CN116747262A

CN116747262A - Substituted flower active substance and preparation method and application thereof

Info

Publication number: CN116747262A
Application number: CN202311015009.5A
Authority: CN
Inventors: 周俊; 王粟萍; 万思迪; 黄灿; 周戟; 董柏秀; 罗慧
Original assignee: Yunnan Yinge Biotechnology Co ltd
Current assignee: Yunnan Yinge Biotechnology Co ltd
Priority date: 2023-08-14
Filing date: 2023-08-14
Publication date: 2023-09-15
Anticipated expiration: 2043-08-14
Also published as: CN116747262B

Abstract

The application provides a substituted flower active substance, a preparation method and application thereof, wherein the active substance extract contains naringin, neohesperidin and synephrine, and the substituted flower active substance extract is prepared by taking substituted flowers or substituted flower extracts as raw materials, extracting with ethanol and purifying with resin; wherein, the content of naringin in the extract is 10-23%, the content of neohesperidin is 30-50%, and the content of synephrine is 1.5-2.5%, calculated by dry mass percent. The preparation process of the substituted flower active extract is simple and environment-friendly, and the obtained substituted flower active extract is simultaneously enriched with three characteristic components in substituted flowers such as naringin, neohesperidin, synephrine and the like, and the total content is not less than 50%.

Description

Substituted flower active substance and preparation method and application thereof

Technical Field

The application relates to the technical field of separation and purification, in particular to a substituted flower active substance and a preparation method and application thereof.

Background

The substituted flower is dried bud of substituted lime of subspecies of Citrus aurantium of Citrus subgenera of Rutaceae, and also called bitter orange flower, citrus aurantium flower and Hui Qing Hua. The main chemical components of the seville orange flower are volatile oil, coumarin, flavone and alkaloids, wherein the main active components are flavone and alkaloid. The flavonoid content is more, mainly is the dihydroflavone such as neohesperidin, naringin, rutin naringin, hesperidin, etc.; the alkaloid contains synephrine as a representative component.

The substituted flowers are used as an economic crop and are planted in a large area in Yunnan Pu' er. At present, the extraction process of the substituted flowers is complex, for example, patent CN115043889A, which is a method for extracting synephrine, hesperidin and naringin from the substituted flowers by enzymolysis, alcohol extraction and alkali extraction.

Therefore, there is a need in the art to develop a preparation method of a substituted flower active substance with simple process and environmental protection, and the obtained substituted flower active substance can simultaneously enrich three characteristic components in substituted flowers such as naringin, neohesperidin and synephrine.

Disclosure of Invention

The application aims to provide a substituted flower active substance, a preparation method and application thereof, wherein the preparation method is simple in process and environment-friendly, and the substituted flower active substance can simultaneously enrich three characteristic components in substituted flowers such as naringin, neohesperidin and synephrine.

In a first aspect of the present application, there is provided a substituted-flower active extract, the substituted-flower active extract containing naringin, neohesperidin and synephrine, the substituted-flower active extract being obtained by ethanol extraction and resin purification from substituted-flower or substituted-flower extract as a raw material; wherein, the content of naringin in the extract of the substituted flower active substance is 10-23%, the content of neohesperidin is 30-50%, and the content of synephrine is 1.5-2.5% based on the dry mass of the extract.

In another preferred example, the extract contains 15-20% of naringin, 35-45% of neohesperidin and 2-2.3% of synephrine, calculated by dry mass percentage of the extract.

In another preferred embodiment, the hydroxy radical scavenging activity of the seville orange flower active extract is IC ₅₀ The value is less than or equal to 160 mug/ml.

In another preferred embodiment, the hydroxy radical scavenging activity of the seville orange flower active extract is IC ₅₀ IC of value Z1 and substituted flower extract ₅₀ The ratio of the values Z0, i.e. Z1/Z0 is less than or equal to 0.7.

In another preferred embodiment, the seville orange flower active extract has an effect of inhibiting the secretion of inflammatory factors.

In another preferred embodiment, the seville orange flower active extract has an inhibitory effect on the secretion of inflammatory factor IL-6.

In another preferred embodiment, the seville orange flower active extract is in a liquid state.

In a second aspect of the present application, there is provided a method of preparing a seville orange flower active extract, the method comprising the steps of:

(1) Extracting: providing a substituted flower, extracting with ethanol for one or more times, mixing the extractive solutions, and concentrating to obtain a substituted flower extract; and

(2) And (3) purifying resin: loading the extract into macroporous resin, eluting the extract with water and then ethanol, collecting ethanol eluate, and concentrating the ethanol eluate under reduced pressure to obtain extract of the active agent of the instead of the flower.

In another preferred embodiment, the method further comprises the step of (3) freeze-drying the extract of the seville orange flower active substance to obtain freeze-dried powder of the seville orange flower active substance.

In another preferred embodiment, the volume ratio of the seville orange flower to the ethanol in step (1) is 1:5-15.

