Application of mulberry bark effective part in whitening and anti-uva damage and preparation method thereof

Abstract

Application of mulberry bark effective part in whitening and anti-UVA damage and preparation method thereof, the effective part is that mulberry bark is extracted with ethanol, the obtained extract is concentrated under reduced pressure, then dissolved with ethanol, then adsorbed with macroporous resin, eluted with different concentrations of ethanol, different concentrations of ethanol eluate is collected respectively, then concentrated under reduced pressure to obtain the effective part. α The protective effect of the effective part on UVA damaged human dermal fibroblasts (HDF- α ) is equivalent to that of the positive control vitamin C (100 μM). The effective part (0.3% emulsion) shows significant whitening effect in ultraviolet irradiation test population, which is equivalent to that of the positive control vitamin C (7% emulsion). The effective part of the application has good application prospect in developing whitening, anti-photo damage or double effect cosmetics.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic application development, specifically relating to a method for preparing an effective component of mulberry bark, and the application of the component or a composition thereof as an active ingredient in the preparation of whitening and anti-photodamage cosmetics. Background Technology

[0002] Currently, ozone layer depletion exacerbates light pollution, significantly increasing ultraviolet (UV) exposure and leading to photodamage such as pigmentation, skin aging, wrinkles, and even skin cancer. It's important to note that the ultraviolet radiation reaching the Earth's surface is primarily composed of UVA (approximately 95%) and UVB. Unlike sunburn caused by UVB, the body lacks a natural warning system for UVA, making its damage often difficult to detect. Therefore, protecting against UVA damage is particularly crucial.

[0003] Tyrosinase inhibitors are widely used in skin-whitening cosmetics. These inhibitors include kojic acid, arbutin, resorcinol and its derivatives, and hydroquinone. However, each has its own side effects or stability issues, affecting the user experience. For example, kojic acid has poor stability and long-term use may pose a carcinogenic risk, while hydroquinone has been banned in several countries. Currently, anti-UV photodamage cosmetics have several problems, such as skin irritation, allergic reactions, photosensitivity, or dry or oily skin. Furthermore, existing cosmetic products typically only provide one effect—either whitening or anti-photodamage—and cannot simultaneously meet consumers' dual needs for whitening and anti-photodamage. Summary of the Invention

[0004] The purpose of this invention is to provide an effective component of mulberry bark that simultaneously possesses whitening and UVA damage-resistant properties, and a method for preparing the same, in order to solve the problems mentioned in the background art.

[0005] Application of effective components of mulberry bark in skin whitening

[0006] The effective fraction contains Diels-Alder adducts, namely Moracenin D, Sanggenon T, Kuwanon G, Mulberrofuran J, Albafuran C, and Kuwanon X.

[0007] Application of effective components of mulberry bark in protecting against UVA damage

[0008] The effective fraction contains Diels-Alder adducts, namely Moracenin D, Sanggenon T, Kuwanon G, Mulberrofuran J, Albafuran C, and Kuwanon X.

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

[0010] The method for preparing the effective component of mulberry bark is characterized in that the effective component is obtained by extracting mulberry bark with ethanol, concentrating the resulting extract under reduced pressure to a dry product, dissolving the dried product in ethanol, adsorbing it with macroporous resin, eluting it with ethanol of different concentrations, and concentrating the 50% ethanol-water eluent under reduced pressure to obtain the effective component of the present invention.

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

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

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

[0014] The effective components are separated and purified, and the specific process is as follows:

[0015] S1, the effective fraction was treated using an LH-20 dextran gel column, and eluted sequentially with 50%, 60%, and 70% methanol-water solutions. During the gradient elution, 19-21 column volumes were eluted with 50% methanol-water, 15-19 column volumes with 60% methanol-water to obtain the first separated fraction, and 14-17 column volumes with 70% methanol-water to obtain the second separated fraction.

[0016] S2, the first separation section is subjected to a second separation using a methanol-water system, and the second separation section is subjected to a third separation;

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

[0018] In the second separation, the first separation fraction is eluted with 50-65% methanol-water at a flow rate of 2-4 ml / min. The eluent contains the Diels-Alder type adducts Moracenin D, Sanggenon T, and Kuwanon G.

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

[0020] The effective components mentioned above are used to prepare anti-UVA damage cosmetics or to prepare cosmetics with both whitening and anti-UVA damage effects.

[0021] The cosmetic composition also includes cosmetic excipients.

