Optimization of extraction and purification process of total flavonoids from hypericum ascyron and application in photodamage protection

Abstract

This invention discloses an optimized extraction and purification process for total flavonoids from Hypericum perforatum and its application in photodamage protection. Belonging to the field of natural product extraction technology, the process includes the following steps: using Hypericum perforatum powder as raw material, ultrasonic-assisted extraction is performed, followed by centrifugation, collection of the supernatant, and repeated extraction of the residue twice. The supernatants are combined to obtain a total flavonoid extract from Hypericum perforatum. The extract is then purified by adsorption using a macroporous adsorption resin, and the eluent is collected, concentrated, and dried to obtain high-purity total flavonoids from Hypericum perforatum. This invention features a stable process, high extraction efficiency, and is environmentally friendly, making it suitable for industrial production. The prepared total flavonoids from Hypericum perforatum exhibit high purity and significant antioxidant and photoprotective effects, and can be widely used in antioxidant, skin protection, and sunscreen products.

Description

Technical Field

[0001] This invention belongs to the field of natural product extraction technology, specifically referring to an optimized extraction and purification process of total flavonoids from Hypericum perforatum and its application in photodamage protection. Background Technology

[0002] Hypericum monogynum Lin is a medicinal plant used to treat hepatitis, acute pharyngitis, conjunctivitis, and bruises. Due to its golden flowers and delicate, thread-like stamens, it is also commonly planted as an ornamental plant in courtyards, parks, and urban roadside greenbelts. Studies have shown that Hypericum flowers contain a large amount of flavonoids, including quercetin-3-O(-6′′-caffeoyl)-β-D-galactoside, 3,8"-bisapigenin, and quercetin-3-O-α-L-rhamnoside. Given the high total flavonoid content in Hypericum flowers and the potential of these components to absorb and resist ultraviolet radiation, optimizing the extraction process to enrich its extracts holds promise for developing it into an ideal candidate raw material for natural sunscreens to prevent ultraviolet damage.

[0003] However, existing technologies, such as the ethanol thermal extraction method for extracting Hypericum flavonoids, have low efficiency and high impurity content; although the ultrasonic method has improved efficiency, the process parameters have not been systematically optimized and the sample solution concentration and pH are poorly matched, making it difficult to achieve efficient enrichment.

[0004] Therefore, it is necessary to develop an optimized extraction and purification process for total flavonoids from Hypericum. Summary of the Invention

[0005] In response to the above situation and to overcome the shortcomings of the existing technology, this invention provides an optimized extraction and purification process for total flavonoids from Hypericum flowers, along with photodamage protection applications. This effectively solves the problems of wasteful Hypericum resources, low added value, insufficient comprehensive utilization, and difficulty in efficient enrichment during purification in the current market.

[0006] The technical solution adopted in this invention is as follows: This invention proposes an optimized extraction and purification process for total flavonoids from Hypericum perforatum, comprising the following steps:

[0007] (1) Using Hypericum powder as raw material, ultrasonic-assisted extraction was performed. After extraction, centrifugation was performed, and the supernatant was collected. The residue was extracted twice, and the supernatants were combined to obtain Hypericum total flavonoid extract.

[0008] (2) The total flavonoid extract of Hypericum perforatum was purified by adsorption using macroporous adsorption resin, the eluent was collected, concentrated and dried to obtain high-purity total flavonoids of Hypericum perforatum.

[0009] Further, the macroporous adsorption resin in step (2) is selected from one of the following: nonpolar resin D101, neutral resin AB-8, HPD-600, S-8 and NKA-9.

[0010] Preferably, the macroporous adsorption resin in step (2) is selected from S-8.

[0011] Furthermore, the macroporous resin described in step (2) has an adsorption capacity of 26.55 ± 0.64 mg / g and a desorption capacity of 26.08 ± 0.72 mg / g.

[0012] Furthermore, in step (2), the purification process parameters for total flavonoids from Hypericum are: 50–90 vol% ethanol, sample concentration of 0.4–1.2 mg / g, and pH value of 2–10.

[0013] Preferably, in step (2), the process parameters for purifying total flavonoids from Hypericum are: using 60 vol% ethanol as the solvent, a sample concentration of 1 mg / ml, and pH 4.

