Method for decolorizing glaucol acid and product and application thereof

Abstract

The application belongs to the technical field of cosmetic raw material preparation, and particularly relates to a method for decolorizing shikonin acid, a product thereof and application thereof. The method comprises the following steps: pretreatment of raw materials: after crushing the medicinal material of arnebia euchroma, the high-pressure instantaneous explosion technology is used for treatment; extraction: after the pretreated material is extracted by using an ethanol aqueous solution, solid-liquid separation is performed to obtain a shikonin acid crude extract of arnebia euchroma; activated carbon decolorization: ethanol is added into the crude extract and uniformly mixed, and then activated carbon is added for decolorization treatment, and after the decolorization is completed, solid-liquid separation is performed; resin purification: the supernatant obtained after the decolorization is loaded onto a resin column, gradient elution is performed, and the ethanol elution section rich in shikonin acid is collected and concentrated and dried. The application realizes an excellent balance between efficient decolorization and high active ingredient reservation. While the deep-colored pigment is removed by strong adsorption, the adsorption loss of the target shikonin acid is significantly reduced.

Description

Technical Field

[0001] This invention belongs to the field of cosmetic raw material preparation technology, specifically relating to a method for decolorizing phenolic acids from Xinjiang shikonin, its products, and applications. Background Technology

[0002] Xinjiang Lithospermum ( Arnebia euchroma Xinjiang limonite is an important medicinal herb in traditional Chinese medicine, and its rhizome is rich in naphthoquinones and phenolic acids. Modern pharmacological studies have shown that the phenolic acid compounds in Xinjiang limonite have significant anti-inflammatory, antibacterial, tissue repair promoting, and antioxidant activities, showing broad application prospects in skin repair, oral health, and wound healing.

[0003] However, the efficient preparation of high-purity phenolic acid extracts from Xinjiang Lithospermum erythrorhizon presents significant challenges. In addition to the target phenolic acids, the raw materials also contain a large amount of fat-soluble pigments, colloids, and other impurities. These coexisting components, especially the dark pigments, not only severely interfere with the subsequent analysis and application of the phenolic acids but also affect the color and purity of the final product. Therefore, effective decolorization becomes one of the key technical steps in the purification process.

[0004] Currently, decolorization of plant extracts commonly employs methods such as adsorption, oxidation, or resin separation. While adsorption can remove some pigments, it suffers from poor selectivity, often resulting in significant loss of phenolic acid active ingredients, making it difficult to balance yield and purity. Some specialized decolorizing resins or oxidation methods may improve decolorization, but they present challenges such as high cost, potential damage to active ingredient structures, or safety hazards. Therefore, developing a simple and safe decolorization and purification method that selectively removes pigments, maximizes the preservation of phenolic acid activity, and achieves high-value utilization of phenolic acids from Xinjiang limonite is crucial. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0006] As one aspect of the present invention, the present invention provides a method for decolorizing Xinjiang shikonin with phenolic acid, which includes the following steps:

[0007] (1) Pretreatment: After the Xinjiang purple herb is crushed, it is treated with high-pressure instantaneous blasting technology;

[0008] (2) Extraction: The pretreated material was extracted with an ethanol-water solution, and the crude extract of phenolic acid from Xinjiang was obtained after solid-liquid separation;

[0009] (3) Activated carbon decolorization: Ethanol is added to the crude extract and mixed evenly, then activated carbon is added for decolorization. After decolorization, solid-liquid separation is performed; wherein, the ethanol concentration is 30-50 wt%.

[0010] The activated carbon is made from a mixture of Masson pine and slash pine, with a mass ratio of Masson pine to slash pine of 1:0.1-10. The raw materials are soaked in a 30-75wt% phosphoric acid aqueous solution and then carbonized at 400-600℃ to form a mesoporous structure. After that, water vapor is introduced at 800-950℃ to form a microporous structure, thus obtaining the activated carbon.

[0011] (4) Resin purification: The supernatant obtained after decolorization is loaded onto a resin column and eluted sequentially with water and ethanol aqueous solution. The ethanol eluent rich in phenolic acid is collected, concentrated and dried to obtain Xinjiang Lithospermum erythrorhizon phenolic acid extract; wherein the resin is a macroporous adsorption resin with a cross-linked styrene-divinylbenzene skeleton modified with acrylic acid and acrylate.

[0012] In a preferred embodiment of the method described in this invention, the activated carbon comprises ZX-767 activated carbon with an average particle size of 150-250 mesh; the resin comprises DM-130 resin column with an average particle size of 20-30 mesh.

[0013] As a preferred embodiment of the method described in this invention, in step (1), the pressure of the high-pressure instantaneous burst is 0.8-1.0 MPa, and the pressure holding time is 10-30 seconds.

[0014] As a preferred embodiment of the method described in this invention, in step (2), the concentration of the ethanol aqueous solution used for extraction is 40-50 wt%; the extraction is carried out at 40-50℃ for 1-2 hours; and the extraction is carried out 1-3 times.

