Application of white mulberry root-bark effective part in whitening and UVA damage resistance and preparation method of white mulberry root-bark effective part

Abstract

The invention relates to application of a white mulberry root-bark effective part in whitening and UVA damage resistance and a preparation method of the white mulberry root-bark effective part. The white mulberry root-bark effective part is obtained by extracting white mulberry root-bark with ethanol, concentrating the obtained extracting solution under reduced pressure, dissolving the concentrated extracting solution with ethanol, adsorbing with macroporous resin, eluting with ethanol with different concentrations, respectively collecting ethanol eluates with different concentrations, and concentrating under reduced pressure. The protection effect of the effective part on UVA damaged human dermal fibroblasts (HDF-alpha) is equivalent to that of positive control vitamin C (100 mu M). The effective part (0.3% emulsion) shows a remarkable whitening effect in ultraviolet radiation tested people, and the effect of the effective part is equivalent to that of positive control vitamin C (7% emulsion). The effective part disclosed by the invention has a better application prospect in developing cosmetics with whitening, light damage resistance or dual effects.

Description

Technical Field

[0001] The invention belongs to the field of cosmetic application development, and specifically relates to a method for preparing an effective part of Morus alba bark, and application of the effective part or a composition using the effective part as an active component in preparing whitening and anti-photodamage cosmetics. Background Art

[0002] Currently, the destruction of the ozone layer has aggravated light pollution and greatly increased ultraviolet (UV) exposure, leading to photodamage lesions such as pigmentation, skin aging, wrinkles and even skin cancer. It is worth noting that the ultraviolet rays that the sun irradiates to the earth's surface are mainly composed of UVA (about 95%) and UVB. Unlike sunburn caused by UVB, the body has no natural warning system for UVA, and the damage caused is often difficult to detect. Therefore, it is particularly important to prevent UVA damage.

[0003] Tyrosinase inhibitors are widely used in whitening cosmetics. These inhibitors include kojic acid, arbutin, resorcinol and its derivatives, and hydroquinone, but they each have side effects or stability issues, which affect the product experience. For example, kojic acid has poor stability and may pose a carcinogenic risk with long-term use, while hydroquinone has been banned in many countries. Currently, there are some problems with anti-UV light damage cosmetics, such as skin irritation, allergic reactions, photosensitivity reactions, or dry or greasy skin. In addition, existing cosmetic products usually only have one effect of whitening or anti-photodamage, and cannot meet consumers' dual needs for whitening and anti-photodamage at the same time. Summary of the invention

[0004] The object of the present invention is to provide an effective part of Morus alba bark having both whitening and UVA damage resistance effects and a preparation method thereof, so as to solve the problems raised in the above background technology.

[0005] Application of effective parts of Morus alba bark in whitening,

[0006] The active fraction comprises Diels-Alder type adducts, and the Diels-Alder type adducts are Moracenin D, Sanggeno T, Kuwanon G, Mulberrofuran J, Albafuran C and Kuwanon X.

[0007] Application of effective parts of Morus alba bark in anti-UVA damage,

[0008] The active fraction comprises Diels-Alder type adducts, and the Diels-Alder type adducts are Moracenin D, Sanggeno T, Kuwanon G, Mulberrofuran J, Albafuran C and Kuwanon X.

[0009] The effective part contains 55-68% Diels-Alder type adduct by mass.

[0010] The preparation method of the effective part of Morus alba bark is characterized in that the effective part is obtained by extracting the Morus alba bark with ethanol, concentrating the obtained extract under reduced pressure to a dry product, dissolving the dry product with ethanol, adsorbing it with a macroporous resin, and eluting it with ethanol of different concentrations, and concentrating the 50% ethanol water eluate under reduced pressure to obtain the effective part of the present invention.

[0011] During the ethanol extraction process, the ethanol concentration is 50-100%, 3-6L of ethanol water is used for each kg of mulberry bark, and the extraction temperature is 20-60°C.

[0012] The concentration of the ethanol used to dissolve the dried product is 90-95%, and the mass ratio of the dried product to the macroporous resin is 1:1.

