Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : July 6, 2021
Accepted : August 25, 2021
Published online : September 30, 2021
doi:10.24659/gsr.8.3_148

Original article

Screening plant extracts for inhibition of hair glycation
Yasunori Okuma 1), Jiro Tanaka 1), Akimasa Mochizuki 1), Masayuki Yagi 2), Yoshikazu Yonei 2)
1) Research Center, Arimino Co., Ltd, Saitama, Japan2) Anti-Aging Medical Research Center and Glycative Stress Research Center,
Graduate School of Life and Medical Sciences, Doshisha University, Kyoto, Japan

Abstract

Objectives: It has been reported that hair glycated over time from the root to the tip exhibits changes in physical properties,
such as a decrease in breaking strength. However, few studies have examined the inhibition of hair glycation. In this study,
we examined the anti-glycation effect of plant extracts to identify materials that inhibit hair glycation.
Methods: A total of 26 plant extracts were used as the cosmetic ingredients. A glycation solution was prepared with 0.1
mol/L phosphate buffer (PB; pH 7.4), 5.0 mol/L glucose, and 5.0 mg/mL keratin; the sample to be tested was then added tothis solution. After thermal reaction, the levels of advanced glycation end products (AGEs) produced in the reaction solution
were measured using the fluorescence method (excitation wavelength 370 nm / detection wavelength 440 nm), and theAGE production inhibitory rate was calculated. In the primary screening, each sample was adjusted to a final concentrationof 0.1 mg/mL in the reaction solution with deionized water, and the AGE production inhibition rate was measured. In thesecondary screening, half maximal inhibitory concentration (IC 50) was calculated and evaluated for samples with an AGE
production inhibition rate of 75 % or more obtained in the primary screening. Next, we obtained hair samples from healthy
women in their 30s who had no history of beauty treatment, and verified the anti-glycation effects of the plant extracts. To
verify the anti-glycation effect, the hair sample was immersed in 0.1 mol/L phosphate buffer (pH 7.4) containing 1. 2 mol/
L of glucose and 0.1 mg/mL of each sample, and reacted at 50 °C for 10 days. Hair protein was extracted, and the levelsof fluorescent AGEs were measured. The physical properties of the hair were measured using a tensile tester at ambient
temperature (25 °C) and humidity (65 % RH).
Results: Following primary screening, 75 % or more of the AGE production inhibitory effect was observed in 9 out of 26
samples. The secondary screening results showed that the IC 50 of rooibos extract had a stronger anti-glycation effect thanthe IC 50 of aminoguanidine. An in vitro hair test showed that rooibos extract inhibited the production of fluorescent AGEs
(production inhibition rate 82.4 %). In addition, the breaking strength of the glycated hair was reduced by 20.8 % comparedto that of the un-glycated hair; however, after treatment with the rooibos extract, the reduction in breaking strength due toglycation could be suppressed to 4.9 %.
Conclusion: Rooibos extract was found to have an inhibitory effect on the production of fluorescent AGEs in hair proteins.
It also showed an inhibitory effect on the decrease in the breaking strength of the hair due to glycation. These results
indicate that the addition of rooibos extract to hair cosmetics may help inhibit the changes in the physical properties of haircaused by glycation.

KEY WORDS: hair, plant extract, Rooibos, glycation, advanced glycation endproducts (AGEs)

Introduction

The non-enzymatic reaction between reducing
sugars, such as glucose and proteins, is called glycation.
Glycation also occurs in vivo, and advanced glycation end
products (AGEs) are produced and accumulated in tissues.
Accumulation of AGEs in vivo has been reported to cause

