Chicory fat-soluble extract as well as preparation method and application thereof

Abstract

The invention discloses a chicory fat-soluble extract as well as a preparation method and application thereof. The active monomer components of the extract comprise lactucin, lactucin, 8-deoxylactucin and esculetin, and the extract has the effect of inhibiting proliferation of tumor cells SH-SY5Y, KGN and pc3, also has certain effects of removing DPPH free radicals and inhibiting ABTS, has the effect of inhibiting macrophage RAW264.7 from releasing inflammatory factors NO and IL-6, has the effect of inhibiting tumor cell proliferation, has the effect of inhibiting tumor cell proliferation, has the effect of inhibiting tumor cell proliferation, has the effect of inhibiting tumor cell proliferation, has the effect of inhibiting tumor cell proliferation, has the effect of inhibiting tumor cell proliferation, has the effect of inhibiting tumor cell proliferation, and has the effect of inhibiting tumor cell proliferation. The invention provides the application of medicines or health-care products for resisting oxidation, delaying senescence, resisting inflammation, preventing and treating cancers and the like, and also can be applied to natural antioxidants. The invention also provides medicines for treating obesity and diseases caused by obesity, such as hyperlipidemia, hypertension, diabetes, fatty liver, coronary heart disease, sleep apnea syndrome and the like, and medicines and health care products related to the diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of medicinal plant extraction, and particularly relates to a chicory fat-soluble extract, a preparation method and application thereof. Background Art

[0002] Chicory (Cichorium intybus L.) is a perennial herb in the Compositae family. Also known as orchid chicory and cassini, it originated in Europe and is now widely cultivated worldwide. Extracts from its roots are often used as a natural food ingredient. Chicory is also a commonly used medicinal herb among the Uyghur people. Its aerial parts and roots have the properties of clearing the liver and promoting bile secretion, strengthening the stomach and aiding digestion, and promoting diuresis and reducing swelling. It is primarily used to treat stomach heat, loss of appetite, and chest and abdominal distension. Studies have shown that chicory contains a variety of compounds, including polysaccharides, terpenes, flavonoids, phenylpropanoids, and phenolic acids. Among these phenolic acids, caffeic acid and its derivatives are the primary components, with chicoric acid being the most abundant. Phenolic compounds are known to possess high antioxidant activity. Literature reports indicate that chicory possesses antioxidant and anti-inflammatory pharmacological activities, affecting related signaling pathways and protecting against oxidative stress-induced damage. Furthermore, various diseases, including tumors, cardiovascular and cerebrovascular diseases, inflammatory diseases, asthma, and rheumatism, are associated with peroxidative damage. Studies have shown that all parts of chicory, especially the leaves, have antioxidant properties. Therefore, this study used modern extraction and separation techniques to obtain extracts from different chicory materials, separated and purified them, and investigated the extracts' anti-tumor, antioxidant, and anti-inflammatory activities. The study also identified the anti-tumor active sites and active monomers in chicory.

[0003] Although chicory has been recorded to have many medicinal effects and functions, there has been no report on the exact active substances and functions of chicory fat-soluble extracts. Chicory fat-soluble extracts have great medical significance, and the market space for this extract is broad and has huge development potential. Summary of the Invention

[0004] The present invention aims to provide a preparation method for extracting fat-soluble components from chicory tea, chicory leaves and chicory aerial stems and branches.

[0005] The present invention also aims to provide the above extract as an antioxidant and its use in the preparation of medicines or functional foods and health products for anti-oxidation, anti-inflammatory, anti-aging and cancer prevention.

[0006] The present invention also aims to provide the use of the above extract in the preparation of medicines for preventing and treating obesity and hyperlipidemia, hypertension, diabetes, fatty liver, coronary heart disease, and sleep apnea syndrome caused by obesity.

