Wang Xiaona, Wang Xiaomei, Yang Suzhen

Shandong Freda Bioeng Co.Ltd., China

Liu Shaoying, Meng Xiangjing

Shandong Provincial Key Laboratory of Biology and Pharmacology, Institute of Biopharmaceuticals of Shandong Province, China

Abstract

Key words

whitening；lotus flavones；skin cream；cytotoxicity；tyrosinase inhibition；DPPH radical scavenging

Introduction

Complexion of human skin is influenced by smoothness level, non-enzymatic glycation and pigmentation, among which melanin content in epidermis is the most important factor.Melanin is synthesized in melanocytes.Melanogenesis is a biochemical process containing a series of reactions.[1,2]Tyrosinase plays an critical role in melanogenesis.Inhibiting tyrosinase activity is a hotspot of skin scientific researches to reduce melanin production.[3,4]UV is also be engaged in melanogenesis by inducing free radicals, which can change cell metabolism and enhance melanin production.So scavenging free radicals is another method to inhibit melanogenesis.[6]

A large number of studies has shown that the flavonoids extracted from lotus have multiple biological activities and good medicinal value, and the flavonoids in lotus leaf are the most abundant, which have excellent antioxidant function.[7-10]At present, the application of lotus leaf flavones in cosmetics is relatively few, and the research on its function mechanism is not thorough enough.This study focused on the high antioxidant and whitening properties of flavone from lotus leaf and its application in cosmetics.

Materials and methods

Materials and instruments

Lotus leaf flavones was extracted via means of selfmade.100 g of dry lotus leaf was crushed, followed by incubated in NaOH solution of pH 9 at 70 °C for 20 h.The extract solution was obtained by pressure filtration.The pH of filtrate was adjusted to 3～5 by HCl solution.

The filtrate flowed through the D101 macroporous resin column, which was then washed by distilled water.60% ethanol was used for elution of flavonoids.Eluant was concentrated and dried in vacuum decompression conditions.Yellow powder gotten from former steps was analysed, in which flavomoids accounted for 85%.

Mouse embryonic fibroblasts (L929 cell strain) was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China).DMEM (High glucose), DMEM (Low glucose), DMEM/F12, fetal bovine serum (FBS), trypsase and other cell culture reagents were purchased from Gibco BRL (Grand Island, NY, USA).Methyl thiazolyl tetrazolium (MTT), dimethyl sulfoxide (DMSO), tyrosine, tyrosinase, 1,1-diphenyl-2 -picrylhydrazyl (DPPH), BHT were purchased from Sigma-Aldrich (St.Louis, MO, USA).Carbon dioxide incubator was purchased from Thermo Fisher (Waltham, MA, USA).Phase contrast Microscope was purchased from Nikon (Tokyo, Japan).Tecan SUNRISE Microplate Reader was purchased from Tecan (Switzerland).VISIA-CRwas purchased from Canfield (USA).

Experimental method

Cytotoxicity assay[5]

Cells were divided into experimental groups, negative control group (C-) and positive control group (C+).Media of experimental groups were DMEM with lotus flavones in it.Concentration of lotus flavones were 0.03%, 0.06%, 0.13%, 0.25%, 0.50%, and 1.00%, respectively.Medium of C0was DMEM.Medium of C+ was DMEM with 20% DMSO in it.Every group contained 6 well of cells.

Cells were cultured at 37 °C with 5% CO2, in medium of DMEM added with 10% FBS.The medium was every third day.When coverage reached 80%, cells were seeded in 96-well plate at a density of 5×104cells every well.After 24 hours, the renewed as described in experimental grouping, 100 μL medium per well.After 24 hours, culture in each well was added into 20 μL MTT at a concentrate of 5 mg/mL.Culture was incubated 4 hours away from light.Medium was discarded.100 μL of DMSO was added into each well.Cell culture plate was placed on shaking table for 20 minutes.The absorbance at 570 nm of each well was measured.The absorbance value was proportional to the survival rate.

Related survival rate against C0(β) was calculated by following equation.

Determination of tyrosinase inhibitory activity

Tyrosinase was assayed by using a previously described method[6]with some modification.Phosphate buffer saline (PBS, 0.05M, pH=6.8) was prepared.Lotus flavones were dissolved in PBS.Concentrations (w/w) of lotus flavones were 0.02%, 0.04%, 0.08%, 0.15%, 0.30%, respectively.L-tyrosine solution (1 mg/mL) and tyrosinase solution (200 units/mL) were prepared with PBS.3-o-ethyl ascorbic acid were dissolved in PBS, getting a series of solution 0.01%, 0.05%, 0.50%, 1.00%, 2.00%, respectively.

Reaction systems were prepared as described in Table 1, and then incubated in 30 °C water for 30 mins.150 μL reaction solution was transfered into 96-well plate.The absorbance at 570 nm of each well was measured by microplate reader.

Tyrosinase inhibitory activity (I) was calculated by following equation.