[0015] As a preferred embodiment of the method described in this invention, in step (3), the amount of activated carbon added is 0.1-1% of the mass of the crude extract; the temperature of the decolorization treatment is 40-60℃ and the time is 0.5-2 hours.

[0016] As a preferred embodiment of the method described in this invention, step (3) further includes, after solid-liquid separation, eluting the separated activated carbon with an ethanol aqueous solution of a concentration of 30-50 wt%, and combining the resulting eluent with the supernatant obtained after decolorization.

[0017] As a preferred embodiment of the method described in this invention, in step (4), the gradient elution specifically involves: first eluting with water until the effluent is nearly colorless, then discarding the water wash; then eluting sequentially with 40-50 wt% ethanol aqueous solution and 80-85 wt% ethanol aqueous solution, and collecting the ethanol eluent.

[0018] As a preferred embodiment of the method described in this invention, the crude extract obtained in step (2) is concentrated and water is added to precipitate impurities before step (3).

[0019] The present invention also provides a Xinjiang shikonin extract obtained by the aforementioned method for decolorizing Xinjiang shikonin.

[0020] The present invention also provides the application of the Xinjiang lithospermum erythrorhizon phenolic acid extract in the preparation of products with skin repair, wound healing promotion, anti-inflammatory or periodontitis improvement functions.

[0021] The products mentioned include cosmetics, topical skin preparations, medical dressings or drugs or oral care products.

[0022] The beneficial effects of this invention are:

[0023] This invention achieves an excellent balance between efficient decolorization and retention of highly active ingredients. It can strongly adsorb and remove dark pigments while significantly reducing the adsorption loss of target phenolic acids, thus resolving the technical contradiction in traditional decolorization methods where decolorization and retention of active substances are difficult to achieve simultaneously. This invention yields a high-purity, high-quality phenolic acid extract. Through the aforementioned selective decolorization steps, combined with gradient elution purification using DM-130 macroporous adsorption resin, the phenolic acid components can be further refined. The final extract has a light color, high phenolic acid purity, and few impurities, laying a solid foundation for the quality of raw materials for the subsequent development of various end products.

[0024] This invention significantly enhances the bioactivity of the extract. It achieves efficient preservation and enrichment of the active structure of phenolic acids. The phenolic acid extract prepared by this invention exhibits excellent multiple effects at the cellular and animal model levels, including promoting skin barrier repair, accelerating wound healing, inhibiting inflammatory response, and improving periodontal tissue health. Its activity is significantly better than that of the control product prepared by conventional decolorization methods.

[0025] This invention features strong process synergy, controllable operation, and industrialization potential. It systematically combines high-pressure explosion pretreatment, activated carbon decolorization under specific conditions, and purification with specialized resin. The steps are synergistically enhanced, and no special or expensive reagents are used, making it suitable for large-scale production. This provides an efficient and reliable technical path for the in-depth development and utilization of Xinjiang's Lithospermum resources. Attached Figure Description

[0026] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, wherein:

[0027] Figure 1 Images of the decolorizing solutions prepared in each embodiment and comparative example.

[0028] Figure 2 The image shows the immunofluorescence results for test case 3.

[0029] Figure 3 The image shows the results of the cell scratch test for Example 4. Detailed Implementation

[0030] To make the above-mentioned objectives, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to specific examples.

[0031] Example 1:

[0032] (1) Raw material pretreatment: Take Xinjiang purple gromwell root dried at 50℃, pulverize it and pass it through a 30-mesh sieve. Put it into an explosion reaction vessel, use high pressure instantaneous explosion technology to maintain it at 0.8 MPa for 20 seconds, and after the explosion is completed, quickly open the explosion valve to rupture the cell wall and release the effective ingredients for subsequent extraction.

[0033] (2) Extraction: 1 kg of pretreated material was added to 10 kg of 50 wt% ethanol aqueous solution and extracted at 45℃ for 1.5 h. The mixture was then filtered through a 0.1 μm ceramic membrane (0.1 MPa, 40℃, Hefei Shijie Membrane Engineering Co., Ltd., equipment model: SJM-FHM-02) and the filtrate was collected. 8 kg of 50 wt% ethanol aqueous solution was added to the filter residue and extracted at 45℃ for 1.5 h. The residue was then filtered again and the filtrate was collected. The filtrates were combined and concentrated to 3 kg using a rotary evaporator at 50℃. 4.5 kg of ultrapure water was added, and the mixture was centrifuged at 8000 rpm and 25℃ for 10 minutes. The supernatant was collected to obtain the crude extract of Xinjiang lithospermic acid.

