Wang Nan, Zhou Zhaoqing, Yin Yuexuan, Zhao Hua

College of Science, Beijing Technology and Business University, China

Wu Jinhao

Beijing Daily Chemical Association, China

Li Ang

Institute of Botany, Chinese Academy of Sciences, China

Tricholoma matsutake Sing (scientific name: Tricholoma matsutake), also called pine mushrooms,[1]belongs to Basidiomycetes Divisio, Basidiomycetes Class, Agaricales,Tricholomataceae, Tricholoma Genus. Tricholoma matsutake mainly grows in Japan, the Korean Peninsula,grows northeast and southwest China. In Japan, it is known as the king of edible fungi, and belongs to the national protection plants in China.[2～4]Tricholoma matsutake is a treasured precious wild fungus, has a high nutritional value, contains a variety of active ingredients,including proteins, polysaccharides, vitamins, etc.[5～7]According to reports, Tricholoma matsutake has effect of whitening, antioxidant and other.[8,9]In recent years,Tricholoma matsutake is widely used in health food and traditional medicine industry. It is also reported on its application in cosmetics,[10]but its efficacy evaluation is rarely reported.

Whitening efficacy evaluation methods can be divided into two types: in vitro and in vivo. In vitro methods include the enzyme inhibition method, cell biology method, animal experiment method[11]and clinical trial method,[12]etc.The biochemical and cell biological methods have many advantages such as short experimental period and high throughput screening, that can be used for the rapid screening of whitening active additives. Human trial method is impersonal more objective and more comprehensive, that can be used for efficacy evaluation of whitening cosmetics.

Using the human trial experiment method to evaluate whitening efficacy of Tricholoma matsutake in cosmetics,the changes of melanin content (MI value), whiteness (L*value) and brightness (ITA° value) were measured before and after use. SAS (Statistics Analysis System) 9.3 was used for statistical analysis.

Experiment

Main materials and instruments

Matsutake fruiting body, produced in Yunnan, Shangri-La; absolute ethyl alcohol; iethyl ascorbic acid; liqaid paraffin; glycerin;

T2002 electronic balance; FW135 pulverizer; RE2000 rotary evaporator; Mexameter MX 18 (CK company,Germany); MPA 9 (CK company, Germany).

Method

preparation of Tricholoma matsutake extract

The fruiting body of Tricholoma matsutake was dried at 50℃, crushed, screened and sealed. Dried powder 5 g, according to the ratio of 1: 20 of the material (g:mL)dissolved in 70% ethanol solution, micro boiling reflux extraction for 2h. The solction was centrifugaliztd (6,000 r·min-1) for 15 min, the supernatant was taken and concentrated, distilled water to dissolve and dilute to 45 mL. Then the Tricholoma matsutake extract was sealed at 4℃ was seaied at to preserve the reserve.

Preparation of test samples

Sample 1: Cream containing extract from Tricholoma matsutake, laboratory production.

Sample 2: Ascorbic acid ethyl ether cosmetic cream,positive control, laboratory production.

Sample 3: Formulation matrix cream, negative control,laboratory production.

The experimental formula is shown in Table 1.

Heat the A phase with mixing to 82℃. Heat the B phase with mixing to 82℃. Add the A phase into the B phase slowly, homogenizing (2,000 r/min) for 5 min.After homogenizing, cool while stirring to 40℃. The C phase and the active and functional components(Tricholoma matsutake extract, ascorbic acid ethyl ether)were added, and stired evenly.

Table1. Formulation of cosmetic cream product

Subject selection

Atotal of 30 women between 20 and 45 years old,who work in indoors, in line with the Helsinki declaration, meet the inclusion, exclusion criteria; were assessed No serious systemic disease; immune deficiency or autoimmune disease; No active allergic disease; No allergic history of skin care cosmetics; No use of steroids within one month.Non pregnancy or lactation; No ethical taboo. According to the Chardon grouping method, the skin color of the volunnteers were between grade Ⅱand grade Ⅳ.

Whitening efficacy evaluation results is affected by the degree of sun exposure and other factors. Therefore,subjects who work in the outdoors were not selected reduce to the impact of external factors on the experimental results.

Experimental index [13～15]

① Skin whiteness (brightness) L* value.

② Skin brightness (dark yellow) ITA° value.

③ MI value of skin melanin content.

Test method

At least one week washout period. The right arms of the volunteers were selected as the test areas, and marked, and the blank areas (without any sample) were set. The subjects were asked to use the samples. Use it every morning and evening for 8 weeks. During the test period, the subjects did not change their daily habits.

The data collection were carried out 6 times, divided into the initial value and the use of samples for 1 weeks,2 weeks, 4 weeks, 6 weeks and 8 weeks. The temperature remained constant at 22℃±1℃ and kept humidity between 50% and 55%, after cleaning the test areas, the volunteers sit in this environment for 20 min.

