Extraction and separation method and application of effective components of safflower seed meal

Abstract

The invention discloses an extraction and separation method and application of effective components of safflower seed meal, and relates to the technical field of traditional Chinese medicine, the key points of the technical scheme are as follows: S1, crude extraction of the effective components of the safflower seed meal: S2, crude separation of the effective components of the safflower seed meal; s3, purifying the effective components of the safflower seed meal to obtain six components which are marked as Fr.A-Fr.F; and S4, separating the Fr.B through a medium-pressure silica gel column with the silica gel particle size of 300-400, a gel column and semi-preparative HPLC (High Performance Liquid Chromatography) to obtain a monomeric compound 1 and a monomeric compound 2. According to the method, a safe, low-toxicity and efficient solvent is adopted, the N-trans-feruloyl methoxy tyramine and Bufoserotonin A are extracted and separated from the safflower seed meal for the first time, a used chromatographic material is safe, the production cost is low, and a good technical scheme is provided for development and utilization of the safflower seed meal.

Description

Technical Field

[0001] The invention relates to the technical field of traditional Chinese medicine, and more specifically, to a method for extracting and separating effective components of safflower seed meal and application thereof. Background Art

[0002] Safflower (Carthamus tinctorius L.) is an annual or biennial herbaceous plant of the Asteraceae family with more than 50 varieties. It is called safflower because of its bright red color. It is also known as grass safflower, chrysanthemum safflower, red orchid, thorny safflower, etc. Its plant height can reach 0.3-2.1 meters. Safflower cultivation originated in South Asia and has a history of at least 4,500 years. It is one of the oldest crops in the world and is currently distributed in more than 60 countries and regions including India, Canada, the United States, Mexico, China, and Iran. The cultivation of safflower in my country can be traced back to the Han Dynasty, with a history of more than 2,100 years. At present, the planting area of ​​safflower in my country is about 49,900 acres, mainly distributed in Xinjiang, Gansu, Sichuan and Yunnan. In addition, it is also planted in Heilongjiang in the northeast, Hebei, Shandong and Henan in the Central Plains, Jiangsu, Zhejiang and Fujian in the coastal areas, and the Qinghai-Tibet Plateau in Yunnan, Guizhou and Sichuan. It is rich in resources and has a wide variety of varieties.

[0003] Safflower is a traditional Chinese medicinal material. Dried flowers are used as medicine. It was first recorded in the Kaibao Materia Medica. It has the functions of breaking blood stasis, promoting blood circulation and relieving pain, and promoting menstruation and reducing swelling. It is clinically used to treat dysmenorrhea, amenorrhea, traumatic injuries and other diseases. Current studies have shown that safflower has anti-tumor, antioxidant, anti-inflammatory, analgesic and cardiovascular system protection effects. In addition, safflower is also used as a colorant and flavoring agent in the food industry and a dye in the textile industry. Safflower seeds are white seeds produced after safflower matures, commonly known as "white flat seeds" in traditional Chinese medicine. Its oil content is 25%-37%, and safflower seed oil is rich in unsaturated fatty acids, of which the linoleic acid content can reach 80%, making it the oil crop with the highest linoleic acid content. In addition, safflower seed oil also contains a variety of active substances such as vitamin E, phytosterols and flavonoids, which have a variety of health benefits such as regulating blood lipids, improving cardiovascular health, improving obesity, and inhibiting tumors.

[0004] Safflower seed meal is the residue of safflower seeds after mechanical pressing or solvent extraction for oil production. Because it contains rich protein, it is often used as an animal feed additive. However, safflower seed meal contains arctiin, which causes diarrhea, and mogroside, which has a bitter taste, which limits its use to a certain extent. In addition, our previous studies have found that safflower seed meal has a good therapeutic effect on ulcerative colitis. Therefore, the development and utilization of the effective ingredients in safflower seed meal has great practical significance. In view of this, the inventors propose a method for extracting and separating the effective ingredients of safflower seed meal and its application. Summary of the invention

[0005] The purpose of the present invention is to provide a method for extracting and separating effective components of safflower seed meal and application thereof, so as to solve the above-mentioned problems and provide a good technical solution for the development and utilization of safflower seed meal.