In another preferred embodiment, in step (1), the concentration of ethanol is 50% v/v to 95% v/v.

In another preferred embodiment, in the step (1), the number of times of ethanol extraction is 1 to 3.

In another preferred embodiment, in the step (1), the extraction time is 1.5h to 2.5h each time.

In another preferred embodiment, in the step (1), the concentration is reduced pressure concentration performed at a certain temperature and vacuum.

In another preferred embodiment, the temperature is 55 to 60 ℃.

In another preferred embodiment, the vacuum degree is 0.05-0.08Mpa.

In another preferred embodiment, in step (2), the macroporous resin is of the type HP20.

In another preferred embodiment, in step (3), the freeze-drying is vacuum freeze-drying.

In another preferred embodiment, in the step (3), the freeze drying time is more than or equal to 24 hours.

In another preferred embodiment, in the step (3), the freezing temperature of the freeze drying is-70 ℃ to-50 ℃.

In another preferred embodiment, in step (3), the vacuum freeze-drying pressure is < 20pa.

In a third aspect of the application there is provided the use of a seville orange flower active extract for the preparation of a product comprising a seville orange flower active extract.

In another preferred embodiment, the product is a pharmaceutical or cosmetic composition that reduces/scavenges DPPH radicals.

In another preferred embodiment, the product is a pharmaceutical or cosmetic composition against ultraviolet damage.

In another preferred example, the product can effectively protect epidermal cells from sunburn, cell activity reduction and epidermal tissue morphology damage caused by ultraviolet radiation.

In another preferred embodiment, the product reduces damage to epidermal cell DNA caused by ultraviolet radiation.

In another preferred embodiment, the product is for inhibiting inflammatory factor secretion.

In another preferred embodiment, the product has an inhibitory effect on the secretion of inflammatory factor IL-6.

In another preferred embodiment, the product is for skin barrier repair.

In another preferred embodiment, the product has an effect on promoting healing of cell scratch damage.

In another preferred embodiment, the cell is a keratinocyte.

In another preferred embodiment, the product has the effect of increasing keratinocyte mobility.

In another preferred embodiment, the daily chemical product is selected from the group consisting of: cosmetic and skin care product.

It is understood that within the scope of the present application, the above-described technical features of the present application and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

Fig. 1 shows the detection spectrum of the extract of the seville orange flower active substance, fig. 1A shows the detection spectrum of naringin and neohesperidin, and fig. 1B shows the detection spectrum of synephrine.

FIG. 2 shows the DPPH radical scavenging test results of the extract of the substituted flowers active substance and the extract of the substituted flowers.

Figure 3 shows the effect of the seville orange flower actives on raw264.7 cell viability.

FIG. 4 shows the inhibition of IL-6 by the substituted flower actives.

Figure 5 shows the effect of the seville orange flower actives on Hacat cell viability.

Figure 6 shows the mobility of the seville flower actives to Hacat cells.

FIG. 7 shows a UVB-EpiKutis histomorphology picture (H & E staining, 40 x magnification).

FIG. 8 shows a UVB-EpiKutis histomorphology (ICH staining, 40 x magnification).

Detailed Description

The inventor of the application, through extensive and intensive research, develops a preparation method of the flower-substituting active substance extract with simple process and environmental protection through a large number of experiments. The application adopts ethanol as an extraction solvent and then uses macroporous resin for purification, can simultaneously enrich three characteristic components in substituted flowers such as naringin, neohesperidin, synephrine and the like, and has the total content not lower than 50 percent.

The application can effectively enrich the characteristic chemical components in the substituted flowers, including naringin, neohesperidin and synephrine by a green, environment-friendly and simple process technology. And the flower-substituting active substance has the effects of resisting oxidation, resisting inflammation, repairing injury and resisting ultraviolet injury in cosmetic application. The present application has been completed on the basis of this finding.

Terminology

As used herein, the term "comprising" or "including" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

Substituted flowers and substituted flower extracts

Dried flower bud of sub-species substituted lime of Citrus aurantium of Citrus subgenera of Rutaceae, also known as bitter orange flower, lime flower and Hui Qing Hua. The main chemical components of the seville orange flower are volatile oil, coumarin, flavone and alkaloids, wherein the main active components are flavone and alkaloid. The flavonoid content is more, mainly is the dihydroflavone such as neohesperidin, naringin, rutin naringin, hesperidin, etc.; the alkaloid contains synephrine as a representative component. Wherein the primary active ingredient

The structures of the naringin, neohesperidin, hesperidin and synephrine are shown as follows:

in the application, the substituted flowers are extracted and concentrated by using an aqueous solvent or an alcohol solvent to obtain the substituted flowers extract.

Preferably, in the present application, the flower substitute raw material is crushed and/or uncrushed.

In the present application, the term "aqueous solvent" refers to a mixed solvent formed from a solvent that is miscible with water (e.g., a C1-C4 alcohol). Preferred alcohols include: methanol, ethanol, propanol, or combinations thereof.

