A composition for scavenging free radicals, its preparation method and application
By combining extracts of blueberry, raspberry, plum, and honeysuckle, the problem of excessive antioxidant usage and insufficient activity in existing technologies is solved, achieving a highly efficient free radical scavenging effect.
Patent Information
- Application Number
- CN202411143253.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-20
- Publication Date
- 2025-12-02
- Estimated Expiration
- 2044-08-20
AI Technical Summary
Existing technologies make it difficult to achieve both high antioxidant activity and reduced dosage by combining natural extracts from different sources.
An antioxidant composition with synergistic effects was prepared by combining extracts of blueberry, raspberry, plum and honeysuckle in a specific ratio and purifying them by ultrasonic-assisted extraction and column chromatography.
It enhances the ability to scavenge DPPH free radicals, hydroxyl free radicals and lipid peroxidation free radicals, thereby achieving enhanced antioxidant activity and reducing the dosage required.
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Figure CN119015345B_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of natural product extraction and application technology, specifically relating to a composition for scavenging free radicals, its preparation method, and its application. Background Technology
[0002] With the accelerated pace of modern life and complex changes in environmental factors, the human body is constantly attacked by free radicals generated by various external environments and internal metabolic processes. Excessive accumulation of these free radicals can lead to cell damage, accelerate the aging process, and is closely related to the development of various chronic diseases, including cardiovascular disease, cancer, and neurodegenerative diseases. Therefore, finding effective free radical scavengers to maintain the body's redox balance has become a key focus of research in the biomedical and health fields in recent years.
[0003] In recent years, the scientific community has begun to focus on the synergistic effect of multiple antioxidants, that is, by combining natural extracts from different sources, the aim is to achieve the superposition and complementarity of antioxidant effects, thereby enhancing the overall antioxidant activity and reducing the amount used. Summary of the Invention
[0004] The purpose of this invention is to provide an antioxidant composition that solves the technical problem of achieving high antioxidant activity against different antioxidant systems while reducing the dosage.
[0005] This invention provides a composition for scavenging free radicals, the formulation comprising the following mass ratio: bilberry (… Water-soaked cranberry L.) extract: raspberry ( Rubus idaeus L.) extract: Prunus armeniaca ( Prunus humble Extract: Honeysuckle fruit ( Blue honeysuckle L.) extract = (1-15): (1-15): (1-15): (1-15): (1-15).
[0006] Further specifying, the formulation consists of the following mass ratio: blueberry extract: raspberry extract: plum extract: honeysuckle extract = 5:8.2:5:3.5.
[0007] To further specify, the method for obtaining the Prunus armeniaca extract is as follows:
[0008] Step 1: Sample extraction method
[0009] After crushing the fruit of Prunus cerasifera, ultrasonic-assisted extraction was performed. The extraction solvent was 70% ethanol (v / v), the material-to-liquid ratio was 1:10 (g:mL), the extraction temperature was set at 40 ℃, the extraction power was 150 W, the extraction time was 30 min, and the extraction was performed twice. The extracts were combined, concentrated under reduced pressure at 48 ℃, and then freeze-dried to obtain crude extract of Prunus cerasifera for later use.
[0010] Step 2: Sample purification method
[0011] The crude extract of *Prunus armeniaca* obtained in step 1 was subjected to column chromatography with XDA-6 resin as the stationary phase. The column was 200 cm high and 10 cm in inner diameter, with a diameter-to-height ratio of 1:10 to 1:15. The loading concentration, calculated as polyphenols, was 10-50 mg / mL, and the loading volume was 1 / 5 to 2 / 5 of the column volume. After loading, the sample was allowed to stand for 30-60 min. Gradient elution was then performed sequentially using 2VB column volumes of distilled water, 1VB column volumes of 10% ethanol aqueous solution, 3VB column volumes of 40% ethanol aqueous solution, and 1VB column volumes of 70% ethanol aqueous solution as mobile phases at a flow rate of 10-15 mL / min. The eluents of different ethanol concentrations were collected, concentrated under reduced pressure, and then frozen under vacuum to obtain lyophilized *Prunus armeniaca* extract powder.
[0012] Methods for obtaining raspberry extract:
[0013] S1: After crushing the raspberry fruit, the raspberry was extracted using an ultrasonic-assisted extraction process. The extraction solvent was 70% ethanol by volume, the material-to-liquid ratio was 1:10 (g:mL), the extraction temperature was set at 40 ℃, the extraction power was 150 W, the extraction time was 30 min, and the extraction was performed twice. The extracts were combined, concentrated under reduced pressure at 48 ℃, and then freeze-dried to obtain crude raspberry extract for later use.
[0014] S2: Sample purification method
[0015] The crude raspberry extract obtained in step S1 was subjected to column chromatography with XDA-6 resin as the stationary phase. The column was 200 cm high and 10 cm in inner diameter, with a diameter-to-height ratio of 1:10 to 1:15. The loading concentration, calculated as polyphenols, was 10-50 mg / mL, and the loading volume was 1 / 5 to 2 / 5 of the column volume. After loading, the sample was allowed to stand for 30-60 min. Gradient elution was then performed sequentially using 2VB column volumes of distilled water, 1VB column volumes of 10% ethanol aqueous solution, 3VB column volumes of 40% ethanol aqueous solution, and 1VB column volumes of 70% ethanol aqueous solution as the mobile phase at a flow rate of 10-15 mL / min. The eluents of different ethanol concentrations were collected, concentrated under reduced pressure, and then frozen under vacuum to obtain lyophilized raspberry extract powder.
