A method for extracting and separating effective components from safflower seed meal and application thereof

Abstract

The application discloses a kind of safflower seed meal effective component extraction separation method and application, it is related to the technical field of traditional Chinese medicine, its technical solution main point is: S1.safflower seed meal effective component is roughly extracted: S2.safflower seed meal effective component is roughly separated;S3.safflower seed meal effective component is purified, obtains 6 components, is recorded as Fr.A-Fr.F;S4.Fr.B is separated to monomer compound 1 and monomer compound 2 by being sequentially passed through silica gel column with 300-400 granularity of medium-pressure silica gel, gel column and semi-preparative HPLC.The application uses a kind of safe, low-toxicity, high-efficiency solvent, N-trans feruloyl methoxy tyramine and Bufoserotonin A are extracted and separated from safflower seed meal for the first time, and the chromatographic material used is safe, and the production cost is low, which provides a good technical scheme for the development and utilization of safflower seed meal.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of traditional Chinese medicine, more particularly, it relates to a method for extracting and separating effective components from safflower meal and application. BACKGROUND

[0002] Safflower (Carthamus tinctorius L.) is an annual or biennial herbaceous plant of the Compositae family, with more than 50 varieties. It is called safflower because of its bright red flowers, and is also known as grass safflower, chrysanthemum safflower, red orchid, and thorny safflower, etc. Its height can reach 0.3-2.1 meters. Safflower originated in South Asia and has a history of at least 4500 years. It is one of the oldest crops in the world, and is now grown in more than 60 countries and regions, including India, Canada, the United States, Mexico, China, and Iran. The cultivation of safflower in China can be traced back to the Han Dynasty, with a history of more than 2100 years. At present, the planting area of safflower in China is about 49900 mu, mainly distributed in Xinjiang, Gansu, Sichuan and Yunnan. In addition, it is also planted in Heilongjiang in the northeast, Jilu in the Central Plains, Jiangzhe in the coastal areas, and Qingzang in the Qinghai-Tibet Plateau. The resources are abundant and the varieties are diverse.

[0003] Safflower is a traditional Chinese medicinal material, and the dried flowers are used as medicine. It was first recorded in the Kaibao Bencao and has the functions of breaking blood stasis, promoting blood circulation, relieving pain, and promoting blood circulation. It is commonly used in the treatment of dysmenorrhea, amenorrhea, and injuries. Current research shows that safflower has anti-tumor, antioxidant, anti-inflammatory, analgesic, and cardiovascular protection effects. In addition, safflower is also used as a coloring agent and flavoring agent in the food industry, and as a dye in the textile industry. Safflower seeds are white seeds that are produced after the maturation of safflower. They are commonly known as "white pingzi" in traditional Chinese medicine. The oil content is 25%-37%, and safflower seed oil is rich in unsaturated fatty acids, with a linoleic acid content of up to 80%. It is the highest oilseed crop in terms of linoleic acid content. In addition, safflower seed oil also contains vitamin E, phytosterols, and flavonoids, and other active substances, which have the functions of regulating blood lipids, improving cardiovascular health, improving obesity, and inhibiting tumors.

[0004] Safflower meal is the residue after mechanical pressing or solvent extraction of safflower seeds to produce oil. Due to its high protein content, it is often used as an animal feed additive. However, safflower meal contains the laxative arctiin and the bitter rhodalin, which to some extent limits its use. In addition, our previous research found that safflower meal has a good therapeutic effect on ulcerative colitis. Therefore, the development and utilization of effective components in safflower meal have great practical significance. In view of this, the present application provides a method for extracting and separating effective components from safflower meal and application. SUMMARY

[0005] The application aims to provide a method for extracting and separating effective components of safflower seed meal and application, and solve the above problems, and provide a good technical solution for development and utilization of safflower seed meal.

[0006] The above technical purpose of the application is achieved by the following technical solution: a method for extracting and separating effective components of safflower seed meal and application, comprising the following steps:

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

[0008] S2. Coarsely separating effective components of safflower seed meal: dispersing the ethanol total extract obtained in step S1 in hot water, sequentially extracting with petroleum ether, ethyl acetate and saturated n-butanol, recovering the solvent to obtain petroleum ether part, ethyl acetate part and n-butanol part;

[0009] S3. Purifying effective components of safflower seed meal: concentrating the ethyl acetate part obtained in step S2, weighing 100 mesh silica gel, wet packing, dry packing, separating through a silica gel chromatographic column, gradient eluting with dichloromethane-methanol with a volume ratio of (100:0)-(0:100), and according to the polarity, a total of 6 components are obtained, which are marked as Fr.A-Fr.F;

[0010] S4. Separating the component Fr.B obtained in step S3 through a medium-pressure silica gel column, gradient eluting with dichloromethane-methanol with a volume ratio of (80:1)-(0:1), and according to the polarity, a total of 12 components are obtained, which are marked as Fr.B1-Fr.B12;

[0011] S5. Separating Fr.B6 through a Sephadex LH-20 gel column to obtain Fr.B6-1-Fr.B6-3; separating Fr.B6-1 through reversed-phase semi-preparative HPLC, using methanol-water as the mobile phase; and eluting gradient is (25:75)-(70:30) to obtain compound 1;

[0012] S6. Separating Fr.B3 through reversed-phase semi-preparative HPLC, using methanol-water as the mobile phase, and eluting gradient is (50:50)-(80:20) to obtain compound 2.