[0007] A chicory fat-soluble extract, wherein the active monomer components of the extract include 8-deoxylactolide, lactucin, lactucin, and esculetin; the molecular formulas thereof are shown in Formula 1, Formula 2, Formula 3, and Formula 4, respectively:

[0008]

[0009] The preparation method of the chicory fat-soluble extract is carried out according to the following steps:

[0010] (1) Dried and crushed chicory leaves and above-ground stems and branches were added with 20-90% ethanol aqueous solution as an extraction solvent at a solid-liquid ratio of 1:(5-20), and extracted by hot reflux extraction for 2-4 times. The extracts were combined and concentrated at a volume ratio of (10-20):1, and allowed to stand for 4-12 hours;

[0011] (2) The concentrated solution prepared in step (1) was filtered, and the filtrate was evaporated to dryness under reduced pressure and then hot-extracted with methanol. The sample was then mixed with silica gel on a 100-200 mesh column and extracted with petroleum ether and ethyl acetate in sequence. The ethyl acetate solution was collected, the ethyl acetate solvent was removed, and the product was dried to obtain a dark yellow solid.

[0012] (3) The dark yellow solid in step (2) was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate mixed solvent with a volume ratio of (0.5-2):1, and eluted in sequence to obtain component A, component B, and component C.

[0013] The material-liquid ratio in step (1) is a mass-to-volume ratio, in units of g / mL.

[0014] Component A in step (3) was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate mixed solvent in a volume ratio of (1-2):1, eluted sequentially, and detected by silica gel TLC. The same fractions were collected and combined, and concentrated to obtain a light yellow solid. The solid was crystallized and recrystallized using a solvent method. The crystallization solvent was petroleum ether, ethyl acetate and a mixed solvent thereof to obtain 8-deoxylactate as a light yellow colloidal solid.

[0015] Component B in step (3) was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate mixed solvent in a volume ratio of (1-2):1, eluted sequentially, and detected by silica gel TLC. The same fractions were collected and combined, and concentrated to obtain a light yellow solid. The solid was crystallized and recrystallized using a solvent method. The crystallization solvent was petroleum ether, ethyl acetate and a mixed solvent thereof to obtain an off-white solid lactucin.

[0016] Component C in step (3) was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate mixed solvent in a volume ratio of 1:(1-2) and eluted in sequence. The mixture was identified by silica gel TLC. The same fractions were collected and combined, and concentrated to obtain a light yellow solid. The solid was subjected to a Sephadex LH-20 column with 80% methanol as the elution solvent and detected by silica gel TLC. This step was repeated 2-6 times. The same fractions were combined and concentrated to dryness to obtain an off-white solid of lactucin and a yellow solid of esculentol.

[0017] The chicory fat-soluble extract is used in the preparation of functional foods with anti-oxidation, anti-inflammatory, anti-aging and cancer prevention effects, and the content of the chicory fat-soluble extract in the functional foods is 10-30 wt%.

[0018] The chicory fat-soluble extract is used in the preparation of health products with antioxidant, anti-inflammatory, anti-aging and cancer prevention effects. The dosage form of the health products is capsules, tablets, oral liquids or granules, and the weight content of the chicory fat-soluble extract in the health products is 10-30%.

[0019] The chicory fat-soluble extract is used in the preparation of functional cosmetics for anti-oxidation, anti-inflammatory and tumor prevention. The functional cosmetics are creams, lotions, essences or masks. The amount of the chicory fat-soluble extract added to the functional cosmetics is 0.5-5 wt%.

[0020] The chicory fat-soluble extract is used in the preparation of drugs for anti-inflammatory, tumor prevention and treatment, and oxidative stress-related disease prevention and treatment.

[0021] Beneficial effects of the invention: The present invention successfully prepares a fat-soluble extract from chicory to obtain 8-deoxylactate, lactucin, lactucin, and esculetin, which have the effects of inhibiting the proliferation of tumor cells SH-SY5Y, HCT116, and PC3, and also have certain effects of scavenging DPPH free radicals and inhibiting ABTS, providing pharmaceutical or health care product uses for anti-oxidation, anti-aging, regulating body immunity, preventing cancer, etc., and can also be used as a natural antioxidant. It also provides drugs for obesity and diseases such as hyperlipidemia, hypertension, diabetes, fatty liver, coronary heart disease, sleep apnea syndrome caused by obesity, as well as pharmaceutical and health care product uses related to the above diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Flowchart for chicory extraction.