[0034] (3) Decolorization with activated carbon: Ethanol was added to the crude extract of shikonin from Xinjiang. The concentration of ethanol in the crude extract of shikonin from Xinjiang was 30 wt%. 0.1 wt% of ZX-767 activated carbon (purchased from Jiangsu Zhuxi Activated Carbon Co., Ltd., the raw material was a mixture of Masson pine and slash pine, the mass ratio of Masson pine to slash pine was 1:0.1-10. The raw material was soaked in 30-75 wt% phosphoric acid aqueous solution and then carbonized at 400-600℃ to form a mesoporous structure. Then, water vapor was introduced at 800-950℃ to form a microporous structure. The activated carbon was stirred at 40℃ for 1 h for decolorization. It was centrifuged at 8000 rpm and 25℃ for 10 minutes. The activated carbon obtained by centrifugation was washed with 300 g of 30 wt% ethanol aqueous solution. The supernatant was rotary evaporated to 4 kg to obtain the decolorized solution.

[0035] (4) Column chromatography: 1 kg of DM-130 resin (purchased from Xi'an Lanxiao Technology New Materials Co., Ltd.), wet loading decolorization solution, first wash with 2 column volumes of water, discard the water elution; then elute with 2 column volumes of 40% ethanol aqueous solution and 2 column volumes of 85% ethanol aqueous solution respectively, combine the eluents of 40% ethanol aqueous solution and 85% ethanol aqueous solution, concentrate and dry using a rotary evaporator at 50℃ to obtain 80.3 g of phenolic acid extract, with a phenolic acid content of 77.9%.

[0036] Example 2:

[0037] (1) Raw material pretreatment: Take Xinjiang purple gromwell root dried at 50℃, pulverize it and pass it through a 30-mesh sieve. Put it into an explosion reaction vessel, use high pressure instantaneous explosion technology to maintain it at 1.0 MPa for 30 seconds, and after the explosion, quickly open the explosion valve to rupture the cell wall and release the effective ingredients for subsequent extraction.

[0038] (2) Extraction: 1 kg of pretreated material was added to 10 kg of 40% ethanol aqueous solution and extracted at 45℃ for 1.5 h. The mixture was then filtered through a 0.1 μm ceramic membrane (0.1 MPa, 40℃, Hefei Shijie Membrane Engineering Co., Ltd., equipment model: SJM-FHM-02). 8 kg of 40% ethanol aqueous solution was added to the filter residue and extracted at 45℃ for 1.5 h, followed by filtration. 8 kg of 40% ethanol aqueous solution was added to the filter residue and extracted at 45℃ for 1.5 h, followed by filtration. The filtrate was collected. The filtrates were combined and concentrated to 3 kg using a rotary evaporator at 50℃. 4.5 kg of ultrapure water was added, and the mixture was centrifuged at 8000 rpm and 25℃ for 10 minutes. The supernatant was collected to obtain the crude extract of Xinjiang lithospermic acid.

[0039] (3) Decolorization with activated carbon: Ethanol was added to the crude extract of shikonin from Xinjiang. The ethanol concentration in the crude extract of shikonin from Xinjiang was 50 wt%. 1 wt% of ZX-767 activated carbon (purchased from Jiangsu Zhuxi Activated Carbon Co., Ltd.) was added. The mixture was stirred at 50℃ for 1.5 h for decolorization. It was then centrifuged at 8000 rpm and 25℃ for 10 minutes. The activated carbon obtained by centrifugation was washed with 375 g of 30 wt% ethanol aqueous solution. The supernatant was rotary evaporated to 4 kg to obtain the decolorized solution.

[0040] (4) Column chromatography: preferably 1 kg of DM-130 resin, wet loading decolorization solution, first wash with 2 column volumes of water, discard the water elution; then elute with 2 column volumes of 40% ethanol aqueous solution and 2 column volumes of 85% ethanol aqueous solution respectively, combine the eluents of 40% ethanol aqueous solution and 85% ethanol aqueous solution, concentrate and dry using a rotary evaporator at 50 ℃ to obtain 82.1 g of phenolic acid extract, with a phenolic acid content of 79.2%.

[0041] Comparative Example 1:

[0042] (1) Raw material pretreatment: Same as in Example 2.

[0043] (2) Extraction: Same as in Example 2.

[0044] (3) Decolorization with activated carbon: The crude extract of phenolic acid from Xinjiang limonite was added to anhydrous ethanol until the total ethanol content was 30%. 1% ZX-200 activated carbon (purchased from Jiangsu Zhuxi Activated Carbon Co., Ltd.) was added, and the mixture was stirred at 50℃ for 1 hour for decolorization. After centrifugation at 8000 rpm and 25℃ for 10 minutes, the activated carbon obtained by centrifugation was eluted with 375 g of 30% ethanol. The supernatant was rotary evaporated to 4 kg to obtain a decolorized solution. At this point, the decolorized solution was dark in color. The extract was concentrated and dried using a rotary evaporator at 50℃ to obtain 149.0 g of extract with a phenolic acid content of 36.3% and a phenolic acid retention rate of 77.2%. The phenolic acid sample was dark in color, indicating poor decolorization effect.

