Rhus chinensis bark extract with whitening and soothing effects as well as preparation method and application of rhus chinensis bark extract

Abstract

The invention relates to a rhus chinensis bark extract with whitening and soothing effects and a preparation method and application thereof, and the preparation method comprises the following steps: crushing rhus chinensis bark, then carrying out heating reflux extraction with ethanol, recovering a solvent from the obtained extracting solution under reduced pressure, and concentrating to obtain an extract; the obtained extract is subjected to MCI small-hole resin sample stirring, MCI column packing and ethanol flushing; an active section is obtained; according to the rhus chinensis bark extract with the whitening and relieving effects and the preparation method and application thereof, technological parameters in the extraction process are optimized, the extraction part of rhus chinensis is optimized, an active segment HDYFM-1 is obtained after an ethanol extract of rhus chinensis bark is separated and purified through MCI small-hole resin, and the rhus chinensis bark extract has the whitening, anti-oxidation and relieving effects; according to the invention, the rhus chinensis barks are used for extraction, which is better than that of the conventional extraction of rhus chinensis leaves in the market, and the composition has excellent effects in whitening, anti-oxidation and soothing effects.

Description

Technical Field

[0001] The invention relates to the field of daily chemicals, in particular to a Rhus chinensis bark extract with whitening and soothing effects, a preparation method and an application thereof. Background Art

[0002] With the development of society, people pay more attention to beauty, and beauty and skin care have gradually become an important part of people's daily life. Environmental pollution, dust and impurities in the air, and external factors such as ultraviolet radiation in modern life will have a negative impact on the skin. Long-term exposure to such an environment, if not properly cared for, the skin is prone to a variety of problems, such as enlarged pores, dull skin, dryness, fine lines, spots, etc.

[0003] Rhus chinensis extract has gradually attracted attention in the cosmetic field in recent years due to its antioxidant, anti-inflammatory and moisturizing properties. It is rich in a variety of active ingredients, such as flavonoids and polyphenols, which can effectively protect the skin from free radical damage and promote skin repair. In addition, its natural origin also meets the preference of modern consumers for natural skin care products.

[0004] Common extraction methods for Rhus chinensis extracts include solvent extraction, ultrasonic extraction and water extraction. Solvent extraction has high efficiency, but there may be residual solvent; ultrasonic extraction can improve the extraction rate, but the equipment cost is high; water extraction has good safety, but the extraction efficiency is low; at the same time, there is a lack of research on the effective parts of the existing Rhus chinensis extracts, and the market needs Rhus chinensis extracts with clear skin care effects. Summary of the invention

[0005] In order to overcome the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a Rhus chinensis bark extract with whitening and soothing effects, a preparation method and an application thereof.

[0006] The technical solution adopted by the present invention to solve the technical problem is: Rhus chinensis bark extract, which is obtained by using Rhus chinensis bark as a raw material and separating by organic solvent chromatography.

[0007] As a further improvement of the present invention: the organic solvent is methanol.

[0008] As a further improvement of the present invention: the organic solvent is ethanol.

[0009] The present invention also includes a method for preparing the Rhus chinensis bark extract, comprising the following steps: The bark of Rhus chinensis is crushed and then extracted with ethanol under heating and reflux. The obtained extract is decompressed to recover the solvent and concentrated to obtain an extract. The obtained extract is mixed with MCI small pore resin, loaded into MCI column, and washed with ethanol to obtain the active segment HDYFM-1.

[0010] As a further improvement of the present invention: after the Rhus chinensis bark is crushed, the ethanol is heated and refluxed with ethanol for extraction, and 90% ethanol is used.

[0011] As a further improvement of the present invention: the extract MCI small pore resin is mixed with 2 times the amount of MCI small pore resin and 5 times the amount of MCI is loaded into the column.

[0012] As a further improvement of the present invention: in the ethanol washing, 10% ethanol, 20% ethanol, 30% ethanol, 50% ethanol and 100% ethanol are used to wash 5 column volumes.

[0013] As a further improvement of the present invention: 100 kg of Rhus chinensis bark is crushed and extracted with twice the amount of 90% ethanol by heating and reflux, and the extraction is repeated three times. The extracts are combined, the solvent is recovered from the obtained extract under reduced pressure, and an extract is obtained after concentration. The obtained extract is mixed with 2 times the amount of MCI small-pore resin, and loaded into a column with 5 times the amount of MCI. 5 column volumes are washed with 10% ethanol, 20% ethanol, 30% ethanol, 50% ethanol, and 100% ethanol, respectively, to obtain active segments 1 to 5, wherein active segment 4 is HDYFM-1.