Data analysis

Statistical analysis was performed by SAS (Statistics among) 9.3. ANCOVA in mixed model was used to examine the difference samples. The general structure of the mixed model used was as follows: Y= Xβ+ZΓ+ε. At the same time,the DUNCAN test method of generalized linear model (GLM)was used to compare the treatment effects of different samples.

Results and discussion

L ＊ values analysis results

The experimental results of L* values were shown in Figure 1:

Figure 1. The change trend of the L＊ values

The multiple comparison results for L* values were shown in Figure 2:

Figure 2. Multiple comparison results of L＊ values during the experimental period

It can be seen from Figure 1 and Figure 2, the L★values between Sample 1, Sample 2, Sample 3, and the skin baseline value were significantly different throughout the experimental period (p＜0.0001, F=15.67, df1=3, df2=591).Duncan multiple comparisons showed no significant difference between sample 1 and sample 2 (same as A),both significantly higher than sample 3 (B), while the skin baseline value (C) was significantly lower than that of the above.

The results showed that all three samples had the effect of enhancing skin whiteness. Sample 1 and sample 2 were significantly higher than sample 3, the effect of the formulation matrix could be eliminated. There was no significant difference between sample 1 and sample 2, indicating that the increase in the L★ value of sample 1 and sample 2 was close.

Therefore, during the experimental period, the creamn cantaining extract from Tricholoma matsutake and positive control ascorbic acid ethyl ether cosmetic cream have similar effects on enhancing skin whiteness, and are more effective than the formulation matrix cream.

ITA° values analysis results

The experimental results of ITA° values were shown in Figure 3.

Figure 3. The change of ITA° values

The multiple comparison results for ITA° values were shown in Figure 4.

Figure 4. Multiple comparison results of ITA° values in the experimental period

It can be seen from Figure 3 and Figure 4, the ITA°values between Sample 1, Sample 2, Sample 3, and the skin baseline value were significantly different throughout the experimental period (p＜0.0001, F=15.67, df1=3, df2=591).Duncan multiple comparisons showed no significant difference between sample 1 and sample 2 (same as A),both significantly higher than sample 3 (B), while the skin baseline value (C) was significantly lower than that of the above.

The results showed that all three samples had the effect of enhancing the skin brightness. Sample 1 and sample 2 were significantly higher than sample 3, the effect of the formulation matrix could be eliminated. There was no significant difference between sample 1 and sample 2,indicating that the increase in the ITA° values of sample 1 and sample 2 was close.

Therefore, during the experimental period, the cream containing extract from Tricholoma matsutake and positive control ascorbic acid ethyl ether cosmetic cream have similar effects on enhancing the skin brightness, and are more effective than the formulation matrix cream.

MI values analysis results

The experimental results of MI values were shown in Figure 5.

Figure 5. The change of MI values

The multiple comparison results for MI values were shown in Figure 6.

Figure 6. Multiple comparison results of MI values in the experimental period

It can be seen from Figure 5 and Figure 6, the MI values between Sample 1, Sample 2, Sample 3, and the skin baseline value were significantly different throughout the experimental period (p＜0.0001, F=15.67, df1=3, df2=591).Duncan multiple comparisons showed no significant difference between sample 1 and sample 2 (same as A),both significantly lower than sample 3 (B), while the skin baseline value (C) was significantly higher than that of the above.

The results showed that all three samples had the effect of reducing the skin melanin content. Sample 1 and sample 2 were significantly lower than sample 3, the effect of the formulation matrix could be eliminated. There was no significant difference between sample 1 and sample 2,indicating that the decrease in the MI values of sample 1 and sample 2 was close.

Therefore, during the experimental period, the the cream containing extract from Tricholoma matsutake and positive control ascorbic acid ethyl ether cosmetic cream have similar effects on reducing the skin melanin content,and are more effective than the formulation matrix cream.

Conclusion

Research on the whitening efficacy of the Tricholoma matsutake cosmetic, and the L* value, ITA value and MI value were measured. The ascorbic acid ethyl ether was used as positive control. The results showed that in the tested area of the Tricholoma matsutake cosmetic, the skin melanin content MI value, skin color L★ value and ITA°value were significantly improved during the experiment period. indicating that the cosmetics containing extract from Tricholoma matsutake have good whitening effect.Duncan multiple comparisons showed that the whitening effect was significantly greater than the formulation matrix, close to the ascorbic acid ethyl ether cosmetic.

The L* value, ITA ° value, and skin melanin content MI value can be used as a means of evaluating the cosmetic whitening effect, and the results judged to be relevant.

The above method can rule out the influence of skin baseline value and formula matrix on the whitening effect of the sample. It makes the evaluation process more scientific and the evaluation result more objective, and provides the theoretical basis and technical support for formulation development.

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