5.0 g of lactic acid bacteria (Lactobacillus acidophilus,
also called strain A, ATCC 4357) and 1 L of deionized water in a UV disinfection chamber before moving
it to a shaking incubator for fermentation for 48 h at 30
C and 125 rpm. After centrifugation at a
high speed, take the supernatant and then sterilize it at 121
C at a concentration of 1.2 kg/cm
in the sterilizer for 1 h. Wait until room temperature is reached, and then ﬁlter it into the UV
Table 1. Butterﬂy pea ﬂower fermentation solution formula.
Approximate Per Liter
Proteose Peptone No.3 10.0 g
Yeast Extract 5.0 g
Dextrose 20.0 g
Polysorbate 80 1.0 g
Ammonium Citrate 2.0 g
Sodium Acetate 5.0 g
Magnesium Sulfate 0.1 g
Dipotassium Phosphate 2.0 g
2.2. Determination of the Total Phenolic Content
The total phenolics content measurement was done in accordance with Taga et al. [
]. The method
was as follows: Add 0.5 mL of 0.3% HCl to standard caffeic acid (0.001 mg/mL to 1.0 mg/mL) solutions
and mix it well. Take 100
L of the mixture, add 2.0 mL of 2% Na
to the mixture, mix it well,
and let it stand for 2 min. Add 0.1 mL of Folin–Ciocalteu agent, and let it stand for 30 min. Repeat
the same procedure for each concentration. Measure the OD765 value of each concentration with a
spectrophotometer and draw a calibration curve. Replace the caffeic acid with the butterﬂy pea extract
and follow the same procedure to determine the total phenolics of the sample .
Sci. Pharm. 2018,86, 53 3 of 9
2.3. Determination of Total Flavonoid Content
The method reported by Jia et al. [
] was revised. Different concentrations of quercetin
) were prepared as standard solutions. The OD values of different concentrations were
measured to draw a calibration curve and the regression equation was obtained. The method used
was as follows: Take 0.5 mL of the sample, add 1.5 mL of pure water, 0.1 mL of 10% aluminum nitrate,
9-hydrate, and 0.1 M of potassium acetate. Then, add 2.8 mL of pure water, mix it well, and let it stand
for 40 min. Measure the OD415 value with the spectrophotometer, and then calculate the ﬂavonoid
content according to the regression equation of the calibration curve.
2.4. Determination of the Ascorbic Acid Ccontent
Extract 1 mL of the extract with 10 mL of 1% metaphosphoric acid, and then add 9 mL of
2,6-dichloroindo-phenol (DPI) to react. Measure the OD515 value with the spectrophotometer,
and draw a calibration curve with different concentrations of ascorbic acid to calculate the ascorbic
acid content in the sample .
2.5. DPPH Free Radical Scavenging Ability
The method was performed as described by Yamaguchi et al. [
] as follows: Mix 100
the butterﬂy pea ﬂower extract well with 400
L of 100 mM Tris-HCl buffer (pH 7.4) and 500
M DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical ethanol solution in a microcentrifuge
tube before placing the tube in a 25
C thermostatic reactor for 20 min. Transfer the tube to a UV-Vis
spectrophotometer to measure the OD517 of the solution. Repeat the procedure three times. Calculate
the DPPH free radical scavenging rate with the following formula:
DPPH free radical scavenging rate (%) = [1 −(OD517 of sample/OD517 of blank solution] ×100% (1)
2.6. Determination of the Reducing Power
Take 2.5 mL of extract, add 2.5 mL of 0.2 M phosphate buffer (pH 6.6) and 2.5 mL of 1% potassium
ferricyanide. Place the solution in a 50
C water bath for 20 min. Add 2.5 mL of 10% TCA to the
solution after cooling, mix it well, and centrifuge at 3000 rpm for 10 min. Take 5 mL of supernatant,
add distilled water and 1 mL of 0.1% ferric chloride, and mix it well. Measure the OD700 value with
the spectrophotometer—the higher the OD value is, the better the reducing power is .
