Keiko Matsui, Chiaki Hara, Makoto Watanabe

Planning & Development Department, Kamogawa Technology Development Group, Japan Kikkoman Biochemifa Company, Japan

Introduction

The term phytochemical refers to a compound or nutrient that originates from plant. Phytochemicals is positioned as the 7th nutrient following the 3 major nutrients (carbohydrate, protein and lipid), 5 major nutrients (+ minerals and vitamins), and 6 major nutrients(+ dietary fiber). They have important roles in regulation functionsin vivo.[1]

In chemical parlance, the term polyphenol refers to multiple phenolic hydroxyl groups in the molecule.Polyphenols often accumulate in plant bodies as bitterness or pigmented compounds. They are phytochemicals with anti-oxidative capacity that most plants possess.Plants contain diverse anti-oxidative substances that allow protection from oxygen and sunlight, particularly in their seeds that pass the genetic information to their descendants.

Proanthocyanidins are a class of polyphenols that are abundant in grape seeds. Among the variety of polyphenols, proanthocyanidins display potent antioxidation activity.

The mortality rate from cardiac diseases in France is markedly lower than that in other Western countries despite the high-fat diet of many French citizens. This is known as the “French paradox” and might be related to the consumption of red wine consumption. Polyphenols in red wine prevent oxidation of low-density lipoprotein cholesterol.[2]Proanthocyanidins are enriched in red wine polyphenols and have been implicated in the French paradox.The anti-oxidative capacity of grape seed polyphenol was studied by determining the oxidation rate of linoleic acidbeta carotene in aqueous solution. The oxidation rate was markedly higher than exemplary commercialized natural anti-oxidative substances (Figure 1).[3]

Gravinol KPA-CU is an extract with increased purity and transparency that is prepared by extracting polyphenols from grape seeds with purified water followed by ethanol-mediated purification. The main component of Gravinol KPA-CU is proanthocyanidins(Figure 2). Proanthocyanidins have oxygen quenching and anti-inflammatory activities,[4]suppress melanin generation by inhibiting tyrosinase activity, and suppress melanocyte proliferation.[5]Recent studies have revealed the strong anti-glycation capacity of proanthocyanidins,with the inhibition of the production of advanced glycation end-products (AGEs).

Figure 1. Relative anti-oxidative activity

Figure 2. Main component, proanthocyanidins

Gravinol KPA-CU is a safe cosmetic material derived from grape. It has all following three strong effect, anti-oxidation,inhibition of tyrosinase activity and anti-glycation and may potentially be exploited to improve the clearness and elasticity skin (i.e., anti-aging). Dermatological effects that have been documented include suppression of pigmentation after oral ingestion[6]and suppression of pigment generation/deposition due to ultraviolet (UV) irradiation.[5]The present human monitoring study was undertaken to explore thein vivousefulness of Gravinol KPA-CU on the skin by in-house volunteers. The results indicate the benefit on improved skin brightness.

Usefulness of Gravinol KPA-CU

The three major characteristics of Gravinol KPA-CU(anti-oxidation, inhibition of tyrosinase activity, and antiglycation) are detailed below.

Anti-oxidation (suppressed generation of active oxygen generation)

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicalscavenging activity was measured to evaluate the antioxidative activity. Forty microliters of the prepared Gravinol KPA-CU was adjusted to be a 0.1% solution. DPPH solution was added and allowed to react for 1 h at room temperature,followed by measurement of absorbance at 517 nm using a multi-well plate reader. The solution for dissolution or dilution of respective samples was used as a blank. The difference in the absorbance of the DPPH radical and non-DPPH radical samples was defined as A, with the difference between the DPPH radical and non-DPPH radical blanks defined as B. DPPH radical-scavenging activity (%) was calculated as:{1- (A/B)} ×100.