[0013] The ethanol elution is performed with pure water, 25% ethanol water and 50% ethanol water in sequence for gradient elution; pure water elution is 8-11 column volumes; 25% ethanol water elution is 12-16 column volumes; 50% ethanol water elution is 9-12 column volumes.

[0014] The effective fraction is separated and purified, and the specific process is:

[0015] S1, treating the effective fraction with an LH-20 dextran gel column, and gradient eluting the effective fraction with 50%, 60%, and 70% methanol water in sequence; in the gradient elution process, eluting with 50% methanol water for 19-21 column volumes, eluting with 60% methanol water for 15-19 column volumes, obtaining a first separation portion; eluting with 70% methanol water for 14-17 column volumes, obtaining a second separation portion;

[0016] S2, performing a second separation on the first separated part using a methanol-water system, and performing a third separation on the second separated part;

[0017] The second and third separations are performed using semi-preparative liquid phase;

[0018] In the second separation, 50-65% methanol water is used at a flow rate of 2-4 ml / min to elute the first separation portion, and the eluent contains the Diels-Alder type adducts Moracenin D, Sanggenon T and Kuwanon G;

[0019] In the third separation, 55-65% methanol water is used at a flow rate of 2-4 ml / min to elute the second separation part, and the eluent contains the Diels-Alder type adducts Mulberrofuran J, Albafuran C and Kuwanon X.

[0020] The effective part is used for preparing anti-UVA damage cosmetics or for preparing whitening and anti-UVA damage dual-effect cosmetics.

[0021] The cosmetic composition further comprises auxiliary materials acceptable in the cosmetic field.

[0022] The dosage form of the cosmetic is emulsion, spray, aerosol, cream, water, gel, oil, patch, film or mud.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1. The present invention provides an effective fraction of Morus alba extract containing 6 Diels-Alder type adducts;

[0025] 2. The effective fraction provided by the present invention shows excellent protective effect on UVA-damaged skin cells, and its activity is equivalent to that of the positive control vitamin C;

[0026] 3. The effective fraction provided by the present invention has good application prospects in the development of whitening, anti-photodamage or dual-effect cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The structural formula and carbon atom numbering diagram of the Diels-Alder type adduct in the effective part of Morus alba bark.

[0028] Figure 2 This is the extracted ion chromatogram (EIC) of UPLC / Q-TOF-MS UV and Diels-Alder type adducts of the effective part of Morus alba bark.

[0029] Figure 3 This graph shows the protective effect of the effective fraction of Morus alba bark (10, 25, 50 mg / ml) on human dermal fibroblasts (HDF-α) damaged by UVA. DETAILED DESCRIPTION

[0030] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Embodiment 1: The preparation method of the effective part of Morus alba bark comprises the following steps:

[0032] S1, crushing the mulberry bark into 100 mesh, soaking and extracting the mulberry bark in a 75% ethanol water system at 25° C. for 3 times to obtain a mulberry bark extract, and combining the 3 extracts and concentrating under reduced pressure to dryness;

[0033] Each kg of Morus alba bark was extracted with 4 L of ethanol water;

[0034] In the three extractions, the first soaking was 48 hours, and the next two soakings were 24 hours;

[0035] S2, dissolving the dried product obtained in step S1 with 95% ethanol, filtering, and collecting the filtrate; mixing the filtrate with D101 macroporous resin to obtain a stirred product, and concentrating under reduced pressure to dryness;

[0036] The mass ratio of macroporous resin to dried product was 1:1;

[0037] S3, applying the mixture from step S2 to a macroporous resin chromatography column, and gradient eluting the mixture with pure water, 25% ethanol water, and 50% ethanol water in sequence;

[0038] The purified water was used for 10 column volumes, the 25% ethanol was used for 14 column volumes, and the 50% ethanol was used for 11 column volumes.

