A safflower extract, a preparation method and application thereof

Abstract

The application belongs to the technical field of safflower and specifically relates to a safflower extract and a preparation method and application thereof, and the safflower extract is obtained by alcohol extraction of safflower kernel skin, and the content of phenylacryl-5-hydroxytryptamine compounds in the extract is 45%-55%. The content of PAHA in the safflower kernel skin extract provided in the application reaches 50%, SKE has good antioxidant activity, can effectively scavenge free radicals and thus delay the oxidation process; the overall antioxidant capacity of HEK293T cells treated by SKE increases, the ability of scavenging free radicals and scavenging peroxides in the body can be enhanced; SKE can effectively slow down the expression increase of inflammatory genes IL-1alpha, IL-6 and COX2 caused by E. coli; SKE has a significant inhibitory effect on specific growth stages of E. coli and Staphylococcus aureus.

Description

Technical Field

[0001] This invention relates to the field of safflower technology, specifically to a safflower extract, its preparation method, and its application. Background Technology

[0002] Safflower (Carthamus tinctorius L.), a member of the Asteraceae family, is an annual herbaceous plant belonging to the Carthamus genus. Also known as safflower or thorny safflower, safflower is rich in various bioactive components, such as flavonoids, alkaloids, sterols, and polyacetylenes. These active components possess anti-inflammatory, antithrombotic, and anticoagulant effects, and can promote blood circulation, remove blood stasis, and protect ischemic myocardium. The safflower filaments and seeds have long been a focus of research by scholars both domestically and internationally. Most research on safflower has focused on the water extract of the safflower filaments, while research on the safflower seed extract after oil extraction is scarce. PAHA (phenylpropanoid amides) is a compound that contains phenylpropanoid amides. 5-hydroxytryptamine (PAHA) is an important class of alkaloids in safflower seeds. Its distribution in plants is relatively narrow and its content is low. Safflower seeds contain a higher PAHA content than other plants, reportedly about 0.5% of the seed mass. The main PAHAs are coumaroyl-5-hydroxytryptamine (CS) and feruloyl-5-hydroxytryptamine (FS). PAHAs are extremely rare in the biological world, and obtaining large quantities and high purity PAHAs is costly and time-consuming. Therefore, most research on PAHA-related bioactivity uses chemically synthesized CS and FS as raw materials, and related systematic studies are limited. Safflower is one of the plants rich in natural PAHAs, and efficient and rapid extraction of high-purity PAHAs from safflower seeds represents a current trend for the high-value utilization of byproducts. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a safflower extract, its preparation method, and its applications.

[0004] A safflower extract, obtained by extracting safflower seed kernel terpene alcohol, wherein the content of phenylacryloyl-5-hydroxytryptamine compounds in the extract is 45%-55%.

[0005] A method for extracting the safflower extract, comprising the following steps:

[0006] Extraction: Add 60% ethanol solution to the safflower seed kernel peel sample for extraction, filter and dry to obtain a paste; the material-to-liquid ratio of safflower seed kernel peel sample to 60% ethanol solution is 1g:10-30mL; the safflower seed kernel peel sample is obtained by crushing the safflower seed kernel peel.

[0007] Extraction: A dissolving solution is added to the paste, and after dissolution, an equal volume of water is added for extraction. The mixture is then dried to obtain the safflower extract, referred to as safflower seed kernel bark extract.

[0008] Preferably, the solution is ethyl acetate.

[0009] Preferably, the ratio of safflower seed kernel peel sample to ethyl acetate is 1g:10-30mL.

[0010] Preferably, in the extraction step, extraction is carried out at 50-60℃ for 30-120 min.

[0011] Preferably, in the extraction step, the extraction temperature is 50-60℃, and a paste is obtained after extraction at 50-60℃.

[0012] Preferably, the safflower extract is obtained by drying after extraction at 50-60°C.

[0013] The application of the safflower extract in enhancing antioxidant effects, wherein the safflower extract enhances the antioxidant effect of safflower seed oil;

[0014] The safflower extract enhances the antioxidant effect of cells.

[0015] The application of the safflower extract in inhibiting the growth of Escherichia coli and Staphylococcus aureus.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] The safflower seed kernel bark extract provided in this application contains 50% PAHA. SKE has good antioxidant activity and can effectively scavenge free radicals, thereby delaying the oxidation process. The overall antioxidant capacity of HEK293T cells treated with SKE is increased, which can enhance the ability to scavenge free radicals and peroxides in vivo. SKE can effectively slow down the increase in the expression of inflammatory genes IL-1α, IL-6 and COX2 caused by Escherichia coli. SKE has a significant inhibitory effect on specific stages of the growth of Escherichia coli and Staphylococcus aureus. Attached Figure Description

[0018] Figure 1 The diagram shows the structure of different parts of a safflower seed: a) is a schematic diagram of the structure of a safflower seed; b) shows the appearance of a safflower seed (left), a longitudinal section (middle), and the separated kernel skin (right).