[0014] Furthermore, in step (1), the parameters for ultrasonic-assisted extraction are: extraction solvent is 30~70 vol% ethanol, liquid-to-solid ratio is 10~30 mL / g, extraction temperature is 40~80℃, and ultrasonic time is 30~70 min.

[0015] Preferably, in step (1), the parameters for ultrasonic-assisted extraction are: extraction solvent is 50 vol% ethanol, liquid-to-solid ratio is 20 mL / g, extraction temperature is 70 °C, and ultrasonic time is 42 min.

[0016] Furthermore, the total flavonoids from Hypericum include hyperoside, quercetin, rutin, quercetin-3-O-β-D-glucuronide, amanthoflavonoids, astilbene, dihydroquercetin, astragaloside, morin, and tetrahydroxyoxanthone.

[0017] Furthermore, the actual average extraction rate of the optimized extraction and purification process for total flavonoids from Hypericum perforatum was 197.73 mg / g.

[0018] In addition, the present invention also provides an application of total flavonoids from Hypericum prepared by an optimized extraction and purification process in photodamage protection.

[0019] Preferably, the application of total flavonoids from Hypericum perforatum in the preparation of drugs or skin care products to protect against UVB-induced skin photodamage.

[0020] The beneficial effects achieved by the present invention using the above solution are as follows:

[0021] (1) The extraction and purification process of total flavonoids from Hypericum provided by the present invention improves the resource utilization rate and added value of Hypericum. The raw materials for the process are widely available and renewable. The extraction efficiency is high, the conditions are mild, the operation is simple, and the stability is strong.

[0022] (2) After purification with S-8 macroporous resin, the purity of total flavonoids was significantly improved, and the product quality was stable. The prepared total flavonoids from Hypericum have strong antioxidant activity and UV protection, and are effective against free radicals including DPPH and ABTS. + Free radicals, O2 - It has a scavenging effect on free radicals, is natural, safe, mild and non-irritating, and can be widely used in the field of photodamage protection, with good economic value and application prospects;

[0023] (3) The formulation and dosage are flexible and easy to adjust, and photodamage protection agents with different protective strengths can be made according to different needs; the source is readily available, and the high-value utilization of resources can be realized. Attached Figure Description

[0024] Figure 1 This invention relates to the adsorption and desorption rates of different types of resins for HMFF in an optimized purification process of total flavonoids from Hypericum perforatum.

[0025] Figure 2 The three-dimensional diagram and its contour plot show the effects of the pairwise interactions of the material-liquid ratio, ethanol concentration, extraction temperature and extraction time on the extraction yield of total flavonoids from Hypericum perforatum proposed in this invention.

[0026] Figure 3 This invention presents a single-factor study on the effect of the mass fraction of total flavonoids after purification on an optimized purification process for total flavonoids from Hypericum perforatum.

[0027] Figure 4 This invention presents a comparative study on the effects of pairwise interactions between ethanol concentration, sample concentration, and pH value on the mass fraction of total flavonoids after purification in an optimized purification process for total flavonoids from Hypericum perforatum.

[0028] Figure 5 For the component analysis of total flavonoids;

[0029] Figure 6 The change in body weight of mice in each group over time;

[0030] Figure 7 Changes in the appearance of mouse skin;

[0031] Figure 8 The effect of HMFF on histopathological changes in mouse skin tissue (HE staining ×20). Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0033] This invention proposes an optimized extraction and purification process for total flavonoids from Hypericum perforatum and its application in photodamage protection.

[0034] Among them, St. John's wort flowers were purchased from Huaxi District, Guiyang City, Guizhou Province; dried to constant weight at 40℃, pulverized, and passed through a 60-mesh sieve. ICR mice were purchased from Changsha Tianqin Biotechnology Co., Ltd.; D101, AB-8, S-8, HPD-600, and NKA-9 macroporous resins were purchased from Anhui Samsung Resin Technology Co., Ltd. Sodium nitrite, aluminum nitrate, and sodium hydroxide were purchased from Shanghai Guoyao Group. Anhydrous ethanol was purchased from Tianjin Fuyu Fine Chemical Co., Ltd.