[0014] As a further improvement of the present invention: 5 column volumes are washed with 10% ethanol, 20% ethanol, 30% ethanol, 50% ethanol and 100% ethanol, active segment 1 is obtained by washing with 10% ethanol, active segment 2 is obtained by washing with 20% ethanol, active segment 3 is obtained by washing with 30% ethanol, active segment 4 is obtained by washing with 50% ethanol, and active segment 5 is obtained by washing with 100% ethanol.

[0015] The invention also includes the application of the Rhus chinensis bark extract in cosmetics.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention provides a Rhus chinensis bark extract with whitening and soothing effects, a preparation method and an application thereof, optimizes the process parameters in the extraction process, optimizes the extraction part of Rhus chinensis, and obtains the active segment HDYFM-1 after separation and purification of the ethanol extract of the Rhus chinensis bark by MCI small pore resin, which has whitening, antioxidant and soothing effects; the present invention uses Rhus chinensis bark for extraction, which is better than the conventional extraction using Rhus chinensis leaves on the market, and has excellent effects in whitening, antioxidant and soothing effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solution, the drawings required for use in the implementation will be briefly introduced below. Obviously, the drawings described below are only some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0018] Figure 1 For example, tyrosinase IC 50 The HDYFM-Y concentration in the assay corresponds to the IC 50 Value and activity graph, in which the X-axis is the HDYFM-Y concentration and the Y-axis is the activity.

[0019] Figure 2 For example, tyrosinase IC 50 The HDYFM-P concentration in the assay corresponds to the IC 50 Value and activity graph, in which the X-axis is the HDYFM-P concentration and the Y-axis is the activity.

[0020] Figure 3 ABST activity and IC 50 The HDYFM-Y concentration in the assay corresponds to the IC 50 Value and activity graph, in which the X-axis is the HDYFM-Y concentration and the Y-axis is the activity.

[0021] Figure 4 ABST activity and IC 50 The HDYFM-P concentration in the assay corresponds to the IC 50 Value and activity graph, in which the X-axis is the HDYFM-P concentration and the Y-axis is the activity.

[0022] Figure 5 DPPH activity and IC of the present invention 50 The HDYFM-Y concentration in the assay corresponds to the IC 50 Value and activity graph, in which the X-axis is the HDYFM-Y concentration and the Y-axis is the activity.

[0023] Figure 6 DPPH activity and IC of the present invention 50 The HDYFM-P concentration in the assay corresponds to the IC 50 Value and activity graph, in which the X-axis is the HDYFM-P concentration and the Y-axis is the activity.

[0024] Figure 7 This is a graph showing the test results of HDYFM-1 in the zebrafish soothing efficacy test according to an embodiment of the present invention.

[0025] Figure 8 This is a bar graph of the relative expression of the il-6 gene of HDYFM-1 in the zebrafish soothing efficacy test of the embodiment of the present invention, in which *p<0.05, ***p<0.001.

[0026] Fig. 9 This is a graph showing the test results of HDYFM-1 in the zebrafish antioxidant efficacy test according to an embodiment of the present invention.

[0027] Fig.10This is a bar graph of the PEG2 content of HDYFM-1 in the cell experiment for testing the soothing effect of the embodiment of the present invention, with **p<0.01.

[0028] Fig.11 This is a bar graph of the relative expression of the TNF-α gene of HDYFM-1 in the soothing efficacy test cell experiment of the embodiment of the present invention, **p<0.01.

[0029] Fig.12 This is a bar graph of the SOD activity of HDYFM-1 in the cell experiment for testing the antioxidant efficacy of an embodiment of the present invention.

[0030] Fig.13 This is a typical graph of ROS fluorescence intensity of the ROS clearance rate test of HDYFM-1 in the antioxidant efficacy test cell experiment of the embodiment of the present invention. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the specific embodiments of the present invention and the corresponding 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 making creative work are within the scope of protection of the present invention.

[0032] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0033] Rhus chinensis Mill. is a deciduous small tree of the Anacardiaceae family. It is up to 10 meters tall, with brown twigs, polymorphic leaves, rounded base, dark green leaves on the surface, pink-green leaves on the back, and sessile. The panicle is broad and branched, and the female inflorescence is shorter and densely pubescent; the bracts are white, the petals are oblong, and roll outward when blooming; the drupes are spherical and red when ripe. It blooms from August to September and bears fruit in October.