[0013] The application is further provided 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 1:15-25 KG / L.

[0014] The application is further provided with: in step S1, the extraction is reflux extraction under the condition of 60 DEG C, and the extraction times are 1-3 times, and each time is 1-3 hours.

[0015] The application is further provided with: in step S2, the mass / volume ratio of the obtained ethanol total extract and hot water is 1:10-20 g / ml.

[0016] The application is further provided with: in step S3, the silica gel particle size in the silica gel chromatographic column is 300-400 mesh; and in step S4, the silica gel particle size in the medium-pressure silica gel column is 300-400 mesh.

[0017] The application is further provided with: in step S3, the mass ratio of ethyl acetate and silica gel is 1:1.

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

[0019] The application is further provided with: 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 and 0:1.

[0020] The application is further provided with: in steps S5 and S6, the flow rate of the mobile phase in the reverse semi-preparative HPLC separation is 3 mL / min.

[0021] Another aspect of the application provides the application of the effective component of safflower meal, the application of the effective component of safflower meal prepared by any of the above methods, and the application of the compound 2 in preparing anti-inflammatory drugs.

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

[0023] (1) The application provides two compounds derived from safflower meal and an extraction and separation method for the two compounds, which are separated and prepared by alcohol extraction, silica gel column chromatography and HPLC, and new compounds are successfully extracted and separated, the operation steps of the method are only four steps, the operation method is simple and fast, and the purity of the compounds separated by the method is higher, all of which are greater than 95.0%;

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

[0025] Figure 1 is the structural formula of N-trans-feruloyl methoxy tyramine and Bufoserotonin A in the embodiment of the present application;

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

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

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

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

[0030] Figure 6 is the cell viability comparison chart of RAW264.7 cells in the embodiment 3 of the present application;

[0031] Figure 7 is the NO inhibition rate comparison chart in the embodiment 3 of the present application. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0033] Extraction and separation of the compound in the embodiment 1

[0034] The extraction and separation method of the compound in the present application has the following specific steps:

[0035] (1) crude extraction of effective components in safflower meal: taking the dried and crushed safflower meal (4 Kg) as raw material, extracting 3 times at 60℃ with 80L 95% ethanol reflux extraction, 2h each time, filtering and combining the filtrate, and concentrating to obtain the total ethanol extract 466g;

[0036] (2) crude separation of effective components in safflower meal: dispersing the total ethanol extract obtained in step (1) in 7L hot water, and sequentially extracting 3 times with petroleum ether, ethyl acetate and saturated n-butanol, recovering the solvent to obtain the petroleum ether part 158g, the ethyl acetate part 120g and the n-butanol part 228g;

[0037] (3) Purification of effective components from safflower seed meal: The ethyl acetate fraction obtained in step (2) was concentrated, 90 g of ethyl acetate was taken, 100 mesh silica gel was weighed according to a proportion of 1:1 to mix with the ethyl acetate, 120 g of silica gel was weighed to mix with the ethyl acetate, and dry loading was performed. The silica gel column (200 mesh-400 mesh) was separated by gradient elution with dichloromethane-methanol (100:0, 70:1, 50:1, 20:1, 10:1, 1:1, 0:100, V / V), 3 column volumes were eluted respectively, thin layer chromatography detection was performed, and similar components were combined. Finally, 6 components were obtained, which were denoted as Fr.A-Fr.F;

[0038] (4) The component Fr.B (70:1 component, 10.7 g) obtained in step (3) was separated by medium pressure silica gel column (300-400 mesh) using dichloromethane-methanol for gradient elution (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 detection, similar components were combined to obtain Fr.B1-Fr.B12. Fr.B6 (30:1 component, 380 mg) was separated by gel column (Sephadex LH-20) using 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 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 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] Identification of the compound of example 2

[0040] Characterization of compound 1 1 H-NMR spectrum and 1 C-NMR spectrum are shown in Figure 2 and Figure 3 respectively;

[0041] Compound 1 is N-trans-feruloyl methoxy tyramine: C 20 H 20 N2O6, EI-MS m / z 407.1220 [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 as follows 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 (101 MHz, DMSO) δ 150.19 (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-l).

[0045] The structural formula of compound 1 and compound 2 are shown as Figure 1

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

[0047] 1. Main materials

[0048] Medicines and reagents: The compounds used in the experiment were prepared by the above method, accurately weighed, and diluted with DMSO to the required solution for each dose group.