physical, physiological, and visual damage 1). Glycation causes
cardiovascular diseases 2), osteoporosis 3), and dementia 4),
and is considered to be one of the risk factors for aging. Evenin the skin, it has been reported that AGEs accumulate inproteins such as keratin in the epidermis 5) and collagen 6) andelastin 7) in the dermis. It is considered that a large accumulationof AGEs in the stratum corneum of the skin causes it toContact address: Yasunori Okuma
Research Center, Arimino Co., Ltd, Saitama, Japan
25-5, Oazamiyako Namegawa-machi, Hiki-gun, Saitama, 355-0812 Japan
TEL: + 81- 493-57- 0621 FAX: + 81- 493-57-0623 e-mail: y-ohkuma@arimino.co. jp
Co-authors: Tanaka J, j-tanaka@arimino.co. jp; Mochizuki A, a-mochizuki@arimino.co. jp;
Yagi M, myagi@mail.doshisha.ac. jp; Yonei Y, yyonei@mail.doshisha.ac. jp_ 14 8 _Glycative Stress Research 2021; 8 (3): 148-155(c) Society for Glycative Stress ResearchGlycative Stress Researchlose its texture 8), and AGEs formed in the dermis cause theyellowing of the skin 9) and also decrease skin firmness andelasticity. Thus, skin glycation is involved in apparent aging,and in recent years, "anti-glycation" has been attracting
attention as an important factor in aging care.
Similarly, hair is an important factor for maintaining
a youthful appearance. Recently, it has been reported thathair is also glycated over time from the root to the tip andexhibits changes in physical properties, such as a decreasein breaking strength 10). Decreased breaking strength ofhair causes deterioration of hair quality, such as hairbreakage and split ends, and accelerates its aging in terms ofappearance. However, few studies have examined potential
therapeutics for the inhibition of glycation, particularly inhair. In this study, we examined the anti-glycation effect ofplant extracts to identify materials that inhibit changes in thephysical characteristics of hair caused by glycation.

Method(1) Reagents
The protein used in the glycation models was woolderived keratin, which was purchased from Nacalai TesqueCo., Ltd. (Kyoto, Japan). Anti-AGE antibody (clone 6D12)
was purchased from Transgenic Co., Ltd. (Fukuoka, Japan).
The Alexa546-labeled secondary antibody was purchasedfrom Thermo Fisher Scientific (Waltham, MA, USA). Can
Get Signal Immunostain Solution was purchased from
Toyobo Co., Ltd. (Osaka, Japan). Other reagents werepurchased from Fujifilm Wako Pure Chemical Industries,
Ltd. (Osaka) and were of analytical grade.

(2) Sample Preparation

Table 1 shows the 26 plant extracts used as cosmetic
ingredients in this study. Aminoguanidine (AG), which isan inhibitory agent for glycation reactions, was used as the
positive control in the investigations of the inhibitory effectsof AGEs.

(3) Verification of the inhibitory effects of the plant
extracts on glycation

The glycation Ker-glucose model was employed to verify
the inhibitory effects of the plant extracts on glycation 11).
A glycation solution was prepared with 0.1 mol/L phosphatebuffer ( PB; pH 7.4), 5.0 mol/L glucose, and 5.0 mg/mL
keratin; the sample to be tested was then added to thissolution. Subsequently, the glycation solution was reacted
at 60 °C for 10 days, and AGE-derived fluorescence wasmeasured using a microplate reader (excitation wavelength
370 nm/detection wavelength 44 0 nm). A reference (ref ) was
prepared with the same amount of purified water in placeof the samples and reacted under the same conditions. As apositive control for fluorescent AGEs, 1.0 mg/mL of AG wasadded in the same amount as the sample. The fluorescenceintensity was calculated as a relative value when the
fluorescence intensity of 5.0 µg/mL quinine sulfate was
1,000. The fluorescence AGE formation inhibitory rate wascalculated using the following formula:

The ratio of inhibitory effect on fluorescent AGE formation (%)= 100 – {(sample Glucose (+) – sample Glucose (–)) /(ref Glucose (+) – ref Glucose (–)) × 100},(4) Primary screening

In the primary screening, each sample was adjusted
to a final concentration of 0.1 mg/mL with purified water,
and the ratio of the inhibitory effect on AGE formation wasmeasured.