[0045] Comparative Example 2:

[0046] (1) Raw material pretreatment: Same as in Example 2.

[0047] (2) Extraction: Same as in Example 2.

[0048] (3) Decolorization with activated carbon: The crude extract of phenolic acid from Xinjiang limonite was added to anhydrous ethanol until the total ethanol content was 30%. 1% ZX-303 activated carbon (purchased from Jiangsu Zhuxi Activated Carbon Co., Ltd.) was added, and the mixture was stirred at 50℃ for 1 hour for decolorization. After centrifugation at 8000 rpm and 25℃ for 10 minutes, the activated carbon obtained by centrifugation was eluted with 375 g of 30% ethanol. The supernatant was rotary evaporated to 4 kg to obtain a decolorized solution. At this point, the decolorized solution was dark in color. The extract was concentrated and dried using a rotary evaporator at 50℃ to obtain 151.7 g of extract with a phenolic acid content of 31.6% and a phenolic acid retention rate of 68.4%. The sample was dark in color, indicating poor decolorization effect.

[0049] Comparative Example 3:

[0050] (1) Raw material pretreatment: Same as in Example 2.

[0051] (2) Extraction: Same as in Example 2.

[0052] (3) Decolorization with activated carbon: 1% ZX-767 activated carbon (purchased from Jiangsu Zhuxi Activated Carbon Co., Ltd.) was added to the crude extract of phenolic acid from Xinjiang Lithospermum erythrorhizon. The mixture was stirred at 50°C for 1 hour for decolorization. It was then centrifuged at 8000 rpm and 25°C for 10 minutes. The activated carbon obtained by centrifugation was washed with 375 g of pure water. The supernatant was concentrated and dried using a rotary evaporator at 50°C to obtain 78.6 g of extract. The phenolic acid content was 16.3% and the phenolic acid retention rate was 18.3%.

[0053] Comparative Example 4:

[0054] (1) Raw material pretreatment: Same as in Example 2.

[0055] (2) Extraction: Same as in Example 2.

[0056] (3) Decolorization with activated carbon: Add anhydrous ethanol to the crude extract of phenolic acid from Xinjiang limonite until the total ethanol content is 10%, add 1% ZX-767 activated carbon (purchased from Jiangsu Zhuxi Activated Carbon Co., Ltd.), stir at 50℃ for 1 hour for decolorization, centrifuge at 8000 rpm and 25℃ for 10 minutes, and wash the activated carbon obtained by centrifugation with 375 g of 10% ethanol. The supernatant is evaporated by rotary evaporation and concentrated and dried at 50℃ using a rotary evaporator to obtain 97.2 g of extract with a phenolic acid content of 20.8% and a phenolic acid retention rate of 28.8%.

[0057] Comparative Example 5:

[0058] (1) Raw material pretreatment: Same as in Example 2.

[0059] (2) Extraction: Same as in Example 2.

[0060] (3) Decolorization with activated carbon: Add anhydrous ethanol to the crude extract of phenolic acid from Xinjiang limonite until the total ethanol content is 60%, add 1% ZX-767 activated carbon (purchased from Jiangsu Zhuxi Activated Carbon Co., Ltd.), stir at 50℃ for 1.5h for decolorization, centrifuge at 8000rpm and 25℃ for 10 minutes, and wash the activated carbon obtained by centrifugation with 375 g of 60% ethanol. The supernatant is concentrated and dried using a rotary evaporator at 50℃ to obtain 121.3 g of extract with a phenolic acid content of 35.4% and a phenolic acid retention rate of 57.8%.

[0061] Comparative Example 6:

[0062] (1) Raw material pretreatment: Same as in Example 2.

[0063] (2) Extraction: Same as in Example 2.

[0064] (3) Decolorization of activated carbon: Add anhydrous ethanol to the crude extract of shikonin from Xinjiang to a total ethanol content of 50%, add 1% ZX-767 activated carbon (purchased from Jiangsu Zhuxi Activated Carbon Co., Ltd.), stir at 50℃ for 1.5h for decolorization, centrifuge at 8000rpm and 25℃ for 10 minutes, and wash the activated carbon obtained by centrifugation with 375g of 30% ethanol. Combine the two supernatants and rotary evaporate to 4 kg.

[0065] (4) Column washing: Take 1 kg of D101 resin, wet loading decolorizing solution, first wash with 2 column volumes of water, discard the water elution; then elute with 2 column volumes of 40% ethanol aqueous solution and 2 column volumes of 85% ethanol aqueous solution respectively, combine the eluents of 40% ethanol aqueous solution and 85% ethanol aqueous solution, concentrate and dry using a rotary evaporator at 50℃ to obtain 81.3 g of phenolic acid extract with a phenolic acid content of 60.5%.

[0066] Comparative Example 7:

[0067] (1) Raw material pretreatment: Same as in Example 2.