[0049] Cell strain: RAW264.7 macrophage cells

[0050] 2. Experimental process

[0051] Cell culture, DMEM high-sugar medium, add 10% fetal bovine serum, 1% antibiotic (100 U / mL penicillin and 100 μg / mL streptomycin), incubate in a 37°C, 5% CO2 incubator. Divide the cells into 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 5 μg / mL LPS, 5-ASA and each compound was dissolved in DMSO to prepare a stock solution of 2 mg / mL, then diluted with DMEM medium to a final concentration of 0, 25, 50, 100 and 200 μg / mL. The treatment group was treated with each compound prepared as described above.

[0052] 2.1 Effect on RAW264.7 cell viability

[0053] After the RAW264.7 cells were resuscitated, they were cultured in cell culture dishes with DMEM complete medium (DMEM + 10% fetal bovine serum FBS + 1% PS), and the culture dishes were placed in a cell incubator (37°C, 5% CO2). The logarithmic growth phase of RAW264.7 cells was inoculated into 96-well plates (2 x 10 4 ​After the cells were completely adhered, the cells were induced by LPS (final concentration 5 μg / mL) to establish the inflammation model, and then the two compounds (final concentration 25 μg / mL) were added to co-incubate for about 24 h. After that, part of the supernatant was taken out, and the NO concentration in the supernatant was determined according to the Griess kit instruction to calculate the NO inhibition rate.

[0054] 2.25 μg / mL LPS induced RAW264.7 cells to produce NO level

[0055] After the RAW264.7 cells were recovered, DMEM complete culture medium (DMEM + 10% fetal bovine serum FBS + 1% PS) was used to culture the cells in a cell culture dish, and the culture dish was placed in a cell incubator (37°C, 5% CO2). The logarithmic growth phase RAW264.7 cells were inoculated into a 24-well plate (8×10 4 cells / well), and the cells were cultured in the incubator overnight. After the cells were completely adhered, the cells were induced by LPS (final concentration 5 μg / mL) to establish the inflammation model, and then the two compounds (final concentration 25 μg / mL) were added to co-incubate for about 24 h. After that, part of the supernatant was taken out, and the NO concentration in the supernatant was determined according to the Griess kit instruction to calculate the NO inhibition rate.

[0056] The research shows that the two compounds can significantly inhibit the NO level in the mouse macrophage RAW246.7 caused by lipopolysaccharide (LPS), and have significant anti-inflammatory activity, as shown in Table 1. Figure 7 and have no significant cytotoxicity, as shown in Table 2. Figure 6 Therefore, the new compounds and derivatives thereof in the present application can be used as a lead for the synthesis of other compounds, and a raw material for the development of new drugs and the research of pharmacological activity, and can also be used for preparing anti-inflammatory drugs and the like.

[0057] Finally, it should be noted that: the above only describes the preferred embodiments of the present application and is not used to limit the present application, although the present application has been described in detail with reference to the foregoing embodiments, and those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments, or make equivalent replacement to some technical features. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for extracting and separating the effective components of safflower seed meal, characterized by: The steps include: S1. Crude extraction of active ingredients 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 active ingredients from flower seed meal: The total ethanol extract obtained in step S1 was dispersed in hot water, sequentially extracted with petroleum ether, ethyl acetate, and saturated n-butanol, and the solvent was recovered to obtain a petroleum ether fraction, an ethyl acetate fraction, and a n-butanol fraction; S3. Purification of the active ingredients of safflower seed meal: The ethyl acetate fraction obtained in step S2 was concentrated, and the sample was weighed and mixed with 100-mesh silica gel. The sample was wet-packed and dry-loaded. The product was separated by silica gel chromatography and eluted with a dichloromethane-methanol gradient at a volume ratio of (100:0) to (0:100). A total of six components were obtained according to their polarity, which were designated as Fr.A-Fr.F. S4. Separate the fraction Fr.B obtained in step S3 by passing it through a medium-pressure silica gel column using a gradient elution method of dichloromethane-methanol at a volume ratio of (80:1) to (0:1). Twelve fractions were obtained based on polarity, designated 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. The structural formula of the compound 1 is 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. The structural formula of compound 2 is 2. The method for extracting and separating the effective components of safflower seed meal according to claim 1, wherein: 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, wherein: In step S1, the extraction is performed under reflux at 60°C for 1-3 times, each time for 1-3 hours.

4. The method for extracting and separating the effective components of safflower seed meal according to claim 1, wherein In step S2, the mass volume ratio of the obtained ethanol total 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, wherein: In step S3, the silica gel in the silica gel chromatography column has a particle size of 200-400 mesh; in step S4, the silica gel in the medium-pressure silica gel column has a particle size of 200-400 mesh.

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

10. Use of the method according to any one of claims 1 to 9 in preparing an effective ingredient of safflower seed meal, characterized in that: The active ingredient is compound 1 or compound 2.

Images