A natural molecule for whitening and resisting photodamage in Morus alba bark and its preparation method

Abstract

A natural molecule in mulberry bark that has whitening and photodamage resistance and a preparation method thereof, the natural molecule being specifically fusinolide-7-O-primulin glycoside. The natural molecule of the present invention is obtained by using mulberry bark as a raw material, extracting with ethanol, performing a first separation on the mulberry bark extract using an ethanol-water system, performing a second separation using a methanol-water system, and performing a third separation using a methanol-water system. The fusinolide-7-O-primulin glycoside of the present invention has good application prospects in the development of whitening, photodamage resistance or dual-effect cosmetics.

Description

Technical Field

[0001] The invention belongs to the field of cosmetic application development, and specifically relates to a natural molecule aesculetin-7-O-primulinatoside in Morus alba bark and a preparation method thereof, as well as application of the aesculetin-7-O-primulinatoside or a combination thereof as active ingredients in the preparation of whitening and anti-photodamage cosmetics. Background Art

[0002] Tyrosinase, also known as polyphenol oxidase, is a key enzyme in the synthesis of melanin in the human body. The color of human skin depends on the content and distribution of melanin in the body. When the melanin level is abnormal, it is often accompanied by lesions such as chloasma, freckles and even pigment tumors. Domestic and foreign scholars use effective tyrosinase inhibitors to control the activity level of tyrosinase, thereby affecting the production of melanin, resulting in a reduction in synthesis and achieving a whitening effect.

[0003] At present, tyrosinase inhibitors are derived from fermentation, artificial synthesis, natural plants, etc., and are widely used in cosmetic products claiming whitening effects. Common inhibitors include kojic acid, arbutin, vitamin C, resorcinol and its derivatives, hydroquinone, etc. However, most of the above inhibitors have certain side effects or stability problems, and are not satisfactory in product applications. For example, kojic acid has low stability and has a carcinogenic risk if used for a long time, while hydroquinone has been listed as a banned additive in many countries. Therefore, finding safe and effective tyrosinase inhibitors is a hot research topic in the field of cosmetic applications and an urgent need of the market.

[0004] Photodamage refers to long-term damage to the skin caused by exposure to ultraviolet (UV) and visible light. This damage can lead to skin aging, pigmentation, wrinkles and skin cancer. It is worth pointing out that the ultraviolet rays that the sun shines on the earth's surface are mainly composed of UVA and UVB. UVA forms 95% of the ultraviolet rays we receive and is more dangerous than UVB. Unlike sunburn caused by UVB, the body has no natural warning system, and the damage caused by UVA is often difficult to detect. Therefore, it is particularly important to prevent UVA damage.

[0005] At present, anti-photodamage cosmetics mainly include sunscreens based on physical isolation, sunscreen sprays, sunscreen lotions and gels, etc. However, these products may cause some problems, such as skin irritation or allergic reactions due to sensitivity to fragrance or preservative ingredients; or photosensitivity reactions, where certain sunscreen ingredients may increase the skin's sensitivity to sunlight, causing sunburn or erythema, or dry or greasy skin, as some products may cause dry or greasy skin.

[0006] Therefore, existing products cannot play a good role in resisting UV damage, and do not have both tyrosinase inhibition and UVA damage resistance. Summary of the invention

[0007] The object of the present invention is to provide a new natural molecular entity having both tyrosinase inhibition and UVA damage resistance and a preparation method thereof, so as to solve the problems raised in the above background technology.

[0008] A natural molecule in Morus alba bark that has whitening and anti-photodamage properties is a coumarin molecule named aesculetin-7-O-primulin glycoside, and its chemical structure is as follows:

[0009] .

[0010] A method for preparing a natural molecule in Morus alba bark having whitening and anti-photodamage properties, the specific steps are as follows:

[0011] S1, extraction of Morus alba bark: soaking Morus alba bark in ethanol to obtain an extract;

[0012] S2, first separation: the extract obtained in step S1 is subjected to a macroporous adsorption resin column, and eluted and separated by an ethanol-water system to obtain a first separation portion;

[0013] S3, second separation: the first separated part obtained in step S2 is separated and purified by using a dextran gel column with a methanol-water system to obtain a second separated part;

[0014] S4, third separation: The second separated portion obtained in step S3 is separated and purified by semi-preparative liquid chromatography using a methanol-water system to obtain the compound aesculetin-7-O-primulinaside.

[0015] In the step S1, the ethanol concentration is 50-100%, the mass ratio of ethanol to mulberry bark is 3-5:1, and the extraction treatment temperature is 20-60°C.