[0039] 11 column volumes of 50% ethanol water eluent contains the effective fraction of the present invention;

[0040] S4, separating and purifying the effective fraction obtained in step S3,

[0041] The effective fraction was gradiently eluted using LH-20 polysaccharide gel column with 50%, 60% and 70% methanol water in sequence;

[0042] 50% methanol water was used for elution for 21 column volumes; 60% methanol water was used for elution for 17 column volumes to obtain the first separation portion; 70% methanol water was used for elution for 15 column volumes to obtain the second separation portion;

[0043] The first separation portion is eluted and separated using 55% methanol at a flow rate of 3.5 ml / min, wherein the eluate contains the Diels-Alder type adducts Moracenin D, Sanggenon T and Kuwanon G;

[0044] The second separation portion is eluted with 51% methanol at a flow rate of 3.5 ml / min+, and the eluate contains the Diels-Alder type adducts Mulberrofuran J, Albafuran C and Kuwanon X;

[0045] The second separation and the third separation were performed using an Agilent 1260 semi-preparative liquid chromatograph and a semi-preparative reverse phase chromatography column (ZORBAX SB-C18, 250 mm×9.4 mm, 5 μm, Agilent Japan).

[0046] The NMR data of the adduct Moracenin D are as follows:

[0047] 1 H-NMR(500MHz,CD3COCD3)δ:13.01,12.99(each 1H,s,OH-5,10"),7.29(1H,1H,d,J=8.2Hz,H-6'),7.25(1H,d,J=8.8Hz,H-14"),6.81(1H,d,J=8.4Hz,H-20"),6.65(1H,dJ=2.0H z,H-3'),6.56(1H,dd,J=8.2,2.0Hz,H-5'),6.20(1H,br.s,H-17”),6.07(1H,br.d,J=8.4Hz,H-19”),6.00(1H,br.s,H-11”),5.9 8(1H,br.s,H-6),5.93(1H,br.d,J=8.8Hz,H-13”),5.20(1H,br.s,H-2”),4.62(1H,br.s,H-4”),4.40(1H,br.d,J=10.2Hz,H-3" ),3.77(1H,br.s,H-5"),2.54(2H,m,H-9),2.11,1.98(2H,m,H-6"),1.69(2H,m,H-10),1.49(3H,s,Me-7"),1.11(6H,s,Me-12and 13); 13C-NMR (125MHz, CD3COCD3) δ: 209.4 (C-8”), 183.5 (C-4), 166.0 (C-10”), 164.8 (C-12”), 161.9 (C-8a), 161.4 (C-4’), 161.2 (C-2, C-7), 15 7.3(C-5),157.0(C-2',C-18”),156.2(C-16”),133.9(C-1”),133.6(C-14”),132.4(C-6’),132.0(C-20”),124.2(C-2”),122.4(C-3,C- 15”),115.7(C-9”),113.3(C-1’),108.1(C-5’),107.9(C-8),107.7(C-19”),107.6(C-13”),105.5(C-4a),103.8(C-3’),103.6(C-17”) ,102.9(C-11”),98.3(C-6),70.2(C-11),47.7(C-4”),43.2(C-10),38.6(C-3”,C-5”,C-6”),29.4(C-12,C-13),23.0(C-7”),21.0(C-9);

[0048] The NMR data was compared with existing data and identified as Moracenin D.

[0049] The NMR data of the adduct Sanggenna T are as follows:

[0050] 1 H-NMR(500MHz,DMSO-d6)δ:13.02,12.98(total 1H,OH-10),12.81,12.44(total1H,OH-5),7.53–7.66(1H,m,H-14”),7.06-7.14(1H,m),6 .65–6.76(1H,m),6.30(1H,br.s),6.18–6.25(1H,m),5.66–6.12(5H,m),5.33,5.47(total 1H,H-2),5.13(1H,m,H-2”),4.77(1H,br.s,H-4”),4.16(1H,br.s,H-3”),3.48(1H,br.s,H-5”),3.13,2 .58(2H,m,H-3),2.45,2.06(2H,m,H-6”),1.93,1.39(6H,m,H2-1”’,2”’,3”’),1.05(6H,s,Me-5”’,6”’); 13C-NMR(125MHz,DMSO-d6)δ:206.8(C-8”),196.7(C-4),164.3(C-10”),164 .1(C-7,C-12”),161.3(C-5),161.1(C-8a),158.5(C-4’),155.7(C-2’),15 5.6(C-16”),156.0(C-18”),135.6(C-1”),132.5(C-14”),128.2(C-20”), 128.1(C-6'),123.8(C-2”),123.7(C-15”),120.9(C-1’),115.4(C-9”),10 8.6(C-6),107.2(C-19”),107.0(C-5’),106.4(C-13’),104.2(C-4a),103 .1(C-17”),102.6(C-3’),101.8(C-11”),95.0,93.9(C-8),73.5,73.6(C-2 ),68.7(C-5”'),44.9(C-4”),43.3,43.0(C-3),41.1(C-3”’),39.5(C-1”’ ),37.3(C-3”,C-5”,C-6”),29.5,29.3(C-5”’,C-6”’),22.0,21.7(C-2”’).