[0035] Example 1

[0036] An Optimized Extraction and Purification Process for Total Flavonoids from Hypericum

[0037] (1) Weigh 1g of Hypericum powder, pass it through a 60-mesh sieve, place it in a 50ml centrifuge tube with a cap, add 30% ethanol extraction solution, and place it in a 100W ultrasonic extractor. Extract at an appropriate extraction temperature and time. After extraction, centrifuge and transfer the supernatant to a 100ml volumetric flask. Repeat the extraction of the residue twice under the same conditions, combine the extracts, and dilute to a final volume to obtain the sample extract.

[0038] (2) HMFF was purified by macroporous resin chromatography. The purification conditions were: extraction solvent 50 vol% ethanol, sample concentration 0.4 mg / g, and pH 2.

[0039] Example 2

[0040] An Optimized Extraction and Purification Process for Total Flavonoids from Hypericum

[0041] (1) Weigh 1g of Hypericum powder, pass it through a 60-mesh sieve, place it in a 50ml centrifuge tube with a cap, add 70% ethanol extraction solution, and place it in a 100W ultrasonic extractor. Extract at an appropriate extraction temperature and time. After extraction, centrifuge and transfer the supernatant to a 100ml volumetric flask. Repeat the extraction of the residue twice under the same conditions, combine the extracts, and dilute to a final volume to obtain the sample extract.

[0042] (2) HMFF was purified by macroporous resin chromatography. The purification conditions were: extraction solvent 90 vol% ethanol, sample concentration 1.2 mg / g, and pH 10.

[0043] Example 3

[0044] An Optimized Extraction and Purification Process for Total Flavonoids from Hypericum

[0045] (1) Weigh 1g of Hypericum powder, pass it through a 60-mesh sieve, place it in a 50ml centrifuge tube with a cap, add 50% ethanol extraction solution, and place it in a 100W ultrasonic extractor. Extract at an appropriate extraction temperature and time. After extraction, centrifuge and transfer the supernatant to a 100ml volumetric flask. Repeat the extraction of the residue twice under the same conditions, combine the extracts, and dilute to a final volume to obtain the sample extract.

[0046] (2) HMFF was purified by macroporous resin chromatography. The purification conditions were: extraction solvent 60 vol% ethanol, loading concentration 1 mg / g, and pH 4.

[0047] like Figure 2 The extraction conditions were: 50 vol% ethanol concentration, liquid-to-solid ratio of 20 mL / g, extraction temperature of 70℃, and extraction time of 42 min. Three parallel validation experiments were conducted under these modified conditions, and the actual average extraction rate was 197.73 ± 1.38 mg / g.

[0048] This study compared the adsorption capacities of five macroporous resins with different polarities. For example... Figure 1 As shown, the non-polar resin D101 and neutral resin AB-8 exhibited weak adsorption capacity for total flavonoids. Polar adsorption resins S-8, HPD-600, and NKA-9 showed high adsorption rates with similar levels. Among them, resin S-8 performed particularly well, with adsorption and desorption capacities of 26.55 ± 0.64 mg / g and 26.08 ± 0.72 mg / g, respectively, significantly higher than HPD-600 and NKA-9. Therefore, S-8 was selected as the optimal macroporous resin. Figure 3 and Figure 4 As shown, its adsorption rate and desorption rate reached 80.95 ± 0.03% and 97.07 ± 0.02%, respectively.

[0049] Example 4

[0050] Application of an optimized extraction and purification process of total flavonoids from Hypericum perforatum in photodamage protection

[0051] A topical oil-in-water cream containing the HMFF extract prepared in Example 3 was prepared by thermal emulsification. The specific steps were as follows: The oil phase component (stearic acid: white petrolatum: glyceryl monostearate: liquid paraffin = 3:3:1:4) was heated to 80°C to melt. Separately, the prescribed amount of HMFF powder was added to the aqueous phase (Tween-80: 1,2-propanediol: distilled water = 1:2:40, containing 0.1 g benzyl alcohol), heated to dissolve, and kept at this temperature. Under continuous stirring and grinding at 80°C, the oil phase was slowly added to the aqueous phase to emulsify until the system was homogeneous. After cooling to room temperature, the product was dispensed. Following this process, a 5 mg / g (low-dose) cream base was prepared.