(5) Secondary screening
In the secondary screening, the half maximal inhibitory
concentration (IC 50) was calculated and evaluated for samples
with a fluorescence AGE production inhibitory rate of 75 %or more obtained in the primary screening. Calibration
curves for IC 50 were constructed by adding individual
samples to a reaction solution at three concentrations (0.1mg/mL, 0.01 mg/mL, 0.001 mg/mL) and calculating theinhibition of AGE formation after the reaction.

(6) Verification of the inhibitory effects on hairglycation

Hair was collected from healthy women in their 30s
who had no history of beauty treatment (bleach, hair color,permanent wave, straightening) with all participants
providing an informed consent in writing. Hair was glycatedin vitro by reacting 1. 2 mol/L of glucose and 0.1 mol/L eachsample in PB at 50 °C for 10 days. As a control (reference:ref ), an equal amount of purified water was added insteadof the sample, and the reaction was performed under thesame conditions.

(7) Measurement of AGEs in hair samples
Each hair sample was immersed in a degreasing agent[chloroform : methanol (MeOH) = 2:1] at room temperaturefor 16 hours. The hair was removed from the degreasing
agent, dried at room temperature for 1 hour or longer, andthen cut to a length of 5 mm or less. Following this, 10 mgof each hair sample was added to 500 μL extraction buffer,
which was exposed to 5 M urea, 2.6 M thiourea, 25 mmol/L
Tris-HCL buffer, and 5 % 3-mercapto-1, 2-propanediol (pH8.5), and then reacted at 50 °C for 1 d with stirring. The
sample was then centrifuged at 10,000 rpm for 1 minuteat 20 °C, and the resulting supernatant was recovered asa protein solution. The fluorescent AGEs of the protein
solution were measured using the fluorescence method in thesame manner as described above (3). Protein concentration
was measured using the Bradford method 12). Fluorescent
AGE content was calculated per 1 mg of the protein content.
The difference between the glucose-added group and thenon-glucose-added group was evaluated as the amount ofchange in AGEs levels per protein.

(8) Hair breaking strength measurement
The tensile strength of the hair was measured using atensile tester (KES-G1-SH single hair tensile tester, Kato
Tech Co., Ltd., Kyoto) at a temperature of 25 °C and 65 %_ 14 9 _Alcohol
Water
Alcohol

Ryoku-cha
Kou-cha

Sasa Veitchii

Aspalathus Linearis

Oenothera biennis

Gynostemma pentaphyllum

Cymbopogon citratus

Artemisia Princeps

Equisetum Arvense

Coix Lacryma-Jobi

Crataegus cuneata

Rosa canina

Matricaria recutita

Aloe Arborescens

Carthamus Tinctorius

Angelica keiskei

Zingiber Officinale

Arctium lappa

Panax Ginseng

Dokudami (Water)

Kuma bamboo grass

Rooibos

Evening primrose

Jiaogulan

Lemon grass

MugwortField Horsetail

hatomugi

Loquat (BG)

Loquat (Alcohol)