[0023] Figure 2 The results show that chicory tea extract has an inhibitory effect on the proliferation of KGN cells.

[0024] Figure 3 The results show that chicory tea extract has an inhibitory effect on the proliferation of PC3 cells.

[0025] Figure 4 The results show that chicory tea extract has an inhibitory effect on the proliferation of SH-SY5Y cells.

[0026] Figure 5 The anti-inflammatory pharmacological effects of chicory leaf extracts CIE-10 and CIE-11 on LPS-induced RAW264.7 cells and their effects on NO release.

[0027] Figure 6 The anti-inflammatory pharmacological effects of chicory leaf extracts CIE-10 and CIE-11 on LPS-induced RAW264.7 cells and their effects on IL-6 release. DETAILED DESCRIPTION

[0028] To facilitate understanding of the present invention, the present invention will be described more fully below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0029] Example 1 Preparation of Chicory Tea Extract

[0030] Weigh 20g of chicory tea (made from artificially grown chicory leaves in Fengqiu County, Henan Province according to the tea making process), and use water decoction or 70% ethanol reflux extraction, with a solid-liquid ratio of 1:10 (mass-to-volume ratio, unit: g / mL), and the extraction time is 30min. Filter, extract the residue once more using the same method, filter, combine the two filtrates, and pass through a D101 macroporous resin column after concentration. The elution solvents are water and 60% methanol in sequence. Collect the extracts of each part, remove the solvent by rotary thin film evaporator to obtain solid samples of each extract. The 60% methanol part collected by elution from the resin column is then reflux extracted with ethyl acetate, methanol, and water in sequence to obtain the corresponding extract sample. Each extraction part is numbered CIE-1 to CIE-9, and its implementation process is shown in Figure 1 a~c.

[0031] Example 2 Preparation of chicory leaf extract

[0032] 20 g of dried chicory leaves collected from artificial cultivation in Fengqiu, Henan Province were added to a 70% ethanol aqueous solution as the extraction solvent at a material-liquid ratio of 1:10 (mass-to-volume ratio, unit: g / mL). Reflux extraction was performed for 30 min, and the residue was filtered. The residue was extracted again using the same method, filtered, and the two filtrates were combined and concentrated to dryness to obtain a solid CIE-10. The solid was then passed through a D101 resin column, and the elution solvents were successively water and 60% methanol. The 60% methanol fraction was collected and evaporated to dryness using a rotary thin film evaporator to obtain the chicory fat-soluble extract CIE-11. The implementation process is shown in FIG. Figure 1 d.

[0033] Example 3 Preparation of chicory aerial part extract

[0034] 20 g of dried chicory stems and branches collected from artificial cultivation in Fengqiu, Henan Province were taken, 70% ethanol aqueous solution was added as the extraction solvent, the material-liquid ratio was 1:10 (mass-to-volume ratio, unit: g / mL), and reflux extraction was used for extraction for 30 min. The residue was filtered and extracted once again by the same method, filtered, and the two filtrates were combined. The filtrate was concentrated to dryness by a rotary thin film evaporator to obtain about 1.5 g of solid matter, which was then passed through a D101 resin column. The elution solvent was successively water and 60% methanol. The 60% methanol fraction was collected and evaporated to dryness by a rotary thin film evaporator to obtain the chicory fat-soluble extract CIE-12. The implementation process is shown in FIG. Figure 1 e.

[0035] Example 4 Preparation of Monomeric Components in Chicory Fat-Soluble Extract

[0036] 260 g of dried artificially cultivated chicory stems collected from Fengqiu, Henan Province were taken, 70% ethanol aqueous solution was added as an extraction solvent, the solid-liquid ratio was 1:10 (mass-to-volume ratio, unit: g / mL), reflux extraction was used for extraction, the extraction time was 30 min, and the residue was filtered. The residue was extracted once again by the same method, filtered, and the two filtrates were combined. The filtrate was concentrated to dryness by a vacuum rotary thin film evaporator to obtain 23 g of solid matter. 10 g of the solid matter was extracted by methanol reflux to obtain a dark yellow solid (methanol part), which was then subjected to silica gel column chromatography. The elution solvent was petroleum ether-ethyl acetate in a volume ratio of 1:0 to 0:1. After detection by TLC, the fractions with fluorescence under UV254 nm were combined and evaporated to dryness by a vacuum rotary thin film evaporator to obtain about 5 g of chicory fat-soluble extract. 4 g of the extract was eluted by silica gel column chromatography using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 1:1 to 1:2 to 1:3 to obtain component A (1 g), component B (1.2 g), and component C (1.4 g).