[0016] In step S2, the ethanol concentration is 25%.

[0017] In step S3, the methanol concentration is 20% and the elution flow rate is 1-10 BV / h.

[0018] In step S4, the methanol concentration is 74% and the flow rate is 6-15 ml / min.

[0019] The esculetin-7-O-primulin glycoside of the present invention is used to inhibit tyrosine kinase activity, with an inhibition rate of 40-80%. The inhibition rate of tyrosinase is in a dose-effect relationship with the concentration. The higher the concentration, the higher the inhibition rate of tyrosinase. The molecule has a protective effect on UVA-damaged skin cells and can be used to resist UVA damage. The irradiation dose is 10J / cm 2Under the condition of (1375-1400UW), after the molecule acted on the cells after ultraviolet irradiation, the cell viability was increased by 80-100% compared with the UVA damage model group.

[0020] The esculetin-7-O-primulina glycoside of the present invention is used to prepare products containing the substance, or products containing the Morus alba extract component of the substance. The preferred product is a daily chemical product, and the daily chemical product also contains additional ingredients that can be used for daily chemical products. Further, the dosage form of the daily chemical product includes but is not limited to emulsions, sprays, aerosols, creams, water preparations, gels, oils, patches, films or mud preparations.

[0021] Preferably, the product has the effects of whitening and resisting photoaging, as well as inhibiting tyrosinase activity. Further, the product has the effect of resisting UVA damage.

[0022] The present invention provides a new natural molecule, which is a coumarin molecule, which not only has significant inhibitory activity on tyrosinase, and its activity is close to that of the positive control phenylethyl resorcinol, but also shows excellent protective effect on UVA damaged skin cells, and its activity is better than that of the positive control vitamin C. The molecule has good application prospects in the development of whitening, anti-photodamage or dual-effect cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the structural formula and carbon atom numbering diagram of aesculetin-7-O-primuloside.

[0024] Figure 2 This is the hydrogen spectrum (500MHz, CDCl3) of esculetin-7-O-primulin glycoside.

[0025] Figure 3 This is the carbon spectrum (150MHz, CDCl3) of 7-O-primulinatoside.

[0026] Figure 4 This is the high-resolution mass spectrum of aesculetin-7-O-primulin glycoside.

[0027] Figure 5 The effects of 7-O-primulin (10, 25, 50 μM) of aesculetin-7-O-primulin (10, 25, 50 μM) on UVA-damaged human dermal fibroblasts (HDF- α )’s protective effect diagram. DETAILED DESCRIPTION

[0028] 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.

[0029] Example 1: A method for preparing the natural molecule aesculetin-7-O-primulinaside, comprising the following steps:

[0030] S1, Morus alba bark extraction: 10 kg of dried Morus alba bark was soaked and extracted three times in 75% ethanol at 25°C, the first soaking time was 48 hours, the second and third soaking times were 12 hours each, the three extracts were combined, and the obtained extract was subjected to reduced pressure distillation to obtain an extract;

[0031] The mass ratio of the ethanol to the mulberry bark is 4:1.

[0032] S2, first separation: the extract obtained in step S1 is separated and purified using a D101 macroporous adsorption resin column, and gradient elution is performed using pure water, 25% ethanol, 50% ethanol, 75% ethanol, and anhydrous ethanol in sequence, and five components AE are collected in sequence;

[0033] The component B eluted with 25% ethanol eluent was concentrated and freeze-dried to obtain the first separation part.

[0034] S3, second separation: 20.4 g of component B eluted with 25% ethanol was purified and separated by chromatography on a LH-20 dextran gel column, and gradient elution was performed with pure water, 20% methanol, 50% methanol and 100% methanol in sequence, and 5 components B1-B5 were collected in sequence; the volume of component B2 eluted with 20% methanol was 3-10 BV, and 4.7-8.6 BV of this part was concentrated and freeze-dried to obtain the second separation part;

[0035] Among them, the elution flow rate is 5BV / h.

[0036] S4, third separation: The second separation part 2.7 g was obtained by concentration and freeze-drying and eluted by Waters 2535 semi-preparative liquid chromatograph with 74% methanol at a flow rate of 10 ml / min to separate and purify the compound quinoline-7-O-primulinaside.

[0037] The structural formula and carbon atom number of the compound aesculetin-7-O-primuloside are as follows Figure 1 shown.

[0038] The characterization of the compound aesculetin-7-O-primulin glycoside is shown in the figure Figure 2-5 shown.