[0019] Figure 2 High-performance liquid chromatograms of standards and safflower seed kernel extract: a) High-performance liquid chromatogram of standards CS and FS; b) High-performance liquid chromatogram of safflower seed kernel extract.

[0020] Figure 3 Comparison of PAHA content in different parts of safflower seeds after oil extraction;

[0021] Figure 4 Mass spectra of the standard and safflower seed kernel extract; a) Mass spectrum of the CS standard; b) Mass spectrum of the FS standard; c) Mass spectrum peak of the safflower seed kernel extract with a retention time of around 13.830 min; d) Mass spectrum peak of the safflower seed kernel extract with a retention time of around 14.411 min.

[0022] Figure 5 Chromatographic analysis of the components of extracts from safflower seed kernels (a) and kernel bark (b);

[0023] Figure 6 Comparison of PAHA content in safflower seed kernel peel extracted with different extraction times;

[0024] Figure 7 The content of CS, FS, and PAHA in safflower seed kernel extract;

[0025] Figure 8 To compare the DPPH radical scavenging effects of PAHA standard and SKE with the same PAHA content, n=3; a), b), c), and d) represent the comparison of the DPPH radical scavenging abilities of SKE at different dilution ratios and corresponding PAHA standard with the same content; 1 represents the cumulative value of the DPPH radical scavenging power of CS and FS standard solutions with the same content in the corresponding SKE; 2 represents the DPPH radical scavenging power of the CS+FS mixed standard solution with the same content in the corresponding SKE; 3 represents the DPPH radical scavenging power of SKE.

[0026] Figure 9 The scavenging curves of VC and safflower seed bark extract (SKE) on DPPH free radicals are shown, n=3; a) DPPH free radical scavenging curve of VC; b) DPPH free radical scavenging curve of SKE.

[0027] Figure 10 The scavenging curves of VC and safflower seed bark extract (SKE) on ABTS free radicals are shown, n=3; a) VC scavenging ABTS free radical curve; b) SKE scavenging ABTS free radical curve;

[0028] Figure 11 The IC50 values ​​of VC and SKE for scavenging DPPH and ABTS free radicals were compared, n=3; a) IC50 values ​​of VC and SKE for scavenging DPPH free radicals, b) IC50 values ​​of VC and SKE for scavenging ABTS free radicals; **** indicates that SKE and VC have a significant difference at the p≤0.0001 level;

[0029] Figure 12 Comparison of FRAP values ​​for VC and SKE;

[0030] Figure 13 To illustrate the inhibitory effect of SKE on the oxidation of safflower seed oil, n=3; a), b), c), d), e), and f) represent comparisons of the peroxide values ​​of safflower seed oil in each group after different storage times; different letters indicate significant differences between groups at the p≤0.05 level;

[0031] Figure 14 The effect of SKE on the viability of HEK293T cells was calculated, n=8; **** indicates that the cell viability of the 20 μg / mL SKE treatment group was significantly different from that of the 0 μg / mL SKE treatment group at the p≤0.0001 level.

[0032] Figure 15 The effect of SKE on the expression levels of antioxidant genes in HEK293T cells was shown in n=3; a), b), c), and d) represent the relative expression levels of Gsh-px, Gsh, Sod, and Cat genes in HEK293T cells in different groups, respectively; **** indicates a significant difference between the 1 μg / mL SKE treatment group and the Control group at the p≤0.0001 level, and * indicates a significant difference between the 1 μg / mL SKE treatment group and the Control group at the p≤0.05 level.

[0033] Figure 16 The effect of different SKE treatment times on the expression levels of antioxidant genes in HEK293T cells is shown in a), b), c), and d), representing the relative expression levels of Gsh-px, Gsh, Sod, and Cat genes in HEK293T cells in different groups. * indicates a significant difference between the 12h treatment group and the Control group at the p≤0.05 level.

[0034] Figure 17 The effect of SKE on ROS levels in HEK293T cells;

[0035] Figure 18 The effects of SEK on GSH-PX activity, MDA content and T-AOC capacity of HEK293T cells;

[0036] Figure 19 The inhibition rate of SKE on bovine serum albumin denaturation, n=3; *** indicates a significant difference between the experimental group and the 10 μg / mL SKE treatment group at the p≤0.001 level; **** indicates a significant difference between the experimental group and the 10 μg / mL SKE treatment group at the p≤0.0001 level.

[0037] Figure 20 The effect of SKE on erythrocyte membrane stability;

[0038] Figure 21 The effect of SKE on the inhibition rate of erythrocyte membrane rupture was calculated, n=3; * indicates a significant difference between the experimental group and the 10 μg / mL SKE treatment group at the p≤0.05 level; *** indicates a significant difference between the experimental group and the 10 μg / mL SKE treatment group at the p≤0.001 level; **** indicates a significant difference between the experimental group and the 10 μg / mL SKE treatment group at the p≤0.0001 level.