[0006] The above technical objectives of the present invention are achieved through the following technical solutions: A method for extracting and separating effective components of safflower seed meal and its application, comprising the following steps:

[0007] S1. Crude extraction of effective components from safflower seed meal: using dried and crushed safflower seed meal as raw material, extracting with ethanol solvent, filtering, combining the filtrate, and concentrating to obtain an ethanol total extract;

[0008] S2. Crude separation of effective ingredients from flower seed meal: The total ethanol extract obtained in step S1 was dispersed in hot water, extracted with petroleum ether, ethyl acetate, and saturated n-butanol in sequence, and the solvent was recovered to obtain a petroleum ether fraction, an ethyl acetate fraction, and an n-butanol fraction;

[0009] S3. Purification of the effective components of safflower seed meal: The ethyl acetate fraction obtained in step S2 was concentrated, and 100-mesh silica gel was weighed and mixed, wet-loaded, and dry-loaded, separated by silica gel chromatography, and eluted with dichloromethane-methanol gradient in a volume ratio of (100:0)-(0:100). A total of 6 components were obtained according to the polarity, which were recorded as Fr.A-Fr.F;

[0010] S4. The component Fr.B obtained in step S3 is separated by a medium-pressure silica gel column in sequence, and gradient elution is performed using dichloromethane-methanol with a volume ratio of (80:1)-(0:1). A total of 12 components are obtained according to the polarity, which are recorded as Fr.B1-Fr.B12;

[0011] S5.Fr.B6 was separated by Sephadex LH-20 gel column with pure methanol as mobile phase to obtain Fr.B6-1-Fr.B6-3; Fr.B6-1 was separated by reverse phase semi-preparative HPLC with methanol-water as mobile phase; the elution gradient was (25:75)-(70:30) to obtain compound 1;

[0012] S6.Fr.B3 was separated by reverse phase semi-preparative HPLC using methanol-water as the mobile phase with an elution gradient of (50:50)-(80:20) to obtain compound 2.

[0013] The present invention is further configured as follows: in step S1, the volume concentration ratio of the ethanol solvent is 90-95%, and the mass volume ratio of the safflower seed meal and the ethanol solvent is 1:15-25 KG / L.

[0014] The present invention is further configured as follows: in step S1, the extraction is reflux extraction at 60°C, the number of extractions is 1-3 times, each time for 1-3 hours.

[0015] The present invention is further configured as follows: in step S2, the mass volume ratio of the obtained total ethanol extract to hot water is 1:10-20 g / ml.

[0016] The present invention is further configured as follows: in step S3, the silica gel particle size in the silica gel chromatographic column is 300-400 mesh; in step S4, the silica gel particle size in the medium-pressure silica gel column is 300-400 mesh.

[0017] The present invention is further configured as follows: in step S3, the mass ratio of ethyl acetate to silica gel is 1:1.

[0018] The present invention is further configured as follows: in step S3, the elution gradient of dichloromethane-methanol is 100:0, 70:1, 50:1, 20:1, 10:1, 1:1, 0:100, and each gradient elution is 2-5 column volumes.

[0019] The present invention is further configured as follows: in step S4, the elution gradient of dichloromethane-methanol is 80:1, 70:1, 65:1, 50:1, 40:1, 30:1, 20:1, 15:1, 10:1, 5:1, 1:1, 0:1.

[0020] The present invention is further configured as follows: in steps S5 and S6, the flow rate of the reverse semi-preparative HPLC separation mobile phase is 3 mL / min.

[0021] Another aspect of the present invention provides the use of the effective ingredients of safflower seed meal, the use of the effective ingredients of safflower seed meal prepared by any of the above methods, and the use of the compound 2 in the preparation of anti-inflammatory drugs.

[0022] In summary, the present invention has the following beneficial effects:

[0023] (1) The present invention provides two compounds derived from safflower seed meal and a method for extracting and separating the two compounds of the present invention, using alcohol extraction, silica gel column chromatography and HPLC for separation, purification and preparation, and successfully extracting and separating new compounds. The method has only four steps, the operation method is simple and fast, and the purity of the compounds separated by the method is high, all greater than 95.0%;

[0024] (2) The compounds of the present invention have significant anti-inflammatory activity, so the two compounds of the present invention and their derivatives can be used as lead compounds for the synthesis of other compounds, as well as raw materials for new drug development and pharmacological activity research. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the structural formula diagram of N-trans-feruloylmethoxytyramine and Bufoseroton in A in the embodiments of the present invention;