[0034] Rhus chinensis is widely distributed in arid and semi-arid regions of Asia, Europe and Africa. It can grow in harsh environments, such as saline-alkali soil, desert edges and riverbanks, showing its strong adaptability and vitality.

[0035] Rhus chinensis has a long history of use in traditional medicine and has been used to treat a variety of conditions, including skin diseases. Its extracts contain a variety of bioactive substances, such as phenolic compounds, flavonoids, and alkaloids, which are believed to have antioxidant, anti-inflammatory, and whitening potential. In particular, its antioxidant components can help protect against free radical damage caused by ultraviolet rays and reduce pigmentation.

[0036] Common extraction methods for Rhus chinensis extracts include solvent extraction, ultrasonic extraction and water extraction. Solvent extraction has high efficiency, but may leave residual solvent; ultrasonic extraction can improve the extraction rate, but the equipment cost is high; water extraction has good safety, but the extraction efficiency is low; at the same time, there is a lack of research on the effective parts of the existing Rhus chinensis extracts. The present invention provides a Rhus chinensis bark extract with whitening and soothing effects and a preparation method and application of the effective parts, optimizes the process parameters in the extraction process, optimizes the extraction parts of Rhus chinensis, and has whitening, anti-oxidation and soothing effects.

[0037] Embodiment 1: Preparation of ethanol extract from Rhus chinensis leaves: 100 g of Rhus chinensis leaves were crushed and extracted with twice the amount of 90% ethanol under heating and reflux, and the extraction was repeated three times. The extracts were combined, and the obtained extracts were decompressed to recover the solvent, and concentrated to obtain the Rhus chinensis leaf ethanol extract HDYFM-Y.

[0038] Embodiment 2: Preparation of Rhus chinensis bark ethanol extract: 100 g of Rhus chinensis bark was crushed and extracted with twice the amount of 90% ethanol under heating reflux, and the extraction was repeated three times. The extracts were combined, and the obtained extracts were decompressed to recover the solvent, and concentrated to obtain the Rhus chinensis bark ethanol extract HDYFM-P.

[0039] Embodiment three: Preparation of active segments HDYFM-Y-1~4 from Rhus chinensis leaves: The Rhus chinensis leaf ethanol extract HDYFM-Y obtained in Example 1 was mixed with 2 times the amount of MCI small pore resin, loaded into a column with 5 times the amount of MCI, and washed with 30% ethanol, 50% ethanol, 70% ethanol, and 100% ethanol for 5 column volumes, respectively, to obtain active segments HDYFM-Y-1~4.

[0040] Embodiment 4: Preparation of active segments HDYFM-P-1~4 from Rhus chinensis bark: The ethanol extract HDYFM-P of Rhus chinensis bark obtained in Example 1 was mixed with 2 times the amount of MCI small pore resin, loaded into a column with 5 times the amount of MCI, and washed with 30% ethanol, 50% ethanol, 70% ethanol, and 100% ethanol for 5 column volumes, respectively, to obtain active segments HDYFM-P-1~4.

[0041] Embodiment five: Preparation of active segment HDYFM-1: 100 kg of Rhus chinensis bark was crushed and then extracted with twice the amount of 90% ethanol under heating and reflux, repeated three times, the extracts were combined, the solvent was recovered from the obtained extract under reduced pressure, and the extract was concentrated to obtain an extract. The obtained extract was mixed with 2 times the amount of MCI small-pore resin, loaded with 5 times the amount of MCI column, and washed with 10% ethanol, 20% ethanol, 30% ethanol, 50% ethanol, and 100% ethanol for 5 column volumes respectively. Active segments 1 to 5 were obtained, among which active segment 4 was HDYFM-1.

[0042] The effect experiment was carried out on the active segment obtained above: Tyrosinase inhibition rate determination: The drug 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, and a blank control without drug and a 377 positive control were set at the same time. The OD value was measured by a microplate reader at room temperature for 5 min, and the detection wavelength was 490 nm. The tyrosinase activity inhibition rate was calculated.