[0039] Figure 22 The effect of SKE on LOX activity;

[0040] Figure 23 The effect of SKE on the expression of inflammatory genes in IPEC-J2 cells;

[0041] Figure 24 To evaluate the inhibitory effect of SKE on inflammation of IPEC-J2 cells induced by *E. coli*, n=3; * indicates a significant difference between each group and the *E. coli* treatment group at the p≤0.05 level; ** indicates a significant difference between each group and the *E. coli* treatment group at the p≤0.01 level; *** indicates a significant difference between each group and the *E. coli* treatment group at the p≤0.001 level; **** indicates a significant difference between each group and the *E. coli* treatment group at the p≤0.0001 level.

[0042] Figure 25 The effect of SKE on the growth curve of Escherichia coli is shown, n=3; * indicates a significant difference between the 200 μg / mL SKE treatment group and the Control group at the p≤0.05 level; # indicates a significant difference between the 100 μg / mL SKE treatment group and the Control group at the p≤0.05 level.

[0043] Figure 26 The figure shows the effect of SKE on the inhibition zone of Escherichia coli. 1 represents control; 2 represents 100 μg / mL SKE treatment; and 3 represents 200 μg / mL SKE treatment.

[0044] Figure 27 The effect of SKE on the growth curve of Staphylococcus aureus is shown, n=3; * indicates a significant difference between the 200 μg / mL SKE treatment group and the Control group at the p≤0.05 level; # indicates a significant difference between the 100 μg / mL SKE treatment group and the Control group at the p≤0.05 level.

[0045] Figure 28The effect of SKE on the inhibition zone of Staphylococcus aureus is shown in Figure 1, where 1 represents control; 2 represents 100 μg / mL SKE treatment; and 3 represents 200 μg / mL SKE treatment.

[0046] Figure 29 Statistics on the size of the inhibition zones of SKE against Escherichia coli and Staphylococcus aureus. Detailed Implementation

[0047] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0048] The extraction method for safflower extract includes the following steps:

[0049] Extraction: After oil extraction, the safflower seed kernels and peels separated are pulverized in small batches multiple times using a high-throughput pulverizer. The conditions are: 1 min, 50-70 Hz, repeated twice. The pulverized powder is passed through a 40-mesh sieve. 10 g of the pulverized powder is accurately weighed and added to 200 mL of 60% ethanol solution. The mixture is extracted at a constant temperature of 50-60℃ using a magnetic stirrer for 30-120 min. After cooling to room temperature, the mixture is filtered. The filtrate is then subjected to small batches of rotary evaporation using a round-bottom flask. The rotary evaporation conditions are: water bath temperature 50-60℃, rotation speed 60-80 rpm, ensuring a gentle boil without backflow, until all the filtrate is evaporated to dryness.

[0050] It should be noted that in the extraction step, constant temperature stirring can be replaced with ultrasonic extraction. Ultrasonic extraction is performed in an ultrasonic cleaner for 30-60 minutes under the following conditions: constant temperature of 30-50℃ and 200-400W.

[0051] Extraction: In a round-bottom flask after evaporation, add 20 mL of ethyl acetate or other dissolving solution and sonicate (80-200W) to dissolve. Add an equal volume of 20 mL of distilled water, transfer to a 250 mL separatory funnel, and shake thoroughly to extract. Allow to stand until complete separation of the upper and lower layers, retaining the upper layer (ethyl acetate phase), and transfer to a 250 mL round-bottom flask. Rotary evaporation is performed under the following conditions: water bath temperature 50-60℃, rotation speed 60-80 rpm. The resulting extract adheres to the wall of the round-bottom flask. Then, add 2 mL of methanol to dissolve all the extract, and then add 1 mL of methanol to wash the flask wall. Collect the methanol solution of the extract in an EP tube, centrifuge, concentrate, and dry to obtain safflower seed kernel bark extract. The purity of PAHA in this extract is 45%-55%.

[0052] Example 1

[0053] Extraction: The safflower seed kernels separated after oil extraction were pulverized in small batches multiple times using a high-throughput pulverizer at 1 min and 60 Hz, repeated twice. The pulverized powder was passed through a 40-mesh sieve. 10 g of the pulverized powder was accurately weighed into a 250 mL Erlenmeyer flask, and 200 mL of 60% ethanol solution was added. The mixture was extracted at 55 °C for 1 h using a magnetic stirrer with constant temperature. After cooling to room temperature, the mixture was filtered. The filtrate was then subjected to small batches of rotary evaporation using a round-bottom flask at a water bath temperature of 50 °C and a rotation speed of 60 rpm, ensuring a gentle boil without backflow, until all the filtrate was evaporated to dryness.

[0054] Extraction: In a round-bottom flask after evaporation, add 20 mL of ethyl acetate or other dissolving solution and sonicate (80-200W) to dissolve. Add an equal volume of 20 mL of distilled water, transfer to a 250 mL separatory funnel, and shake thoroughly to extract. Allow to stand until complete separation of the upper and lower layers, retaining the upper layer (ethyl acetate phase), and transfer to a 250 mL round-bottom flask. Rotary evaporation is performed under the following conditions: water bath temperature 50℃, rotation speed 60 rpm. The resulting extract adheres to the wall of the round-bottom flask. Then, add 2 mL of methanol to dissolve all the extract, followed by adding 1 mL of methanol to wash the flask wall. Collect the methanol solution of the extract in an EP tube, centrifuge, concentrate, and dry to obtain safflower seed kernel bark extract. The purity of PAHA in this extract is 45%-55%.