[0016] Further specifying the method for obtaining honeysuckle berry extract:
[0017] Step 1: Sample extraction method
[0018] After crushing the honeysuckle fruit, it was extracted using an ultrasonic-assisted extraction process. The extraction solvent was 70% ethanol (v / v), the solid-liquid ratio was 1:10 (g:mL), the extraction temperature was set at 40 ℃, the extraction power was 150 W, the extraction time was 30 min, and the extraction was performed twice. The extracts were combined, concentrated under reduced pressure at 48 ℃, and then freeze-dried to obtain crude honeysuckle fruit extract for later use.
[0019] Step 2: Sample purification method
[0020] The crude extract of honeysuckle fruit obtained in step 1 was subjected to column chromatography with XDA-6 resin as the stationary phase medium. The column was 200 cm high and 10 cm in inner diameter, with a diameter-to-height ratio of 1:10 to 1:15. The loading concentration, calculated as anthocyanins, was 10-50 mg / mL, and the loading volume was 1 / 5-2 / 5 of the column volume. After loading, the sample was allowed to stand for 30-60 min. Gradient elution was then performed sequentially using 2VB column volumes of distilled water, 1VB column volumes of 10% ethanol aqueous solution, 3VB column volumes of 40% ethanol aqueous solution, and 1VB column volumes of 70% ethanol aqueous solution as mobile phases at a flow rate of 10-15 mL / min. The eluents of different concentrations of ethanol were collected, concentrated under reduced pressure, and then frozen under vacuum to obtain lyophilized honeysuckle fruit extract powder.
[0021] Methods for obtaining blueberry extract:
[0022] S1: After crushing the blueberry fruit, the extraction was carried out using an ultrasonic-assisted extraction process. The extraction solvent was 70% ethanol by volume, the solid-liquid ratio was 1:10 (g:mL), the extraction temperature was set at 40 ℃, the extraction power was 150 W, the extraction time was 30 min, and the extraction was carried out twice. The extracts were combined, concentrated under reduced pressure at 48 ℃, and then freeze-dried to obtain the crude blueberry extract for later use.
[0023] S2: Sample purification method
[0024] The crude extract of Bilberry obtained in step S1 was subjected to column chromatography with XDA-6 resin as the stationary phase. The column was 200 cm high and 10 cm in inner diameter, with a diameter-to-height ratio of 1:10 to 1:15. The loading concentration, calculated as anthocyanins, was 10-50 mg / mL, and the loading volume was 1 / 5-2 / 5 of the column volume. After loading, the sample was allowed to stand for 30-60 min. Gradient elution was then performed sequentially using 2VB column volumes of distilled water, 1VB column volumes of 10% ethanol aqueous solution, 3VB column volumes of 40% ethanol aqueous solution, and 1VB column volumes of 70% ethanol aqueous solution as mobile phases at a flow rate of 10-15 mL / min. The eluents of different concentrations of ethanol were collected, concentrated under reduced pressure, and then frozen under vacuum to obtain lyophilized Bilberry extract powder.
[0025] This invention provides the use of the above-described composition in the preparation of a drug for scavenging free radicals.
[0026] Further specifying, the free radical is any one or more of DPPH, hydroxyl radical and lipid peroxidation LOP.
[0027] Further specifying, the dosage form of the drug includes any one of tablets, capsules, granules, powders, liquid preparations, and pills.
[0028] Beneficial effects: Bilberry ( Water-soaked cranberry L.) extract: raspberry ( Rubus idaeus L.) extract: Prunus armeniaca ( Prunus humilis Extract: Honeysuckle fruit ( Blue honeysuckle L.) extract = (1-15): (1-15): (1-15): (1-15). After appropriate compatibility, it can not only improve the ability to scavenge DPPH free radicals, but also enhance the ability to scavenge hydroxyl free radicals and lipid peroxidation free radicals of different antioxidant systems. This indicates that the combination of these fruit extracts has a synergistic effect on antioxidant activity. Attached Figure Description
[0029] Figure 1 Graphs showing the effect of DPPH free radical scavenging on monomers and optimized combinations;
[0030] Figure 2 The result graph shows the concentration range of the composition;
[0031] Figure 3 The graph shows the scavenging effects of single components and their combinations on different free radical systems. Detailed Implementation
[0032] Example 1. Method for extracting berry extract
[0033] 1. Sample extraction method
[0034] Fresh fruits of four berries (bilberry, honeysuckle berry, European plum, and raspberry) were directly crushed and then extracted using an ultrasonic-assisted extraction process. The extraction solvent was 70% ethanol (v / v), the solid-liquid ratio was 1:10 (g:mL), the extraction temperature was set at 40 ℃, the extraction power at 150 W, and the extraction time at 30 min. Extraction was performed twice, and the extracts were combined. The extract was concentrated under reduced pressure at 48 ℃ and then freeze-dried to obtain the crude fruit extract for later use.