[0052] Example 5

[0053] Application of an optimized extraction and purification process of total flavonoids from Hypericum perforatum in photodamage protection

[0054] A topical oil-in-water cream containing the HMFF extract prepared in Example 3 was prepared by thermal emulsification. The specific steps were as follows: The oil phase component (stearic acid: white petrolatum: glyceryl monostearate: liquid paraffin = 3:3:1:4) was heated to 80°C to melt. Separately, the prescribed amount of HMFF powder was added to the aqueous phase (Tween-80: 1,2-propanediol: distilled water = 1:2:40, containing 0.1 g benzyl alcohol), heated to dissolve, and kept at this temperature. Under continuous stirring and grinding at 80°C, the oil phase was slowly added to the aqueous phase to emulsify until the system was homogeneous. After cooling to room temperature, the product was dispensed. Following this process, a 10 mg / g (medium-dose) cream base was prepared.

[0055] Example 6

[0056] Application of an optimized extraction and purification process of total flavonoids from Hypericum perforatum in photodamage protection

[0057] A topical oil-in-water cream containing the HMFF extract prepared in Example 3 was prepared by thermal emulsification. The specific steps were as follows: The oil phase component (stearic acid: white petrolatum: glyceryl monostearate: liquid paraffin = 3:3:1:4) was heated to 80°C to melt. Separately, the prescribed amount of HMFF powder was added to the aqueous phase (Tween-80: 1,2-propanediol: distilled water = 1:2:40, containing 0.1 g benzyl alcohol), heated to dissolve, and kept at this temperature. Under continuous stirring and grinding at 80°C, the oil phase was slowly added to the aqueous phase to emulsify until the system was homogeneous. After cooling to room temperature, the product was dispensed. Following this process, a 20 mg / g (high-dose) cream base was prepared.

[0058] Comparative Example 1

[0059] A topical oil-in-water cream containing HMFF extract was prepared using a superheated emulsification method. The specific steps are as follows: The oil phase component (stearic acid: white petrolatum: glyceryl monostearate: liquid paraffin = 3:3:1:4) was added to the aqueous phase (Tween-80: 1,2-propanediol: distilled water = 1:2:40, containing 0.1 g benzyl alcohol) and heated to 80°C to melt. After cooling to room temperature, it was dispensed.

[0060] Experimental Example 1

[0061] The structure of the purified HMFF was initially determined using ultra-high performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry (UPLC-Q-TOF-MS / MS). 10.0 mg of sample powder was accurately weighed, dissolved in 1 mL of methanol, filtered, and then used for chromatographic analysis. Chromatographic separation was performed using a Waters Acquity UPLC HSS T3 C18 reversed-phase column (100*2.1 mm, 1.8 μm), with a mobile phase consisting of 0.1% formic acid / acetonitrile (A) – 0.1% formic acid / water (B). The flow rate was constant at 0.3 mL / min, the column temperature at 40℃, and the injection volume at 10 μL. Gradient elution was performed, as shown in Table 1.

[0062] Table 1. Gradient elution program for mobile phase

[0063]

[0064] Qualitative analysis of the HMFF fractions purified under optimal conditions was performed using UPLC-Q-TOF-MS / MS. Raw data were processed using Compound Discoverer 3.2 (Thermo Fisher Scientific, USA) software and compared against databases including ChemSpider, CHEBI, CHEMBL, natural product libraries, and flavonoid libraries. Further matching of the MS / MS spectra was evaluated using the Best Match algorithm of mzClou and mzVault, with a screening threshold set at a fragment ion matching degree greater than 80%. Ultimately, various flavonoid compounds, including flavonoids, flavonoid glycosides, and flavonols, were identified. Figure 5 The document lists the retention time, theoretical mass-to-charge ratio, measured mass-to-charge ratio, characteristic fragment ions, and identification scores for each compound. The flavonoids in the purified Hypericum flowers were identified as hyperoside, quercetin, rutin, quercetin-3-O-β-D-glucuronide, amanthoflavonoids, astilbene, dihydroquercetin, astragaloside, morin, and tetrahydroxyoxanthone.