2.7. Whitening Effect and Moisture Retention Assay
Instrument: three-in-one skin analyzer (SSC3) and NF333 spectrophotometer (Figure 1);
Brand: SSC3, Courage-Khazaka Electronic Gmbh (CK), Köln, Germany;
NF333: NIPPON DENSHOKU, Tokyo, Japan.
This research protocol was approved by Human Research Ethics Committee of Research
Ethics Ofﬁce of National Taiwan University, protocol number: 201705ES002 (4 October 2018).
Sampling: The butterﬂy pea ﬂower fermentation solutions were diluted with distilled water
in the following concentrations. Two milliliters of 1.0%, 2.5%, 5.0%, 7.5%, and 10.0% diluted
solutions were used.
•Testing spots: forehead and cheek.
Testing age/skin condition: 18- to 20-year-old females. They are all obtained and indicated
this issue for publication.
•Testing environment: thermostatic indoor temperature at 22 °C.
Testing area: divided into an experimental area and a control area. The right site was the
experimental area (for the cleansing mousse with the butterﬂy pea ﬂower fermentation
Sci. Pharm. 2018,86, 53 4 of 9
solution), and the left side was the control area (for the cleansing mousse without the
butterﬂy pea ﬂower fermentation solution).
•Retention time: the entire face was cleaned and then we waited for 30 min until testing.
Figure 1. Three-in-one skin analyzer and NF333 spectrophotometer.
The total phenolics were 185.3 mg/100 g and 239.6 mg/100 g in butterﬂy pea ﬂower extract for
the cold water extraction and hot water extraction, respectively. The ﬂavonoid concentration was
106.9 mg/100 g and 128.3 mg/100 g in butterﬂy pea ﬂower extract in the cold water extraction and
hot water extraction, respectively. The ascorbic acid content was 10.36 mg/100 g in the butterﬂy pea
ﬂower cold water extraction. Because hot water destroys ascorbic acid, the ascorbic acid content was
not measured for the hot water extraction, and thus was indicated as “not detected” (N.D., see Table 2).
The results showed that hot water can extract more total phenolics and ﬂavonoids. However, ascorbic
acid is susceptible to high temperatures, so it cannot be extracted by hot water.
Table 2. Contents of total phenolics, ﬂavonoids, and ascorbic acid in the butterﬂy pea ﬂower sample.
Sample Extraction Method Total Phenolics
Butterﬂy pea ﬂower
Cold water extraction 185.3 ±0.096 106.9 ±0.23 10.36 ±0.028
Hot water extraction 239.6 ±0.081 128.3 ±0.054 Not Detected
3.1. DPPH Free Radical Scavenging Ability of the Butterﬂy Pea Flower Extract
DPPH free radicals are stable free radicals containing an odd number of electrons. When they
are combined with other free radicals or reduced by antioxidants, DPPH free radicals are scavenged
DPPH-H + A), and their color changes from purple to light yellow, which, in turn,
reduces their absorbance. The lower the absorbance is, the stronger a sample’s DPPH scavenging ability
is and the stronger its antioxidation ability is. DPPH ethanol solution has very strong absorbance at
517 nm visible light. Figure 2shows the DPPH free radical scavenging ability of the butterﬂy pea ﬂower
cold water extraction and hot water extraction. In terms of antioxidative power, the DPPH free radical
scavenging ability of the 1 mg/mL butterﬂy pea ﬂower hot water extraction was about 6.79% and that
of the 10 mg/mL extract was about 65.23%. When the concentration of the butterﬂy pea ﬂower hot
water extraction increased to 100 mg/mL, the DPPH free radical scavenging ability increased to 75.69%,
which is equivalent to 76% of 1 mg/mL BHT. As for the butterﬂy pea ﬂower cold water extraction,
the DPPH free radical scavenging ability was about 5.39% and 58.23% for 1 mg/mL and 10 mg/mL of
extract, respectively. When the concentration of the cold butterﬂy pea ﬂower extract was increased
to 100 mg/mL, the DPPH free radical scavenging ability increased to 63.25%, which is equivalent to
64% of 1 mg/mL BHT. The results showed that the butterﬂy pea ﬂower hot water extraction had a
better DPPH free radical scavenging ability than the cold water extraction. When the concentration
of the butterﬂy pea ﬂower extracts increased from 1 mg/mL to 10 mg/mL, the DPPH free radical
Sci. Pharm. 2018,86, 53 5 of 9
scavenging ability increased. This was true for the butterﬂy pea ﬂower cold water extraction and hot
water extraction. However, when the concentration was higher than 10 mg/mL, the DPPH free radical
scavenging ability did not increase linearly, indicating that the DPPH free radical scavenging abilities
of the butterﬂy pea ﬂower extracts were about 60% to 70% of 1 mg/mL BHT (Figure 2).