[0035] 2. Sample purification methods
[0036] (1) Prunus cerasifera polyphenol extract: The crude extract of Prunus cerasifera obtained in step 1 above was subjected to column chromatography with XDA-6 resin as the stationary phase and different concentrations of ethanol-water solvent as the eluent, using a gradient elution method. The chromatography column was 200 cm high and 10 cm in inner diameter, with a diameter-to-height ratio of 1:10 to 1:15. The loading concentration, calculated as polyphenols, was 10-50 mg / mL, and the loading volume was 1 / 5-2 / 5 of the column volume. After loading, the column was allowed to stand for 30-60 min. Gradient elution was performed sequentially using 2VB column volumes of distilled water, 1VB column volumes of 10% ethanol-water solution, 3VB column volumes of 40% ethanol-water solution, and 1VB column volumes of 70% ethanol-water solution as the mobile phase, with a flow rate of 10-15 mL / min. The ethanol eluents of different concentrations were collected, concentrated under reduced pressure, and frozen under vacuum to obtain lyophilized Prunus cerasifera polyphenol extract powder.
[0037] (2) Raspberry polyphenol extract: The crude raspberry extract obtained in step 1 above was subjected to column chromatography with XDA-6 resin as the stationary phase and different concentrations of ethanol-water solvent as the eluent, using a gradient elution method. The chromatography column was 200 cm high and 10 cm in inner diameter, with a diameter-to-height ratio of 1:10 to 1:15. The loading concentration, calculated as polyphenols, was 10-50 mg / mL, and the loading volume was 1 / 5-2 / 5 of the column volume. After loading, the sample was allowed to stand for 30-60 min. Gradient elution was performed sequentially using 2VB column volumes of distilled water, 1VB column volumes of 10% ethanol-water solution, 3VB column volumes of 40% ethanol-water solution, and 1VB column volumes of 70% ethanol-water solution as the mobile phase, with a flow rate of 10-15 mL / min. The ethanol eluents of different concentrations were collected, concentrated under reduced pressure, and frozen under vacuum to obtain lyophilized raspberry polyphenol extract powder.
[0038] (3) Honeysuckle berry anthocyanin extract: The crude honeysuckle berry extract obtained in step 1 above was subjected to column chromatography with XDA-6 resin as the stationary phase and different concentrations of ethanol-water solvent as the eluent, using a gradient elution method. The chromatography column was 200 cm high and 10 cm in inner diameter, with a diameter-to-height ratio of 1:10 to 1:15. The loading concentration, calculated as anthocyanins, was 10-50 mg / mL, and the loading volume was 1 / 5-2 / 5 of the column volume. After loading, the column was allowed to stand for 30-60 min. Gradient elution was performed sequentially using 2VB column volumes of distilled water, 1VB column volumes of 10% ethanol-water solution, 3VB column volumes of 40% ethanol-water solution, and 1VB column volumes of 70% ethanol-water solution as the mobile phase, with a flow rate of 10-15 mL / min. The ethanol eluents of different concentrations were collected, concentrated under reduced pressure, and frozen under vacuum to obtain lyophilized honeysuckle berry anthocyanin extract powder.
[0039] (4) Vaccinium buergerianum anthocyanin extract: The crude Vaccinium buergerianum extract obtained in step 1 above was subjected to column chromatography with XDA-6 resin as the stationary phase and different concentrations of ethanol-water solvent as the eluent, using a gradient elution method. The chromatography column was 200 cm high and 10 cm in inner diameter, with a diameter-to-height ratio of 1:10 to 1:15. The loading concentration, calculated as anthocyanins, was 10-50 mg / mL, and the loading volume was 1 / 5-2 / 5 of the column volume. After loading, the column was allowed to stand for 30-60 min. Gradient elution was then performed sequentially using 2VB column volumes of distilled water, 1VB column volumes of 10% ethanol-water solution, 3VB column volumes of 40% ethanol-water solution, and 1VB column volumes of 70% ethanol-water solution as the mobile phase, with a flow rate of 10-15 mL / min. The eluents of different concentrations of ethanol were collected, concentrated under reduced pressure, and then frozen under vacuum to obtain lyophilized Vaccinium buergerianum anthocyanin extract powder.
[0040] 3. Detection of anthocyanin content
[0041] Anthocyanin content was detected using a pH differential method. Preparation of the standard curve: 10.0 mg of cyanidin-3-glucoside standard was taken and diluted to 100 mL with 70% ethanol solution, and mixed thoroughly to obtain a 100 ug / mL cyanidin-3-glucoside standard solution. 1.0 mL of the standard solution was serially diluted with 70% ethanol solution to prepare working solutions of 0, 10, 20, 30, 40, and 50 ug / mL. Two 1.0 mL aliquots of the standard solution were transferred and diluted to 10.0 mL with buffer solutions of pH 1.0 and pH 4.5 respectively. After standing in the dark for 110 min to reach equilibration, the absorbance of the samples was measured at 525 nm and 700 nm. The absorbance was calculated using formula (2-1). With absorbance as the ordinate and cyanidin-3-glucoside content as the abscissa, the anthocyanin standard curve equation was obtained by regression: y = 0.0054x - 0.0009, R 2 =0.9986.