[0065] Experiment Example 2

[0066] DPPH free radical scavenging experiment

[0067] The in vitro antioxidant activity of HMFF was evaluated using the DPPH free radical scavenging method. Samples were accurately weighed and prepared into a series of concentration solutions of 5, 25, 50, 100, 250, 500, and 1000 μg / mL. 100 μL of each concentration sample solution was added to 100 μL of 0.2 mmol / L DPPH ethanol solution, mixed well, and reacted at room temperature in the dark for 30 min. The absorbance was measured at 517 nm and recorded as A1. Similarly, the DPPH solution was replaced with an equal volume of anhydrous ethanol, and the absorbance was measured as the sample background value and recorded as A2. Anhydrous ethanol was used instead of the sample solution, and the absorbance was measured as a blank control group and recorded as A0. Ascorbic acid (VC) solution was also used as a positive control. The DPPH free radical scavenging rate was calculated using the following formula:

[0068]

[0069] The DPPH radical scavenging performance of the samples at various concentrations showed a dose-dependent trend, with the scavenging rate increasing at higher concentrations and then plateauing. The scavenging rate of the positive control VC remained at a consistently high level. The IC50 of the HMFF extract was calculated. 50 The value was 55.23 μg / mL, while the IC50 of VC was... 50 The value was 1.47 μg / mL. The results indicate that the extract possesses a certain degree of free radical scavenging ability.

[0070] Experimental Example 3

[0071] ABTS + Free radical scavenging experiment

[0072] ABTS + The determination of free radical scavenging activity was based on and improved from a literature method. The ABTS stock solution was prepared by mixing an equal volume of 7 mmol / L ABTS solution with 2.45 mmol / L potassium persulfate solution and incubating at room temperature in the dark for 16 h. Before use, the stock solution was diluted with anhydrous ethanol until its absorbance at 734 nm was 0.70 ± 0.02, thus obtaining the ABTS working solution (prepared fresh). Purified HMFF samples were prepared into a series of concentrations of 1, 2, 4, 6, 8, 10, and 12 μg / mL. 100 μL of each concentration sample solution was mixed with 100 μL of ABTS working solution, and reacted at room temperature in the dark for 10 min. The absorbance was then measured at 734 nm and recorded as A1. Simultaneously, a background control group (using an equal volume of deionized water instead of the ABTS working solution, absorbance recorded as A2) and a blank control group (using an equal volume of deionized water instead of the sample solution, absorbance recorded as A0) were set up. Ascorbic acid (VC) solution was used as a positive control. ABTS free radical scavenging rate was calculated using the following formula:

[0073]

[0074] The results showed that both exhibited dose-dependent scavenging effects within a concentration range of 1–12 μg / mL. The scavenging rate of the HMFF extract gradually increased from 5.95% to 69.50%, while the scavenging ability of VC was significantly stronger, rapidly increasing from 4.82% to 99.89% within the same concentration range. The IC50 of the HMFF extract for scavenging ABTS+ free radicals was calculated. 50 The value was 6.09 μg / mL, and the IC50 of VC was... 50 The value was approximately 5.84 μg / mL. The results indicate that the extract possesses a certain ABTS free radical scavenging ability.

[0075] Experiment Example 4

[0076] O2 - Free radical scavenging experiment

[0077] A modified pyrogallol autoxidation method was used to evaluate the sample's resistance to superoxide anion radicals (O2). - The scavenging effect of purified Hypericum flowers was assessed. The purified Hypericum flowers were prepared into concentration gradients of 15.625, 31.25, 62.5, 125, 250, 500, and 1000 μg / mL. 200 μL of sample solution was incubated with 700 μL Tris-HCl buffer (10 mmol / L, pH 8.2) in a 25°C water bath for 20 min. 100 μL of 50 mmol / L pyrogallol solution was added, and the reaction was incubated in a 25°C water bath for 4 min. The reaction was then terminated with 10 μL of 1.6 mol / L HCl solution, and the absorbance was measured at 325 nm (A1). Three control groups were simultaneously set up: ① Sample background group (A2, using ultrapure water instead of pyrogallol); ② Reagent blank group (A0, using ultrapure water instead of the sample); ③ System control group (A, using 10 mmol / L HCl instead of pyrogallol). Ascorbic acid (VC) was used as a positive control.