Figure 2. DPPH free radical scavenging ability of the butterﬂy pea ﬂower extracts.
3.2. Reducing Power of the Butterﬂy Pea Flower Extracts
The higher the OD
absorbance is, the better the reducing power is. In this study, 1 mg/mL,
10 mg/mL, 25 mg/mL, 50 mg/mL, and 100 mg/mL butterﬂy pea ﬂower cold water and hot water
extractions were used to determine the reducing power of butterﬂy pea ﬂower extract, and this was
compared with the standard BHT. The results showed that the OD700 values of the butterﬂy pea ﬂower
hot water extraction were about 0.5–2.3, and those of the butterﬂy pea ﬂower cold water extraction
were about 0.4–2.0, indicating that the butterﬂy pea ﬂower hot water extraction had a higher reducing
power. At the concentration of 100 mg/mL, the reducing power of the butterﬂy pea ﬂower hot water
extraction was about 96% of the standard BHT. However, that of the butterﬂy pea ﬂower cold water
extraction was only about 83% of the standard BHT (Figure 3).
Figure 3. Reducing power of the butterﬂy pea ﬂower extract.
3.3. Moisturizing Effect of the Butterﬂy Pea Flower Fermentation Solution
The butterﬂy pea ﬂowers were fermented by lactic acid bacteria. The fermentation solution
was diluted with distilled water to 1.0%, 2.5%, 5.0%, 7.5% and 10.0% diluents. The diluents were
Sci. Pharm. 2018,86, 53 6 of 9
the experimental group, whereas distilled water was the control group. After the assessment by the
moisture instrument, the moisture improvement rates after 14 days were 7.87% for the 1.0% diluent,
11.20% for the 2.5% diluent, 15.15% for the 5.0% diluent, 17.91% for the 7.5% diluent, and 18.11% for
the 10.0% diluent. The moisture improvement rates after 28 days were 10.20% for the 1.0% diluent,
14.28% for the 2.5% diluent, 15.91% for the 5.0% diluent, 19.63% for the 7.5% diluent, and 20.32% for
the10.0% diluent (see Table 3and Figure 4).
Moisture values of the fermentation solutions at different concentrations after 14 days and 28
days of use.
Concentration of Butterﬂy Pea
Flower Fermentation Solution
Day 14 Day 28
Control (%) Experimental (%) Control (%) Experimental (%)
1.0% 12.7 13.7 12.2 13.5
2.5% 12.5 13.9 12.25 14
5.0% 13.2 15.2 12.25 14.2
7.5% 13.4 15.8 12.12 14.5
10.0% 13.8 16.3 12.3 14.8
Moisture improvement rates (%) of the fermentation solutions at different concentrations
after 14 days and 28 days of use.
3.4. Whitening Effect of the Butterﬂy Pea Flower Fermentation Solution
The whitening improvement rate after 28 days was 7.99% for the 1.0% diluent; 10.53% for the 2.5%
diluent; 18.50% for the 5.0% diluent; 22.21% for the 7.5% diluent; and 29.97% for the 10.0% diluent
(see Table 4and Figure 5).
Whitening values of the fermentation solution at different concentrations after 28 days of use.
Concentration of Butterﬂy Pea
Flower Fermentation Solution
Control (%) Experimental (%)
1.0% 64.63 69.79
2.5% 63.87 70.60
5.0% 62.84 74.47
7.5% 65.02 79.46
10.0% 64.21 83.45
3.5. Formula Design and Product Development
With the rise in environmental awareness, many people have started to pay attention to the
safety of skincare products. We have all heard about the serious pollution and damage imposed
upon the environment by skincare products made of chemical raw materials, such as environmental
hormones. Such skincare products also harm the human body to various degrees. Therefore, natural
and environmentally-friendly products have become preferred by consumers.