[0042] A=(A525nm–A700nm)pH1.0–(A525nm–A700nm)pH4.5 (1-1)
[0043] Sample determination: Weigh 0.1000 g of lyophilized powder of crude berry extract and anthocyanin extract respectively, dissolve in 10.0 mL of distilled water to prepare a 10 mg / mL solution, accurately transfer 1.0 mL of sample solution to replace the standard solution and perform 3 parallel tests according to the above operation, determine the content according to the standard curve, calculate the anthocyanin content according to formula (1-2), calculate the anthocyanin purity according to formula (1-3), and express the results as average values.
[0044] Anthocyanin content (mg / g) = (C×DF) / (M×1000) (1-2)
[0045] In equation (1-2):
[0046] C — Anthocyanin content (ug / g) obtained from the standard curve.
[0047] DF – the dilution factor of the solution
[0048] M — Sample mass (g)
[0049] 1000 – This is the conversion factor for converting the content from ug to mg.
[0050] Anthocyanin purity (%) = anthocyanin content × 1000 / sample mass × 100 (1-3)
[0051] 1000 – This is the conversion factor for converting the content from mg to g.
[0052] 100 — Anthocyanin content in 100g of sample
[0053] 4. Detection of polyphenol content
[0054] Weigh 0.1000 g of the lyophilized powder of each berry crude extract and polyphenol extract, dissolve it in 10.0 mL of distilled water, and set aside. The total polyphenol content was determined using the Folin-Ciocalteu method.
[0055] Accurately weigh 0.110 g of gallic acid. First, add a small amount of distilled water until the gallic acid is completely dissolved. Then, add distilled water to bring the volume to 1000 mL and mix well to obtain a gallic acid standard solution with a concentration of 100 μg / mL. Measure 0, 0.20, 0.40, 0.60, 0.80, 1.00, and 1.2 mL of this standard solution into 10 mL colorimetric tubes. Add 5 mL of distilled water, shake well, and then add 1.0 mL of Folin-phenol reagent. After 4 min, add 3.0 mL of 7.5% sodium carbonate solution. Incubate at 25 ℃ for 2 h and measure the absorbance of the solution at 765 nm (A). 765 Three parallel experiments were conducted, and a standard curve was plotted with absorbance as the ordinate and gallic acid content (μg) as the abscissa. The regression equation for the polyphenol standard curve was: y = 0.0027x + 0.0257, R0 2 =0.9979.
[0056] The sample determination was performed according to the above method, repeated three times. The total polyphenol content was calculated as the gallic acid equivalent per gram of sample.
[0057] The calculation formula is as follows:
[0058] Polyphenol content (mg / g) = (C×DF) / (M×1000) (2-1)
[0059] In the formula:
[0060] C represents the content (μg) obtained from the standard curve;
[0061] M: Sample mass (g);
[0062] DF – the dilution factor of the solution
[0063] 1000 – is the conversion factor for converting the content from ug to mg.
[0064] Polyphenol purity (%) = polyphenol content × 1000 / sample mass × 100 (2-2)
[0065] 1000 – This is the conversion factor for converting the content from mg to g.
[0066] 100 — the polyphenol content in 100 g of sample
[0067] The 40% ethanol aqueous solution eluent, which yielded the largest amount and highest purity of anthocyanins (polyphenols), was used as the research subject for compounding. The anthocyanin purity of Bilberry (D) was 54.63%, that of Honeysuckle berry (L) was 52.63%, that of Prunus persica polyphenols (O) was 33.95%, and that of Raspberry polyphenols (S) was 42.30%.
[0068] Example 2. Method for preparing the complex
[0069] Four extract combinations with different ratios that synergistically enhance antioxidant effects were obtained through compounding studies. The results are shown in Table 2. The table shows the IC50 values. 50 The smaller the value, the stronger the antioxidant capacity; the smaller the CI value, the better the synergistic effect.
[0070] The ratio of Prunus armeniaca extract to Cassia alatus extract to Lonicera japonica extract to Raspberry extract is 10:12:1:9. The actual concentrations used are shown in Table 2.
[0071] Example 3.
[0072] The ratio of Prunus armeniaca extract to Cassia alatus extract to Lonicera japonica extract to Raspberry extract is 14:10:6:2. The actual concentrations used are shown in Table 2.
[0073] Example 4.
[0074] The ratio of Prunus armeniaca extract to Cassia alatus extract to Lonicera japonica extract to Raspberry extract is 12:14:11:11. The actual concentrations used are shown in Table 2.
[0075] Example 5.
[0076] The ratio of Prunus armeniaca extract to Cassia alatus extract to Lonicera japonica extract to Raspberry extract is 6:11:9:15. The actual concentrations used are shown in Table 2.
[0077] Example 6.
[0078] The ratio of Prunus armeniaca extract to Cassia alatus extract to Lonicera japonica extract to Raspberry extract is 2:13:14:4. The actual concentrations used are shown in Table 2.
[0079] Example 7.
[0080] The ratio of Prunus armeniaca extract to Cassia alatus extract to Lonicera japonica extract to Raspberry extract is 3:9:7:12. The actual concentrations used are shown in Table 2.
[0081] Example 8.