Japanese hawthorn

Rose hip

Chamomile

Aloe

Safflower

Ashitaba

GingerEdible burdock

Asian Ginseng

Green tea

Black tea

Oolong tea

Aminoguanidine

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

_ 15 0 _

26

Ref

–

Camellia Sinensis

Eriobotrya Japonica

Houttuynia Cordata

Dokudami (Alcohol)–

Theaceae

Araliaceae

Asteraceae

Zingiberaceae

Apiaceae

Asteraceae

Asphodelaceae

Asteraceae

Rosaceae

Rosaceae

Rosaceae

Poaceae

Equisetaceae

Asteraceae

Poaceae

Cucurbitaceae

Onagraceae

Fabaceae

Poaceae

Saururaceae–

Oolong-cha

Otane ninjin

Gobou

ShougaAshitaba

Benibana

Aloe

Kamiture

Rose Hip

Sanzasi

Biwa

Hatomugi

SuginaYomogiLemon grass

Amatyaduru

Mematuyoigusa

Rooibos

Kuma-zasa

Dokudami–

Camellia Sinensis Leaf Extract

Panax Ginseng Root Extract

Arctium Lappa Root Extract

Zingiber Officinale（Ginger）Rhizome Extract

Angelica Keiskei Leaf/Stem Extract

Carthamus Tinctorius (Safflower) Flower Extract

Aloe Arborescens Leaf Extract

Chamomilla Recutita (Matricaria) Flower Extract

Rosa Canina Fruit Extract

Crataegus Oxyacantha Fruit Extract

Eriobotrya Japonica Leaf Extract

Coix Lacryma-Jobi Ma-yuen Seed Extract

Equisetum Arvense Extract

Artemisia Princeps Leaf Extract

Cymbopogon Schoenanthus Leaf/Stem Extract

Gynostemma Pentaphyllum Leaf Extract

Oenothera Biennis (Evening Primrose) Seed Extract

Aspalathus Linearis Leaf Extract

Sasa Veitchii Leaf Extract

Houttuynia Cordata Extract–

Alcohol

Alcohol

Alcohol

Alcohol

Alcohol

Alcohol

Butylene Glycol

Alcohol

Butylene Glycol

Alcohol

Alcohol

Alcohol

Alcohol

Butylene Glycol

Butylene Glycol

Alcohol

Alcohol

Butylene Glycol

Water

Water

Butylene Glycol

Water

3

Rubus Suavissimus (Raspberry) Leaf Extract

Butylene Glycol

Ten-cha

Dokudami (BG)

Rosaceae2

Extractants

Rubus Suavissimus

I NCI

Chinese blackberry

Japanese name1

Family Name

Scientific Name

Sample NameID

Table 1. Sample profile.

Screening plant extracts for inhibition of hair glycation

Glycative Stress Research

Resultrelative humidity. The cross-sectional area of the hair wasmeasured using a hair diameter measuring device (SK2000
Kato Tech Co., Ltd.), and the breaking strength of the hair
was calculated using the following formula:(1) Primary screening results

Fig. 1 shows the inhibitory rate of fluorescent AGE
formation in each sample. All 26 samples inhibited theformation of fluorescent AGEs. In addition, 75 % or moreof the inhibitory effect of fluorescent AGE production wasobserved in 9 of the 26 samples.

Breaking strength = Breaking load / Hair cross-sectional area.

(9) Immunofluorescence staining
Immunofluorescence staining was performed using the
ABC method with the monoclonal antibody clone 6D12 8).
The glycated hair was cut to a thickness of 5 μm using a
microtome (REM-710 Yamato Scientific Co., Ltd., Asaka,
Saitama, Japan), and the sections were fixed on a glass slide.
The primary antibody was diluted 1/200 in Can Get Signal
Immunostain Solution A and reacted with the hair overnightat 4 °C. Then, the secondary antibody was diluted in thesame manner as the primary antibody and reacted with thehair at room temperature for 1 hour. After washing with PB,
fluorescence images were obtained using a fluorescencemicroscope (BZ -X810, Keyence, Osaka). The exposure time
was set as the time during which autofluorescence was notdetected in the unglycated hair.

(2) Secondary screening results

The IC 50 values are shown in Fig. 2; Rooibos extract
had the highest anti-glycation activity, followed by the green
tea and chamomile extracts. In particular, rooibos extract
showed a high anti-glycation effect, which was twice ashigh as that of the green tea extract and more than 20 times
higher than that of AG. Among the IC 50 results, the upper
rooibos extract, middle tencha extract (Chinese blackberry),and lower kocha extract (black tea) were used to verify theanti-glycation effect on hair.

Inhibition of AGEs (%)
0

20

40

60

80

100

Chamomile (17)108.27

Rooibos (6)101.26

Field horsetail (11)92.43

Mugwort (10)92.31

Green tea (24)86.94

Evening primrose (7)86.09

Oolong tea (26)85.93

Chinese blackberry (1)85.88Black tea (25)

77.63

Ashitaba (20)74.03

Loquat (BG) (13)

72.43

Dokudami (BG) (2)71.96

Lemon grass (9)71.04

Loquat (Alcohol) (14)67.99

Safflower (19)66.80Japanese hawthorn (15)

63.33

Dokudami (Alcohol) (3)60.98

Aloe (18)58.56

Kuma bamboo grass (5)57.53

Dokudami (Water) (4)44.07

Rose hip (16)42.43

Asian Ginseng (23)40.19

Jiaogulan (8)38.69Ginger (21)29.79

Aminoguanidine (Ref)hatomugi (12)Edible burdock (22)

120

26.39
11.20
9.78

Fig. 1. Inhibitory effect of plant extracts on AGEs in the Ker-Glucose reaction model.