Excessive active oxygen, such as radicals, produced in the body trigger the formation of pigmented spots and wrinkles in the skin, arteriosclerosis, diabetes mellitus,cancer, and various other age-related changes. Generation of active oxygen is also induced by the ultraviolet rays in sunlight, smoking, physical stress, pharmaceutical chemicals, or consumption of certain foods. Quenching the active oxygen may be important to prevent various age-related changes.

When radical-scavenging activity was measured using DPPH, Gravinol KPA-CU displayed strong radical-scavenging activity at low doses, with an antioxidative effect equal to or better than that of cosmetic raw materials used as skin whitening agents (Figure 3).The proanthocyanidins constituent of the grape seed polyphenols may have been the basis of this effective radical-scavenging activity at low content, since proanthocyanidins have an anti-oxidative effect that is stronger than vitamin C, vitamin E, catechin, and gallic acid. Thus, proanthocyanidins could prove capable of suppressing the initial stimuliin vivothat may trigger the serial reactions that lead to skin pigmentation and aging.

Figure 3. Anti-oxidation activity（concentration of each test section was 0.1%）

Inhibition of tyrosinase activity

Inhibition was evaluated using tyrosinase derived from mushrooms. Twenty microliters of the prepared sample received 100 μL of 1/15 M phosphate buffer and 40 μL of 40 U/mL mushroom-derived tyrosinase. After thorough mixing, 50 μL of 2.5 mM L-3,4-dihydroxyphenylalanine(L-DOPA) was added and allowed to react for 15 min.Then, the absorbance at 490 nm was measured using a multi-well plate reader. The solution for dissolution and dilution of the respective samples were used as the blank.Difference in measurements between the L-DOPA group and non-L-DOPA group in the samples was defined as A,and the difference between the L-DOPA group and non-L-DOPA blanks was defined as B. Inhibition of tyrosinase activity (%) was calculated as: {1- (A/B)} ×100.

Pigmentation in the skin, such as skin dullness or pigmented spots, is caused by accumulation of melanin in the keratinocytes. Tyrosinase is a key enzyme in the melanin production. In the body, the amino acid tyrosine is converted by tyrosinase to DOPA and DOPA quinone, which are eventually excreted or accumulated as mature melanin. Since the inhibition of tyrosinase activity effectively suppresses melanin production,kojic acid, ellagic acid, and arbutin, etc., which has such functionality, are often used as skin whitening materials.

Gravinol KPA-CU inhibited tyrosinase activity in a dose-dependent manner (Figure 4). It has previously been demonstrated that proanthocyanidins have stronger suppressive activity against melanin production as its degree of polymerization increases. Grape seed-derived polyphenols contain an abundance of highly polymerized proanthocyanidins that seem to effectively inhibit tyrosinase activity. A similar efficacy may be presently apparent using Gravinol KPA-CU.

Figure 4. Inhibition of tyrosinase activity

Anti-glycation

Inhibition of AGEs production was measured using human serum albumin to evaluate the anti-glycation effect of Gravinol KPA-CU. Twenty microliters of sample prepared at a 0.1% concentration received 500 μL of 0.1 M phosphate buffered saline (PBS), 20 μL of human serum albumin, and 100 μL of 2 M glucose. After thorough mixing and incubation at 60°C for 40 h, the solution was dispensed in 200 μL aliquots in wells of a multi-well plate and the fluorescence intensity was measured using excitation and emission wavelengths of 370 nm and 440 nm, respectively.Distilled water was used as control. The difference in the results between the glucose and non-glucose sample groups was defined as A, and the difference between the glucose and non-glucose blanks was defined as B.Inhibitory activity (%) against AGEs production was calculated as: {1- (A/B)} ×100.

AGEs, an abbreviation for advanced glycation end products, are produced by non-enzymatic glycation of protein. Accumulation of AGEs in the body is manifest as various age-related changes. In the skin, these include skin dullness and hardening. AGEs accumulation in vessels causes arteriosclerosis and exacerbates diabetes mellitus.Suppression of AGEs production is important to prevent age-related changes.