Representative aqueous solvents include (but are not limited to): mixed solvent of water and alcohol such as methanol and/or ethanol in any proportion (e.g. 0.1-99.5% alcohol + balance water). Typically, the aqueous solvent contains 5-90% alcohol and the balance water.

It should be understood that any solvent that can extract the substituted flowers can be used to prepare the substituted flower extract in the present application.

Extract of active matter of seville orange flower

The "substituted flower active extract" in the application can be a substituted flower active extract which is not subjected to freeze drying, and can also be a substituted flower active freeze-dried powder which is subjected to freeze drying.

The naringin content in the extract of the substituted flower active substance prepared by the application is 10-23%, the neohesperidin content is 30-50%, and the synephrine content is 1.5-2.5% based on the dry mass of the extract.

Preferably, the active extract of the seville orange flower simultaneously enriches three characteristic components in seville orange flower such as naringin, neohesperidin and synephrine, and the total content is not less than 50%.

In the application, the extract of the substituted flower active substance has stronger activity of scavenging hydroxyl free radicals. For example, the extract of the present application has a higher hydroxyl radical scavenging activity than an unpurified extract of a seville orange flower.

Therefore, compared with the non-purified substituted flower extract, the substituted flower active extract provided by the application has unexpectedly stronger oxidation resistance, and can be used for various occasions needing oxidation resistance, such as cosmetics, foods, medicines and the like.

In addition, the extract of the substituted flower active substance provided by the application has the effect of inhibiting the secretion of inflammatory factors unexpectedly, so that the extract can be used for various occasions needing anti-inflammatory performance, such as cosmetics, foods, medicines and the like.

Preparation of extract of active substance of seville orange flower

In the application, the substituted flowers are taken as raw materials, ethanol is taken as an extraction solvent, and macroporous resin is used for purification to obtain substituted flower active substance extract, and the preparation method specifically comprises the following steps:

(1) Extracting: providing a substituted flower, extracting with ethanol for one or more times, mixing the extractive solutions, and concentrating to obtain a substituted flower extract;

Preferably, the method further comprises the step of (3) freeze-drying the seville orange flower active extract to obtain seville orange flower active freeze-dried powder.

Preferably, the volume ratio of the substituted flowers to the ethanol in the step (1) is 1:5-15.

Preferably, in step (1), the concentration of ethanol is 50% v/v to 95% v/v.

Preferably, in the step (1), the number of times of ethanol extraction is 1 to 3.

Preferably, in the step (1), the extraction time is 1.5h to 2.5h each time.

In an embodiment of the present application, in step (1), the concentration is reduced pressure concentration performed at a certain temperature and vacuum.

Preferably, the temperature is 55-60 ℃.

Preferably, the vacuum degree is 0.05-0.08Mpa.

Preferably, in step (2), the macroporous resin is of the type HP20.

Preferably, in step (3), the freeze-drying is vacuum freeze-drying.

Preferably, in the step (3), the freeze drying time is more than or equal to 24 hours.

Preferably, in the step (3), the freezing temperature of the freeze drying is-70 ℃ to-50 ℃.

Preferably, in step (3), the vacuum freeze-drying pressure is less than 20pa.

Use of substituted flower actives

The seville orange flower actives of the present application may be used to prepare products containing seville orange flower actives.

In the present application, the product is a pharmaceutical composition or a cosmetic composition for reducing/scavenging DPPH radicals.

In the present application, the product is a pharmaceutical composition or a cosmetic composition against ultraviolet damage.

Preferably, the product can effectively protect epidermal cells from sunburn, cell activity reduction and epidermal tissue morphology damage caused by ultraviolet radiation.

Preferably, the product can reduce the damage of epidermal cell DNA caused by ultraviolet radiation.

In the present application, the product may be used to inhibit inflammatory factor secretion.

Preferably, the product has an inhibitory effect on the secretion of inflammatory factor IL-6.

In the present application, the product is used for skin barrier repair.

Preferably, the product has an effect of promoting healing of cell scratch damage.

Preferably, the cell is a keratinocyte.

Preferably, the product has the effect of increasing keratinocyte mobility.

Preferably, the daily chemical product is selected from the group consisting of: cosmetic and skin care product.

Compared with the prior art, the application has the main advantages that:

1. the content and the yield of naringin, neohesperidin and synephrine in the extract of the substituted flowers active substance prepared by the application are high, and the extract of the substituted flowers active substance simultaneously enriches three characteristic components in the substituted flowers such as naringin, neohesperidin and synephrine, and the total content is not less than 50%.

2. The flower-replacing active substance extract prepared by the application has the effects of better free radical removal, anti-inflammatory, injury repair and ultraviolet injury resistance.

3. The preparation method of the application can obtain the extract of the substituted flower active substance only through simple processes of alcohol extraction, concentration and resin purification.