[0022] The dosage forms of the cosmetics are emulsions, sprays, aerosols, creams, liquids, gels, oils, patches, films, or mud-like agents.

[0023] Compared with the prior art, the beneficial effects of the present invention are:

[0024] 1. This invention provides an effective fraction of mulberry bark extract containing 6 Diels-Alder adducts;

[0025] 2. The effective component provided by this invention shows superior protective effect against UVA-damaged skin cells, with activity comparable to that of the positive control vitamin C;

[0026] 3. The effective components provided by this invention have good application prospects in the development of whitening, anti-photodamage, or dual-effect cosmetics. Attached Figure Description

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

[0028] Figure 2 The UPLC / Q-TOF-MS ultraviolet and Diels-Alder type adduct extraction ion chromatograms (EIC) of the effective parts of mulberry bark.

[0029] Figure 3 The protective effect of effective fractions of mulberry bark (10, 25, 50 mg / ml) on UVA-damaged human dermal fibroblasts (HDF-α) is shown in the figure. Detailed Implementation

[0030] The technical solutions of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are merely some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0031] Example 1: A method for preparing the effective components of mulberry bark, comprising the following steps:

[0032] S1. The mulberry bark is pulverized to 100 mesh, and the mulberry bark is soaked and extracted three times at 25°C using a 75% ethanol-water system to obtain mulberry bark extract. The three extracts are combined and concentrated under reduced pressure to dry matter.

[0033] Each kg of mulberry bark is extracted using 4 L of ethanol-water immersion.

[0034] In the three extractions, the first soaking lasted 48 hours, and the subsequent two soakings lasted 24 hours.

[0035] S2, dissolve the dried material obtained in step S1 in 95% ethanol, filter, and collect the filtrate; mix the filtrate with D101 macroporous resin to obtain a stirred mixture, and concentrate and dry under reduced pressure.

[0036] The mass ratio of macroporous resin to dried material is 1:1;

[0037] S3, the mixture from step S2 is loaded onto a macroporous resin chromatography column and eluted sequentially with pure water, 25% ethanol-water, and 50% ethanol-water.

[0038] Elution was performed with pure water for 10 column volumes, with 25% ethanol and water for 14 column volumes, and with 50% ethanol and water for 11 column volumes.

[0039] Eleven column volumes of 50% ethanol-water eluent contained the effective components described in this invention.

[0040] S4. The effective components obtained in step S3 are separated and purified.

[0041] The effective fraction was eluted sequentially with 50%, 60%, and 70% methanol-water using an LH-20 dextran gel column.

[0042] The fraction was eluted with 50% methanol and water for 21 column volumes; eluted with 60% methanol and water for 17 column volumes to obtain the first separation fraction; and eluted with 70% methanol and water for 15 column volumes to obtain the second separation fraction.

[0043] The first separation fraction was eluted with 55% methanol at a flow rate of 3.5 ml / min. The eluent contained the Diels-Alder type adducts Moracenin D, Sanggenon T, and Kuwanon G.

[0044] The second separation fraction was eluted with 51% methanol at a flow rate of 3.5 ml / min. The eluent contained the Diels-Alder type adducts Mulberrofuran J, Albafuran C, and Kuwanon X.

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

[0046] The NMR data for 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 for the adduct Sanggenon 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, compared with existing data, identified the compound as Sanggenon T.

[0052] The NMR data for 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, compared with existing data, identified the compound as Kuwanon G.

[0055] The NMR data for 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, compared with existing data, identified the compound as Mulberrofuran J.

[0058] The NMR data for 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, compared with existing data, identified the compound as Albafuran C.

[0061] The NMR data for 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, compared with existing data, identified the compound as Kuwanon X (Sanghuangtong G).

[0064] Characterization of the effective components of mulberry bark using UPLC / Q-TOF-MS ultraviolet and extractive ion chromatograms (EIC) of Diels-Alder type adducts is as follows: Figure 2 As shown, Figure 2 The display shows that the m / z value at 14.25 min is 709 [MH]. - The molecular ion peak is consistent with that of Moracenin D; the m / z at 14.48 min is 711 [MH]. - The molecular ion peak is consistent with Sanggenon T; the m / z at 21.31 min is 691 [MH]. - The molecular ion peak is consistent with that of Kuwanon G; the m / z at 12.91 min is 579 [MH]. - The molecular ion peak is consistent with that of Mulberrofuran J; the m / z at 14.76 min is 579 [MH]. - The molecular ion peak is consistent with that of Albafuran C; the m / z at 10.37 min is 581 [MH]. - The molecular ion peak is consistent with that of Kuwanon X.