Determination of DPPH scavenging[11]

Lotus flavones were dissolved in alcohol to escalating concentrations of 0.2-3.0 mg/mL.DPPH was dissolved in alcohol at a concentration of 200 μmol/L.DPPH solution was added into 96-well plate, in a volume of 180 μL per well.After that, lotus flavones solution was added, in a volume of 20 μL per well.The absorbance at 517 nm was taken at once, and recorded as zero line (A1).The 96-well plate was incubated in 37 °C for 30 mins.Then the absorbance at 517 nm was taken again, and recorded as A2.In the blank group, Lotus flavones was replaced by alcohol (A0), while 0.001%, 0.004, 0.007, 0.010, 0.013, 0.016 mg/mL Vitamin C was used as positive control.

Table 1.Tyrosinase inhibition reaction system

DPPH scavenging activity (γ) was calculated by following equation.

Detection of skin pigment

The formula of skin cream is shown in Table 2.The raw materials in part A were mixed, and heated to 80 °C.Then solid melted into liquid.The raw materials in part B were mixed in another beaker, and heated to 85 °C.Part A and part B were both added into the emulsification pot and then homogenized for 8 mins.The homogeneous mixture was cooled to 50 °C with continuous stirring.Part C and part D were added into the mixture and stired evenly.The mixture were cooled to room temperature and skin cream was abtained.Another skin cream containing no part C was prepared as blank control.

60 female participants were devided into experimental group and control group randomly, 30 participants in each group.They usde face cream once a day.After face was cleaned and dried every moring, the face cream was applied evenly on face.The pigment was quantitatively determined on day 0, day 7, day 14, day 21, day 28.No other skin whitening product was used during experiment.The face cream of experimental group contained lotus flavones, while the face cream of control group contained dilluted water instead of lotus flavones.

Table 2.Skin cream formula

Results and discussion

Cytotoxicity

Cells were seeded in 96-well plate.24 hours later, lotus flavones were added into cell culture.Another 24 h later, MTT test was carried out.The results are shown in Figure 1.Within the range of 0.03% to 1.00%, the concentration of lotus flavone is negatively correlated with cell survival rate.Cell survival rate was higher than negative control group when 0.03%-0.13% lotus flavones existed in culture media.It was revealed that lotus flavones of 0.03% to 0.13% increased cell proliferation.When lotus flavones concentration was 0.25%, cell survival rate was lower than negative control group.Cells with 1.00% lotus flavones showed a survival rate of 87%, which is statistically significant different with negative control group (P＜0.05).According to toxicity assessment in ISO 10993-5:2009, test sample is nontoxic when it leads to a cell survival rate more than 70% and its 1/2 concentration leads to a survival rate with no significant differences from the negative control.Lotus flavones during 0.03% to 1.00% leaded to high cell survival rates of more than 70%.

Figure 1.Effects of lotus leaf flavones on cell survival rate

Tyrosinase inhibition effect

Tyrosinase inhibition of lotus flavones is shown in Figure 2.Within the range of 0.01% to 2.00%, 3-o-ethyl ascorbic acid had a tyrosinase inhibition effect which was concentration dependent (Figure 2b).Within the range of 0.02% to 0.30%, lotus flavones also had a tyrosinase inhibition effect which was concentration dependent as well (Figure 2a).Data showed that lotus flavones are more effective than 3-o-ethyl ascorbic acid on tyrosinas inhibition effect.Even 0.02% lotus flavones leaded to a statistically significant difference.

Figure 2. Tyrosinase inhibitory effects

DPPH scavenging

DPPH is one type of free radicals, which is soluble in ethanol.The solution has a characteristic light absorption at 517 nm, which value is proportional to concentration of DPPH.The ability of lotus flavones to scavenge DPPH is shown in Figure 3.Even as low as 0.001～0.010 g/L, lotus flavones leaded to an evident decline of DPPH concentration.The DPPH scavenging rate is positively correlated with concentration of lotus flavones.When the concentration of lotus flavones was above 0.010 g/L, DPPH scavenging rate was no longer changed.In positive group, DPPH was scavenged by Vc.It is revealed in Figure 3 that lotus flavones scavenged a little less DPPH than Vc.

Figure 3. DPPH radical scavenging activity of lotus flavonoids

It can be seen that lotus leaf flavonoids have a significant DPPH free radical scavenging effect, which should be added to cosmetics to work as antioxidant effect.

Inhibiting the synthesis of skin pigment

Lotus flavones was used as material in face cream.The kin pigment was dectected once a week.Results were showed in Figure 4.At the start time of experiment, skin pigment was detected, which value was set as 100%.1 week, 2 weeks, 3 weeks, 4 weeks later, skin pigment got less and less.It was revealed that 1% lotus flavones could lead to a decline of skin pigment.

Figure 4. The effect of skin cream containing flavonoids on the skin whitening test

Conclusion

(1) Lotus leaf flavonoids have fine biological compatibility.In MTT test, cell survival rate was close to the control group.In the range of 0.03% to 1.00%, lotus leaf flavonoids showed no cytotoxicity, and should be used in cosmetics for skin caring；

(2) Lotus leaf flavonoids have ability to inhibit tyrosinase.As little as 0.02% can significantly reduce tyrosinase activity；

(3) Lotus leaf flavonoids are excellent antioxidants, with which DPPH can be scavenged up to 80%；

(4) Skin cream containing lotus leaf flavonoids has a whitening effect on human facial skin.