[0026] Figure 2 is the compound 1 in Example 1 of the present invention 1 H-NMR spectrum;

[0027] Figure 3 is the compound 1 in Example 1 of the present invention 1 C-NMR spectrum;

[0028] Figure 4 is the compound 2 in Example 1 of the present invention 1 H-NMR spectrum;

[0029] Figure 5 is the compound 2 in Example 1 of the present invention 1 H-NMR spectrum;

[0030] Figure 6 This is a comparison chart of the cell viability of RAW264.7 cells in Example 3 of the present invention;

[0031] Figure 7 This is a comparison chart of NO inhibition rate in Example 3 of the present invention. DETAILED DESCRIPTION

[0032] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Example 1 Extraction and separation of compounds

[0034] The extraction and separation method of the compound of the present invention comprises the following specific steps:

[0035] (1) Crude extraction of effective components from safflower seed meal: Using dried and crushed safflower seed meal (4 kg) as raw material, reflux extraction was performed with 80 L of 95% ethanol at 60°C for 3 times, each time for 2 h, and the filtrates were filtered, combined, and concentrated to obtain 466 g of ethanol total extract;

[0036] (2) Rough separation of effective components of safflower seed meal: The ethanol total extract obtained in step (1) was dispersed in 7 L of hot water, extracted three times with petroleum ether, ethyl acetate, and saturated n-butanol in sequence, and the solvent was recovered to obtain 158 g of petroleum ether fraction, 120 g of ethyl acetate fraction, and 228 g of n-butanol fraction;

[0037] (3) Purification of the effective components of safflower seed meal: Concentrate the ethyl acetate fraction obtained in step (2), take 90 g of ethyl acetate, weigh 100 mesh silica gel in a ratio of 1:1 and mix with the sample, weigh 120 g of silica gel and mix with the sample, dry-load the sample, separate through a silica gel chromatography column (200 mesh-400 mesh), and use dichloromethane-methanol gradient elution (100:0, 70:1, 50:1, 20:1, 10:1, 1:1, 0:100, V / V), elute for 3 column volumes respectively, detect by thin layer chromatography, combine components with similar polarity, and finally obtain 6 components, which are recorded as Fr.A-Fr.F;

[0038] (4) The component Fr.B (70:1 component, 10.7 g) obtained in step (3) was separated by a medium pressure silica gel column (300∽400 mesh) and eluted with dichloromethane-methanol gradient (80:1, 70:1, 65:1, 50:1, 40:1, 30:1, 20:1, 15:1, 10:1, 5:1, 1:1, 0:1, v / v). After thin layer chromatography, similar components were combined to obtain Fr.B1-Fr.B12. Fr.B6 (30:1 component, 380 mg) was separated by a gel column (Sephadex LH-20) with pure methanol as the mobile phase to obtain Fr.B6-1-Fr.B6-3. Fr.B6-1 was separated by reverse phase semi-preparative HPLC using a methanol-water gradient (flow rate: 3 mL / min; 25:75-70:30, v / v) as the mobile phase to obtain compound 1 (22.5 mg). Fr.B3 (65:1 component, 50 mg) was separated by reverse phase semi-preparative HPLC using a methanol-water gradient (flow rate: 3 mL / min; 50:50-80:20, v / v) as the mobile phase to obtain compound 2 (10.5 mg).

[0039] Example 2 Identification of compounds

[0040] Compound 1 1 H-NMR spectra and 1 The C-NMR spectra are shown in Figure 2 and Figure 3 As shown;

[0041] Compound 1 is N-trans-feruloylmethoxytyramine: C 20 H 20 N2O6, EI-MS m / z407.1220[M+Na] + . 1HNMR(600MHz,MeOD)δ7.37(d,J=15.6Hz,1H),7.09(d,J=2.1Hz,1H),6.99(dd,J=8.2,2.1Hz,1H),6.87(d,J=2.5Hz,1H),6.76 (d,J=8.2Hz,1H),6.73(d,J=8.4Hz,1H),6.69(dd,J=8.4,2.5Hz,1H),3.87(s,2H),3.59–3.56(m,1H),2.14(t,J=7.7Hz,1H).; 13 C NMR(151MHz,MeOD)δ181.70(C,C-2),169.12(C,C-7),154.96(C,C-22),149.63(C,C-1 3,14),142.18(CH,C-9),134.49(C,C-28),133.74(C,C-20),127.59(C,C-10),123.43( C,C-11),118.15(CH,C-8),116.72(CH,C-12),113.04(CH,C-23,24),112.00(CH,C-21 ),111.41(CH,C-17),73.86(C,C-3),64.42(CH,C-4),56.33(-OCH3),38.24(CH2,C-5).