[0051] The NMR data were compared with existing data and the compound was identified as Sanggeno T.

[0052] The NMR data of the adduct Kuwanon G are as follows:

[0053] 1H-NMR(500MHz,CD3COCD3)δ:13.01,12.97(each 1H,s,OH-5,10”),7.29(1H,d,J=8.2Hz,H-6'),7.25(1H,d,J=8.8Hz,H-14"),6.78(1H,d,J=8.4Hz,H-20"),6.66(1H,d,J=1.9Hz,H-3'),6.56(1H,dd,J=8.2,1.9Hz,H-5'),6.20(1H,br.s,H-17”),6.06(1H,br.d,J=8.4Hz,H-19”),6.01(1H,br.s,H-11”),5.98(1H,br.s,H-6),5.93(1H,br.d,J=8.8Hz,H-13”),5.21(1H,br.s,H-2”),5.18(1H,t,J=7.2Hz,H-10),4.62(1H,br.s,H-4”),4.42(1H,br.d,J=10.0Hz,H-3"),3.77(1H,br.s,H-5”),3.08–3.25(2H,m,H-9),2.11,1.98(2H,m,H-6"),1.60(3H,s,Me-12),1.51(3H,s,Me-7"),1.47(3H,s,Me-13); 13 C-NMR(125MHz,CD3COCD3)δ:209.4(C-8”),183.2(C-4),166.0(C-10”),164.8(C-12”),162.1(C-8a),161.5(C-4'),161.3(C-2),161.2(C-7),157.3(C-5),157.0(C-2',C-18”),156.2(C-16”),133.9(C-1”),133.6(C-14”),132.2(C-11,C-6'),132.1(C-20”),124.3(C-2”),122.8(C-10),122.6(C-15”),121.4(C-3),115.7(C-9”),113.3(C-1'),108.1(C-5'),108.0(C-8),107.8(C-19”),107.6(C-13”),105.5(C-4a),103.7(C-3'),103.6(C-17”),102.9(C-11”),98.3(C-6),47.6(C-4”),38.6(C-3”,C-5”,C-6”),25.8(C-12),24.5(C-9),23.1(C-7”),17.7(C-13)。

[0054] The NMR data were compared with existing data and the compound was identified as Kuwanon G.

[0055] The NMR data of the adduct Mulberrofuran J are as follows:

[0056] 1 H-NMR(500MHz,CD3COCD3)δ:13.11(1H,s,OH-10”),9.07,8.49,8.17,8.07×2,7.86(6H,OH),7.72(1H,d,J=8.2Hz,H-14”),7.34(1H ,d,J=8.4Hz,H-4),6.91(1H,d,J=2.2Hz,H-7),6.88(1H,d,J=8.8Hz,H-20”),6.84(1H,d,J=2.2Hz,H-3),6.81,6.65(1H,s,H-2'and H-6'),6.76(1H,dd,J=8.4,2.2Hz,H-5),6.21(1H,d,J=1.7Hz,H-17”),6.10(1 H,dd,J=8.2,2.4Hz,H-13”),6.03(1H,dd,J=8.8,1.7Hz,H-19”),5.95(1H,d,J= 2.4Hz,H-11”),5.38(1H,s,H-2”),4.94(1H,br.s,H-4”),4.52(1H,br.dJ=9.5 Hz,H-3”),3.66(1H,br.s,H-5”),2.51,2.20(2H,m,H-6”),1.75(3H,s,Me-7”); 13C-NMR (125MHz, CD3COCD3) δ: 209.8 (C-8”), 166.0 (C-10”), 164.7 (C-12”), 158.3 (C-7a), 157.2 (C-16”, C-18”), 15 6.6(C-2),156.5(C-3',5'),155.3(C-6),134.2(C-1”),133.9(C-20”),130.5(C-14”,C-1’),125.1(C-2”),122.5 (C-15”),121.8(C-4,C-3a),117.3(C-9”),115.9(C-4’),113.1(C-5),107.6(C-13”),107.4(C-19”),104.7(C-17 ”),103.7(C-2’,6’),102.8(C-3),101.7(C-11”),98.3(C-7),46.7(C-4”),38.8(C-3”,C-5”,C-6”),23.4(C-7”).