[0068] (2) Extraction: 1 kg of the blasted material was added to 10 kg of 50% ethanol and extracted at 45°C for 1.5 h, followed by filtration. The second filter residue was then added to 8 kg of 50% ethanol and extracted at 45°C for 1.5 h, followed by filtration. The third filter residue was then added to 8 kg of 50% ethanol and extracted at 45°C for 1.5 h. The combined filtrates were concentrated to 3 kg without ethanol and then 4.5 kg of ultrapure water was added. The mixture was centrifuged at 8000 rpm and 25°C for 10 minutes. The supernatant was the crude extract of phenolic acid from Xinjiang Lithospermum erythrorhizon. The extract was concentrated and dried using a rotary evaporator at 50°C to obtain 170.1 g of phenolic acid extract with a phenolic acid content of 41.2% and a phenolic acid retention rate of 100%.

[0069] Comparative Example 8:

[0070] (1) Raw material pretreatment: Same as in Example 2.

[0071] (2) Extraction: Same as in Example 2.

[0072] (3) Decolorization with activated carbon: The crude extract of phenolic acid from Xinjiang Lithospermum erythrorhizon was added to anhydrous ethanol until the total ethanol content was 50%. 1% ZX-767 activated carbon (purchased from Jiangsu Zhuxi Activated Carbon Co., Ltd.) was used for decolorization by stirring at 50℃ for 1.5h. The mixture was then centrifuged at 8000rpm and 25℃ for 10 minutes. The activated carbon obtained by centrifugation was eluted with 375 g of 30% ethanol. The supernatant was concentrated and dried using a rotary evaporator at 50℃ to obtain 167.5 g of phenolic acid extract with a phenolic acid content of 40.8% and a phenolic acid retention rate of 97.5%.

[0073] Comparative Example 9:

[0074] (1) Raw material pretreatment: Same as in Example 2.

[0075] (2) Extraction: Same as in Example 2.

[0076] (3) Decolorization with activated carbon: Same as comparative example 1. Add anhydrous ethanol to the crude extract of shikonin from Xinjiang until the total ethanol content is 30%, add 1% ZX-200 activated carbon (purchased from Jiangsu Zhuxi Activated Carbon Co., Ltd.), stir at 50℃ for 1 hour for decolorization, centrifuge at 8000 rpm and 25℃ for 10 minutes, and wash the activated carbon obtained by centrifugation with 375 g of 30% ethanol. Combine the two supernatants and rotary evaporate to 4 kg of decolorized liquid.

[0077] (4) Column washing: Take 1 kg of DM-130 resin and wet loading decolorizing solution. First, wash with water using 2 column volumes of water and discard the water-eluted portion. Then, elute with 2 column volumes of 40% ethanol aqueous solution and 2 column volumes of 85% ethanol aqueous solution respectively. Combine the eluents of 40% ethanol aqueous solution and 85% ethanol aqueous solution and concentrate and dry them using a rotary evaporator at 50℃ to obtain 73.4 g of phenolic acid extract with a phenolic acid content of 67.4% and a phenolic acid retention rate of 70.6%.

[0078] Comparative Example 10:

[0079] (1) Raw material pretreatment: Same as in Example 2.

[0080] (2) Extraction: Same as in Example 2.

[0081] (3) Decolorization with activated carbon: Same as comparative example 2. Add anhydrous ethanol to the crude extract of shikonin from Xinjiang until the total ethanol content is 30%, add 1% ZX-303 activated carbon (purchased from Jiangsu Zhuxi Activated Carbon Co., Ltd.), stir at 50℃ for 1 hour for decolorization, centrifuge at 8000 rpm and 25℃ for 10 minutes, and wash the activated carbon obtained by centrifugation with 375 g of 30% ethanol. Combine the two supernatants and rotary evaporate to 4 kg of decolorized liquid.

[0082] (4) Column washing: Take 1 kg of DM-130 resin and wet loading decolorizing solution. First, wash with water using 2 column volumes of water and discard the water-eluted portion. Then, elute with 2 column volumes of 40% ethanol aqueous solution and 2 column volumes of 85% ethanol aqueous solution respectively. Combine the eluents of 40% ethanol aqueous solution and 85% ethanol aqueous solution and concentrate and dry them using a rotary evaporator at 50℃ to obtain 70.5 g of phenolic acid extract with a phenolic acid content of 61.3% and a phenolic acid retention rate of 61.7%.

[0083] Test Example 1:

[0084] The color of the sample solutions obtained in Examples 1-2 and Comparative Examples 1-8 were evaluated using colorimetric tubes and a UV-Vis spectrophotometer.

[0085] Instruments: Glass colorimetric tubes, T600 UV-Vis spectrophotometer (Purkinje Company).

[0086] Detection:

[0087] 1. Take 10 ml of each of the decolorizing solutions obtained after treatment in each example and comparative example, add them to a glass colorimetric tube, and take pictures under the same background.