[0055] Example 2

[0056] The difference between Example 2 and Example 1 is that in the extraction step, constant temperature stirring is replaced with ultrasonic extraction. Ultrasonic extraction is performed in an ultrasonic cleaner for 1 hour under the following conditions: constant temperature of 35°C and 300W. All other conditions and steps are the same as in Example 1. The purity of PAHA in the safflower seed bark extract obtained by ultrasonic assisted extraction is 55%.

[0057] Comparative Example 1

[0058] The difference between Comparative Example 1 and Example 1 is that the sample powder is safflower kernel cake powder, while the other steps and conditions are the same as in Example 1.

[0059] Comparative Example 2

[0060] The difference between Comparative Example 2 and Example 1 is that the sample powder is safflower seed shell, while the other steps and conditions are the same as in Example 1.

[0061] Comparative Example 3

[0062] The difference between Comparative Example 3 and Example 1 is that the solution was ethanol, 80% ethanol and ethyl acetate, and the extraction steps were as follows:

[0063] One-step extraction: Add ethanol and sonicate to dissolve + isooctane, retain the ethanol phase (lower layer), and evaporate to dryness;

[0064] Two-step extraction: Add 80% ethanol and sonicate to dissolve + n-hexane, retain the ethanol phase (lower layer), and evaporate to dryness;

[0065] Three-step extraction: Add ethyl acetate and sonicate to dissolve + distilled water, retain the ethyl acetate phase (the upper layer), evaporate to dryness, and the resulting extract adheres to the wall of the round bottom flask.

[0066] All other steps and conditions are the same as in Example 1.

[0067] Comparative Example 4

[0068] The difference between Comparative Example 3 and Example 1 is that the solution is 80% ethanol and ethyl acetate, and the extraction steps are as follows:

[0069] One-step extraction: Add 80% ethanol and sonicate to dissolve + n-hexane, retain the ethanol phase (lower layer), and evaporate to dryness;

[0070] Two-step extraction: Add ethyl acetate and sonicate to dissolve + distilled water, retain the ethyl acetate phase (the upper layer), evaporate to dryness, and the resulting extract adheres to the wall of the round bottom flask.

[0071] All other steps and conditions are the same as in Example 1.

[0072] Effect verification

[0073] The extracts were analyzed by high-performance liquid chromatography (HPLC). By comparing the retention times of extracts such as safflower seed husk extract and standards CS and FS under the same HPLC conditions, the presence of CS and FS in the extracts was preliminarily determined. Figure 2 As shown, by comparing the high performance liquid chromatography (HPLC) chromatograms of the standard and extracts such as safflower seed kernel bark, it can be seen that the peaks of the safflower seed kernel bark extract with retention times of 13.830 min and 14.411 min are consistent with the retention times of the standard CS and FS. Therefore, it is preliminarily determined that the substance with a retention time of 13.830 min in the safflower seed kernel bark extract is coumaroyl-5-hydroxytryptamine (CS), and the substance with a retention time of 14.411 min is feruloyl-5-hydroxytryptamine (FS).

[0074] The PAHA active ingredient in safflower kernel cake powder, safflower seed shell, and safflower seed husk was identified by high performance liquid chromatography. The contents of coumaroyl-5-hydroxytryptamine (CS) and feruloyl-5-hydroxytryptamine (FS) in the extracts were simultaneously calculated based on the standard curve equation of the standard products, and the total PAHA content was obtained. Figure 3The results showed that the PAHA content in safflower seed cake powder was 0.095±0.03 mg / g, the PAHA content in safflower seed shell was 6.862±0.165 mg / g, and the PAHA content in safflower seed kernel skin was 25.746±2.718 mg / g. This indicates that PAHA in safflower seeds is mainly concentrated in the kernel skin, while the PAHA content in the safflower seed kernel itself is extremely low. Figure 3 ,5a), the PAHA content in safflower seed skin is about 270 times that in safflower seed kernel.

[0075] To further confirm that the peaks at retention times of 13.830 min and 14.411 min shown in the HPLC separation analysis indeed correspond to CS and FS, we performed mass spectrometry analysis on the safflower seed kernel bark extract for identification. Figure 3 As shown, Figure 4 a and Figure 4 c has the same molecular ion peak with an m / z of 323. According to the literature, the relative molecular mass of coumaroyl-5-hydroxytryptamine (CS) is 322. Therefore, the peak substance with a retention time of 13.830 min in the safflower seed bark extract is identified as CS. Figure 4 b and Figure 4 d has the same molecular ion peak, with a mass-to-nucleus ratio of 353. The relative molecular mass of feruloyl-5-hydroxytryptamine (FS) is 352. Therefore, the peak substance with a retention time of 14.411 min in the safflower seed bark extract can be identified as FS.