[0082] The ratio of Prunus armeniaca extract to Cassia alatus extract to Lonicera japonica extract to Raspberry extract is 7:15:4:6. The actual concentrations used are shown in Table 2.
[0083] Example 9.
[0084] The ratio of Prunus armeniaca extract to Cassia alatus extract to Lonicera japonica extract to Raspberry extract is 8:4:10:1. The actual concentrations used are shown in Table 2.
[0085] Example 10.
[0086] The ratio of Prunus armeniaca extract to Cassia alatus extract to Lonicera japonica extract to Raspberry extract is 1:5:5:10. The actual concentrations used are shown in Table 2.
[0087] Example 11.
[0088] The ratio of Prunus armeniaca extract to Cassia alatus extract to Lonicera japonica extract to Raspberry extract is 15:8:13:7. The actual concentrations used are shown in Table 2.
[0089] Example 12.
[0090] The ratio of Prunus armeniaca extract to Cassia alatus extract to Lonicera japonica extract to Raspberry extract is 9:6:15:13. The actual concentrations used are shown in Table 2.
[0091] Example 13.
[0092] The ratio of Prunus armeniaca extract to Cassia alatus extract to Lonicera japonica extract to Raspberry extract is 11:1:8:5. The actual concentrations used are shown in Table 2.
[0093] Example 14.
[0094] The ratio of Prunus armeniaca extract to Cassia alatus extract to Lonicera japonica extract to Raspberry extract is 5:7:2:3. The actual concentrations used are shown in Table 2.
[0095] Example 15.
[0096] The ratio of Prunus armeniaca extract to Cassia alatus extract to Lonicera japonica extract to Raspberry extract is 13:3:3:14. The actual concentrations used are shown in Table 2.
[0097] Example 16.
[0098] The ratio of Prunus armeniaca extract to Cassia alatus extract to Lonicera japonica extract to Raspberry extract is 4:2:12:8. The actual concentrations used are shown in Table 2.
[0099] Example 17.
[0100] The ratio of Prunus armeniaca extract to Cassia alatus extract to Lonicera japonica extract to Raspberry extract is 5:8.2:5:3.5. The actual concentrations used are shown in Table 2.
[0101] Detection methods for DPPH, hydroxyl radicals, and lipid peroxidation radicals:
[0102] 1. Weigh 0.1000 g of the positive control vitamin C powder and the lyophilized powder of four kinds of crude berry extracts and extracts, dissolve them in 10.0 mL of distilled water to prepare a 10 mg / mL solution for later use.
[0103] The method for DPPH free radical scavenging rate involves accurately weighing DPPH reagent powder and preparing an 8.62 × 10⁻² mmol / L DPPH ethanol solution in a brown bottle immediately before use. Add 2.0 mL of DPPH reagent and 2.0 mL of different test sample solutions to a test tube, mix thoroughly, and incubate in the dark for 30 min. Measure the absorbance value A1 of the mixture at a wavelength of 517 nm. Repeat the same procedure, replacing the test sample with 2.0 mL of ethanol and measuring the absorbance value A0. Alternatively, without DPPH solution, replace the test sample with 2.0 mL of ethanol and add different concentrations of test sample solutions, measuring the absorbance value A2 using the same procedure. Each test sample is tested in triplicate, with ascorbic acid as a positive control. The DPPH free radical scavenging ability of the sample is calculated using the following formula:
[0104] Clearance rate (%) = ;
[0105] The method for hydroxyl radical scavenging involves mixing 1.0 mL of the test sample solution with 0.2 mL of 20 mmol / L sodium salicylate solution, totaling 3.0 mL. After thorough mixing, the mixture is incubated at 37 °C for 1 h. The absorbance (A1) of the mixture is then measured at 510 nm. The 1.0 mL of the test sample solution is replaced with distilled water, and the absorbance (A0) is measured using the same method. The 0.2 mL of sodium salicylate solution is replaced with the same volume of distilled water, and the absorbance (A2) is measured using the same method. Each test sample is tested in triplicate, with ascorbic acid as a positive control. The result is calculated using the following formula.
[0106] Clearance rate (%) = ;
[0107] Methods for determining lipid peroxidation free radicals
[0108] The detection was performed using a lipid peroxidation kit (LOP). The calculation formula is as follows:
[0109] Clearance rate (%) = .
[0110] 2. Synergistic Effect Analysis Method
[0111] Synergy analysis is based on the Chou-Talalay method, and the metric used is the Combination Index (CI), calculated as follows:
[0112] CI= (1)
[0113] In the formula:
[0114] D1 and D2 represent the effect levels achieved (e.g., 50% inhibition rate, IC50, etc.). 50 or EC 50 When using compound A and compound B in combination therapy, the actual concentrations of these compounds were determined using the IC50 assay in this experiment. 50 .
[0115] (Dx)1 and (Dx)2 are the concentrations required to achieve the same effect level when compound A and compound B are used alone, respectively.
[0116] CI<1: indicates synergism.
[0117] CI = 1: indicates additive effect;
[0118] CI>1: indicates an antagonistic effect.
[0119] 3.IC 50 value
[0120] IC calculation using GraphpadPrism 8.2.0 software 50 value
[0121] Table 1. Synergistic effect of four berry extract combinations in scavenging DPPH free radicals.