[0088] 2. Add 3 mL of decolorizing solution to a quartz cuvette, subtract the solvent blank, and measure the absorbance at an absorption wavelength of 450 nm.

[0089] Figure 1 Images of the decolorizing solutions prepared in each example and comparative example. From left to right, they are the decolorizing solutions of Example 1, Example 2, Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6, Comparative Example 7, and Comparative Example 8. Figure 1 In this study, the colorimetric concentration of all samples was 1%.

[0090] Table 1

[0091]

[0092] Test Example 2:

[0093] The total phenolic acid content of Examples 1-2 and Comparative Examples 1-8 was tested using the Folin-Ciocalteu method.

[0094] Instruments: T600 UV-Vis spectrophotometer (Purchip Technology Co., Ltd.), LC-0004 0.0001 g electronic analytical balance (Sartorius Scientific Instruments Co., Ltd.).

[0095] Preparation of Folin reagent: Weigh 10g sodium tungstate and 2g phosphomolybdic acid, add 5mL of 85% phosphoric acid solution and 75mL of distilled water, heat under reflux for 2h, cool and make up to 100mL, and store in a brown bottle.

[0096] Preparation of Na2CO3 solution: Prepare an aqueous solution of Na2CO3 with a concentration of 75 g / L.

[0097] Preparation of standards: Accurately weigh approximately 10 mg of caffeic acid standard, dissolve it in anhydrous ethanol, and dilute to 50 mL. Pipette 0, 0.25, 0.5, 1.0, 2.0, and 2.5 mL of the above solution into 10 mL volumetric flasks, and dilute to volume with distilled water to obtain caffeic acid standard solutions with concentrations of 0, 5.0, 10.0, 20.0, 40.0, and 50.0 µg / mL.

[0098] Standard curve construction: Take 1 mL of each of the above standard solutions, add 5 mL of distilled water, 1 mL of Folin-Ciocalteu reagent, and 3 mL of Na₂CO₃ solution, and let stand for 2 hours. Then, measure the absorbance of the series of standard solutions at the maximum absorption wavelength of 750 nm. Plot a standard curve with absorbance as the ordinate and caffeic acid concentration as the abscissa, and perform linear regression analysis.

[0099] Sample preparation: Shake the sample well, accurately weigh 0.15~0.2g of sample into a 50ml volumetric flask, add an appropriate amount of pure water, sonicate for 20mins, cool to room temperature and then make up to volume.

[0100] Sample testing: Accurately transfer 1.0 mL of the above solution, add 5 mL of distilled water, 1 mL of Folin reagent and 3 mL of Na2CO3 solution, let stand for 2 h, and then measure the absorbance of the series of standard solutions at the maximum absorption wavelength of 750 nm.

[0101] Phenolic acid content = (C × 50 × V) / (M × 1000) × 100%

[0102] Where: C: concentration obtained from the absorbance-concentration curve (mg / ml); M: sample weight (g); V: volume used when measuring the absorbance of the sample.

[0103] Table 2

[0104]

[0105] Test Example 3: Evaluation of Repair Promotion Effect

[0106] Human immortalized keratinocytes (HaCaT) in the logarithmic growth phase were harvested, digested, resuspended, and seeded into 24-well cell culture plates containing climbing slides, with three replicates per group. The cells were incubated at 37°C in a 5% CO2 environment. When the cells reached approximately 50% confluence, samples from each example and comparative example were added, with the phenolic acid content adjusted to 200 μg / mL. Groups BC served as the blank control group.

[0107] After culturing for another 3 days, the supernatant was discarded, and cells were fixed. The relative fluorescence intensity of ZO-1 protein (Zonula Occludens-1) in each group was detected by immunofluorescence antibody incubation and fluorescence microscopy. Relative fluorescence intensity (IOD) R The calculation formula is:

[0108] IOD R =(IOD T -IOD B ) / IOD B *100%

[0109] Among them: IOD T The fluorescence intensity of the experimental group; IOD B The fluorescence intensity represents the BC group. See the immunofluorescence results image below. Figure 2 .

[0110] Table 3

[0111]

[0112] In the examples, the Xinjiang shikonin extracts processed using different decolorization techniques all significantly promoted the expression of ZO-1 protein in keratinocytes, demonstrating a good repair effect. Furthermore, the effects of the Xinjiang shikonin extracts in the examples were significantly better than those in the comparative examples.

[0113] Test Example 4: Evaluation of Wound Healing Promotion Effect

[0114] Human immortalized keratinocytes (HaCaT) in the logarithmic growth phase were harvested, digested, and resuspended. The cells were seeded into 6-well cells on day 2, with three replicates per group. Once the cells had reached confluence, scratching was performed using a sterile pipette tip to create uniform scratches in the cells, simulating wound formation. The medium was then replaced with serum-free medium containing samples from each example and comparative study, with the phenolic acid concentration adjusted to 200 μg / mL. BC served as the blank control group. Cell migration was observed under a microscope at 0 h and 24 h after scratching, and the migration rate and relative migration rate were calculated. (See attached table). Figure 3 .