[0043] Tyrosinase activity inhibition rate (%) = (1 – sample well OD 490 nm / Experimental control well OD 490 nm ) × 100%; Table 1 Tyrosinase activity inhibition rate results for each active segment serial number concentration Tyrosinase inhibition rate HDYFM-Y 100 mg / ml -15.21±0.81 HDYFM-Y-1 100 mg / ml 27.89±1.66 HDYFM-Y-2 100 mg / ml 17.9±0.68 HDYFM-Y-3 100 mg / ml 4.21±0.23 HDYFM-Y-4 100 mg / ml 11.15±0.25 HDYFM-P 100 mg / ml 43.06±0.99 HDYFM-P-1 100 mg / ml 31.74±0.73 HDYFM-P-2 100 mg / ml 38.02±1.57 HDYFM-P-3 100 mg / ml 5.19±0.98 HDYFM-P-4 100 mg / ml 9.17±0.12 HDYFM-1 100 mg / ml 53.76±0.13 Tyrosinase IC 50 Determination: Add 20 ml of the drug solution to be tested to a 96-well culture plate, divided into five experimental groups (gradient of 1000 mg / ml, 250 mg / ml, 62.5 mg / ml, 15.625 mg / ml, 3.90625 mg / ml), set 3 replicate wells, and set up a background group without tyrosinase (add the same concentration of the drug to be tested). Add 105 ml PBS buffer to the experimental group, add 130 ml PBS buffer to the background group and mix well. Add 25 ml tyrosinase solution (200 U / mL) to the experimental group, mix well, add 50 ml substrate L-dopa, react at 25 ºCC for 5 min, and measure the absorbance (OD value) with an enzyme reader. The detection wavelength is 490 nm. IC was calculated using GraphPad Prism 10. 50 value.

[0044] Table 2 IC values ​​of active segments of HDYFM-1 and HDYFM-P tyrosinase 50 Measurement value statistics table serial number <![CDATA[IC 50 Value]]> HDYFM-1 79.16 mg / ml HDYFM-P 83.64 mg / ml ABST activity and IC 50 Determination: 20 ml of the sample solution to be tested (divided into six concentrations (gradient of 1000 mg / ml, 200 mg / ml, 40 mg / ml, 8 mg / ml, 1.6 mg / ml, 0.32 mg / ml, 3 replicates in each group) and two blank groups (ultrapure water with the same concentration of DMSO as the extract) and the positive control water-soluble vitamin E solution were added to a 96-well plate. One blank group and sample group added ABTS, and one group did not. The blank group without ABTS was called the blank background reaction group, and the sample group without ABTS was called the sample background reaction group. The remaining experimental groups were added with 180 ml of the diluted ABTS working solution and placed at room temperature in the dark for 5 minutes. The absorbance was measured at 734 nm to calculate the ABTS free radical scavenging activity. Using GraphPad Prism10, the IC 50 value.

[0045] Table 3 Active segments HDYFM-1 and HDYFM-P ABST IC 50 Measurement value statistics table serial number <![CDATA[IC 50 Value]]> HDYFM-1 122.2 mg / ml HDYFM-P 213.7 mg / ml DPPH activity and IC 50 Determination: Add 20 uL of the sample solution to be tested to a 96-well plate, divided into six concentrations (gradient of 1000 mg / ml, 200 mg / ml, 40 mg / ml, 8 mg / ml, 1.6 mg / ml, 0.32 mg / ml), set up 3 replicate wells, and a blank control (anhydrous ethanol with the same concentration of DMSO added to the extract). Continue to add 180 ml of DPPH solution to the 96-well plate, and then place it in a 37 °C incubator for 1 h. Measure the absorbance at 517 nm and calculate the DPPH free radical scavenging activity. Use GraphPad Prism10 to calculate the IC 50 value.

[0046] Table 4 Active segments HDYFM-1 and HDYFM-P IC 50 Measurement value statistics table serial number <![CDATA[IC 50 Value]]> HDYFM-1 154.9 mg / ml HDYFM-P 221.4 mg / ml According to the tyrosinase inhibition results, the tyrosinase inhibition rate of the crude extract of HDYFM-P (43.059±0.989%) was better than that of the crude extract of HDYFM-Y (-15.211±0.805), so the parts of Rhus chinensis bark were selected for further development and efficacy testing. The tyrosinase inhibition rate of the active segment HDYFM-1 (53.76±0.126%) and the tyrosinase inhibition rate IC 50 (IC 50 :79.16 mg / ml), ABST clearance IC 50 (IC 50 :122.2 mg / ml)、DPPH clearance rate IC 50 (IC 50 :154.9 mg / ml) were superior to the tyrosinase inhibition rate of HDYFM-P crude extract (43.06±0.99%) and tyrosinase inhibition rate IC 50 (IC 50 : 83.64 mg / ml), ABST clearance IC 50 (IC 50 :213.7 mg / ml)、DPPH scavenging rate IC 50 (IC 50 : 221.4 mg / ml). Therefore, the active segment HDYFM-1 was selected for further efficacy testing.