[0076] The PAHAs obtained by the three extraction methods were subjected to chromatographic identification and statistical analysis, such as... Figure 6 As shown, the PAHA content in safflower seed kernel peel obtained from three extractions was 15.122±2.856 mg / g, the PAHA content in the safflower seed kernel peel obtained from two extractions was 16.510±3.155 mg / g, and the PAHA content in the safflower seed kernel peel obtained from one extraction was 25.746±2.718 mg / g. It is evident that the more extractions, the lower the PAHA extraction rate. The PAHA content from three extractions was nearly 40% lower than that from one extraction. As the number of extractions decreased, the loss of PAHA content gradually decreased, and the final PAHA content retained in the extract was higher. Figure 6 ).

[0077] By comparing the CS+FS peak area ratio under the same conditions with different extraction times ( Figure 2-8The data (area%) shows that CS+FS constitutes the vast majority of the extract, exceeding 91%, indicating that the main detectable substance is PAHA, with other substances being present in smaller quantities. Furthermore, the PAHA percentages obtained from different extraction times are not significantly different, at 93.5% (1 extraction), 91.5% (2 extractions), and 90.5% (3 extractions). Therefore, with increasing extraction times, both the main component and various impurities decrease by almost the same proportion, or rather, the reduction in the main component is slightly greater. Purity is not significantly improved; instead, the total amount of substances is lost more, and the amount of the target product also decreases.

[0078] To verify the stability of PAHA content in the extract obtained by a single extraction method, we conducted repeated extraction experiments. The results showed that all five repeated experiments yielded high PAHA yields, with the PAHA content consistently around 50% of the extract content. Figure 7 ).

[0079] To verify whether PAHA is the antioxidant in SKE, we prepared a CS standard solution with the same content as CS in SKE, a FS standard solution with the same content as FS in SKE, and a PAHA (CS+FS) standard solution with the same content as PAHA in SKE, based on the HPLC analysis results of SKE. We also prepared the corresponding SKE solution and performed DPPH free radical scavenging experiments. Figure 8 It can be seen that in the four sets of experiments, the higher the dilution factor of the test solution, or the lower the concentration, the lower its ability to scavenge free radicals. Both the SKE solution and the PAHA (CS+FS) standard solution with the same concentration as SKE showed considerable scavenging ability against DPPH free radicals. The cumulative free radical scavenging ability of the two single standards was slightly higher than that of their mixture and also slightly higher than that of the SKE solution, but the difference was not statistically significant. Therefore, the substance providing antioxidant capacity in the SKE solution is mainly PAHA (CS+FS).

[0080] Vitamin C (VC) is a globally recognized substance with excellent antioxidant effects, and its value as an important reference for evaluating other antioxidants is evident. This study compares and analyzes the scavenging abilities of VC and SKE solutions against DPPH free radicals. Figure 9 It can be seen that the scavenging ability of VC for DPPH free radicals gradually increases with increasing VC concentration. SKE also has the function of scavenging DPPH free radicals, and its scavenging ability is positively correlated with the concentration of SKE; as the SKE concentration increases, its scavenging ability for DPPH free radicals gradually increases. At the same concentration, the scavenging rate of SKE for DPPH free radicals is lower than that of VC, indicating that SKE's ability to scavenge DPPH free radicals is weaker than that of VC.

[0081] By comparing and analyzing the scavenging abilities of VC and SKE solutions against ABTS free radicals, Figure 10 It can be seen that the scavenging ability of VC for ABTS radicals gradually increases with increasing VC concentration. SKE also has the function of scavenging ABTS radicals, and its scavenging ability is positively correlated with the concentration of SKE. The scavenging ability of SKE for ABTS radicals gradually increases with increasing SKE concentration. At the same concentration, the scavenging rate of SKE for ABTS radicals is lower than that of VC, and the ability of SKE to scaveng ASTS radicals is weaker than that of VC.

[0082] IC 50 IC50 represents the sample concentration required to remove 50% of free radicals. 50 The smaller the value, the stronger its ability to scavenge free radicals. According to... Figure 9 and Figure 10 The IC50 values ​​of SKE and VC for scavenging DPPH and ABTS free radicals were obtained from the fitting equations of the fitted curves. 50 Value, see Figure 11 The experimental results show that in the DPPH radical scavenging experiment, the IC50 of VC is [missing information]. 50 The value was 0.0253 mg / mL, and the IC50 of SKE was... 50 The concentration of SKE was 0.0578 mg / mL, and its scavenging ability against DPPH free radicals was about half that of VC. In the ABTS free radical scavenging experiment, the IC50 of VC was... 50 The value was 0.0342 mg / mL, and the IC50 of SKE was... 50 At a concentration of 0.0619 mg / mL, SKE's ability to scavenge ABTS free radicals is up to half that of VC.