[0037] Component A was eluted by silica gel column chromatography using a mixed solvent of petroleum ether and ethyl acetate in a volume ratio of 1:1 to 1:2. The same fractions were collected and combined by silica gel TLC and concentrated to obtain 60 mg of a light yellow solid, which was identified as a relatively pure monomer by silica gel TLC. The product was purified by mass spectrometry and nuclear magnetic resonance (NMR) 1 H-NMR, 13 C-NMR) and identified it as 8-deoxylactate (Compound 1). 1H NMR (300MHz, MeOD) δ: 6.43 (1H, brs, H-3), 6.12 (1H, d, J = 3.33Hz, H-13a), 5.58 (1H, d, J = 3.33Hz, H-13b), 4.93 (1H, d, J = 1.92Hz, overl apped,H-15a),4.45(1H,d,J=18.33Hz,H-15b),3.84(1H,d,J=10.29Hz,H-5),3.65(1H,m,H-6),3.05(1H,m,H-7),2.47(3H,s,H-14).

[0038] 13 C NMR(75MHz,MeOD)δ:131.55(C-1),195.98(C-2),131.74(C-3),174.76(C-4),49.77(C-5),84.27(C-6),52.18(C-7),23.90( C-8),36.79(C-9),154.49(C-10),139.23(C-11),168.49(C-12),117.80(C-13),20.62(C-14),61.68(C-15).ESI-MS:[M+H] + m / z 261.09.

[0039] Component B was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate mixed solvent with a volume ratio of 1:1 to 1:2 for sequential elution. The same fractions were collected and combined for detection by silica gel TLC, and concentrated to obtain a light yellow solid. The solid was crystallized and recrystallized using a solvent method. The crystallization solvent was petroleum ether, ethyl acetate and a mixed solvent thereof to obtain 40 mg of an off-white solid, which was identified as a single component by silica gel TLC. The component was detected by nuclear magnetic resonance ( 1 H-NMR, 13 C-NMR) and identified it as lactucin (Compound 2).

[0040] 1 H-NMR (500 MHz, CD3OD)δ H(ppm):6.45(1H,s,H-3),3.86(1H,d,J=11.1Hz,H-5),3.78(1H,t,J=10.0Hz,H-6),3.44(1H,m,H-7),4.9 2(1H,m,H-8),2.86(1H,t,J=10.4Hz,H-9a),2.47(1H,d,J=7.0Hz,overlapped,H-9b),5.95(1H,d,J=3.2 Hz,H-13a),5.31(1H,d,J=2.9Hz,H-13b),2.44(3H,s,H-14),4.87(1H,m,overlapped,H-15a),4.43(1H, d,J=18.3Hz,H-15b),3.66(2H,s,H-2'),7.16(2H,d,J=8.0Hz,H-4',8'),6.79(2H,d,J=7.9Hz,H-5',7'); 13 C-NMR (125 MHz, CD3OD)δ C (ppm):133.14(C-1),195.47(C-2),132.14(C-3),174.7(C-4),48.28(C-5),81.0 8(C-6),54.04(C-7),69.69(C-8),43.57(C-9),146.55(C-10),136.55(C-11),16 8.74(C-12),120.83(C-13),20.05(C-14),61.72(C-15),171.76(C-1'),40.2(C- 2'),124.29(C-3'),130.16(C-4',8'),115.19(C-5',7'),156.59(C-6'); ESI-MS:

[0041] [M+H2O+H] + m / z 429.44.