[0122]
[0123] The optimal combination can be obtained by constraining the objective function to a maximum clearance rate of 100%.
[0124] The results are shown in Table 1. The four berry extracts, when combined in different proportions, showed a synergistic effect in scavenging DPPH free radicals, and the scavenging ability varied with the different proportions.
[0125] Depend on Figure 1 It is evident that, among the four berry anthocyanins and polyphenols, any single extract (D, L, S, O) exhibits a lower IC50 value for scavenging DPPH free radicals. 50 The values were all significantly higher than those of the vitamin C positive control group, indicating that the antioxidant activity of the single-component compounds was not as good as that of vitamin C; after optimization of the combination (combinations 1, 2, and 7 in Table 1, which are the preferred combinations in Table 1), the IC50 values of each group were significantly higher than those of the vitamin C positive control group, indicating that the antioxidant activity of the single-component compounds was not as good as that of vitamin C; 50 The values were all significantly lower than those in the vitamin C-positive control group, indicating that the antioxidant activity was enhanced after the optimized combination; and the optimal combination selected by optimization had an IC50 value. 50 The concentration of the target (0.0206 mg / mL) was significantly lower than that of the other combinations (groups 1-15), indicating that after optimization, a lower dose can be used to achieve better results.
[0126] Depend on Figure 2 It is evident that within the range of 0-0.30 mg / mL, the ability of the compound combinations (combinations 1, 2, and 7 in Table 1) to scavenge DPPH free radicals increased with increasing sample concentration. When the sample concentration reached 0.24 mg / mL, the DPPH free radical scavenging ability of each group exceeded 90%, and when it reached 0.30 mg / mL, the DPPH free radical scavenging ability of each group approached 100%. Since theoretically, the maximum scavenging rate of the sample against DPPH free radicals is 100%, further increasing the concentration has no effect on increasing the scavenging rate and only wastes raw materials. However, below 0.01 mg / mL, the scavenging rate of the compound combinations against free radicals was below 20%, and the scavenging effect was not significant. Therefore, it is recommended that the compound concentration be 0.01~0.30 mg / mL.
[0127] With a fixed composition concentration of 0.24 mg / mL (of which, the preferred combination concentration in Table 1 is 0.217 mg / mL), the preferred combination was studied for scavenging hydroxyl radicals (·OH) and resisting lipid peroxidation (LOP) to analyze the ability of the compound combination to scavenge free radicals in different antioxidant systems.
[0128] Depend on Figure 3 It is evident that, after formulation and combination, the ability of each group (combinations 1, 2, and 7 in Table 1, representing the preferred combinations in Table 1) to scavenge hydroxyl radicals and lipid peroxidation radicals, while slightly weaker than that of vitamin C, was significantly superior to that of the individual components of the four berries. This indicates that appropriate formulation not only enhances the ability to scavenge DPPH radicals but also strengthens the scavenging ability of hydroxyl radicals and lipid peroxidation radicals from different antioxidant systems. This demonstrates that the purified active substances from these fruits, when combined, have a synergistic effect on antioxidant activity.
[0129] Comparative Example 1.
[0130] Prunus cerasifera extract: Raspberry extract = 1:1, concentrations are shown in Table 3.
[0131] Comparative Example 2.
[0132] The ratio of Prunus armeniaca extract to Cunninghamia lanceolata extract is 1:1, and the concentrations are shown in Table 3.
[0133] Comparative Example 3.
[0134] The ratio of Prunus armeniaca extract to Bilberry extract is 1:1, and the concentrations are shown in Table 3.
[0135] Comparative Example 4.
[0136] 1. Sample extraction method
[0137] Fresh cranberry berries were directly crushed and then extracted using an ultrasonic-assisted extraction process. The extraction solvent was 70% ethanol (v / v), the solid-liquid ratio was 1:10 (g:mL), the extraction temperature was set at 40 ℃, the extraction power at 150 W, and the extraction time at 30 min. Extraction was performed twice, and the extracts were combined. The extract was concentrated under reduced pressure at 48 ℃ and then freeze-dried to obtain crude cranberry extract for later use.
[0138] 2. Sample purification methods
[0139] Cranberry polyphenol extract: The crude cranberry extract obtained in step 1 above was subjected to column chromatography with XDA-6 resin as the stationary phase and different concentrations of ethanol-water solvent as the eluent, using a gradient elution method. The chromatography column was 200 cm high and 10 cm in inner diameter, with a diameter-to-height ratio of 1:10 to 1:15. The loading concentration, calculated as polyphenols, was 10-50 mg / mL, and the loading volume was 1 / 5-2 / 5 of the column volume. After loading, the sample was allowed to stand for 30-60 min. Gradient elution was then performed sequentially using 2VB column volumes of distilled water, 1VB column volumes of 10% ethanol-water solution, 3VB column volumes of 40% ethanol-water solution, and 1VB column volumes of 70% ethanol-water solution as the mobile phase, at a flow rate of 10-15 mL / min. The eluents of different concentrations of ethanol were collected, concentrated under reduced pressure, and then frozen under vacuum to obtain lyophilized cranberry polyphenol extract powder.