The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

EXAMPLE 1 preparation of substituted flower actives

In the experiment, the substituted flowers are used as raw materials, ethanol is used as an extraction solvent, and macroporous resin is used for purification to obtain substituted flower active substance extract, and the preparation steps are as follows:

(1) Extracting: mixing the substituted flower raw material (dried flower) with 70% ethanol solution, and extracting under normal pressure and heating and reflux for 2 times, wherein the feed liquid ratio of the first reflux extraction is 1:10, heating at 85 ℃ for 2 hours; the feed liquid ratio of the second reflux extraction is 1:8, heating at 85 ℃ for 1h; filtering the feed liquid by adopting filter cloth to obtain crude extract.

(2) Concentrating: concentrating the crude extract under reduced pressure at 55-60deg.C and vacuum degree of 0.05-0.08MPa to 1.5 times of the feed amount to obtain the extract.

(3) And (3) purifying resin: loading the seville orange flower extract on HP20 macroporous resin, eluting with water eluting volume of 3BV; then 70% ethanol is used for eluting, and the volume of ethanol elution is 3BV. Collecting ethanol eluent, and concentrating under reduced pressure to obtain extract of the active substance of the seville orange flower.

(4) And (3) freeze drying: freeze-drying the extract of the substituted flower active substance for more than 24 hours in a vacuum freeze-dryer, wherein the freeze-drying temperature is (-70 ℃ to-50 ℃) and the pressure is less than 20pa, and preparing the freeze-dried powder of the substituted flower active substance;

the content and the yield of naringin, neohesperidin and synephrine in the prepared extract are shown in table 1, the detection spectrum of the extract is shown in fig. 1, fig. 1A is the detection spectrum of naringin and neohesperidin, and fig. 1B is the detection spectrum of synephrine.

Table 1: content and yield of ingredients in the substituted flower active substance

* The yield is calculated based on the dry weight of the corresponding components in the feed. The naringin content in the flower substitute raw material is as follows: 4.14%, neohesperidin content: 9.09%, and 0.54% of synephrine.

Comparative example 1 preparation of substituted flower extract C1

In this experiment, the experimental method was the same as in example 1, except that: and (3) in the step (1), extracting the seville orange flowers at normal pressure by taking water or ethanol with different concentrations as an extracting agent, concentrating in the step (2) to obtain seville orange flower extract, and obtaining seville orange flower extract freeze-dried powder in the step (4) from the seville orange flower extract.

Wherein the ethanol concentration of the extractant is 50%, 70% and 85%, respectively, and the yields and contents (percentage based on dry weight) of naringin and neohesperidin in the prepared seville orange flower extract are shown in Table 2. The naringin content in the raw materials is as follows: 2.81%, neohesperidin content: 9.88%.

Table 2: content and yield of each component in the substituted flower extract C1

Comparative example 2 preparation of extract C2 of substituted flowers active substance using resin purified substituted flowers extract of different types

In this experiment, the experimental method was the same as in example 1, except that: and (3) purifying the substituted flower extract obtained in the step (2) by using different types of resins, wherein the loading amount and the resin ratio of each group are fixed. The yields and the contents of naringin and neohesperidin in the prepared extract are shown in table 3, and the naringin content in the raw materials is as follows: 2.81%, neohesperidin content: 9.88%.

Table 3: content and yield of each component in the extract C2 of the substituted flower active substance

The resin is preferably: HP20 > HPD300 > AB-8 > D101

Results safety and efficacy testing

1. Phototoxicity test

Phototoxicity test was performed by taking the freeze-dried powder of the substituted flower active substance prepared in example 1.

1.1 Main reagent and instrument

DMEM modified basal medium, new born calf serum, 0.25% trypsin, PBS buffer, neutral red reagent, chlorpromazine hydrochloride, absolute ethanol, acetic acid, etc.

1.2 Test procedure

1.2.1 Cell plating

3T3 cells were grown at 2X 10 ⁴ cell/well inoculation in 96-well plate at 37deg.C, 5% CO ₂ Culturing under the condition.

1.2.2 Adding the test substance

Culturing 24h, adding the solutions of the blank group, positive group, negative group, and sample group, respectively, placing at 37deg.C and 5% CO ₂ Incubation in incubator.

Positive group: samples of different concentrations of chlorpromazine hydrochloride were prepared with PBS buffer.

Sample group: the freeze-dried powder of the seville orange flower active substance is prepared into samples with different concentrations by PBS buffer solution.

Blank group: cell-free multiplexed wells were selected and added to the PBS solution.

Negative control group: multiple wells containing cells were selected and added to the PBS solution.

Test substances with different concentrations were prepared with PBS buffer according to experimental design, and the concentration distribution is shown in Table 4:

table 4: concentration profile of the test substance

1.2.3 Light exposure

After the test object 1h was added, the treatment plate was subjected to light exposure treatment at an irradiation dose of 5J/cm ² The control plate was left to react in the dark at room temperature for the same period of time, after the light exposure was completed, the test solution was removed, 100. Mu.L of DMEM-modified complete medium was added to each well at 37℃with 5% CO ₂ Culturing was continued under the conditions.