[0078] O2⁻ clearance rate is calculated according to the following formula:

[0079]

[0080] The scavenging activity of purified HMFF against superoxide anions was evaluated using the pyrogallol autoxidation method. The results showed that within the concentration range of 15.625–1000 μg / mL, the scavenging rate of HMFF against superoxide anions gradually increased from 36.22% to 92.28%, exhibiting a clear concentration-dependent relationship. VC achieved a scavenging rate of 93.76% at 62.5 μg / mL, and the scavenging rate plateaued at 100% at 125 μg / mL. The calculated IC50 of HMFF was 98.47 μg / mL, while that of VC was 34.12 μg / mL. These results indicate that the HMFF extract possesses strong superoxide anion scavenging activity, approximately one-third that of VC, suggesting its potential for further research as a natural antioxidant. This experiment provides in vitro experimental evidence for the antioxidant effect of HMFF.

[0081] Experimental Example 5

[0082] Acute UV damage test in hairless mice

[0083] Four-week-old ICR mice were acclimatized for one week and then anesthetized by intraperitoneal injection of 3% chloral hydrate solution according to their body weight. After anesthesia, the backs of the mice were routinely shaved and treated with depilatory cream to create hairless experimental areas. Subsequently, 60 skin-prepared ICR mice were randomly divided into 6 groups (n=10 per group): blank control group (A), ultraviolet damage model group (B), cream matrix control group (C), Example 4 (D), Example 5 (E), and Example 6 (F). To construct a skin photodamage model, except for group A (blank control), all other groups of mice were exposed to UVB (313 nm) for 1.5 hours daily for 2 weeks.

[0084] Before daily irradiation, mice in groups C, D, E, and F that required drug administration were pre-treated with 0.5 g of the corresponding ointment or base on their backs, and irradiated 10 minutes later; group B (model group) was irradiated directly without application. During the experiment, the weight of all mice was measured and recorded every other day.

[0085] Changes in body weight are the most direct indicator of whether mice are growing normally. Experimental results are as follows: Figure 6 As shown, A. Blank control; B. Ultraviolet model group; C. Cream base control group; D. Example 4; E. Example 5; F. Example 6.

[0086] The initial body weights of the six groups of mice were similar, with no significant differences between groups (P>0.05). Starting from day 2, the mice's body weight increased to varying degrees. The weight increase was most significant in the control group and mice in Example 6, followed by Example 5. The weight increase was the slowest in mice in Example 4, while the weight of the matrix group and the model group showed almost no increase. At the end of the experiment, there was a significant difference in body weight between the model group and the drug-treated group (P<0.01). The body weight of mice in Examples 4, 5, and 6 was significantly higher than that of the model group (P<0.01), but the degree of increase was lower than that of the control group.

[0087] Figure 7 The skin condition of mice in each group was shown on day 14 after UVB irradiation. A. Blank control; B. UV model group; C. Cream base control group; D. Example 4; E. Example 5; F. Example 6.

[0088] The control group mice had smooth, delicate skin on their backs with clear texture and healthy skin condition. The UVB model group showed severe skin damage, with large areas of deep purplish-red erythema on their backs, accompanied by significant edema, rough surface, and localized ulceration, demonstrating a strong photodamage effect. The cream-based control group mice still showed significant inflammatory reactions, with widespread erythema, slightly lighter in color than the model group, accompanied by a small amount of crusting, and a rough skin surface, indicating that the cream base had only a weak effect in alleviating UVB damage. In Example 4, the redness and peeling of the mouse skin were slightly improved, but the effect was not significant. In Example 5, the crusting of the mouse skin was significantly reduced, with only a small amount of peeling and mild redness observed. In Example 6, the mouse skin had only a few wrinkles, close to the normal control group, suggesting that high-dose drugs have a strong protective and repairing effect on UVB-induced skin damage.

[0089] Experimental Example 6

[0090] Skin tissue samples from the backs of mice in each group were fixed with 4% paraformaldehyde, sectioned, and stained. Their morphology and structure were observed under an optical microscope. HE staining directly reflected the damage to skin structure caused by UVB irradiation and the repair status in each group. Figure 8 A. Blank control; B. Ultraviolet model group; C. Cream matrix control group; D. Example 4; E. Example 5; F. Example 6. Among them: (a) Epidermal loss; (b) Epidermal thickening; (c) Reduction of hair follicles and sebaceous glands; (d) Necrotic fragments; (e) Inflammatory cell infiltration.