[0115] The formula for calculating mobility (MR) is: MR = (S0 - S) 24 ) / S0*100%

[0116] Where: S0 is the area of ​​the scratched region at 0h; S 24 The area of ​​the scratched region after 24 hours; the relative migration rate (RMR) is calculated using the formula: RMR% = MR 样品组 / MR 空白组 *100%.

[0117] Table 4

[0118]

[0119] The experimental results above show that the Xinjiang shikonin extracts obtained using different decolorization processes in the examples all significantly promoted the migration of keratinocytes and exhibited good wound healing effects. Furthermore, the effects of the Xinjiang shikonin extracts in the examples were significantly better than those in the comparative example.

[0120] Test Example 5: Evaluation of Anti-inflammatory Effect

[0121] Human immortalized oral epithelial cells CA9-22 in logarithmic growth phase were digested, resuspended, and seeded into 24-well cell culture plates, with three replicates per group. When the cells reached approximately 50% confluence, samples containing 5 µg / mL LPS were added, along with samples from each example and comparative study. The phenolic acid content was adjusted to 150 μg / mL in all samples. After culturing for another 24 hours, the supernatant was collected, and the IL-6 levels in each group were detected by ELISA.

[0122] Table 5

[0123]

[0124] Conclusion: The Xinjiang shikonin extracts obtained using different decolorization processes in the examples all significantly inhibited LPS-induced IL-6 production, demonstrating good anti-inflammatory effects. Furthermore, the effects of the Xinjiang shikonin extracts in the examples were significantly better than those in the comparative examples.

[0125] Test Example 6: Evaluation of the effect of improving periodontitis

[0126] Eight-week-old male C57BL / 6 mice were selected to establish a periodontitis model. The modeling method was as follows: 10 mice were inoculated with... 9 CFU / ml of P. gingivalis (ATCC 33277) was administered, followed by ligation of the maxillary second molar with non-absorbable surgical sutures for combined modeling, and then 10 doses were injected the next day. 9A mouse model of periodontitis was established by inoculating *P. gingivalis* at CFU / ml three times. Successfully modeled mice were then randomly divided into groups of six (n=6 per group) based on body weight. In the control group, the sutures were cut and removed immediately after modeling. The experimental groups were given gels containing 2% of different test samples, while the control group received an equal amount (1g each time) of excipient gel. Gel administration was once daily for 7 consecutive days, starting 7 days after modeling. After 7 days of administration, the sulcus bleeding index (SBI) of the mice in each group was assessed, and samples were then collected for testing. The gel formulations containing 2% of different test samples are shown in Table 6, and the preparation process is as follows:

[0127] 1. Premix: Thoroughly dry mix carbomer 940 and xanthan gum, then add glycerin and butylene glycol, and premix and grind into a fine paste.

[0128] 2. Preparation of aqueous base system: Add the prescribed amount of purified water to the mixing tank, stir at a low speed of 300 r / min, add the above glycerol butylene glycol-thickener premix paste, raise the speed to 800 r / min and stir for 10 min until it is evenly dispersed and free of lumps.

[0129] 3. Thickener swelling: Control the temperature at 40±5℃, and stir at 1000r / min for 30~40min until the thickener is completely swollen into a transparent, particle-free thin gel. Allow it to cool naturally to below 30℃.

[0130] 4. Addition of the preservative system: Stir at a low speed of 300 r / min, add 1,2-hexanediol and p-hydroxyacetophenone in sequence, and stir for 10 min until completely dissolved and the system is homogeneous.

[0131] 5. Neutralization and gel formation, pH adjustment: Stir at 300 rpm, add triethanolamine dropwise at a rate of 1 ml / min, and continue stirring for 15 min after the addition is complete to crosslink and form a gel. Adjust the pH of the system to 6.0~7.0.

[0132] 6. Addition and homogenization of active ingredients: Add shikonin, stir at 600 rpm for 20 min for initial dispersion, transfer to a homogenizer and homogenize at 20,000 rpm for 5 min until the ingredients are evenly dispersed.

[0133] 7. Vacuum degassing: Transfer the gel into a vacuum degassing machine and degas for 10-15 minutes at a vacuum of -0.08 to -0.1 MPa to remove air bubbles from the system.

[0134] Table 6

[0135]

[0136] The sulcus bleeding index (SBI) uses the Mazza criteria (1981). A blunt-tipped WHO probe is gently inserted 1 mm below the gingiva to observe the presence and amount of gingival bleeding. Based on the bleeding level, it is graded into six levels: 0, 1, 2, 3, 4, and 5. Scoring criteria: 0 = healthy gums; 1 = mild inflammation, slight discoloration, mild edema, no bleeding on probing; 2 = mild gingival inflammation, discoloration, no edema or swelling, punctate bleeding on probing; 3 = moderate gingival inflammation, discoloration, mild edema, bleeding on probing, bleeding extending along the gingival margin; 4 = severe gingival inflammation, not only discoloration but also significant swelling, bleeding on probing, blood overflowing the gingival margin; 5 = discoloration, significant swelling, sometimes ulceration, bleeding on probing or spontaneous bleeding. Probing and visual inspection are performed to observe the color of the gums and to gently probe the gingival sulcus with a periodontal probe to check for bleeding. The score is the sum of the scores for each tooth divided by the number of teeth examined.