1.2.4 Detection of

18-22 h, removing culture medium in the holes, adding 100 μl of neutral red working solution into each hole, standing at 37deg.C and 5% CO ₂ After incubation in an incubator for 3 hours, the neutral red dye solution was removed, 150 μl of neutral red desorption solution was precisely added to each well, and the mixture was shaken on a shaker for 10 min to form a uniform solution, and absorbance was measured at 540nm wavelength.

Calculation of cell viability under treatment of each sample

Fitting a concentration-response curve with the sample concentration as x-axis and the cell viability as y-axis and calculating IC of +Irr group and-Irr group, respectively ₅₀ Reference is made to EU directive EU 67/548/EEC annex VB.41 "in vitro 3T3 neutral Red uptake phototoxicity test method" photo-stimulus factor (PIF) values.

1.2.5 Analysis of results

1.2.5.1 Cell viability

The cell activities of the positive control group and the negative control group are shown in Table 5, and the cell activities of the freeze-dried powder sample group of the substituted flower active substances are shown in Table 6.

Table 5: cell viability of positive control group and negative control group

Table 6: cell viability of freeze-dried powder sample group of substituted flower active substance

1.2.5.2 phototoxicity results evaluation is shown in Table 7.

Table 7: phototoxicity results

As shown in the results of table 7, the freeze-dried powder of the seville orange flower active substance prepared in this experimental example 1 had no toxicity.

2. DPPH radical scavenging test

The freeze-dried powder of the flower-substituted active substance prepared in example 1 was used for DPPH radical scavenging ability test.

2.1 Main reagent and instrument

DPPH (1, 1-diphenyl-2-picrylhydrazine), VC (ascorbic acid), methanol, enzyme-labeled instrument.

2.2 Preparation method

DPPH methanol solution: accurately weighing 7.88 mg of DPPH, fixing the volume to 100 mL by methanol, completely dissolving, and preserving in dark place;

VC solution (0.1 mg/mL): accurately weighing 5 mg of VC, and fixing the volume to 100 mL by methanol to completely dissolve;

sample: the freeze-dried powder of the seville orange flower extract and the freeze-dried powder of the seville orange flower active substance in example 1 were weighed and prepared into a sample solution of 5 mg/mL by methanol.

2.3 Detection flow

Experimental group: samples of 25. Mu.L, 50. Mu.L, 100. Mu.L, 250. Mu.L, 400. Mu.L and 500. Mu.L were taken in deep well plates, distilled water was used to make up to 500. Mu.L, 2mL of DPPH methanol solution was added, mixed well, reacted in the absence of light for 30 min, and absorbance was measured at 520 nm.

Positive control group: respectively taking 25 mu L, 50 mu L, 100 mu L, 250 mu L, 400 mu L and 500 mu L of VC solution in a deep hole plate, supplementing the solution to 500 mu L by distilled water, adding 2mL of DPPH methanol solution, uniformly mixing, carrying out light shielding reaction for 30 min, and measuring the absorbance at 520 and nm.

Blank group: the same volume of sample solution as the experimental group is respectively taken from a deep pore plate, distilled water is added to 500 mu L, 2mL methanol solution is added, the mixture is uniformly mixed, the reaction is carried out for 30 min in a dark place, and the absorbance value is measured at 520 nm.

Control group: 500. mu.L of distilled water was added to 2mL of DPPH methanol solution, and after mixing, the mixture was reacted in a dark place for 30 minutes, and the absorbance was measured at 520. 520 nm.

2.4 Calculation formula

DPPH clearance (%) = (a0—a1+a2)/a0×100% (where A0 is the absorbance of the control group, A1 is the absorbance of the experimental group, and A2 is the absorbance of the blank group)

The results are shown in figure 2, and show that the hydroxy radical scavenging activity of the freeze-dried powder of the seville orange flower active substance prepared by the application is IC ₅₀ IC of the extract of seville orange flower with a value of 148.2. Mu.g/ml ₅₀ IC with a value of 254.4 μg/ml compared to the extract of instead of flowers ₅₀ The activity of the agent is improved by 1.7 times, which indicates that the agent has obvious free radical scavenging effect.

3. Anti-inflammatory efficacy test

The anti-inflammatory efficacy test is carried out on the freeze-dried powder of the substituted flower active substance prepared in the example 1, and the results are shown in fig. 3 and 4.

Sample group: the freeze-dried powder of the seville orange flower active substance is prepared into tested samples with different concentrations by using a basic culture medium.

Blank control group: and selecting a basic culture medium.

Positive control group: dexamethasone was formulated at 100 μg/ml with basal medium.

3.1 Main reagent and instrument

DMEM high sugar medium, fetal bovine serum, MTT, PBS, dexamethasone, and the like.