[0140] The ratio of Prunus armeniaca extract to cranberry extract is 1:1, and the concentrations are shown in Table 3.
[0141] Comparative Example 5.
[0142] The ratio of Prunus armeniaca extract to Lonicera japonica extract is 1:1, and the concentrations are shown in Table 3.
[0143] Comparative Example 6.
[0144] 1. Sample extraction method
[0145] Fresh blackcurrant fruits were directly crushed and then extracted using an ultrasonic-assisted extraction process. The extraction solvent was 70% ethanol (v / v), the solid-liquid ratio was 1:10 (g:mL), the extraction temperature was set at 40 ℃, the extraction power at 150 W, and the extraction time at 30 min. Extraction was performed twice, and the extracts were combined. The extract was concentrated under reduced pressure at 48 ℃ and then freeze-dried to obtain crude blackcurrant extract for later use.
[0146] 2. Sample purification methods
[0147] Blackcurrant anthocyanin extract: The blackcurrant extract obtained in step 1 above was subjected to column chromatography with XDA-6 resin as the stationary phase and different concentrations of ethanol-water solvent as the eluent, using a gradient elution method. The chromatography column was 200 cm high and 10 cm in inner diameter, with a diameter-to-height ratio of 1:10 to 1:15. The loading concentration, calculated as anthocyanins, was 10-50 mg / mL, and the loading volume was 1 / 5-2 / 5 of the column volume. After loading, the column was allowed to stand for 30-60 min. Gradient elution was then performed sequentially using 2VB column volumes of distilled water, 1VB column volumes of 10% ethanol-water solution, 3VB column volumes of 40% ethanol-water solution, and 1VB column volumes of 70% ethanol-water solution as the mobile phase, at a flow rate of 10-15 mL / min. The eluents of different concentrations of ethanol were collected, concentrated under reduced pressure, and then frozen under vacuum to obtain a lyophilized powder of the blackcurrant anthocyanin extract.
[0148] Prunus cerasifera extract: blackcurrant extract = 1:1, concentrations are shown in Table 3.
[0149] The purity of cranberry polyphenols (M) was 42.87%; the purity of blackcurrant anthocyanins (J) was 42.62%. The CI values are shown in Table 2.
[0150] Table 2 Synergistic effects of different berry extract combinations
[0151]
[0152] Note: In the table, "O" represents Prunus persica polyphenol extract; "S" represents raspberry polyphenol extract; "H" represents red bean and blueberry polyphenol extract; "D" represents blueberry anthocyanin extract; "M" represents cranberry polyphenol extract; "L" represents honeysuckle anthocyanin extract; and "J" represents blackcurrant anthocyanin extract.
[0153] Two substances were mixed in a 1:1 ratio, with a total concentration range of (0-0.6 mg / mL). Each combination had seven concentration ranges: 0 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, and 0.6 mg / mL. Free radical scavenging experiments were conducted using these combinations. The specific concentration ratios for each pair are shown in Table 3. Based on the concentration and scavenging rate, the IC50 was calculated. 50 Value, based on IC 50 The synergistic effect was analyzed to determine the synergistic effect index (CI).
[0154] Table 3 Concentration ratios of different berry extract combinations
[0155]
[0156] As shown in Table 2, not all combinations of compounds with antioxidant activity exhibit synergistic antioxidant effects. Some compounds show antagonistic interactions; for example, the 1:1 combination of prunol and blackcurrant anthocyanins shows antagonistic effects in scavenging DPPH free radicals. Furthermore, the same combination of two compounds produces different synergistic effects depending on the antioxidant detection system. For instance, the 1:1 combinations of prunol and red bean polyphenols, and prunol and cranberry polyphenols, show synergistic effects in scavenging DPPH free radicals, but antagonistic effects in scavenging hydroxyl free radicals. This indicates that synergistic antioxidant effects cannot be achieved simply by combining substances with antioxidant activity.