[0057] The NMR data were compared with existing data and the compound was identified as Mulberrofuran J.

[0058] The NMR data of the adduct Albafuran C are as follows:

[0059] 1 H-NMR(500MHz,CD3COCD3)δ:13.09(1H,s,OH-10”),9.07(s,OH),8.38(2H,s,OH-3',5'),8.26(s,OH),8.11(s,OH),7.84(s,OH),7.43(1H,s ,H-4),7.42(1H,d,J=8.8Hz,H-14”),6.98(1H,s,H-3),6.91(1H,d,J=8.3Hz,H-20”),6.79(2H,d,J=2.1Hz,H-2',6'),6.74(1H,s,H-7),6.3 3(1H,t,J=2.1Hz,H-4'),6.20(1H,d,J=2.4Hz,H-17”),6.09(1H,dd,J=8.3,2.4Hz,H-19”),5.94(1H,d,J=2.4Hz,H-11”),5.89(1H,dd,J=8. 8,2.4Hz,H-13”),5.42(1H,s,H-2”),4.46(1H,s,H-4”),4.30(1H,s,H-3”),3.66(1H,s,H-5”),2.73,2.20(2H,m,H-6”),1.79(3H,s,Me-7”);13 C-NMR (125MHz, CD3COCD3) δ: 209.4 (C-8”), 166.0 (C-10”), 164.7 (C-12”), 159.8 (C-3’, C-5’), 157.3 (C-16”), 156.6 (C-18”),155.3(C-2),155.2(C-7a),154.1(C-6),134.4(C-1”),133.9(C-14”,C-20”),133.4(C-1’),128.3(C-5),12 6.1(C-2”),122.6(C-4),121.6(C-3a,C-15”),115.8(C-9”),107.4(C-13”,C-19”),103.8(C-17”),103.7(C-2',C-6 '),103.4(C-4'),102.8(C-11”),102.3(C-3),97.9(C-7),51.3(C-4”),43.4(C-3”,C-5”),37.5(C-6”),23.5(C-7”).

[0060] The NMR data were compared with existing data and the compound was identified as Albafuran C.

[0061] The NMR data of the adduct Kuwanon X are as follows:

[0062] 1H-NMR(500MHz,CD3COCD3)δ:13.12(1H,s,OH-10”),9.08,8.49,8.33,8.04,7.88,7.84,7.82(each 1H,OH),7.72(1H,d,J=8.9Hz,H-14”),7.33(1H,d,J=8.6Hz,H-6),7.17(1H,d,J=16.5Hz,H-β) ,6.87(1H,d,J=8.2Hz,H-20”),6.69(1H,d,J=16.5Hz,H-α),6.48(1H,s,H-2'),6.40(1H,d,J= 2.4Hz,H-3),6.34(1H,dd,J=8.6,2.4Hz,H-5),6.32(1H,s,H-6'),6.21(1H,d,J=1.8Hz,H-17” ),6.09(1H,dd,J=8.9,2.4Hz,H-13”),6.04(1H,dd,J=8.2,1.8Hz,H-19”),5.96(1H,d,J=2.4Hz H-11”),5.36(1H,s,H-2”),4.91(1H,br.s,H-4”),4.49(1H,br.d,J=10.0H z,H-3”),3.67(1H,m,H-5”),2.12–2.22(2H,m,H-6”),1.74(3H,s,Me-7”); 13 C-NMR (125MHz, CD3COCD3) δ: 210.0 (C-8”), 166.0 (C-10”), 164.7 (C-12”), 159.0 (C-4), 158.0 (C-16”), 157.2 (C-3’), 156 .7(C-2,C-5'),156.4(C-18”),138.8(C-1’),134.0(C-14’),133.9(C-1”),128.0(C-β),125.9(C-α),125.6(C-2”,C-6), 123.5(C-20”),122.0(C-15”),117.2(C-1),116.0(C-9”),115.7(C-4’),108.4(C-5),107.6(C-13”),107.4(C-19”),106 .3(C-2'),105.6(C-6'),103.8(C-17”),103.5(C-3),102.8(C-11”),46.8(C-4”),38.8(C-3”,C-5”,C-6”),23.4(C-7”).