[0039] The present invention's aesculetin-7-O-primulin glycoside (hereinafter referred to as "Ehr824") is a white powder. High resolution mass spectrometry (HRESIMS) shows that its molecular formula is C 20 H 24 O 13 ([M + Na] + m / z 495.1108, calcd: 495.1109). Proton spectrum ( 1 HNMR) as shown in Table 1 shows a group of coumarin 3,4-position characteristic signals [ δ H 6.27 (1H, d, J = 9.5 Hz, H-3), 7.90(1H, d, J = 9.5, Hz, H-4)], two aromatic hydrogen proton signals [ δ H 7.06 (1H, s, H-5), 7.14 (1H, s,H-8)], and two terminal hydrogen proton signals [ δ H 4.92 (1H, d, J = 7.4 Hz, H-1ˈ), 4.14 (1H, d, J =7.6, Hz, H-1ˈˈ)]. The above signals indicate that the compound is a coumarin glycoside compound. Its carbon spectrum ( 13 C NMR) and DEPT (distortionless polarization transfer technique) data showed that the compound has 2 methylene groups, 13 methine groups, and 5 quaternary carbons. Further comparison revealed that the above data of Ehr824 were similar to those of Scopoletin7-O- β -D-Xylopyranosyl-(1→6)- β -D-glucopyranoside is very similar ( Chem. Pharm. Bull. 2003, 51, 1106-1108), the only difference is that Ehr824 lacks a 6-methoxy signal. 13 The change in C NMR chemical shift suggested that the 6-position of Ehr824 was replaced by hydroxyl group, which was the main factor in the formation of Scopoletin 7-O- β -D-Xylopyranosyl-(1→6)- β -Methoxy group of D-glucopyranoside. In HMBC ( 1In the heteronuclear multi-carbon correlation spectrum of H, the correlation between H-1ˈ and C-7 indicates that the glucose group is located at the C-7 position of the coumarin aglycone; the HMBC correlation between H-1ˈ and C-6ˈ confirms that the 6th position of glucose is connected to the 1st position of xylose. In the ROSEY (Overhausenhanced Spectroscopy in Rotating Coordinate System) spectrum, the ROSEY correlation between H-1ˈ and H-8 again verifies that the glucose group is located at the C-7 position. Through the above analysis, Ehr824 (6,7-dihydroxycoumarin-7- O - β -D-Xylosyl-(1→6)- β -D-glucoside) chemical structure Figure 1 shown.

[0040] Table 1 Esculetin-7-O-primulin glycoside 1 H-NMR and 13 C-NMR data (DMSO- d 6)

[0041]

[0042] The tyrosinase inhibitory activity of aesculetin-7-O-primulosidase of the present invention was tested, and the testing method was as follows:

[0043] The new natural molecule to be tested was mixed with L-Dopa, and tyrosinase (final concentration 25 U / mL) was added to start the reaction. Three replicate wells were set up, and a blank control without the new natural molecule to be tested and a positive control of phenylethylresorcinol were set up at the same time. The reaction was carried out at room temperature for 25 min. The OD value was measured by a microplate reader with a detection wavelength of 490 nm, and the enzyme activity inhibition rate was calculated using the following formula:

[0044] Tyrosinase activity inhibition rate (%) = (1-sample well OD490 nm / experimental control well OD490 nm) × 100%.

[0045] IC 50 (50% inhibitory concentration) was calculated according to the Reed & Muench method.

[0046] After testing, aesculetin-7-O-primrose glycoside showed a concentration-dependent inhibitory effect on tyrosinase within the tested concentration range, with outstanding activity and a half inhibitory concentration IC 50 The value was 0.51±0.01 μM, which was close to the positive control phenethyl resorcinol (IC 50 : 0.16±0.01 μM), as shown in Table 2.

[0047] Table 2 Inhibition of samples on tyrosinase

[0048]

[0049] The data in Table 2 were analyzed in three parallel experiments for each group, n=3, and the data were expressed as Mean ± SEM.

[0050] The effect of aesculetin-7-O-primulin on human dermal fibroblasts (HDF- α ) The protective effect is tested, and the test method is as follows:

[0051] Step 1: HDF-α cells were cultured at 1×10 5 / mL density was transferred into 96-well plates and cultured overnight.

[0052] Step 2: After the cells in the 96-well plate have 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.