[0065] The specific process for preparing the ion extraction sample is as follows: take 2.0 mg of sample (effective part of mulberry bark), dissolve it in 1.0 mL of methanol, filter it through a filter membrane, place it in a 1.5 mL injection bottle, and inject 3 μL.

[0066] The detection conditions for ultra-high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry 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: Phase A is methanol, and 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: Dual ESI ion source; negative ion scanning mode; nebulizer pressure 40 psi; drying gas flow rate 9 L / min; drying gas temperature 350 °C; ion source voltage 3500 V; capillary voltage (Fragmentor) 135 V; aperture voltage (Skimmer) 65 V; quadrupole voltage 750 V; scan range 50-1700 Da.

[0074] The protective effect of the effective components of mulberry bark obtained in this embodiment against UVA-damaged human dermal fibroblasts (HDF-α) is as follows:

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

[0076] The detection method is as follows:

[0077] (1) HDF-α cells were fed at a rate of 1×10 5 Transferred at a density of / mL into 96-well plates and incubated overnight;

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

[0079] (3) Five replicates were set up for each group: a UVA injury group (model group), a UVA injury post-drug treatment group, and a normal control group (NC group). Among them:

[0080] Normal control group (NC): D-Hanks were aspirated after being placed in a light-proof environment for 2 hours, and 100 μL of LMEM medium was added to each well. The mixture was then incubated for another 24 hours.

[0081] UVA damage group (model group): The UV lamp irradiation dose was adjusted to 10 J / cm2 (1375-1400 UW) and the irradiation time was 2 h. After UV irradiation, D-Hanks were aspirated, and 100 μL of LDM medium was added to each well. The mixture was then incubated for another 24 h.

[0082] UVA-damaged drug-treated group: The UV lamp irradiation dose was adjusted to 10 J / cm² (1375-1400 UW), and the irradiation time was 2 h. After UV irradiation, D-Hanks were aspirated, and 100 μL of the effective fraction solution was added to each well at concentrations of 20, 50, and 100 mg / ml (final concentrations: 10, 25, and 50 mg / ml), and incubation was continued for 24 h.

[0083] Positive control vitamin C group: The UV lamp irradiation dose was adjusted to 10 J / cm2, and the irradiation time was 2 h. After UV irradiation, D-Hanks were aspirated, and 100 μL of positive drug solution was added to each well, with a concentration of 200 μM (final concentration: 100 μM), and incubation was continued for 24 h.

[0084] (4) Aspirate the supernatant, wash twice with PBS, and use the MTS kit to detect cell viability;

[0085] Thaw the MTS in advance. The ratio of MTS to culture medium is 1:4. Perform the operation in the dark with the lights off. Wrap the 15ml centrifuge tubes with aluminum foil to protect them from light. Add 100vl of reagent to the incubator and incubate for 2-4 hours. After the incubation is complete, wrap the tubes with aluminum foil and go to measure the data.

[0086] (5) Use an ELISA reader to detect OD 490nm and calculate cell viability (Survival rate) using the following formula.

[0087]

[0088] Test results as follows Figure 3 As shown, the effective fraction of mulberry bark exhibited significant protective effects against UVA-damaged human dermal fibroblasts (HDF-α) at ​​concentrations of 25 and 50 mg / ml. Specifically, the protective effect of the effective fraction of mulberry bark against UVA-damaged human dermal fibroblasts (HDF-α) at ​​50 mg / ml was comparable to that of the positive control vitamin C (100 μM).

[0089] Each of the above detection experiments was conducted in five parallel trials, n=5; data are expressed as mean ± SEM. One-way ANOVA 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 components obtained in this embodiment on the subjects subjected to ultraviolet irradiation was tested as follows:

[0091] A 0.3% emulsion of the effective components of mulberry bark was used;

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

[0093] The study included 20 participants, 3 males and 17 females, aged 23 to 57 years, with a mean age of 45.70 ± 9.34 years.