[0063] The NMR data were compared with existing data and the compound was identified as Kuwanon X (mulberry flavone G).

[0064] UPLC / Q-TOF-MS UV and Diels-Alder type adducts characterization of the effective parts of Morus alba bark Figure 2 As shown, Figure 2 The display shows that the m / z at 14.25min is 709 [MH] - The molecular ion peak is consistent with Moracenin D; m / z is 711 [MH] at 14.48 min - The molecular ion peak is consistent with Sanggenon T; m / z is 691 [MH] at 21.31 min - The molecular ion peak is consistent with Kuwanon G; m / z is 579 [MH] at 12.91 min - The molecular ion peak is consistent with Mulberrofuran J; m / z is 579 [MH] at 14.76 min - The molecular ion peak is consistent with Albafuran C; m / z is 581 [MH] at 10.37 min - The molecular ion peak was consistent with Kuwanon X.

[0065] The specific process of preparing the extracted ion sample is to take 2.0 mg of the sample (effective part of Morus alba bark), dissolve it in 1.0 mL of methanol, filter it with a filter membrane, place it in a 1.5 mL injection bottle, and inject 3 ul.

[0066] The ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry detection conditions are:

[0067] Instrument: Agilent UPLC / 6540Q-TOF;

[0068] Chromatograph: Agilent 1290UPLC;

[0069] Detector: Diode Array Detector;

[0070] Chromatographic column: Extend-C18 column (5μm, 4.6×150mm).

[0071] Mobile phase: Mobile phase A is methanol, and mobile phase B is water;

[0072] Elution gradient: 0 min, 40% A; 10 min, 60% A; 40 min, 80% A; 50 min, 100% A. Flow rate: 1 mL / min.

[0073] Mass spectrometry conditions: ion source Dual ESI; negative ion scanning mode; nebulizer gas pressure 40psi; drying gas flow rate 9L / min; drying gas temperature 350℃; ion source voltage 3500V; capillary voltage (Fragmentor) 135V; cone voltage (Skimmer) 65V; quadrupole voltage 750V; scanning range 50-1700Da.

[0074] The protective effect of the effective fraction of Morus alba obtained in this example on human dermal fibroblasts (HDF-α) damaged by UVA was detected as follows:

[0075] Human dermal fibroblasts (HDF-α), DMEM culture medium, PBS, D-Hanks, vitamin C (positive drug), and MTS kit were purchased from Sigma.

[0076] The detection method is as follows:

[0077] (1) HDF-α cells were cultured at 1×10 5 / mL density was transferred into a 96-well plate and cultured overnight;

[0078] (2) After the cells in the 96-well plate adhered, the culture medium was aspirated, 100 μL PBS was added to each well and washed twice, and then 100 μL D-Hanks was added to each well;

[0079] (3) A UVA damage group (model group), a UVA damage group with medication, and a normal control group (NC group) were set up, with five parallel groups in each group.