3.2 Maximum safe concentration determination by MTT method

Raw264.7 cells grown in log phase were grown at 8X 10 ⁴ cell/ml density was inoculated into 96-well plates, a blank control group was set, and at 37℃5% CO ₂ After culturing under the condition of 24h, adding test substances with different concentrations into cells to serve as a sample group, replacing the samples with a culture medium to serve as a negative control group, culturing for 24h, adding 20 mu L of MTT solution into each hole, continuously culturing for 4h, discarding liquid in the holes, adding 150 mu L of DMSO solution into each hole, oscillating for 10 min at room temperature, and measuring the absorbance at 490 nm.

According to the following steps: cell viability% = (sample OD value-blank OD value)/(negative control OD value-blank OD value) ×100%. Cell viability was calculated at each concentration.

3.3 Test procedure

Placing into Raw264.7 cell well plate in logarithmic growth phase, 37 deg.C, 5% CO ₂ After culturing for 24 hours under the condition, adding test substances with different concentrations, setting a negative control group and a positive control group, adding LPS for stimulation after 1 hour, placing the test substances back into an incubator for incubation for 24 hours, respectively collecting cell supernatants corresponding to each sample group, and measuring the concentration of IL-6 in the cell supernatants by using an ELISA kit.

3.4 Experimental results

3.4.1 The MTT assay measures cell viability as shown in FIG. 3. As shown in the figure, the sample concentration is less than or equal to 0.0625%, and the activity of Raw264.7 cells is not obviously affected.

3.4.2 The ELISA method is used for determining the inhibition effect of the frozen powder of the substituted flower active substance on IL-6, as shown in figure 4, compared with a blank control group (BC group), the secretion of the IL-6 is obviously increased (# # P < 0.001) after LPS stimulation (NC group), the concentration of the IL-6 in a positive control group (PC group) and an experimental group is obviously lower than that in the NC group, the secretion of the IL-6 is obviously inhibited by positive control dexamethasone (100 mug/ml) (P < 0.001), and the secretion of the IL-6 can be obviously inhibited/lightened by 0.0625 percent of frozen powder of the substituted flower active substance.

4. Cell scratch repair

In this experiment, the effect of the seville orange flower actives on the rate of scratch healing was examined based on scratch damage combined with LPS-stimulated keratinocytes (Hacat).

Test group 1: the freeze-dried powder of the seville orange flower extract prepared in example 1 was prepared with PBS buffer at 50. Mu.g/mL as a test sample.

Test group 2: the frozen powder of the flower-substituted active substance prepared in example 1 was prepared with PBS buffer at 50. Mu.g/mL as a test sample.

Blank control group: basal medium was added to the group without LPS stimulation.

Negative control group: LPS stimulated group was added to basal medium.

Positive control group: centella asiatica extract was formulated with PBS buffer at 100. Mu.g/mL as positive control.

4.1 Main reagent and instrument

MEM medium, fetal bovine serum (BI), MTT (thiazole blue, solaba), PBS (pH 7.4, BI), lipopolysaccharide (LPS), positive control, etc

4.2 MTT method for detecting cell viability

Hacat cells grown in log phase were grown at 8X 10 ⁴ cell/ml density was inoculated into 96-well plates, a blank control group was set, and at 37℃5% CO ₂ After culturing under the condition of 24h, adding test substances with different concentrations into cells to serve as a sample group, replacing the samples with a culture medium to serve as a negative control group, culturing for 24h, adding 20 mu L of MTT solution into each hole, continuously culturing for 4h, discarding liquid in the holes, adding 150 mu L of DMSO solution into each hole, oscillating for 10 min at room temperature, and measuring the absorbance at 490 nm.

4.3 Experimental procedure

Inoculating Hacat cells in a growth log phase into a 6-well plate, taking out the 6-well plate after 24 hours, discarding the medium in the hole, adding 2mLMEM basic medium into each hole, and scratching by using a transverse line of a gun head perpendicular to the back surface after 24 hours, wherein each hole is scratched by 3 scratches; cells were washed 3 times with PBS, the scraped cell debris was washed clean, then the scratches were recorded by microscopic photography, and 1.99mL of MEM basal medium containing the sample and 10. Mu.L of LPS at a concentration of 200. Mu.g/mL were added to each well with centella asiatica extract as a positive control, 2 multiplex wells per group. Microscopic photographing is carried out 24 hours after dosing, and scratch change conditions are recorded.

4.4 Data processing and analysis

Cell scratches were analyzed using Image Pro Plus, cell scratch areas were calculated for 0h and 24h, and scratch healing rates were calculated according to the formula, comparing the differences in scratch healing rates between groups. The scratch healing rates of each group were collated, plotted using GraphPad prim, and statistically analyzed using T-Test.