[0047] Zebrafish soothing effect test: 1) Randomly select zebrafish and place them in a 6-well plate, with 15 zebrafish in each well.

[0048] 2) A zebrafish skin inflammation model was established by administering SLS in water.

[0049] 3) The water-soluble samples were administered, and a normal control group and a model control group were set up at the same time. The capacity of each well was 3 mL.

[0050] 4) Incubate at 28°C in the dark for 18 h.

[0051] 5) Ten zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. Advanced image processing software was used to analyze and collect data. The number of neutrophils in the zebrafish skin (N) was analyzed and the formula was used to calculate and determine whether the sample had a soothing effect.

[0052] Soothing effect (%) = (N (model control group) - N (sample group))*100% / (N (model control group) N´ (normal control group)); Table 5. Test concentrations corresponding to efficacy and test results serial number Detection concentration% effect% P-value Test results HDYFM-1 0.008 61 < 0.001 Significant 1) Randomly select zebrafish and place them in a 6-well plate, with 30 zebrafish in each well.

[0053] 2) A zebrafish skin inflammation model was established by administering SLS in water.

[0054] 3) Water-soluble administration samples, normal control group and model control group were set up at the same time, with a volume of 3 ml per well. Three biological replicates were performed.

[0055] 4) Incubate at 28°C in the dark for 18 h.

[0056] 5) Total RNA was extracted from zebrafish in each experimental group, cDNA was synthesized, and the gene expression of β-actin and target genes was detected by q-PCR.

[0057] 6) Use β-actin as the internal reference for gene expression and calculate the relative RNA expression of the target gene.

[0058]

[0059] Table 6 IL-6 gene relative expression level and test results corresponding to the test concentration serial number Detection concentration% Relative expression of IL-6 gene P-value Test results HDYFM-1 0.005 0.567 < 0.001 Significant Zebrafish Antioxidant Efficacy Test: 1) Randomly select zebrafish and place them in a 6-well plate, with 15 zebrafish in each well.

[0060] 2) The oxidative stress model of zebrafish was established by administering water-soluble menadione.

[0061] 3) Water-soluble samples were administered, and a normal control group and a model control group were set up at the same time. The capacity of each well was 3 mL.

[0062] 4) Incubate at 28°C in the dark for 22 h.

[0063] 5) The zebrafish were stained with a specific ROS fluorescent reagent. After staining, 10 zebrafish were randomly selected from each experimental group and photographed under a fluorescence microscope. Advanced image processing software was used to analyze and collect data, and the fluorescence intensity (S) of the zebrafish yolk sac was analyzed. The antioxidant effect of the sample was calculated according to the formula to determine whether it had antioxidant effect.

[0064]

[0065] Table 7 Test concentrations corresponding to efficacy and test results serial number Detection concentration% effect% P-value Test results HDYFM-1 0.008 24 < 0.001 Significant Soothing effect test cell experiment: 1. PGE2 content 1) Seed RAW264.7 cells into 6-well plates (3 × 105 cells / well) and incubate at 37 °C, 5% CO2 for 24 h.

[0066] 2) After the incubation, remove the culture medium, wash 1-2 times with D-Hanks, replace the culture medium with fresh medium for the normal control group, replace the culture medium with fresh medium containing the modeling agent (0.5 μg / mL LPS) for the model control group, and replace the culture medium with fresh medium containing the sample and the modeling agent (0.5 μg / mL LPS) for the sample group. Continue to incubate at 37°C, 5% CO2 for 24 h.

[0067] 3) Collect the cell culture supernatant into a 1.5 mL sterile centrifuge tube and perform the test according to the instructions of the ELISA test kit.

[0068]

[0069] 2. TNF-α gene expression 1) Seed HaCaT cells into 6-well plates (6 × 105 cells / well) and incubate at 37 °C, 5% CO2 for 24 h.