[0042] Component C was eluted sequentially by silica gel column chromatography using a petroleum ether-ethyl acetate mixed solvent in a volume ratio of 1:2 to 1:3. The mixture was identified by silica gel TLC. The same fractions were collected and combined, and concentrated to obtain components C1 and C2. The two were then eluted by a dextran gel column Sephadex LH-20 using methanol as the elution solvent. The same fractions were collected and combined, and concentrated to dryness to obtain 35 mg of an off-white solid (compound 3) and 20 mg of a light yellow solid (compound 4). The components were identified as a single component by silica gel TLC. Compound 3 was analyzed by mass spectrometry and nuclear magnetic resonance (NMR). 1 H-NMR, 13 C-NMR) identified it as lactucin.

[0043] 1 H-NMR (300 MHz, DMSO-d4) δ H (ppm):6.29(1H,d,J=1.4Hz,H-3),3.83(1H,d,J=12.0Hz,H-5),3.76(1H,m,H-6),3.08(1 H,m,H-7),3.76(1H,m,H-8),2.77(1H,dd,J=10.6,13.6Hz,H-9a),2.29(1H,dd,J=2.3,13 .5Hz,H-9b),6.14(1H,dd,J=1.6,3.1Hz,H-13a),6.02(1H,dd,J=1.5,3.3Hz,H-13b),2.3 5(3H,s,H-14),4.68(1H,dd,J=2.0,18.8Hz,H-15a),4.27(1H,dd,J=1.9,19.0Hz,H-15b); 13 C-NMR (75 MHz, DMSO-d4) δ C (ppm):132.77(C-1),194.85(C-2),132.32(C-3),175.42(C-4),48.34(C-5),81.22(C-6),56.86(C-7),66.96(C-8) ,48.8(C-9),147.07(C-10),138.48(C-11),169.25(C-12),121.98(C-13),21.53(C-14),61.76(C-15); ESI-MS:[MH] - m / z 275.71.

[0044] Compound 4 was characterized by mass spectrometry and nuclear magnetic resonance ( 1 H-NMR, 13 C-NMR) identified it as esculetin.

[0045] 1 H-NMR (300 MHz, CD3OD)δ H (ppm):7.78(1H,d,J=9.4Hz,H-4),6.93(1H,s,H-5),6.75(1H,s,H-8),6.17(1H,d,J=9.4Hz,H-3); 13 C-NMR (75MHz, CD3OD)δ C(ppm):162.89(C-2),150.64(C-7),149.11(C-9),144.67(C-4),143.19(C- 6),111.61(C-5),111.40(C-10),111.09(C-3),102.23(C-8); ESI-MS:[MH] - m / z 177.06.

[0046] Example 5 Test on the proliferation inhibition activity of tumor cells

[0047] Human granulosa cell tumor KGN, human neuroblastoma SH-SY5Y, and human prostate cancer cell pc3 were cultured in DMEM with 10% fetal bovine serum and 5×10 3 Cells were added to a 96-well plate at 100 μL per well. A zero-adjustment well (no cells), a negative control group (containing cells), and a drug-treated group were set up. The cells were cultured in an incubator at 37°C with 5% CO2 for 24 hours, and the original culture medium was aspirated and discarded. The zero-adjustment group and the negative control group were added with culture medium only, and the drug-treated group was added with DMEM culture medium containing the chicory extracts CIE-2, 3, 7, and 8 (50, 100, and 200 μg / mL) prepared in Examples 1 to 3, and a series of concentrations of the monomer compounds obtained in Example 4; the culture was continued for 24 or 48 hours, the supernatant was aspirated, 100 μL of DMEM culture medium was added, and then 20 μL of 5 mg / mL MTT reagent was added. The culture was continued for 4 hours, the supernatant was aspirated, 100 μL of DMSO was added, and the cells were shaken for 10 minutes. The absorbance (A) was measured at 490 nm using a microplate reader, and the inhibitory effect of the drug on cell proliferation was calculated based on the absorbance value. 3 to 6 replicate wells were set for each drug concentration. Drug inhibition rate on cell proliferation (%) = (A 对照 -A 实验 )×100% / (A 对照 -A 空白 The experimental results are shown in Figures 2-4 .