[0094] The testing method was conducted according to the specific requirements of the "Cosmetic Safety Technical Specifications" (2015 edition). The unexposed areas of the subjects' thighs or backs were selected as test sites. 24 hours prior to the test, the minimum erythema dose (MED) of the subjects' skin to ultraviolet radiation was predicted. The subjects were then irradiated once daily at 0.75 times the MED dose using a xenon arc lamp from a sunlight simulator at the same irradiation point for four consecutive days. The four days following the irradiation end were designated as the skin melanization period, during which no treatment was performed. On the fifth day after the irradiation end, the skin color of each test area was visually assessed and measured using a skin color instrument. Test areas with poor consistency (areas where the ITA° value differed from the mean of all test areas by more than 5) were removed. Starting that day, the corresponding test substance was applied to each melanized test area according to a randomized table, and the test substance was applied continuously for four weeks. Visual assessments and instrumental measurements of skin color were performed at 1, 2, 3, and 4 weeks after application, and the results were recorded in Table 1 below.

[0095] Table 1

[0096]

[0097]

[0098] The table shows that after 1, 2, 3, and 4 weeks of application of the mulberry bark emulsion, the skin ITA° value before and after application was significantly different from that of the negative control (P < 0.05); after 1, 2, 3, and 4 weeks of application, the MI value before and after application was significantly different from that of the negative control (P < 0.05); after 1, 2, 3, and 4 weeks of application, the visual skin tone grade before and after application was significantly different from that of the negative control (P < 0.05); and compared with the negative control, the regression coefficients of ITA°, MI, and visual skin tone grade of the test product were all significantly different (P < 0.05), indicating that the test product has the effect of removing freckles and whitening skin.

[0099] The table shows the parameters for ITA° characterizing human skin color; a higher ITA° value indicates a lighter skin tone. MI value measures the melanin content in the skin; a higher MI value indicates a higher melanin content. A positive control of vitamin C (7%) was used. Data were provided by Huante Biotechnology. ITA° / MI / skin color grade data are averages. The comparison between the tested product and the negative result was statistically significant (P = 0.000). Independent samples t-tests or rank-sum tests were used for comparison. Regression coefficients for each parameter over time were calculated, with a significance level of P < 0.05.

Claims

1. A method for preparing the effective components of mulberry bark, characterized in that... The effective components of mulberry bark are obtained by extracting mulberry bark with ethanol, concentrating the resulting extract under reduced pressure to a dry matter, dissolving the dry matter in ethanol, adsorbing it with macroporous resin, and then eluting it with ethanol of different concentrations. The 50% ethanol-water eluent, after being concentrated under reduced pressure, contains the effective components of mulberry bark. During the ethanol extraction process, the ethanol concentration is 50-100%, and 3-6L of ethanol-water is used for extraction per kg of mulberry bark, with an extraction temperature of 20-60℃. The ethanol concentration used to dissolve the dried material is 90-95%, and the mass ratio of the dried material to the macroporous resin is 1:

1. Ethanol elution was performed sequentially with pure water, 25% ethanol-water, and 50% ethanol-water; pure water elution was performed for 8-11 column volumes; 25% ethanol-water elution for 12-16 column volumes; and 50% ethanol-water elution for 9-12 column volumes. The effective components of mulberry bark are separated and purified, and the specific process is as follows: S1, the effective fraction of mulberry bark was treated using an LH-20 dextran gel column, and the effective fraction of mulberry bark was eluted sequentially with 50%, 60%, and 70% methanol-water solutions; during the gradient elution process, 19-21 column volumes were eluted with 50% methanol-water solution and 15-19 column volumes were eluted with 60% methanol-water solution to obtain the first separated fraction. Elute with 70% methanol and water for 14-17 column volumes to obtain the second separation fraction; S2, the first separation section is subjected to a second separation using a methanol-water system, and the second separation section is subjected to a third separation; The second and third separations are performed using a semi-preparative liquid phase. In the second separation, the first separation fraction is eluted with 50-65% methanol-water at a flow rate of 2-4 ml / min. The eluent contains Diels-Alder type adducts Sanggenon T and Kuwanon G. In the third separation, the second separation fraction is eluted with 55-65% methanol-water at a flow rate of 2-4 ml / min. The eluent contains Diels-Alder type adducts Mulberrofuran J, Albafuran C, and Kuwanon X. The effective component of the mulberry bark contains 55-68% Diels-Alder type adduct by weight; The Diels-Alder type adduct includes Sanggenon T, Kuwanon G, Mulberrofuran J, Albafuran C, and Kuwanon X.

2. The effective components of mulberry bark prepared by the method described in claim 1.

3. The application of the effective components of mulberry bark as described in claim 2 in the preparation of anti-UVA damage cosmetics or cosmetics with both whitening and anti-UVA damage effects.

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