[0070] 2) After the incubation, the culture medium was removed, and the cells were washed with D-Hanks for 1-2 times. Then, the sample group and the model control group were exposed to ultraviolet light (80 mJ / cm 2 ).

[0071] 3) After modeling, the normal control group and the model control group were replaced with fresh culture medium, and the sample group was added with fresh culture medium containing samples, and incubated at 37°C, 5% CO2 for 24 h.

[0072] 4) Extract total RNA from each experimental group, synthesize cDNA, and use q-PCR to detect β-actin and gene expression of target genes.

[0073] 5) Use β -actin was used as an internal reference for gene expression to calculate the relative RNA expression of the target gene.

[0074]

[0075] Table 8 PGE2 content detection concentration corresponding efficacy and test results serial number Detection indicators Detection concentration% effect% P-value Test results HDYFM-1 PGE2 content 0.003125 58 < 0.01 Significant Table 9 TNF-α gene expression detection concentration test results serial number Detection indicators Detection concentration% Relative gene expression P-value Test results HDYFM-1 TNF-α gene expression 0.0015625 0.324 < 0.01 Significant Antioxidant efficacy test cell experiment: 1. SOD activity test 1) Seed cells in a 6-well plate (6 × 105 cells / well) and incubate at 37 °C, 5% CO2 for 24 h.

[0076] 2) Modeling: After incubation, the culture medium was removed, the cells were gently rinsed once or twice with D-Hanks, and then the modeling process was performed. The model control group and the sample group were irradiated with UVB at 120 mJ / cm 2 , while the normal control group was placed in the dark.

[0077] 3) Sample administration: The normal control group and the model control group were replaced with fresh culture medium, and the sample group was replaced with fresh culture medium containing samples, and incubated at 37 ℃, 5% CO2 for 24 h.

[0078] 4) After the incubation, remove the culture medium, wash three times with D-Hanks, collect the cells, and perform the assay according to the instructions of the SOD assay kit. Calculate the antioxidant effect of the sample according to the following formula.

[0079]

[0080] Table 10 Test concentrations corresponding to efficacy and test results serial number Detection concentration% effect% P-value Test results HDYFM-1 0.0015625 33 < 0.01 Significant 2. ROS clearance test 1) Seed cells in a 12-well plate (1×105 cells / well) and incubate at 37°C, 5% CO2 for 24 h.

[0081] 2) Sample administration: Remove the culture medium and gently rinse the cells once or twice with D-Hanks. Replace the culture medium with fresh medium for the normal control group and the model control group, and replace the culture medium with fresh medium containing the sample for the sample group, and continue to incubate at 37 ℃, 5% CO2 for 24 h.

[0082] 3) Modeling: After incubation, the culture medium was removed, the cells were gently rinsed once or twice with D-Hanks, and then the modeling process was performed. The model control group and the sample group were irradiated with UVA (9J / cm 2 ), and the normal control group was placed in the dark.

[0083] 4) After modeling, the cells were stained with the reactive oxygen species detection kit, photographed with a fluorescence microscope, and the average fluorescence intensity (S) of each group of cells was analyzed by Image J. The ROS clearance rate of the sample group was calculated according to the formula.

[0084] Table 11 Test concentrations corresponding to efficacy and test results serial number Detection concentration% ROS clearance rate % P-value Test results HDYFM-1 0.0015625 73 < 0.001 Significant The Rhus chinensis bark extract lotion prepared by using the Rhus chinensis bark extract of the present application was tested for its freckle removal and whitening efficacy, and the test method is as follows: 1.1 Test product: The final cosmetic product made with the Rhus chinensis bark extract HDYFM-1 prepared in this application as the only whitening ingredient in the formula.

[0085] 1.2 Negative control: blank control of blackened area.

[0086] 1.3 Positive control: 7% ascorbic acid (vitamin C) preparation.

[0087] Subjects: A total of 20 subjects, including 3 males and 17 females, aged 23 to 57 years old, with an average age of 45.70±9.34 years old, met the voluntary inclusion criteria.

[0088] Test method: Test 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 were selected as the test site. The minimum erythema dose (MED value) of the subject's skin to ultraviolet radiation was predicted 24 hours before the test, and the subject was irradiated once a day at a dose of 0.75 times the MED at the same irradiation point using a xenon arc lamp of a daylight simulator 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 was visually evaluated and instrumentally tested, 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) were eliminated. Starting from the same day, the corresponding test substance was applied to each melanization test area according to the random table, and the test substance was applied continuously for 4 weeks. The skin color was visually evaluated and instrumentally tested 1 week, 2 weeks, 3 weeks and 4 weeks after application, and the results were recorded.