[0048] Example 6 DPPH free radical scavenging test

[0049] Each extract of Example 5 was accurately weighed and prepared into 50% ethanol solutions with concentrations of 1.56, 3.12, 6.25, 12.5, 25.0, and 50.0 μg / mL, respectively. 1 mL of each 50% ethanol sample solution was taken, and 2 mL of 0.1 mM DPPH 50% ethanol solution was added. The mixture was thoroughly mixed and allowed to react at room temperature in the dark for 30 minutes. The absorbance at 525 nm was measured. 1 mL of 50% ethanol was used instead of the sample as a blank. Vitamin C (Vc) was used as a positive control drug. The DPPH free radical scavenging ability of the compound was calculated using the following formula: scavenging rate (%) = (A0-A1) / A0×100, where A0 is the blank absorbance and A1 is the sample absorbance. The half-scavenging rate IC of Vc on DPPH free radicals was calculated: 50 The extract had an IC of 7.653 μg / mL. 50 See Table 1. DPPH free radical scavenging test results indicate that chicory extract has a certain free radical scavenging effect. It can be used in medicine, healthcare, and the food industry for applications such as anti-oxidation, anti-aging, immunity enhancement, and cancer prevention.

[0050] Example 7 ABTS test

[0051] Prepare the ABTS reagent as follows: Weigh 38.6 mg of ABTS into a 20 mL brown vial and add 10 mL of purified water to make a 7 mM stock solution. Weigh 43.0 mg of K₂S₂O₄ into a 15.9 mL of purified water to make a 10 mM stock solution. Mix the two stock solutions in a 1:1 volume ratio in a brown vial to create the ABTS working solution. Let it sit for 24 hours before use. Dilute the ABTS working solution with purified water to a desired concentration and measure its absorption spectrum in the 200-1000 nm range on a spectrophotometer. The maximum absorption wavelength is 734 nm. Measure the absorbance at 734 nm and select the diluted ABTS working solution with an absorbance around 0.8 for the test reagent.

[0052] Each chicory extract was accurately weighed and diluted with distilled water to create a stock solution of a specific concentration. 50 μL of this stock solution was transferred to a 1.5 mL centrifuge tube and diluted with 150 μL of water. This solution was then diluted with water in sequentially increasing proportions to obtain a series of sample solutions at 15.62, 31.25, 62.5, 125, 250, and 500 μg / mL. In a 96-well microplate, 200 μL of ABTS working solution was added to each well, followed by 10 μL of the chicory sample solution. Three replicate wells were set up for each concentration, which served as sample wells. A zero well was then set with water, and a control well (200 μL of ABTS working solution + 10 μL of water) was set up. The absorbance was measured at 734 nm. Vitamin C was used as a positive control. The formula for calculating the ABTS free radical scavenging rate is as follows: Scavenging rate (%) = (A1-A2)*100 / (A1-A0), where A0 is the zero well, A1 is the control well, and A2 is the sample well. The IC50 inhibition rate of Vc on ABTS is calculated. 50 was 1.512 μg / mL, and the IC 50 See Table 1.

[0053] Example 8 Folin phenol test

[0054] The Folin-Ciocalteu (FC) method is a commonly used method for determining total phenols contents (TPC). Under alkaline conditions, the amount of Folin-Ciocalteu reagent reduced by phenols can be used to calculate the total phenols content of the substance being measured.

[0055] The specific steps are as follows: Weigh 10.08g of Na2CO3 into a 250mL Erlenmeyer flask and dissolve it in 100mL of distilled water to prepare a 10% Na2CO3 aqueous solution. Take 2.4mL of Folin phenol liquid reagent and dilute it with purified water at a 1:1 volume ratio to obtain 4.8mL of Folin phenol working solution for testing.

[0056] Accurately weigh 14.4 mg of gallic acid, dissolve in water, and transfer to a 25 mL volumetric flask. Adjust volume to a 5.76 mg / mL stock solution. Add 100 μL of the gallic acid stock solution to 100 μL of the folin phenol working solution, mix thoroughly, and incubate in the dark for 6 minutes. Then, add 600 μL of a 10% Na₂CO₃ solution and incubate in the dark for 45 minutes. Dilute the solution in the appropriate dilution ratio with water. Measure the absorption spectrum of each solution and determine the appropriate test solution concentration. The maximum absorption wavelength was measured at 665 nm.