The results are expressed as means. 0.1 mg/mL plant extracts were introduced into glycation models containing 5.0 mg/mL
Ker and 1.2 mol/L glucose (n = 3). After 10 days of incubation at 60°C, fluorescent AGEs were measured by excitation at 370
nm and emission at 440 nm. AGEs, advanced glycation end products; Ker, keratin.

_ 151 _

Screening plant extracts for inhibition of hair glycation

IC 50 (mg /mL)
0.0

Rooibos (6)

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.018

Green tea (24)0.039

Chamomile (17 )0.041

Mugwort (10 )0.044

Chinese blackberry (1)0.045

Oolong tea (26)0.045

Evening primrose (7 )0.048

Field horsetail (11)0.049Black tea (25)0.054

Aminoguanidine (Ref )0.632

Fig. 2. IC 50 of plant extracts on AGEs in the Ker-Glucose reaction model.

The results are expressed as means. 0.1 mg/mL, 0.01 mg/mL, and 0.001 mg/mL plant extracts were introduced
into glycation models containing 5.0 mg/mL Ker and 1.2 mol/L glucose (n = 3). After 10 days of incubation at
60°C, fluorescent AGEs were measured by excitation at 370 nm and emission at 440 nm. IC50, half maximal
inhibitory concentration; AGEs, advanced glycation end products; Ker, keratin.

Discussion(3) Anti-glycation effects on hair.

Fig. 3 shows the verification results for the antiglycation effect of the screened samples on hair. The amountof AGEs accumulated in the hair increased with the numberof days of in vitro glycation. Rooibos extract strongly
inhibited the production of fluorescent AGEs (inhibitory
rate, 82.4 %). In addition, tencha extract and kocha extract
also showed a higher production inhibitory effect than AG,
and the strength of the effect showed the same trend as the
IC 50 value.

(4) Breaking strength measurements

Fig. 4 shows the results the breaking strength of hair.
The breaking strength of the glycated hair was 20.8 % lower
than that of the unglycated hair. On the other hand, theaddition of rooibos extract inhibited the decrease in breaking
strength due to glycation to 4.9 %.

(5) Fluorescence analysis results

Fig. 5 shows an image of immunofluorescent stainingof hair. The accumulation of AGEs in hair due to in vitro
glycation was observed by fluorescence. It was observed
not only in the cuticle on the surface but also in the medulaand cortex. The addition of rooibos extract inhibited theformation of AGEs in the entire hair.

Screening method using a keratin-glucose glycation
reaction model

To date, anti-glycation effects on proteins such as bovine
serum albumin (BSA) and human serum albumins (HSA)
have been reported for more than 500 types of materials 13-17).
However, it has been reported that the amount of AGEs
produced differs depending on the type of protein, and theanti-glycation effect differs depending on the target protein 16).
Therefore, in this study, we first screened for keratin, aprotein that constitutes hair. After that, the anti-glycation
effects of the plant extracts on keratin were confirmed byscreening and verified further using actual hair. Rooibos
extract strongly inhibited the production of fluorescent
AGEs in both keratin and hair. This result indicates that thekeratin-glucose glycation reaction model may be useful for
screening materials for hair anti-glycation activities.

Relationship between glycation and hair breaking
strength

Glycation is caused not only by reducing sugars, butalso by various aldehydes derived from lipids and alcohols 18).
These aldehydes react with proteins to produce AGEs andcarbonized proteins, causing various disorders in the body.
Recently, the effects of these aldehydes on hair have beenreported. One is that glyoxal and glyoxylic acid cause_ 152 _Glycative Stress Research

2.00

Blank (Water)
Rooibos (6)Chinese blackberry (1)Black tea (25)

AGEs per protein(count / mg protein)

1.50

Aminoguanidine (Ref)

1.00

0.50

0.00

0

1

5

10

Incubation period (days)

Fig. 3. AGEs in hair protein.