4.5 Experimental results

4.5.1 The MTT assay measures cell viability as shown in FIG. 5. As shown in the figure, when the sample concentration is less than or equal to 312.5 mug/ml, the cell viability is basically close to 100%, so that the seville orange flower extract and seville orange flower active substances have no obvious influence on the viability of Hacat cells.

4.5.2 As shown in fig. 6, BC is a blank control group, NC is a negative control group, PC is a positive control group-centella asiatica extract, 1 is a test group 1 generation flower extract lyophilized powder, and 2 is a test group 2 generation flower active substance lyophilized powder.

Cell mobility in the negative control group (NC group, LPS-stimulated) was significantly reduced (#P < 0.05) relative to the blank control group (BC group) based on the scratch damage combined with LPS-stimulated keratinocyte model test; compared with a negative control group (NC), the positive control group (centella asiatica extract) can remarkably improve the mobility of keratinocytes (P is less than 0.01), and the freeze-dried powder of the substituted flower extract (wherein the naringin concentration is 3.83 mug/mL, the neohesperidin concentration is 8.32 mug/mL) and the freeze-dried powder of the substituted flower active substance (wherein the naringin concentration is 9.19 mug/mL and the neohesperidin concentration is 19.71 mug/mL) have remarkably improved cell mobility (P is less than 0.001) at the concentration of 50 mug/mL, so that the modified flower extract has the effect of promoting the healing of scratches and LPS-stimulated keratinocyte models.

5. Efficacy test of 3D epidermis model (UVB-EpiKutis)

5.1 Preparation of 3D epidermis model

Culturing human keratinocytes to a cell density of about 60%, adding the keratinocytes into TU culture medium according to a proper density, culturing in an incubator for 24h, changing liquid, culturing for 4 days by changing TA1 culture solution, culturing by changing TA2 culture solution, and constructing the epidermis model after 8 days.

5.2 Testing

A blank control group (BC), a negative control group (NC), a positive control group (PC) and a sample group (seville orange flower extract lyophilized powder) were respectively set.

The blank control group is: PBS buffer

The concentration of the positive control group was: formulation of VC in PBS buffer ve=100 μg/ml: the concentration of the particles was 7. Mu.g/ml,

the negative control group (NC) was: PBS buffer

The sample group is: the freeze-dried powder of the seville orange flower extract is prepared by PBS buffer solution, and the concentration of the sample is 0.2%.

Each group of samples were sampled at 25. Mu.l, smeared on the surface of the model, and after 24 hours of incubation, the negative control group, the positive control group and the sample group were irradiated with a solar ultraviolet simulator at a dose of 600mJ/cm ² Culturing for 24h after irradiation, and performing histopathological detection on the four groups of 3D skin models after the experiment is finished.

5.3 Correlation index detection

5.3.1 HE staining detection of photodamaged 3D skin pathological tissue

Four groups of skin tissues were fixed with paraformaldehyde, dehydrated with ethanol, transparent with xylene and paraffin-embedded, and then routinely sectioned, stained with hematoxylin-eosin (HE), and finally observed under a microscope for tissue morphology, viable cell layer thickness and sunburn cell number.

5.3.2 IHC for detecting cell DNA damage

Four groups of skin tissues were fixed with paraformaldehyde, dehydrated with ethanol, transparent with xylene and paraffin embedded, and after conventional sections, stained by Immunohistochemistry (IHC), the positive cell rate of cyclobutane thymine dimer (CPD) was observed under a microscope.

5.4 Test results

5.4.1 Tissue morphology, living cell layer thickness, sunburn cell count

As shown in fig. 7, table 8 and table 9, from the aspect of tissue morphology, the NC group living cell layer was reduced in thickness, the cell arrangement was disordered, the four-layer structure was unclear, and sunburn cells were increased after UVB stimulation; the thickness of the living cells in the sample group (the freeze-dried powder of the substituted flower active substance) is increased, the cells are orderly arranged, the four layers of structures are clear, and the sunburn cells are reduced; the results show that the sample group has a certain effect on resisting the UVB-induced epidermal damage.

TABLE 8 thickness of viable cell layer

Remarks: by usingtWhen the test method is used for statistical analysis, the significance of the NC group is represented as # compared to the BC group,Pvalue < 0.05 is expressed as #,Pvalue < 0.01 is denoted as #; compared with the NC group, the significance of the PC group and the sample group is expressed by the x,Pvalue < 0.05 is expressed as,Pvalue < 0.01 is expressed as.

TABLE 9 sunburn cell count

Remarks: by usingtWhen the test method is used for statistical analysis, the significance of the NC group is represented as # compared to the BC group,Pvalue < 0.05 meansIs #,Pvalue < 0.01 is denoted as #; compared with the NC group, the significance of the PC group and the sample group is expressed by the x,Pvalue < 0.05 is expressed as,Pvalue < 0.01 is expressed as.