[0080] Normal control group (NC): protected from light by tin foil, placed in room environment for 2 hours, then aspirated D-Hanks, added 100 μL DMEM medium to each well, and continued incubation for 24 hours;

[0081] UVA damage group (model group): UV lamp irradiation dose was adjusted to 10 J / cm2 (1375-1400 UW), irradiation time was 2 h, and UV irradiation was performed; after UV irradiation, D-Hanks was aspirated, 100 μL DMEM medium was added to each well, and incubation was continued for 24 h;

[0082] UVA damage and drug addition group: adjust the UV lamp irradiation dose to 10J / cm2 (1375-1400UW), irradiation time to 2h, and perform UV irradiation. After UV irradiation, aspirate D-Hanks, add 100μL of effective part solution to each well, with concentrations of 20, 50, and 100mg / ml (final concentrations: 10, 25, and 50mg / ml), and continue incubation for 24h;

[0083] Positive control vitamin C group: adjust the UV lamp irradiation dose to 10J / cm2, irradiation time to 2h, and perform UV irradiation. After UV irradiation, aspirate D-Hanks, add 100μL of positive drug solution to each well, the concentration is 200μM (final concentration: 100μM), and continue incubation for 24h;

[0084] (4) Aspirate the supernatant, wash twice with PBS, and perform cell viability detection using the MTS kit;

[0085] MTS was thawed in advance, MTS: culture medium = 1:4, and the operation was carried out in a dark place with lights off. A 15 ml centrifuge tube was wrapped with tin foil to avoid light, and 100 vl of reagent was added to the incubator for incubation for 2-4 hours. After the incubation was completed, the tube was wrapped with tin foil and the data was measured;

[0086] (5) OD 490 nm was detected using an ELISA reader, and cell viability (Survival rate) was calculated using the following formula.

[0087]

[0088] Test results such as Figure 3 As shown, the effective fraction of Morus alba showed significant protective effect on human dermal fibroblasts (HDF-α) damaged by UVA at concentrations of 25 and 50 mg / ml. Among them, the protective effect of the effective fraction of Morus alba on human dermal fibroblasts (HDF-α) damaged by UVA at 50 mg / ml was equivalent to that of the positive control vitamin C (100 μM).

[0089] Five parallel experiments were set up for each group of the above test experiments, n = 5; data are expressed as Mean ± SEM. One-way analysis of variance and Tukey's multiple comparison test were used to evaluate statistical significance. ns (P>0.05), **P<0.01, ****P<0.0001 (vs. UVA group); NS (P>0.05) (vs. VC group)

[0090] The whitening effect of the effective part obtained in this example on the ultraviolet irradiated test population was detected as follows:

[0091] Use 0.3% emulsion of the effective part of Morus alba bark;

[0092] The negative control was the blackened area blank control; the positive control was 7% ascorbic acid (vitamin C) emulsion;

[0093] There were 20 subjects in total, including 3 males and 17 females, ranging in age from 23 to 57 years old, with an average age of 45.70±9.34 years old.

[0094] The test method is tested in accordance with the specific requirements of the "Technical Specifications for Safety of Cosmetics" (2015 edition). The non-exposed areas of the subject's thigh or back are selected as the test site. The minimum erythema dose (MED value) of the subject's skin to ultraviolet radiation is predicted 24 hours before the test, and the same irradiation point is irradiated once a day with a xenon arc lamp of a daylight simulator at a dose of 0.75 times the MED, for 4 consecutive days. The 4 days after the end of irradiation are the skin melanization period, and no treatment is performed. On the 5th day after the end of irradiation, the skin color of each test area is visually evaluated and instrumentally tested for skin color, and the test areas with poor consistency (areas where the difference between the ITA° value and the mean of all test areas is greater than 5) are eliminated. Starting on the same day, the corresponding test substance is applied to each melanization test area according to the random table, and the test substance is applied continuously for 4 weeks. The skin color is visually evaluated and instrumentally tested 1 week, 2 weeks, 3 weeks and 4 weeks after application, and recorded in the following Table 1.

[0095] Table 1

[0096]

[0097]

[0098] From the table, it can be seen that after applying the effective part emulsion of Morus alba bark of the test sample for 1 week, 2 weeks, 3 weeks and 4 weeks, the difference in skin ITA° value before and after application was significantly different from that of the negative control (P < 0.05); after applying the test product for 1 week, 2 weeks, 3 weeks and 4 weeks, the difference in MI before and after application was significantly different from that of the negative control (P < 0.05); after applying the test sample for 1 week, 2 weeks, 3 weeks and 4 weeks, the difference in visual skin color grade before and after application was significantly different from that of the negative control (P < 0.05); compared with the negative control, the regression coefficients of ITA°, MI and visual skin color grade of the test sample were significantly different (P < 0.05), indicating that the test product has the effect of removing spots and whitening.