The mechanism of AGEs production involves complex processes such as Amadori rearrangement and protein condensation polymerization. Presently, the addition of a low concentration Gravinol KPA-CU to the reaction system between human serum albumin and glucose suppressed AGEs production. As well, a stronger anti-glycation effect was clearly evident, compared to commercial plant extracts that are marketed as having an anti-glycation effect or compared to the cosmetic raw materials used as skin whitening agents (Figure 5).

Figure 5. Anti-glycation activity (concentration of each test section was 0.1%)

The mechanism of action of most skin whitening materials involves inhibition of tyrosinase activity and, for some materials, together with an anti-oxidative effect. It can be said that Gravinol KPA-CU is an unprecedented skin whitening and anti-aging material because of its anti-glycation effect in addition to the other former activities. Thus, Gravinol KPACU achieves skin whitening via multiple routes.

Skin whitening effect of Gravinol KPA-CU

Gravinol KPA-CU is expected to improve pigmented spots and pigmentation through its active oxygen quenching capability and by the suppression of melanin production by the inhibition of tyrosinase activity of the proanthocyanidins, active ingredient. The improved pigmentation of human skin was measuredin vivoto evaluate skin whitening. Eleven healthy males and females in their 20s to 50s participated. All provided informed written consent. They received a topical preparation consisting of a cream containing 1% Gravinol KPA-CU.They applied the cream on the lateral forearm twice daily in the morning and evening. A cream lacking Gravinol KPA-CU was used as the placebo in the control group.The active cream contained no skin whitening component other than Gravinol KPA-CU, UV scattering agent, or UV absorbing agent. Skin brightness (L? value) was measured with a CR-300 chroma meter (KONICA MINOLTA Co.Ltd.). In photographs taken of the forearms, a color scale(Casmatch, Kyowa Tokei Kogyo Co., Ltd.) was applied to adjust the color so that the skin color was imaged equally before and after the test.

Gravinol KPA-CU cream increased the L* value 4 weeks after application compared to placebo, and significantly improved skin brightness (p= 0.085) at week 8 (Figure 6). Skin color markedly brightened visually between week 0 and 8 weeks of application (Figure 7).

Pigmented spots on the skin of the face can be esthetically unappealing. Active oxygen produced by UV stimulation is considered a cause. Proanthocyanidins in grape seed polyphenols appear to have a strong antioxidant and radical scavenging activity that quench active oxygen. Furthermore, it appeared that the melanin level decreased resulting in brighter skin, as suppression of melanocyte proliferation has been reported to be a mechanism of improvement of pigmentation in addition to suppression of melanin production.[7]

In the remainder of the document, future studies,we address the correlation between the difference in the degree of polymerization of proanthocyanidins and its improvement of pigmentation in human skin.

Figure 6. Skin whitening effect

Figure 7. Pictures of skin before and after application

Conclusion

Gravinol KPA-CU displays anti-oxidative, tyrosinase inhibition, and anti-glycation (AGEs production inhibition). These attributes may be exploited for use as a potent skin whitening material to reduce age-related changes in skin multilaterally. It is readily formulated in skin lotions, essences, emulsions, and creams. We recommend a content of 0.1% to 1% to make its excellent functions be utilized.

Gravinol KPA-CU is a cosmetic raw material derived from grape which has long time human's eating experience and high safety, and has already been used as a raw material for cosmetics for approximately a dozen years in Japan and other countries. It has been previously employed for practical use according to Raw Material Specification/Accompanying Specification of Quasi-Drug and therefore can be formulated into quasi-drugs. This clear and slightly reddish brown liquid is accepted as a favorable characteristic of a plant-derived formulation.

In future studies, we would like to obtain data on the functionality and efficacy of Gravinol KPA-CU, and explore further applications, with the expectation that it will be applied in new cosmetic products.