Skin pigmentation

Glycation – an important factor for skin pigmentation, dark skin tone

Glycation is also called Maillard reaction

The Maillard reaction is a chemical reaction between carbohydrates and proteins and results in brown end-products, melanoidins. These end-products are important in food industry for flavoring and coloring purposes. The Maillard reaction also occurs in the human body. It is a step in the formation of advanced glycation end-products (AGEs).ts)

 

The influence of AGEs (Advanced Glycation End Products):

 

1. Glycation of collagen and elastin

As age increases, AGEs accumulate in human skin and lead to the damage to skin cells and aging. AGEs damage elastin and collagen and cause the loss of skin elasticity. AGEs also coaggregate with collagen (glycation of collagen and elastin) that will result to secretion of collagen-breakdown enzymes to digest collagen, ultimately leads to the apoptosis of fibroblast. AGEs will turn human skin into saggy, wrinkled, dull, pigmentation and speckled skin tone.

 

2. Diabetes and aging

Report of AGEs in diabetes and metabolic diseases:The content of carboxymethyllysine(CML) and pentsidine (AGEs) in skin were analyzed. There were strong correlation between AGEs and ages of subjects of both groups,  indicating age-related chemical modification of collagen by Maillard reaction and the process is accelerated in diabetes.


Sakura extract: inhibition of AGEs

Sakura (cherry blossom) is the flower of genus Prunus. ORYZA and Kyoto Pharmaceutical University discovered the physiological functions of sakura extract and applied sakura extract to health food for beauty and cosmetics. Food-grade of Yaezakura (multi-layered cherry blossom) was cultivated in Japan and was harvested raw materials for extraction.

Functional ingredients:

1. SAKURA EXTRACT: inhibition of AGEs production

【Experiment I】
Bioactive components of SAKURA EXTRACT and SAKURA EXTRACT were added to buffer solution containing D-glucose and bovine serum albumin at 60°C and left to stand for 2 days. The AGEs levels were measured via photometer.

【Result】

(1) SAKURA EXTRACT and the major bioactive components significantly inhibited the production of AGEs in a dose-dependent manner.

Inhibition of AGEs production ratio (%) IC50
10 (μg/mL) 30(μg/mL) 100(μg/mL) 300(μg/mL) (μg/m)
SAKURA EXTRACT -10.8±0.4 -9.9±0.6 15.1±0.7** 42.6±3.2** >300
1-caffeoyl-O-β-D-glucopyranoside 10.7±0.1** 19.5±0.3** 25.0±0.3** 30.0±0.4** >300
quercetin 3-O-β-D-glucopyranoside 27.6±0.5** 49.8±0.7** 74.2±1.1** 100.8±0.6** 30

◎Each value was shown in the mean and standard error of three cases. With asterisks were significantly different from untreated samples by Dunnett’s multiple comparison test. *: p <0.05, **: p <0.01

(2)SAKURA EXTRACT (100µg/mL) significantly inhibited the production of AGEs. Meanwhile, the major bioactive components, 1-caffeoyl-O-β-D-glucopyranoside and quercetin 3-O-β-D-glucopyranoside significantly inhibited the production of AGEs at concentration as low as 10µg/mL.

(3)SAKURA EXTRACT showed strong inhibitory potency in AGEs production. 1-caffeoyl-O-β-D-glucopyranoside and quercetin 3-O-β-D-glucopyranoside contribute 93.3% in the inhibition of AGEs production

Inhibition of AGEs production ratio (%) IC50 Purity(%) Contribution(%)
10 (μg/mL) 30(μg/mL) (μg/m)
SAKURA EXTRACT -10.8±0.4 15.1±0.7** >300 100.0 100
1-caffeoyl-O-β-D-glucopyranoside 10.7±0.1** 25.0±0.3** >300 10.2 72.3
quercetin 3-O-β-D-glucopyranoside 27.6±0.5** 74.2±1.1** 30 1.15 21.0

◎Each value was shown in the mean and standard error of three cases. With asterisks were significantly different from untreated samples by Dunnett’s multiple comparison test. *: p <0.05, **: p <0.01

2.SAKURA EXTRACT: Inhibition of fibroblasts apoptosis (Caspase Assays)

【Experiment II】
Accumulation of AGEs in skin triggers skin damage and apoptosis of fibroblasts. The effect of SAKURA EXTRACT and its bioactive components on carboxylmethyl lysine (CML)-collagen induced fibroblasts apoptosis was examined.