[0083] Fe 3+ The principle behind the Ferricion Reducing Antioxidant Power (FRAP) method for determining total antioxidant capacity is that, under acidic conditions, antioxidants can reduce Fe... 3+ -TPTZ produces blue-violet Fe 2+ -TPTZ, and then the absorbance was measured at 593 nm, which can be used as an indicator of the total antioxidant capacity of the sample. According to Figure 12 The results show that the FRAP values ​​of VC and SKE increase with increasing concentration, and the total antioxidant capacity of SKE is about half that of VC at all concentrations.

[0084] Safflower seed oil is rich in the polyunsaturated fatty acid linoleic acid, which is easily oxidized. Butylated hydroxytoluene (BHT) is a widely used antioxidant to prevent food oxidation; it is readily soluble in oils and fats and is of significant reference value for evaluating the antioxidant effects of other antioxidants in food. To determine the effect of the SKE solvent EtOH on the oxidation of safflower seed oil, safflower seed oil samples with the same EtOH content were stored and measured under the same conditions. This experiment evaluated the oxidation status of safflower seed oil by adding different amounts of SKE and antioxidants, and measuring the peroxide value of the safflower seed oil every 30 days, thereby exploring the antioxidant effect of SKE. Figure 13 It can be seen that before the experiment, the oxidation status of safflower seed oil in each group was consistent. With increasing time, the peroxide value of safflower seed oil in each group increased. The control group and the EtOH group had the highest peroxide values, and the addition of EtOH had no effect on the oxidation of safflower seed oil. The BHT group had the lowest peroxide value, and the peroxide values ​​of the 0.05% SKE group and the 0.15% SKE group were comparable. Due to its higher concentration, the 0.2% SKE group showed a slightly higher peroxide value compared to the other SKE groups. Overall, the order of peroxide values ​​among the groups was: Control = EtOH > 0.2% SKE > 0.15% SKE = 0.05% SKE > 0.2% BHT. This indicates that SKE has an inhibitory effect on the oxidation of safflower seed oil and can delay its oxidation, but its antioxidant capacity is slightly weaker than that of BHT.

[0085] SKE's excellent antioxidant properties were verified through DPPH and ABTS free radical scavenging assays. Considering the issues of organismal metabolism, absorption, and bioavailability, we selected HEK293T cells as a model to conduct in vivo antioxidant studies. To determine the concentration range within which SKE is non-toxic to HEK293T cells, the CCK-8 assay was used to determine the effect of different concentrations of SKE on cell viability. Figure 14 It was found that SKE concentrations of 0.1-10 μg / mL had no significant effect on cell viability, while a concentration of 20 μg / mL significantly reduced cell viability. To ensure normal cell viability, SKE concentrations of 1 μg / mL and 5 μg / mL were selected for subsequent screening of optimal treatment times.

[0086] HEK293T cells were treated with different concentrations of SKE for 12 hours, and then the expression of related genes in each group was measured. GSH-PX, GSH, SOD, and CAT are antioxidant genes in organisms; increased expression of antioxidant genes enhances the body's antioxidant capacity. Results are as follows: Figure 15As shown, 1 μg / mL SKE significantly increased the relative expression levels of antioxidant genes GSH-PX, GSH, and CAT in HEK293T cells, while 5 μg / mL SKE had no significant effect on the relative expression of antioxidant genes in HEK293T cells. Therefore, 1 μg / mL SKE was selected for subsequent experiments.

[0087] After determining the optimal concentration of SKE for HEK293T cells, the optimal duration of SKE action on HEK293T cells was further determined by detecting the relative expression levels of antioxidant genes in HEK293T cells. Results are as follows: Figure 16 As shown, treatment of HEK293T cells with 1 μg / mL SKE for 12 h significantly increased the relative expression levels of antioxidant genes GSH-PX and SOD, while treatment with 1 μg / mL SKE for 24 h had no significant effect on the expression of antioxidant genes. Therefore, treatment of HEK293T cells with 1 μg / mL SKE for 12 h was selected as the subsequent experimental condition.

[0088] Reactive oxygen species (ROS) levels reflect the state of oxidative stress in cells; when an organism experiences oxidative stress, the ROS content increases. The oxidative stress state of cells was evaluated by detecting the overall fluorescence of ROS-specific probes loaded in untreated and SKE-treated HEK293T cells using a fluorescence microplate reader. Figure 17 It can be seen that, compared with the control group, after 1 μg / mL SKE was applied to HEK293T cells for 12 h, there was no significant change in the ROS level in the cells. SKE did not cause oxidative stress in the cells. However, even when HEK293T cells did not experience oxidative stress, SKE could not reduce the ROS content in HEK293T cells.