Sci. Pharm. 2018,86, 53 7 of 9
Whitening improvement rates of the fermentation solutions at different concentrations after
28 days of use.
The above experiments conﬁrmed that the butterﬂy pea ﬂower fermentation solution has
whitening, moisture retention, and anti-aging effects. It is suitable for use as a raw material in skin care
products. Therefore, we have preliminarily designed a mask formula containing sodium bicarbonate,
hot spring water, and butterﬂy pea fermentation solution, and have looked for a manufacturer to
develop the product through industry–academia collaboration.
Good essence is the soul of a skin care product. Exquisite ingredients produce remarkable effects.
From design to material selection, we failed more than 10 times before coming up with the mask
formula presented in Table 5. After 30 days of use, the hot spring mask containing 6% butterﬂy pea
ﬂower fermentation solution can improve moisture retention by 20.35% (Table 6) and enhance the
whitening effect by 18.34% (Table 7). With respect to skin irritation, there was no irritation after 24 h of
patch testing. In an accelerated aging test, there was no deterioration after the sample was tested in a
thermostat chamber at 40 ±2◦C with a relative humidity of 75 ±5%.
Table 5. Butterﬂy pea ﬂower brightening mask containing hyaluronic acid and hot spring water.
No. Chinese Name %
1 Pure water To 100.00
2 Hot spring water 6.00
3 Fermented butterﬂy pea ﬂowers 6.00
4 Glycerin 2.00
5 Propylene glycol 0.80
6 Cucumis sativus (Cucumber) fruit extract 0.60
7 Saccharomyces lysate extract 0.45
8 Acrylates/ C10-30 alkyl acrylate crosspolymer 0.12
9 Phenoxyethanol 0.17
10 Xanthan gum 0.06
11 Chlorphenesin 0.20
12 Arginine 0.03
13 Sodium hyaluronate 0.30
14 Hydrolyzed hyaluronic acid 0.15
15 Allantoin 0.10
Ammonium acryloyldimethyltaurate/VP copolymer
17 Aloe barbadensis leaf juice 0.10
18 Panthenol 0.05
19 Glycyrrhiza glabra (Licorice) root extract 0.05
20 Fragrance 0.01
21 Capryl glycol 0.10
Sci. Pharm. 2018,86, 53 8 of 9
Average moisture retention effect of 20 subjects using the butterﬂy pea ﬂower hot spring
Control (%) Experimental (%)
Average whitening effect of 20 subjects using the butterﬂy pea ﬂower hot spring water mask.
Control (%) Experimental (%)
The experimental data show that the organic butterﬂy pea ﬂower extract or fermentation solution
not only did not cause redness, itching, allergy, or irritation to the skin but also improved moisture
retention and had whitening effects, and these effects increased as its concentration increased. As a
result, the butterﬂy pea ﬂower fermentation solution can be added to cosmetic formulas as a natural
raw material of skin care products. The outcome of the industry–academia collaboration can be added
to teaching materials of various courses, such as cosmetics preparation, projects, and on-campus
internships, and can enhance the students
interests and skills in cosmetics preparation and achieve
the effects of diverse learning. The teachers and students will also fulﬁll their local social responsibility,
assist in the innovation and development of the local industry, and be pioneers of the University Social
Responsibility (USR) Project. The university can also reuse the resources and make green living a
reality in order to develop a green economy, implement ecological concepts, and make life full of joy
Conceptualization, P.H.H.; determination of total phenolics, total ﬂavonoids, ascorbic
acid, DPPH free radical scavenging ability, and reducing power, L.H.C.; whitening effect and moisture retention
assay, P.H.H.; methodology, P.Y.C.; validation: I.C.C.; writing—original draft preparation, L.H.C. and P.H.H.;
writing—review and editing, L.H.C. and P.H.H.; supervision, P.H.H. and P.Y.C.
This research was funded by Cardinal Tien College of Healthcare and Management, grant number
Conﬂicts of Interest:
The authors declare no conﬂict of interest, and the funder had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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