【Result】
(1) SAKURA EXTRACT, 1-caffeoyl-O-β-D-glucopyranoside and quercetin 3-O-β-D-glucopyranoside decreased caspase activity, meaning fibroblasts apoptosis were suppressed.

Inhibition rate of apoptosis (%)
1 (μg/mL) 3

(μg/mL)

10(μg/mL) 100

(μg/mL)

SAKURA EXTRACT 61.8±2.6 77.1±4.2*
1-caffeoyl-O-β-D-glucopyranoside 26.2±0.5* 37.6±1.2 72.2±2.7*
quercetin 3-O-β-D-glucopyranoside 44.2±1.5* 39.0±1.1* 121.5±5.4*
Aminoguanidine (positive control) 104.8±3.4*

◎Each value was shown in the mean and standard error of three cases. With asterisks were significantly different from untreated samples by Dunnett’s multiple comparison test. *: p <0.05, **: p <0.01

(2)quercetin 3-O-β-D-glucopyranoside is the major active compound in suppression of fibroblasts apoptosis..

3. SAKURA EXTRACT: Promotion of collagen formation in fibroblasts

【Experiment III】

collagen formation

Fibroblasts were cultured in collagen containing medium and treated with glyoxal, a glycation inducer. Regularly, fibroblasts produces collagen lattice when cultured in collagen containing medium. Collagen lattice formation was enhanced in the presence of SAKURA EXTRACT of 100 µg/mL and 1000 µg/mL. SAKURA EXTRACT was suggested to prevent glycation of fibroblasts.

【Result】

collagen formation and glycation inhibition

Fibroblasts were cultured in collagen containing medium and treated with glyoxal, a glycation inducer. Glyoxal inhibits the formation of collagen lattice. In the presence of SAKURA EXTRACT, inhibition by glyoxal was not observed. Growth of fibroblasts and collagen lattice formation were enhanced in the presence of SAKURA EXTRACT of 100 µg/mL and 1000 µg/mL. SAKURA EXTRACT was suggested to prevent glycation of fibroblasts and to be effective in maintaing extracellular collagen matrix.

4. SAKURA EXTRACT: skin whitening effect

【Experiment IV】
SAKURA EXTRACT was added to B16 melanoma cell culture and incubated. Then cells were crushed by hypersonication followed by absorbance measurement (melanin formation).

【Result】
(1) SAKURA EXTRACT demonstrated inhibition of melanin formation in a dose-dependent manner. SAKURA EXTRACT inhibits melanin formation even under low concentration.
(2) Arbutin (positive control) showed highest inhibition rate. SAKURA EXTRACT demonstrated inhibition of melanin formation similar to L-Ascorbic Acid 2-Glucoside (vitamin C).

Effect of SAKURA EXTRACT on melanin production ratio (%) in B16 melanoma cells

0 (μg/mL) 1(μg/mL) 3(μg/mL) 10(μg/mL)
SAKURA EXTRACT 100±1.7 96.2±2.8 94.9±1.2 90.1±3.5
Arbutin (positive control) 100±3.7 95.5±1.5 87.9±1.3 84.6±0.5
L-Ascorbic Acid 2-Glucoside 100±3.2 97.9±0.3 94.6±1.0 90.2±0.5

melanin production ratio(% of Controll)
(3)Inhibition of tyrosinase activity:SAKURA EXTRACT showed inhibitory effect on tyrosinase activity in a dose-dependent manner, suggesting its skin whitening effect.

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