[0089] Malondialdehyde (MDA) is cytotoxic. During cellular oxidative stress, lipid peroxides decompose to produce MDA, which is often used as a marker of oxidative stress. Under oxidative stress, cellular antioxidant capacity decreases, and correspondingly, GSH-PX activity and T-AOC also decrease. Therefore, the antioxidant level of cells can be comprehensively evaluated by detecting MDA content, GSH-PX activity, and T-AOC. MDA content, GSH-PX activity, and T-AOC in HEK293T cells of each group were measured using kits. Results are presented below. Figure 18It can be seen that, compared with the control group, the MDA content of the 1 μg / mL SKE group was reduced, the GSH-PX activity was slightly increased, and the T-AOC was also increased. These results show that the overall antioxidant capacity of HEK293T cells after SKE treatment is increased, which can enhance the ability to scavenge free radicals and peroxides in vivo and resist oxidative stress.

[0090] Inhibitory effect of SKE on bovine serum albumin denaturation

[0091] External factors such as stress, temperature, and pH can all cause changes in protein structure, leading to protein denaturation. Inflammation can cause the destruction of proteins in tissues. Therefore, inhibiting protein denaturation can, to some extent, suppress inflammation. The degree of denaturation of bovine serum albumin was measured after adding different concentrations of SKE. The results were obtained by... Figure 19 It can be seen that the inhibition rate of protein denaturation is positively correlated with the increase of SKE concentration. As the SKE concentration increases, its inhibition rate of bovine serum albumin denaturation also gradually increases. When the SKE concentration is 150 μg / mL, its protein denaturation inhibition rate can reach 73.554±2.868%.

[0092] SKE's protective effect on erythrocyte membranes

[0093] During inflammation, enzymes are released from lysosomes. Lysosomes contain many enzymes that participate in and exacerbate inflammation; therefore, lysosomal membrane stability can inhibit the release of these enzymes. Human erythrocyte membranes are structurally similar to lysosomes; therefore, inhibiting erythrocyte lysis in environments prone to lysosomal membrane rupture can be considered an anti-inflammatory mechanism. For ethical reasons, we used erythrocytes from New Zealand rabbits instead of human erythrocytes in our experiments. Solutions of New Zealand rabbit erythrocytes with different concentrations of SKE were heated, centrifuged, and the supernatant was measured. A darker color and a higher OD value indicated more severe erythrocyte rupture, while a lighter color and a lower OD value indicated less severe erythrocyte membrane rupture. Results were obtained by… Figure 20-21 It can be seen that as the concentration of SKE increases, the protective effect of SKE on the erythrocyte membrane gradually strengthens. This is also evident in the experimental graph, which shows a decrease in the number of ruptured erythrocyte membranes and a lighter liquid color. This indicates that the higher the concentration of SKE, the lower the degree of erythrocyte membrane rupture and the better the stability of the erythrocyte membrane. When the SKE concentration is 150 μg / mL, its inhibition rate on erythrocyte membrane rupture reaches 63.70 ± 3.37%, and the experimental graph clearly shows a lighter solution color, indicating a significant reduction in the degree of erythrocyte membrane rupture. Therefore, SKE can effectively prevent erythrocyte membrane rupture and reduce the inflammatory response caused by the outflow of intracellular substances.

[0094] Inhibitory effect of SKE on LOX activity

[0095] Arachidonic acid (AA) is an essential fatty acid for the human body, participating in the entire process of inflammation development and progression in ulcerative colitis (UC). AA is mainly metabolized through two pathways: cyclooxygenase (COX) and lipoxygenase (LOX). LOX is a key enzyme in the AA metabolic pathway, and its metabolite, leukotriene B4 (LTB4), is an important mediator of inflammation. Therefore, inhibiting LOX enzyme activity can reduce inflammation to some extent. The activity of LOX enzyme was measured by adding different mass concentrations of SKE to LOX enzyme solutions. The results were obtained by... Figure 22 It can be seen that as the mass concentration of SKE increases, its inhibition rate on LOX activity also gradually increases, and the two are positively correlated. As the mass concentration of SKE increases, the inhibition rate of SKE on LOX enzyme activity gradually increases. When the mass concentration of SKE is 150 μg / mL, the inhibition rate on LOX activity can reach 93.413±0.848%.

[0096] Inhibitory effect of SKE on the expression of inflammatory factor genes in IPEC-J2 cells

[0097] IPEC-J2 cells were treated with 50 μg / mL SKE for 12 h, and the relative expression levels of inflammatory genes in IPEC-J2 cells were detected by gene expression assays. Figure 23 The results showed that SKE did not increase the expression of inflammatory genes in IPEC-J2 cells. Therefore, treatment of IPEC-J2 cells with 50 μg / mL SKE for 12 h did not produce toxic effects. Subsequent experiments can use 50 μg / mL SKE to treat IPEC-J2 cells for 12 h.

[0098] Toxigenic Escherichia coli produces lipopolysaccharide (LPS), which triggers cellular inflammatory responses. IPEC-J2 cells were pretreated with SKE, then stimulated with E. coli, and finally the expression levels of IPEC-J2 cell-related inflammatory genes were detected. Results were obtained from... Figure 24 It can be seen that Escherichia coli causes a significant increase in the expression of inflammatory genes in IPEC-J2 cells, while IPEC-J2 cells pretreated with SKE can effectively reduce the increase in the expression of inflammatory genes IL-1α, IL-6, and COX2 caused by Escherichia coli.