[0053] Step 3, set up a UVA damage group (model group), a UVA damage group with medication, and a normal control group (NC group), with five parallel groups in each group; among which:

[0054] Normal control group (NC): protected from light by tin foil, placed in indoor environment for 2 hours, aspirated D-Hanks, added 100 μL DMEM medium to each well, and continued incubation for 24 hours;

[0055] UVA damage group (model group): UV lamp irradiation dose was adjusted to 10J / cm 2 (1375-1400UW), irradiation time is 2h, and UV irradiation is performed; after UV irradiation, D-Hanks is aspirated, 100μL DMEM medium is added to each well, and incubation is continued for 24h;

[0056] UVA damage and drug addition group: UV lamp irradiation dose was adjusted to 10J / cm 2 (1375-1400UW), irradiation time is 2h, and ultraviolet irradiation is performed; after ultraviolet irradiation, D-Hanks is aspirated, and 100μL of aesculetin-7-O-primulin solution is added to each well, with concentrations of 20μM, 50μM, and 100μM, respectively. The final experimental concentrations are 10μM, 25μM, and 50μM, respectively, and incubation is continued for 24h;

[0057] Positive drug control experimental group: UV lamp irradiation dose was adjusted to 10J / cm 2 , irradiation time is 2h, UV irradiation is performed; after UV irradiation, D-Hanks is aspirated, 100μL of positive drug solution is added to each well, the concentration is 200μM, the final experimental concentration is 100μM, and incubation is continued for 24h;

[0058] Step 4, aspirate the supernatant, wash twice with PBS, and perform cell viability detection using the MTS kit;

[0059] MTS was thawed in advance, MTS: culture medium = 1:4, and the light was turned off during operation. A 15 ml centrifuge tube was wrapped with tin foil to avoid light, 100 vl of reagent was added and incubated in the incubator for 2-4 hours. After the culture was completed, the tube was wrapped with tin foil to measure the data.

[0060] Step 5, use a microplate reader to perform OD 490nm detection and calculate cell viability (Survival rate) using the following formula;

[0061] .

[0062] The test results are as follows Figure 5 As shown in Figure 2, aesculetin-7-O-primulin glycoside at 10 μM, 25 μM, and 50 μM concentrations had an inhibitory effect on UVA-damaged human dermal fibroblasts (HDF- α ) have significant protective effects. The protective effect of aesculetin-7-O-primulin glycoside reached saturation at a concentration of 10 μM. At a concentration of 10 μM, it had no significant protective effects on human dermal fibroblasts (HDF- α ) was significantly better than the positive control vitamin C (100 μM).

[0063] Figure 5 In the experiment, NC was the blank control group; UVA was the UVA damage model group; VC was the positive control vitamin C group. Five parallel experiments were set up in each group, n = 5, and the data were expressed as Mean ± SEM. One-way ANOVA and Tukey's multiple comparison test were used to evaluate statistical significance.

[0064] ****P<0.05 (vs. VC group); ****P<0.0001 (vs. UVA group).

Claims

1. A natural molecule in Morus alba bark that has the function of whitening and resisting photodamage is a coumarin molecule named as aesculetin-7-O-primulin glycoside, and its chemical structure is as follows: 。 2. The method for preparing a natural molecule for whitening and resisting light damage in Morus alba bark as claimed in claim 1, characterized in that The specific steps of this method are as follows: S1, extraction of Morus alba bark: soaking and extracting Morus alba bark with ethanol to obtain an extract; S2, first separation: the extract obtained in step S1 is subjected to a macroporous adsorption resin column, and eluted and separated by an ethanol-water system to obtain a first separation portion; S3, second separation: the first separated part obtained in step S2 is separated and purified by using a dextran gel column with a methanol-water system to obtain a second separated part; S4, third separation: the second separated part obtained in step S3 is separated and purified by semi-preparative liquid chromatography using a methanol-water system to obtain a compound aesculetin-7-O-primuloside; In the step S1, the ethanol concentration is 50-100%, the mass ratio of ethanol to Morus alba bark is 3-5:1, and the extraction temperature is 20-60°C; In step S2, the ethanol concentration is 25%; In step S3, the methanol concentration is 20% and the elution flow rate is 1-10 BV / h; In step S4, the methanol concentration is 74% and the flow rate is 6-15 ml / min.

3. The method for preparing a natural molecule for whitening and resisting light damage in Morus alba bark as claimed in claim 2, characterized in that In the step S1, the ethanol concentration is 75%, the mass ratio of ethanol to mulberry bark is 4:1, and the extraction treatment temperature is 25°C.

4. The aesculetin-7-O-primulosidoside as claimed in claim 1 is used to prepare tyrosinase inhibitors, whitening cosmetics based on tyrosinase inhibition, anti-UVA damage preparations, anti-UVA light damage cosmetics or whitening and anti-light damage dual-effect cosmetics.

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