[0057] Take 400 μL of the gallic acid stock solution and dilute it 2-fold with water. Then, dilute it 2-fold to produce a series of gallic acid standard solutions with concentrations of 0.09, 0.18, 0.36, 0.72, and 1.44 mg / mL. Add the folin working solution and 10% Na2CO3. After reacting in the dark, measure the absorbance and plot a standard curve.

[0058] 50 μL each of the chicory sample solution and the folin phenol working solution were transferred to a 1.5 mL centrifuge tube. After incubation in the dark for 6 minutes, 300 μL of 10% Na₂CO₃ solution was added. This was repeated three times, and the reaction was in the dark for approximately 45 minutes. 100 μL of the reaction solution from the centrifuge tube was added to a 96-well plate, with three replicates per sample, and water used as the zero well. The absorbance was measured at a wavelength of 665 nm. The phenol content in the chicory sample was calculated using a standard curve. The results are shown in Table 1.

[0059] Table 1 Antioxidant activity of chicory extract (IC 50 ) and phenol content (%)

[0060]

[0061] Example 9 Pharmacological Effects of Chicory Leaf Extract on LPS-Induced Inflammation Model of RAW264.7 Cells

[0062] RAW264.7 cell culture and drug addition: RAW264.7 cells were cultured normally in a 5% CO2, 37℃ cell culture incubator. Cells in logarithmic growth phase were plated in 24-well plates, with 2.5*10 cells per well. 5 1 mL was added to each well and incubated overnight to allow cells to adhere. Chicory extract samples (CIE-10 and CIE-11) were precisely weighed and dissolved in phosphate-buffered saline (PBS) to prepare a 10 mg / mL stock solution. The stock solution was stored in a -20°C freezer. After rewarming, the stock solution of each extract was diluted with cell culture medium to the desired dosing concentration. A 2 mg / mL stock solution of lipopolysaccharide (LPS) was prepared in PBS and diluted with cell culture medium to the desired dosing concentration. The 24-well cell culture plate was removed from the cell culture incubator, and the original culture medium was discarded. Culture medium containing various concentrations of drugs and 5 μg / mL LPS was added to each well of the drug-treated group (CIE-14 / 15). Culture medium containing 5 μg / mL LPS was added to the LPS-induced group. Only culture medium was added to the negative control wells. Three replicates were set up for each drug concentration, and then incubated in the cell culture incubator for another 24 h. The 24-well cell culture plate was removed from the incubator, and the cell supernatant was aspirated into a 1.5 mL centrifuge tube. The cells were centrifuged at room temperature (3000 rpm, 10 min), and 650 μL of the supernatant in the centrifuge tube was aspirated into a new 1.5 mL centrifuge tube and stored at -20°C.

[0063] Griess method to test NO content: Take 50 μL of the cell supernatant obtained by culturing RAW264.7 cells and treating with drugs (warmed to room temperature before experimental operation) and add it to a 96-well plate, then add 50 μL of Griess A reagent, then add 50 μL of Griess B reagent, incubate at 37°C for 20 minutes, test the absorbance value of each well at 450 nm on a microplate reader, and set up 3 replicates for each sample. The NO content in the cell supernatant was calculated based on the NaNO2 calibration curve. Data processing was performed using Excel 2019, and graphing was performed using Graphpad Prism 10.1.2 ( Figure 5 ).

[0064] ELISA method for IL-6 content: Take the cell supernatant obtained by culturing RAW264.7 cells and adding drugs (warm to room temperature before the experiment), operate according to the instructions of the ELISA kit, and finally test at two wavelengths of 450nm and 570nm on a microplate reader. According to the standard curve and the absorbance value of each sample (A 450nm -A 570nm ), calculate the IL-6 concentration in the sample solution to be tested. Data processing was performed using Excel 2019, and graphing was performed using Graphpad Prism 10.1.2 ( Figure 6 ).