[0099] Inhibitory effect of SKE on the growth of Escherichia coli

[0100] SKE was added to the E. coli culture medium and the culture was shaken. The OD value of the bacterial solution was measured at different time points. The results are as follows: Figure 25As shown, the logarithmic growth phase of *E. coli* in both the control and experimental groups was very rapid. Since the *E. coli* inoculated in this experiment were fresh suspensions each time, the *E. coli* could quickly enter the logarithmic growth phase and then enter the stationary phase after 8 hours. In the initial stage of the logarithmic growth phase, *E. coli* in both the control and experimental groups grew rapidly, and SKE did not significantly inhibit the growth rate of *E. coli*. In the later stage of the logarithmic growth phase, the growth rate of *E. coli* in the control and experimental groups showed a difference, with *E. coli* treated with higher concentrations of SKE showing a significantly slower growth. Once *E. coli* entered the stationary phase, between 8 and 12 hours, both the 100 μg / mL SKE group and the 200 μg / mL SKE group showed significant inhibitory effects on *E. coli* growth, and this inhibition was dose-dependent; the higher the concentration of SKE, the more pronounced the inhibitory effect on *E. coli* growth.

[0101] Filter paper soaked in SKE solution was placed on a solid culture medium coated with E. coli culture, and the E. coli was cultured under suitable conditions. The results are as follows: Figure 26 As shown, control, 100 μg / mL SKE, and 200 μg / mL SKE all exhibited significant inhibition zones against the growth of *E. coli*. The control group (DMSO) itself has an inhibitory effect on *E. coli*. The size of the inhibition zones in the three groups was measured: control group (10.75 ± 0.2 mm), 100 μg / mL SKE group (11.58 ± 0.31 mm), and 200 μg / mL SKE group (12.08 ± 0.12 mm). However, there was no statistically significant difference in the size of the inhibition zones among the three groups.

[0102] Inhibitory effect of SKE on the growth of Staphylococcus aureus

[0103] SKE was added to Staphylococcus aureus culture medium and the culture was shaken. The OD value of the bacterial solution was measured at different time points. Figure 27 As shown, *Staphylococcus aureus* entered a stationary growth phase after 16 hours. During the logarithmic growth phase of *Staphylococcus aureus*, both the 100 μg / mL SKE group and the 200 μg / mL SKE group significantly inhibited the growth of *Staphylococcus aureus* in a dose-dependent manner; the higher the concentration of SKE, the more pronounced the inhibitory effect on *Staphylococcus aureus*. When *Staphylococcus aureus* entered the growth plateau phase, there was no difference in the OD values ​​of the control group, the 100 μg / mL SKE group, and the 200 μg / mL SKE group.

[0104] Filter paper soaked in SKE solution was placed on a solid culture medium coated with Staphylococcus aureus culture, and Staphylococcus aureus was cultured under suitable conditions. Figure 28As shown, control, 100 μg / mL SKE, and 200 μg / mL SKE all exhibited significant inhibition zones against the growth of Staphylococcus aureus. The control group, consisting of DMSO, inherently inhibits the growth of Staphylococcus aureus. The size of the inhibition zones in the three groups was measured: 11.33 ± 0.12 mm in the control group, 11.92 ± 0.24 mm in the 100 μg / mL SKE group, and 11.5 ± 0.2 mm in the 200 μg / mL SKE group. However, there was no statistically significant difference in the size of the inhibition zones among the three groups.

[0105] according to Figure 29 Based on the experimental results of the effect of SKE on the growth curves of Escherichia coli and Staphylococcus aureus, it can be seen that SKE has a significant inhibitory effect on specific stages of the growth of Escherichia coli and Staphylococcus aureus, but its inhibition zone effect is not obvious.

[0106] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0107] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0108] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A safflower extract, characterized in that, It is obtained by extracting safflower seed kernel terpene alcohol, and the content of phenylacryloyl-5-hydroxytryptamine compounds in this extract is 45%-55%; The extraction method for the safflower extract includes the following steps: Extraction: Add ethanol solution to safflower seed kernel peel sample for extraction to obtain paste; the material-to-liquid ratio of safflower seed kernel peel sample to ethanol solution is 1g:10-30mL; extract at 50-60℃ for 30-120 min; Extraction: Add a dissolving solution to the paste, and after dissolving, add an equal volume of water to the dissolving solution for a single extraction to obtain the safflower extract. The dissolving solution is ethyl acetate. The safflower extract is used to inhibit the growth of Escherichia coli and Staphylococcus aureus.

2. The safflower extract according to claim 1, characterized in that, The ratio of safflower seed kernel peel sample to ethyl acetate was 1g:10-30mL.

3. The safflower extract according to claim 1, characterized in that, In the extraction step, a paste is obtained after extraction at 50-60℃.

4. The safflower extract according to claim 1, characterized in that, The safflower extract was obtained after extraction at 50-60℃.

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