[0042] Compound 2 1 H-NMR spectra and 1 The C-NMR spectra are shown in Figure 4 and Figure 5 As shown;

[0043] Compound 2 is Bufoserotonin A:C 11 H 13 N3O2, EI-MS m / z 220.2520[M+H] + . 1 H NMR (400MHz, DMSO) δ10.49(s,1H),7.12(d,J=8.7Hz,2H),7.04(dd,J=7.1,2.6Hz,1H),6.84(d ,J=2.4Hz,1H),6.59(dd,J=8.6,2.3Hz,1H),6.51–6.40(m,1H),3.77(d,J=23.1Hz,3H),3.45–

[0044] 3.39(m,2H),2.77(t,J=7.4Hz,2H); 13C NMR(101MHz,DMSO)δ150.19(C,C-5),130.82(C,C-9),127.89(C,C-6),123.09(CH,C-10), 111.66(CH,C-8),111.28(C,C-11),110.84(CH,C-2),102.24(CH,C-3),25.46(CH2,C-1).

[0045] The structural formulas of compound 1 and compound 2 are as follows Figure 1 shown.

[0046] Example 3 Anti-inflammatory activity test of the compound

[0047] 1. Main Materials

[0048] Drugs and reagents: The compounds used in the experiment were prepared by the above method, accurately weighed, and diluted with DMSO to the required solutions for each dosage group as shown below.

[0049] Cell line: RAW264.7 macrophages

[0050] 2. Experimental Procedure

[0051] Cell culture, DMEM high glucose medium, added 10% fetal bovine serum, 1% antibiotics (100U / mL penicillin and 100μg / mL streptomycin), placed in 37°C, 5% CO2 incubator culture. The cells were divided into a blank group, LPS model group, 5-aminosalicylic acid (5-ASA) positive drug group and treatment group. The blank group was cultured in DMEM medium, the model group was treated with 5μg / mL LPS, 5-ASA and each compound were dissolved in DMSO, prepared into a 2mg / mL stock solution, and then diluted with DMEM medium to a final concentration of 0, 25, 50, 100 and 200μg / mL. The treatment group was treated with each compound prepared as described above.

[0052] 2.1 Effect on RAW264.7 cell viability

[0053] After RAW264.7 cells were revived, they were cultured in a cell culture dish with DMEM complete medium (DMEM + 10% fetal bovine serum FBS + 1% PS), and the dish was placed in a cell culture incubator (37°C, 5% CO2). RAW264.7 cells in the logarithmic growth phase were inoculated into 96-well plates (2×10 4 / well) and cultured in an incubator for 24 hours. After the cells were completely attached, LPS (final concentration 5 μg / mL) was used to induce a cell inflammation model, and the compound (final concentration 25, 50, 100, 200 μg / mL) was added for about 24 hours of co-incubation. After taking out the above-mentioned part of the supernatant, CCK-8 reagent with a volume of 10% of the culture medium volume was added to the 96-well plate, and the absorbance value was measured according to the instructions of the kit and the cell viability was calculated.

[0054] Effect of 2.25 μg / mL LPS on NO production in RAW264.7 cells

[0055] After RAW264.7 cells were revived, they were cultured in a cell culture dish with DMEM complete medium (DMEM + 10% fetal bovine serum FBS + 1% PS), and the dish was placed in a cell culture incubator (37°C, 5% CO2). RAW264.7 cells in the logarithmic growth phase were inoculated into a 24-well plate (8×10 4 / well) and cultured overnight in an incubator. After the cells were completely attached, LPS (final concentration 5 μg / mL) was used to induce the cell inflammation model, and the two compounds (final concentration 25 μg / mL) were added and co-incubated for about 24 hours. Part of the cell supernatant was taken out, and the NO concentration in the cell supernatant was determined according to the Griess kit instructions, and the NO inhibition rate was calculated.