[0065] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A chicory fat-soluble extract, characterized in that The active monomer components of the extract include 8-deoxylactate, lactucin, lactucin, and esculetin; the molecular formulas thereof are shown in Formula 1, Formula 2, Formula 3, and Formula 4, respectively:

2. The method for preparing the chicory fat-soluble extract according to claim 1, characterized in that: Follow these steps: (1) Dried and crushed chicory leaves and above-ground stems and branches were added with 20-90% ethanol aqueous solution as an extraction solvent at a solid-liquid ratio of 1:(5-20), and extracted by hot reflux extraction for 2-4 times. The extracts were combined and concentrated at a volume ratio of (10-20):1, and allowed to stand for 4-12 hours; (2) The concentrated solution prepared in step (1) was filtered, and the filtrate was evaporated to dryness under reduced pressure and then hot-extracted with methanol. The sample was then mixed with silica gel on a 100-200 mesh column and extracted with petroleum ether and ethyl acetate in sequence. The ethyl acetate solution was collected, the ethyl acetate solvent was removed, and the product was dried to obtain a dark yellow solid. (3) The dark yellow solid in step (2) was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate mixed solvent with a volume ratio of (0.5-2):1, and eluted in sequence to obtain component A, component B, and component C.

3. The method for preparing the chicory fat-soluble extract according to claim 2, wherein: The material-liquid ratio in step (1) is a mass-to-volume ratio, in units of g / mL.

4. The method for preparing the chicory fat-soluble extract according to claim 2, characterized in that: Component A in step (3) was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate mixed solvent in a volume ratio of (1-2):1, eluted sequentially, and detected by silica gel TLC. The same fractions were collected and combined, and concentrated to obtain a light yellow solid. The solid was crystallized and recrystallized using a solvent method. The crystallization solvent was petroleum ether, ethyl acetate and a mixed solvent thereof to obtain 8-deoxylactate as a light yellow colloidal solid.

5. The method for preparing the chicory fat-soluble extract according to claim 2, wherein: Component B in step (3) was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate mixed solvent in a volume ratio of (1-2):1, eluted sequentially, and detected by silica gel TLC. The same fractions were collected and combined, and concentrated to obtain a light yellow solid. The solid was crystallized and recrystallized using a solvent method. The crystallization solvent was petroleum ether, ethyl acetate and a mixed solvent thereof to obtain an off-white solid lactucin.

6. The method for preparing the chicory fat-soluble extract according to claim 2, characterized in that: Component C in step (3) was subjected to silica gel column chromatography using a petroleum ether-ethyl acetate mixed solvent in a volume ratio of 1:(1-2) and eluted in sequence. The mixture was identified by silica gel TLC. The same fractions were collected and combined, and concentrated to obtain a light yellow solid. The solid was subjected to a Sephadex LH-20 column with 80% methanol as the elution solvent and detected by silica gel TLC. This step was repeated 2-6 times. The same fractions were combined and concentrated to dryness to obtain an off-white solid of lactucin and a yellow solid of esculentol.

7. Use of the chicory fat-soluble extract according to claim 1 in the preparation of functional foods for anti-oxidation, anti-inflammatory, anti-aging and cancer prevention, characterized in that: The content of the chicory fat-soluble extract in the functional food is 10-30 wt%.

8. The use of the chicory fat-soluble extract according to claim 1 in the preparation of health products for anti-oxidation, anti-inflammatory, anti-aging and cancer prevention, characterized in that: The dosage form of the health care product is capsule, tablet, oral liquid or granule, and the weight content of the chicory fat-soluble extract in the health care product is 10-30%.

9. The use of the chicory fat-soluble extract according to claim 1 in the preparation of functional cosmetics for antioxidant, anti-inflammatory and tumor prevention, characterized in that: The functional cosmetics are face cream, lotion, essence or face mask, and the chicory fat-soluble extract is added in an amount of 0.5 to 5 wt % in the functional cosmetics.

10. Use of the chicory fat-soluble extract according to claim 1 in the preparation of drugs for anti-inflammatory, prevention and treatment of tumors and oxidative stress-related diseases.

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