The results are expressed as means. Hair samples were introduced into PB containing 1.2 mol/L glucose
and 0.1 mg/mL samples. After 10 days of incubation at 50 °C, fluorescent AGEs were measured byexcitation at 370 nm and emission at 440 nm. The degree of AGE content was calculated per 1 mg ofprotein content. AGEs, advanced glycation end products; PB, phosphate buffer.

25.00

Stress (kgf / mm 2 )

20.00

15.00

10.00

5.00

0.00

Gulcose -Gulcose +Rooibos -Fig. 4. Tensile strength of hair.

Gulcose +Rooibos +Results are expressed as mean ± SD. * p < 0.05, Tukey (Kramer test). Hair samples were introduced into
PB containing 1.2 mol/L glucose and 0.1 mg/mL samples. After 10 days of incubation at 50 °C, the tensile
strength was measured using a high-sensitivity hair rheology analyzer at breaking strength, when the hair
was torn off. PB, phosphate buffer; SD, standard deviation.

_ 153 _

Screening plant extracts for inhibition of hair glycation

Blank (Water)

Rooiboss 0.1 mg / mL

Fig. 5. Immunofluorescent staining of hair.

Hair samples were introduced into PB containing 1.2 mol/L glucose and 0.1 mg/mL samples.
After 10 days of incubation at 50 °C, AGEs were immunostained with an anti-AGE antibody.
AGEs, advanced glycation end products; PB, phosphate buffer.

disturbances in the keratin structure of hair and reduce thetensile strength of hair 19). In addition, glyoxylic acid-treated
yak hair rearranges the secondary structure distribution,changes the conformation of disulfide bridges, reduces
serine residues, and forms imines 20). Glyoxal and glyoxylic
acid contain aldehyde groups as well as glucose. Therefore,
it can be concluded that the above reaction is similar toglycation. This way, the effect of glycation on hair extends tocomplex and detailed structures, and as a result, changes inphysical properties (such as a decrease in breaking strength)
occur. In the future, it will be necessary to consider thedetailed mechanisms.

Anti-glycation effect of rooibos extract

The rooibos plant (Aspalathus Linearis) is endemic to asmall part of the western coast of the Western Cape provinceof South Africa. Rooibos tea, which is produced by thefermentation of harvested leaves, is widely used as a healthdrink in Africa, Europe, and Japan. Rooibos tea has beenreported to have biological effects, such as providing relief
from digestive diseases such as constipation, stress relief, andallergic symptoms 21). In terms of nutrition, it is caffeine-freeand contains a large amount of minerals such as potassium,
sodium, and magnesium.
Furthermore, it contains various polyphenols, such as
rutin, luteolin, quercetin, aspalathin 22). Luteolin is a flavone
and has been reported to have an anti-glycation effect on
BSA 23). Quercetin, a flavonol, has been reported to enhance
the inhibitory effect of sulforaphane on the formation of
AGEs 24). These reports suggest that polyphenol components
in rooibos inhibit glycation in hair. Among the various
components contained in rooibos extract, the component withthe anti-glycation effect on hair requires further verificationin the future.

Conclusion
In this study, we found that rooibos extract is a novel
ingredient that prevents hair glycation. The use of haircosmetics containing rooibos extract can prevent glycationand maintain beautiful and strong hair.

Conflict of Interest Declaration
Research support was provided by Arimino Co., Ltd.

Acknowledgments
A part of this paper was presented at the 18th Meetingof the Society for Glycative Stress Research (August 31,
2019, Kyoto) and IFSCC Mexico Conference 2021 (October
18-28, 2021, web lecture). The publication of this study was
supported by the Research Foundation of Crude Drug gained
from the same source of Medicine & Food. (IDF#21007;
Chuo-ku, Tokyo).

_ 15 4 _Glycative Stress Research

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