[0089] Statistical methods: Statistical analysis software was used for statistical analysis of data. Measurement data were expressed as mean ± standard deviation, and normal distribution test was performed. If the data met the normal distribution requirements, the paired t test was used for comparison before and after use, otherwise the two related sample rank sum test was used; the comparison of rank data before and after use was done with two related sample rank sum test; the comparison between the test product and the control group was done with independent sample t test or rank sum test.

[0090] At the same time, the regression coefficient of each parameter changing with time was calculated, and the significance level was P < 0.05.

[0091] The test results of ITA° and MI value products and controls are as follows: Test products:

[0092] Negative Control:

[0093] Positive Control:

[0094] Volunteer visual skin color level self-assessment test product and control test results: Test products:

[0095] Negative Control:

[0096] Positive Control:

[0097] Analysis of the time difference between the test product and the negative control before and after application:

[0098] Note: “NS” means no significant difference when P≥0.05; “S” means significant difference when P<0.05.

[0099] Regression coefficient analysis:

[0100] Note: “NS” means no significant difference when P≥0.05; “S” means significant difference when P<0.05.

[0101] Test conclusion: The test results of the freckle removal and whitening efficacy of cosmetics showed that after applying the test product for 1, 2, 3 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, 2, 3 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 product for 1, 2, 3 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 product were significantly different (P < 0.05), indicating that the test product has freckle removal and whitening efficacy.

[0102] In summary, after reading the present invention document, ordinary technicians in this field can make various other corresponding transformation schemes based on the technical scheme and technical concept of the present invention without creative mental work, which all fall within the scope of protection of the present invention.

Claims

1. An extract of Rhus chinensis bark having whitening and soothing effects, characterized in that: The Rhus chinensis bark extract is obtained by using Rhus chinensis bark as a raw material and separating by organic solvent chromatography.

2. The Rhus chinensis bark extract according to claim 1, characterized in that The organic solvent is ethanol.

3. The method for preparing the Rhus chinensis bark extract with whitening and soothing effects according to claim 1, characterized in that: The following steps are involved: The bark of Rhus chinensis is crushed and then extracted with ethanol under heating and reflux. The obtained extract is decompressed to recover the solvent and concentrated to obtain an extract. The obtained extract is mixed with MCI small pore resin, loaded into MCI column, and washed with ethanol to obtain the active segment HDYFM-1.

4. The method for preparing the Rhus chinensis bark extract according to claim 3, wherein: After the bark of Rhus chinensis is crushed, the bark is subjected to ethanol heating reflux extraction, and the ethanol used is 60% to 90%.

5. The method for preparing the Rhus chinensis bark extract according to claim 3, wherein: The obtained extract is mixed with MCI small pore resin in an amount of 1 to 3 times the amount of MCI small pore resin, and loaded into the column in an amount of 2 to 8 times the amount of MCI.

6. The method for preparing the Rhus chinensis bark extract according to claim 5, characterized in that: The obtained extract was mixed with MCI small pore resin with 2 times the amount of MCI small pore resin and loaded into the column with 5 times the amount of MCI.

7. The method for preparing the Rhus chinensis bark extract according to claim 3, characterized in that: In the ethanol wash, 5 column volumes were washed with 10% ethanol, 20% ethanol, 30% ethanol, 50% ethanol, and 100% ethanol.

8. The method for preparing the Rhus chinensis bark extract according to claim 3, characterized in that: The following steps are involved: 100 kg of Rhus chinensis bark was crushed and extracted with 1 to 3 times the amount of 60% to 90% ethanol by heating and reflux, and the process was repeated three times. The extracts were combined, and the solvent was recovered under reduced pressure. The extracts were concentrated to obtain an extract. The extract was mixed with 2 times the amount of MCI small-pore resin, and loaded with 5 times the amount of MCI column. Five column volumes were washed with 10% ethanol, 20% ethanol, 30% ethanol, 50% ethanol, and 100% ethanol, respectively, to obtain active segments 1 to 5, among which active segment 4 was HDYFM-1.

9. Use of the Rhus chinensis bark extract according to claim 1 in cosmetics.

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