[0091] The epidermis of the skin tissue in the blank control group was intact, with a clear structure and normal stratum corneum; the collagen fibers in the dermis were neatly arranged, and skin appendages such as hair follicles and sebaceous glands were normally distributed in the dermis. Figure 8A). The model group showed severe acute photodamage. Extensive epidermal loss, structural damage, significant reduction in hair follicles and sebaceous glands, and numerous necrotic fragments were observed locally, accompanied by marked inflammatory cell infiltration. Figure 8 B). The stromal group showed some relief from the damage compared to the model group, but it was still significant. Extensive thickening of the epidermis was observed, with localized epidermal loss. Hair follicles and sebaceous glands were significantly reduced, accompanied by a small amount of inflammatory cell infiltration. Figure 8 C). With increasing HMFF dosage, the epidermal thickness of the mouse skin lesions gradually decreased, inflammatory cell infiltration decreased, and the boundary between the dermis and epidermis became clearer, showing a dose-dependent improvement effect. In Example 6, the skin structure remained essentially normal, indicating that HMFF has a protective effect against photodamage to the skin. Figure 8 DF).

[0092] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0093] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

[0094] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention; the actual structure is not limited thereto. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the invention, such designs should fall within the protection scope of the present invention.

Claims

1. An optimized extraction and purification process for total flavonoids from Hypericum perforatum, characterized in that: Includes the following steps: (1) Using Hypericum powder as raw material, ultrasonic-assisted extraction was performed. After extraction, centrifugation was performed, and the supernatant was collected. The residue was extracted twice, and the supernatants were combined to obtain Hypericum total flavonoid extract. (2) The total flavonoid extract of Hypericum perforatum was purified by adsorption using macroporous adsorption resin, the eluent was collected, concentrated and dried to obtain high-purity total flavonoids of Hypericum perforatum.

2. The optimized extraction and purification process for total flavonoids from Hypericum perforatum according to claim 1, characterized in that, The macroporous adsorption resin mentioned in step (2) is selected from one of the following: non-polar resin D101, neutral resin AB-8, HPD-600, S-8 and NKA-9.

3. The optimized extraction and purification process for total flavonoids from Hypericum perforatum according to claim 2, characterized in that, The macroporous adsorption resin used in step (2) is S-8.

4. The optimized extraction and purification process for total flavonoids from Hypericum perforatum according to claim 3, characterized in that, In step (2), the adsorption capacity of the S-8 macroporous adsorption resin is 26.55±0.64 mg / g, and the desorption capacity is 26.08±0.72 mg / g.

5. The optimized extraction and purification process for total flavonoids from Hypericum perforatum according to claim 4, characterized in that, The macroporous adsorption resin described in step (2) has an adsorption rate of 80.95±0.03% for total flavonoids from Hypericum perforatum and a desorption rate of 97.07±0.02%.

6. The optimized extraction and purification process for total flavonoids from Hypericum perforatum according to claim 1, characterized in that, In step (2), the process parameters for purifying total flavonoids from Hypericum are: 50-90 vol% ethanol, sample concentration of 0.4-1.2 mg / g, and pH value of 2-10.

7. The optimized extraction and purification process for total flavonoids from Hypericum perforatum according to claim 1, characterized in that, In step (1), the parameters for ultrasonic-assisted extraction are: extraction solvent is 30-70 vol% ethanol, liquid-to-solid ratio is 15-25 mL / g, extraction temperature is 60-80℃, and ultrasonic time is 30-50 min.

8. The optimized extraction and purification process for total flavonoids from Hypericum perforatum according to claim 1, characterized in that, The total flavonoids from Hypericum include hyperoside, quercetin, rutin, quercetin-3-O-β-D-glucuronide, amanthoflavonoids, astilbene, dihydroquercetin, astragaloside, morin, and tetrahydroxyoxanthone.

9. The application of total flavonoids from Hypericum flowers prepared by the optimized extraction and purification process according to claim 1 in photodamage protection.

10. The application according to claim 9, characterized in that, Application of total flavonoids from Hypericum perforatum in the preparation of drugs or skin care products to protect against UVB-induced skin photodamage.

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