[0056] The study showed that the two compounds could significantly inhibit the NO level in mouse macrophage RAW246.7 cells induced by lipopolysaccharide (LPS) and had significant anti-inflammatory activity. Figure 7 As shown; and no significant cytotoxicity, such as Figure 6 Therefore, the new compound and its derivatives of the present invention can be used as the lead compound for the synthesis of other compounds, as well as the raw materials for the development of new drugs and the study of pharmacological activities, and can also be used to prepare anti-inflammatory drugs.

[0057] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for extracting and separating effective components of safflower seed meal, characterized in that: The steps include: S1. Crude extraction of effective components from safflower seed meal: using dried and crushed safflower seed meal as raw material, extracting with ethanol solvent, filtering, combining the filtrate, and concentrating to obtain an ethanol total extract; S2. Crude separation of effective ingredients from flower seed meal: The total ethanol extract obtained in step S1 was dispersed in hot water, extracted with petroleum ether, ethyl acetate, and saturated n-butanol in sequence, and the solvent was recovered to obtain a petroleum ether fraction, an ethyl acetate fraction, and an n-butanol fraction; S3. Purification of the effective components of safflower seed meal: The ethyl acetate fraction obtained in step S2 was concentrated, and 100-mesh silica gel was weighed and mixed, wet-loaded, and dry-loaded, separated by silica gel chromatography, and eluted with dichloromethane-methanol gradient in a volume ratio of (100:0)-(0:100). A total of 6 components were obtained according to the polarity, which were recorded as Fr.A-Fr.F; S4. The component Fr.B obtained in step S3 is separated by a medium-pressure silica gel column in sequence, and gradient elution is performed using dichloromethane-methanol with a volume ratio of (80:1)-(0:1). A total of 12 components are obtained according to the polarity, which are recorded as Fr.B1-Fr.B12; S5.Fr.B6 was separated by Sephadex LH-20 gel column with pure methanol as mobile phase to obtain Fr.B6-1-Fr.B6-3; Fr.B6-1 was separated by reverse phase semi-preparative HPLC with methanol-water as mobile phase; the elution gradient was (25:75)-(70:30) to obtain compound 1; S6.Fr.B3 was separated by reverse phase semi-preparative HPLC using methanol-water as the mobile phase with an elution gradient of (50:50)-(80:20) to obtain compound 2.

2. The method for extracting and separating the effective components of safflower seed meal according to claim 1, characterized in that: In step S1, the volume concentration ratio of the ethanol solvent is 90-95%, and the mass volume ratio of the safflower seed meal and the ethanol solvent is 1:15-25KG / L.

3. The method for extracting and separating the effective components of safflower seed meal according to claim 1, characterized in that: In step S1, the extraction is performed under reflux at 60°C for 1 to 3 times, each time for 1 to 3 hours.

4. The method for extracting and separating the effective components of safflower seed meal according to claim 1, characterized in that: In step S2, the mass volume ratio of the obtained total ethanol extract to hot water is 1:10-20 g / ml.

5. The method for extracting and separating the effective components of safflower seed meal according to claim 1, characterized in that: In step S3, the silica gel particle size in the silica gel chromatographic column is 200-400 mesh; in step S4, the silica gel particle size in the medium-pressure silica gel column is 200-400 mesh.

6. The method for extracting and separating the effective components of safflower seed meal according to claim 1, characterized in that: In step S3, the mass ratio of ethyl acetate to silica gel is 1:

1.

7. The method for extracting and separating the effective components of safflower seed meal according to claim 1, characterized in that: In step S3, the elution gradient of dichloromethane-methanol is 100:0, 70:1, 50:1, 20:1, 10:1, 1:1, 0:100, and each gradient elution is 2-5 column volumes.

8. The method for extracting and separating the effective components of safflower seed meal according to claim 1, characterized in that: In step S4, the elution gradient of dichloromethane-methanol is 80:1, 70:1, 65:1, 50:1, 40:1, 30:1, 20:1, 15:1, 10:1, 5:1, 1:1, 0:

1.

9. The method for extracting and separating the effective components of safflower seed meal according to claim 1, characterized in that: In steps S5 and S6, the flow rate of the mobile phase for reverse phase semi-preparative HPLC separation was 3 mL / min.

10. Use of the method according to any one of claims 1 to 9 for preparing effective ingredients of safflower seed meal, characterized in that: The application of the compound 2 in the preparation of anti-inflammatory drugs.

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