[0137] Table 7

[0138]

[0139] In the examples, the Xinjiang shikonin extracts with different decolorization processes can all significantly improve gingival sulcus bleeding, and the effects of the Xinjiang shikonin extracts in the examples are significantly better than those in the comparative examples.

[0140] Fixed tissue samples were removed and fixed in foam according to groups, then placed in a 34mm scanning tube. The scanning parameters were set as follows: X-ray tube current 145mA, voltage 55kV, slice thickness 10μm, and high resolution. The trabecular bone parameters in the bifurcation region of the maxillary second molar were analyzed and measured using the instrument's built-in analysis software, and bone volume density (BVD) was calculated.

[0141] Table 8

[0142]

[0143] In the examples, the Xinjiang shikonin extracts with different decolorization processes can all significantly improve alveolar bone resorption caused by periodontitis, and the effects of the Xinjiang shikonin extracts in the examples are significantly better than those in the comparative examples.

[0144] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for decolorizing phenolic acids from Xinjiang shikonin, characterized in that, Includes the following steps: (1) Pretreatment: After the Xinjiang purple herb is crushed, it is treated with high-pressure instantaneous blasting technology; (2) Extraction: The pretreated material was extracted with an ethanol-water solution, and the crude extract of phenolic acid from Xinjiang was obtained after solid-liquid separation; (3) Activated carbon decolorization: Ethanol is added to the crude extract and mixed evenly, then activated carbon is added for decolorization. After decolorization, solid-liquid separation is performed; wherein, the ethanol concentration is 30-50 wt%. The activated carbon is made from a mixture of Masson pine and slash pine, with a mass ratio of Masson pine to slash pine of 1:0.1-10. The raw materials are soaked in a 30-75wt% phosphoric acid aqueous solution and then carbonized at 400-600℃ to form a mesoporous structure. After that, water vapor is introduced at 800-950℃ to form a microporous structure, thus obtaining the activated carbon. (4) Resin purification: The supernatant obtained after decolorization is loaded onto a resin column and eluted sequentially with water and ethanol aqueous solution. The ethanol eluent rich in phenolic acid is collected, concentrated and dried to obtain Xinjiang Lithospermum erythrorhizon phenolic acid extract; wherein the resin is a macroporous adsorption resin with a cross-linked styrene-divinylbenzene skeleton modified with acrylic acid and acrylate.

2. The method for decolorizing Xinjiang shikonin according to claim 1, characterized in that, The activated carbon includes ZX-767 activated carbon with an average particle size of 150-250 mesh; the resin includes DM-130 resin column with an average particle size of 20-30 mesh.

3. The method according to claim 1 or 2, characterized in that, In step (1), the pressure of the high-pressure instantaneous burst is 0.8-1.0 MPa, and the pressure holding time is 10-30 seconds; In step (2), the concentration of the ethanol aqueous solution used for extraction is 40-50 wt%; the extraction is carried out at 40-50℃ for 1-2 hours; the extraction is carried out 1-3 times.

4. The method according to claim 1 or 2, characterized in that, In step (3), the amount of activated carbon added is 0.1-1% of the mass of the crude extract; the temperature of the decolorization treatment is 40-60℃ and the time is 0.5-2 hours.

5. The method according to claim 1 or 2, characterized in that, In step (3), after solid-liquid separation, the activated carbon obtained by separation is eluted with an aqueous ethanol solution of 30-50 wt%, and the resulting eluent is combined with the supernatant obtained after decolorization.

6. The method according to claim 1 or 2, characterized in that, In step (4), the gradient elution is specifically as follows: first, wash with water until the effluent is nearly colorless, and discard the water washing solution; then, elute sequentially with 40-50 wt% ethanol aqueous solution and 80-85 wt% ethanol aqueous solution, and collect the ethanol eluent.

7. The method according to claim 1 or 2, characterized in that, Before proceeding to step (3), the crude extract obtained in step (2) is concentrated and water is added to precipitate impurities.

8. The Xinjiang shikonin extract obtained by the method for decolorizing Xinjiang shikonin according to claim 1.

9. The use of the Xinjiang lithospermum erythrorhizon phenolic acid extract according to claim 8 in the preparation of products with skin repair, wound healing promotion, anti-inflammatory or periodontitis improvement functions.

10. The application according to claim 9, characterized in that: The products include cosmetics, topical skin preparations, medical dressings or drugs or oral care products.

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