[0099] In the table, ITA° is a parameter that characterizes the color of human skin. The larger the ITA° value, the lighter the skin color. MI value is a parameter that measures the melanin content in the skin. The larger the MI value, the higher the melanin content. The positive control is vitamin C (7%). The data is provided by Huante Biotechnology. ITA° / MI / skin color grade data are average values. a test product VS negative, and P = 0.000, there is a significant difference. The comparison adopts independent sample t test or rank sum test. At the same time, the regression coefficient of each parameter changing over time is calculated, and the significance level is P < 0.05.

Claims

1. Application of effective parts of Morus alba bark in whitening, The active fraction comprises Diels-Alder type adducts, and the Diels-Alder type adducts are Moracenin D, Sanggeno T, Kuwanon G, Mulberrofuran J, Albafuran C and Kuwanon X.

2. Application of effective parts of Morus alba bark in anti-UVA damage, The active fraction comprises Diels-Alder type adducts, and the Diels-Alder type adducts are Moracenin D, Sanggeno T, Kuwanon G, Mulberrofuran J, Albafuran C and Kuwanon X.

3. The effective part of Morus alba bark according to claim 1, characterized in that The effective part contains 55-68% Diels-Alder type adduct by mass.

4. A method for preparing an effective fraction of Morus alba bark, characterized in that The effective part is prepared by extracting the mulberry bark with ethanol, concentrating the obtained extract under reduced pressure to a dry product, dissolving the dry product with ethanol, adsorbing it with a macroporous resin, and eluting it with ethanol of different concentrations. The effective part of the present invention is obtained by concentrating the 50% ethanol water eluate under reduced pressure.

5. The method for preparing the effective fraction of Morus alba bark as claimed in claim 4, characterized in that During the ethanol extraction process, the ethanol concentration is 50-100%, 3-6 L of ethanol water is used for extraction per kg of mulberry bark, and the extraction temperature is 20-60°C.

6. The method for preparing the effective fraction of Morus alba bark according to claim 4, characterized in that The concentration of the ethanol used to dissolve the dried product is 90-95%, and the mass ratio of the dried product to the macroporous resin is 1:

1.

7. The method for preparing the effective fraction of Morus alba bark as claimed in claim 4, characterized in that The ethanol elution is performed by gradient elution with pure water, 25% ethanol water and 50% ethanol water in sequence; pure water elution is 8-11 column volumes; 25% ethanol water elution is 12-16 column volumes; 50% ethanol water elution is 9-12 column volumes.

8. The method for preparing the effective fraction of Morus alba bark as claimed in claim 4, characterized in that The effective fraction is separated and purified, and the specific process is: S1, treating the effective fraction with an LH-20 dextran gel column, and gradient eluting the effective fraction with 50%, 60%, and 70% methanol water in sequence; during the gradient elution, eluting with 50% methanol water for 19-21 column volumes, and eluting with 60% methanol water for 15-19 column volumes, to obtain a first separation portion; 70% methanol water was used for elution for 14-17 column volumes to obtain the second separation portion; S2, performing a second separation on the first separated part using a methanol-water system, and performing a third separation on the second separated part; The second and third separations are performed using semi-preparative liquid phase; In the second separation, 50-65% methanol water is used at a flow rate of 2-4 ml / min to elute the first separation portion, and the eluent contains the Diels-Alder type adducts Moracenin D, Sanggenon T and Kuwanon G; In the third separation, 55-65% methanol water is used at a flow rate of 2-4 ml / min to elute the second separation part, and the eluent contains the Diels-Alder type adducts Mulberrofuran J, Albafuran C and Kuwanon X.

9. The effective parts of the mulberry bark are used to prepare anti-UVA damage cosmetics or to prepare whitening and anti-UVA damage dual-effect cosmetics.

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