5.4.2 CPD positive cell rate

As shown in fig. 8 and table 10, from the tissue morphology, the NC group CPD positive cells were stained deeper and the positive cell rate was higher after UVB stimulation; CPD positive cells in the sample group (the freeze-dried powder of the substituted flower active substance) are shallow in staining, the positive cell rate is reduced, and the result shows that the sample group has a certain effect on resisting the UVB-induced DNA damage of the epidermal cells.

TABLE 10 CPD positive cell Rate

Remarks: by usingtWhen the test method is used for statistical analysis, the significance of the NC group is represented as # compared to the BC group,

Pvalue < 0.05 is expressed as #,Pvalue < 0.01 is denoted as #; compared with the NC group, the significance of the PC group and the sample group is expressed by the x,Pvalue < 0.05 is expressed as,Pvalue < 0.01 is expressed as.

Discussion of the application

Patent CN115043889a is a method for extracting synephrine, hesperidin and naringin from seville orange flower, and the technological route of the patent is as follows: enzymolysis to obtain enzymolysis filtrate and enzymolysis filter residue, ethanol extracting the filter residue to obtain crude extract and crude extract, mixing the enzymolysis filtrate and the crude extract, and ultrafiltering to obtain permeate and retentate; purifying the permeate by ion exchange resin and silica gel to obtain synephrine; crystallizing the trapped fluid to obtain naringin; extracting the crude extract residue with alkali water, and crystallizing to obtain hesperidin. The process flow is complex, the alkali used in the extraction process is not environment-friendly, the obtained component has no test method, and the data reliability is not high.

Patent CN110496163a is a preparation method of effective part or component of flower bud or flower or branch or leaf or fruit of citrus plant and its application in preparing medicine, and the technological route of the patent is: distilling volatile oil by steam method, extracting residue with (water-ethanol), purifying with macroporous resin, and eluting with different ethanol concentrations. The resin type used is macroporous resin, and the resin type is AB-8, D101 and HPD400. The obtained product contains naringin, the detection method is not described, and the obtained component is uncontrollable.

The application can obtain the substituted flower active extract by simple processes of alcohol extraction, concentration and resin purification, and the obtained substituted flower active extract is enriched with three characteristic components in substituted flowers such as naringin, neohesperidin and synephrine, the total content is not less than 50%, and the content yields of naringin, neohesperidin and synephrine are high. In addition, the extract of the substituted flower active substance prepared by the application has better effects of free radical scavenging, anti-inflammatory and anti-ultraviolet injury. The application can utilize the substituted flowers in a high-valued way, promote the market application of products downstream of the substituted flowers, increase the income for the planting of the substituted flowers and have good social and economic benefit prospects.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

1. The extract is characterized by comprising naringin, neohesperidin and synephrine, wherein the extract is prepared by taking seville orange flower or seville orange flower extract as a raw material, extracting with ethanol and purifying with resin; wherein, the content of naringin in the extract of the substituted flower active substance is 10-23%, the content of neohesperidin is 30-50%, and the content of synephrine is 1.5-2.5% based on the dry mass of the extract.

2. The seville orange flower active extract of claim 1, wherein the seville orange flower active extract has an IC of hydroxy radical scavenging activity ₅₀ The value is less than or equal to 160 mug/ml.

3. The seville orange flower active extract of claim 1, wherein the seville orange flower active extract has an IC of hydroxy radical scavenging activity ₅₀ IC of value Z1 and substituted flower extract ₅₀ The ratio of the values Z0, i.e. Z1/Z0 is less than or equal to 0.7.

4. A method for preparing the extract of flower-replacing active substances according to claim 1, wherein the method comprises the steps of:

(1) Extracting: providing a substituted flower, extracting with ethanol for one or more times, combining the extracting solutions, and concentrating to obtain a substituted flower extract, wherein the extracting time is 1.5-2.5 h each time; and

(2) And (3) purifying resin: loading the seville orange flower extract obtained in the step (1) into macroporous resin, eluting the seville orange flower extract with water and then ethanol, collecting ethanol eluent, and concentrating the ethanol eluent under reduced pressure to obtain seville orange flower active extract, wherein the model of the macroporous resin is HP20; the content of naringin in the extract of the substituted flower active substance is 10-23%, the content of neohesperidin is 30-50%, and the content of synephrine is 1.5-2.5% based on the dry mass of the extract.

5. The method of claim 4, wherein the volume ratio of said seville orange flower to said ethanol in step (1) is 1:5-15.

6. The process according to claim 4, wherein in step (1), the concentration of ethanol is 50% v/v to 95% v/v.

7. Use of a seville orange flower active extract according to claim 1 for the preparation of a product containing seville orange flower active extract.

8. The use according to claim 7, wherein the product is a pharmaceutical or cosmetic composition for reducing/scavenging DPPH radicals.

9. Use according to claim 7, wherein the product is a pharmaceutical or cosmetic composition against ultraviolet damage.

10. The use according to claim 7, wherein the product is a pharmaceutical or cosmetic composition for inhibiting secretion of inflammatory factors.