Claims
1. A composition for scavenging free radicals, characterized in that, The composition comprises the following mass ratio: Bilberry ( Vaccinium uliginosum L.) extract: raspberry ( Rubus idaeus L.) extract: Prunus armeniaca ( Prunus humilis Extract: Honeysuckle fruit ( Lonicera caerulea L.) extract = (1-15): (1-15): (1-15): (1-15); Methods for obtaining Prunus armeniaca extract: Step 1: Sample extraction method After crushing the fruit of Prunus cerasifera, ultrasonic-assisted extraction was performed. The extraction solvent was 70% ethanol (v / v), the material-to-liquid ratio was 1:10 (g:mL), the extraction temperature was set at 40 ℃, the extraction power was 150 W, the extraction time was 30 min, and the extraction was performed twice. The extracts were combined, concentrated under reduced pressure at 48 ℃, and then freeze-dried to obtain crude extract of Prunus cerasifera for later use. Step 2: Sample purification method The crude extract of Prunus armeniaca obtained in step 1 was subjected to column chromatography with XDA-6 resin as the stationary phase. The column was 200 cm high and 10 cm in inner diameter, with a diameter-to-height ratio of 1:10 to 1:
15. The loading concentration, calculated as polyphenols, was 10-50 mg / mL, and the loading volume was 1 / 5 to 2 / 5 of the column volume. After loading, the sample was allowed to stand for 30-60 min. Gradient elution was then performed sequentially using 2VB column volumes of distilled water, 1VB column volumes of 10% ethanol aqueous solution, 3VB column volumes of 40% ethanol aqueous solution, and 1VB column volumes of 70% ethanol aqueous solution as the mobile phase at a flow rate of 10-15 mL / min. The eluents of different concentrations of ethanol were collected, concentrated under reduced pressure, and then frozen under vacuum to obtain lyophilized Prunus armeniaca extract powder. Methods for obtaining raspberry extract: S1: After crushing the raspberry fruit, the raspberry was extracted using an ultrasonic-assisted extraction process. The extraction solvent was 70% ethanol by volume, the material-to-liquid ratio was 1:10 (g:mL), the extraction temperature was set at 40 ℃, the extraction power was 150 W, the extraction time was 30 min, and the extraction was performed twice. The extracts were combined, concentrated under reduced pressure at 48 ℃, and then freeze-dried to obtain crude raspberry extract for later use. S2: Sample purification method The crude raspberry extract obtained in step S1 was subjected to column chromatography with XDA-6 resin as the stationary phase. The column was 200 cm high and 10 cm in inner diameter, with a diameter-to-height ratio of 1:10 to 1:
15. The loading concentration, calculated as polyphenols, was 10-50 mg / mL, and the loading volume was 1 / 5 to 2 / 5 of the column volume. After loading, the sample was allowed to stand for 30-60 min. Gradient elution was then performed sequentially using 2VB column volumes of distilled water, 1VB column volumes of 10% ethanol aqueous solution, 3VB column volumes of 40% ethanol aqueous solution, and 1VB column volumes of 70% ethanol aqueous solution as the mobile phase at a flow rate of 10-15 mL / min. The eluents of different concentrations of ethanol were collected, concentrated under reduced pressure, and then frozen under vacuum to obtain lyophilized raspberry extract powder. Methods for obtaining honeysuckle fruit extract: Step 1: Sample extraction method After crushing the honeysuckle fruit, it was extracted using an ultrasonic-assisted extraction process. The extraction solvent was 70% ethanol (v / v), the solid-liquid ratio was 1:10 (g:mL), the extraction temperature was set at 40 ℃, the extraction power was 150 W, the extraction time was 30 min, and the extraction was performed twice. The extracts were combined, concentrated under reduced pressure at 48 ℃, and then freeze-dried to obtain crude honeysuckle fruit extract for later use. Step 2: Sample purification method The crude extract of honeysuckle fruit obtained in step 1 was subjected to column chromatography with XDA-6 resin as the stationary phase medium. The column was 200 cm high and 10 cm in inner diameter, with a diameter-to-height ratio of 1:10 to 1:
15. The loading concentration, calculated as anthocyanins, was 10-50 mg / mL, and the loading volume was 1 / 5-2 / 5 of the column volume. After loading, the sample was allowed to stand for 30-60 min. Gradient elution was then performed sequentially using 2VB column volumes of distilled water, 1VB column volumes of 10% ethanol aqueous solution, 3VB column volumes of 40% ethanol aqueous solution, and 1VB column volumes of 70% ethanol aqueous solution as mobile phases at a flow rate of 10-15 mL / min. The eluents of different concentrations of ethanol were collected, concentrated under reduced pressure, and then frozen under vacuum to obtain lyophilized honeysuckle fruit extract powder. Methods for obtaining blueberry extract: S1: After crushing the blueberry fruit, the extraction was carried out using an ultrasonic-assisted extraction process. The extraction solvent was 70% ethanol by volume, the solid-liquid ratio was 1:10 (g:mL), the extraction temperature was set at 40 ℃, the extraction power was 150 W, the extraction time was 30 min, and the extraction was carried out twice. The extracts were combined, concentrated under reduced pressure at 48 ℃, and then freeze-dried to obtain the crude blueberry extract for later use. S2: Sample purification method The crude extract of Bilberry obtained in step S1 was subjected to column chromatography with XDA-6 resin as the stationary phase. The column was 200 cm high and 10 cm in inner diameter, with a diameter-to-height ratio of 1:10 to 1:
15. The loading concentration, calculated as anthocyanins, was 10-50 mg / mL, and the loading volume was 1 / 5-2 / 5 of the column volume. After loading, the sample was allowed to stand for 30-60 min. Gradient elution was then performed sequentially using 2VB column volumes of distilled water, 1VB column volumes of 10% ethanol aqueous solution, 3VB column volumes of 40% ethanol aqueous solution, and 1VB column volumes of 70% ethanol aqueous solution as mobile phases at a flow rate of 10-15 mL / min. The eluents of different concentrations of ethanol were collected, concentrated under reduced pressure, and then frozen under vacuum to obtain lyophilized Bilberry extract powder.
2. The composition according to claim 1, characterized in that, The formulation consists of the following mass ratio: Bilberry extract: Raspberry extract: Prunus cerasifera extract: Honeysuckle berry extract = 5:8.2:5:3.
5.
3. The composition according to claim 1, characterized in that, The dosage form of the composition includes any one of tablets, capsules, granules, powders, liquid preparations, and pills.