METHOD FOR SEPARATING AND PREPARING MULTIPLE ISOMER COMPOUNDS FROM SEABUCKTHORN POMACE

Abstract

The invention provides a method for separating and preparing triterpenoid acids in seabuckthorn pomace, which mainly comprises extracting and purifying triterpenoid acids, and purifying and separating using a chromatographic method under specific conditions. The present invention effectively separates two groups of isomers of triterpene acid from seabuckthorn pomace simultaneously at the first time through specific methods and conditions, and provides possibility for further research and utilization of seabuckthorn pomace.

Claims

1. A method for separating and preparing 4 isomers from seabuckthorn pomace, characterized in that, the method comprises: (1) taking seabuckthorn pomace, extracting with alcohol and then precipitating with water, loading the precipitate on a C18 column, and eluting with 20%±5, 60%±5, and 80%±5 of aqueous methanol solutions and pure methanol in turn, and collecting a first yellow band eluted with pure methanol; (2) loading the yellow band on C18 column to perform chromatography with isocratic elution of an aqueous methanol solutions of 80-90:20-10 v/v and a flow rate of 14-18 mL/min, and collecting an eluent at 29-50 min, and removing a solvent to obtain a fraction F4-2; (3) loading the fraction F4-2 on C18 column to perform chromatography with an elution procedure of: A-water, B-acetonitrile, 65%-72% B at 0-17.5 min, 72%-73% B at 17.5-20 min, 73%-77% B at 20-40 min, 77%-100% B at 40-60 min, and a flow rate of 14-18 mL/min; collecting 4th to 7th chromatographic peaks in turn from 7 obvious strong peaks; removing a solvent, and obtaining compounds 3-6 with the following structures, respectively: ##STR00011##

2. The method according to claim 1, characterized in that: in step (1), 85% v/v or more of ethanol is used for the extracting with alcohol and the precipitating with water.

3. The method according to claim 1, characterized in that: in step (1), 95% v/v or more of ethanol is used for the extracting with alcohol and the precipitating with water.

4. The method according to claim 1, characterized in that: during performing chromatography in steps (2) and (3), a detection wavelength is 210 nm.

5. The method according to claim 1, characterized in that: the chromatographic column used in step (2) is selected from Xaqua C18, 250×21.2 mm, 5 μm.

6. The method according to claim 1, wherein the chromatographic column used in step (3) is selected from Kromasil C18, 250×21.2 mm, 5 μm.

7. The method according to claim 1, wherein in step (2), the isocratic elution of aqueous methanol solutions of 85:15 v/v is adopted.

8. The method according to claim 1, wherein in steps (2) and (3), the elution flow rate is 16 mL/min.

9. A method for separating and preparing 2 isomers from seabuckthorn pomace, characterized in that, the method comprises: (1) taking seabuckthorn pomace, extracting with alcohol and then precipitating with water, loading the precipitate on a C18 column, and eluting with 20%±5, 60%±5, and 80%±5 of aqueous methanol solutions and pure methanol in turn and collecting a first yellow band eluted with pure methanol; (2) loading the yellow band on C18 column to perform chromatography with isocratic elution of an aqueous methanol solutions of 80-90:20-10 v/v and a flow rate of 14-18 mL/min, and collecting an eluent at 29-50 min, and removing a solvent to obtain a fraction F4-2; (3) loading the fraction F4-2 on C18 column to perform chromatography with an elution procedure of: A-water, B-acetonitrile, 65%-72% B at 0-17.5 min, 72%-73% B at 17.5-20 min, 73%-77% B at 20-40 min, 77%-100% B at 40-60 min, and a flow rate of 14-18 mL/min; collecting 2.sup.th to 3.sup.th chromatographic peaks in turn from 7 obvious strong peaks; removing a solvent, and obtaining compounds 1 and 2 with the following structures, respectively: ##STR00012##

10. A method for separating and preparing 6 isomers from seabuckthorn pomace, characterized in that, the method comprises: (1) taking seabuckthorn pomace, extracting with alcohol and then precipitating with water, loading the precipitate on a C18 column, and eluting with 20%±5, 60%±5, and 80%±5 of aqueous methanol solutions and pure methanol in turn and collecting a first yellow band eluted with pure methanol; (2) loading the yellow band on C18 column to perform chromatography with isocratic elution of a aqueous methanol solutions of 80-90:20-10 v/v and a flow rate of 14-18 mL/min, and collecting an eluent at 29-50 min, and removing a solvent to obtain a fraction F4-2; (3) loading the fraction F4-2 on C18 column to perform chromatography with an elution procedure of: A-water, B-acetonitrile, 65%-72% B at 0-17.5 min, 72%-73% B at 17.5-20 min, 73%-77% B at 20-40 min, 77%-100% B at 40-60 min, and a flow rate of 14-18 mL/min; collecting 2.sup.th to 7.sup.th chromatographic peaks in turn from 7 obvious strong peaks; removing a solvent, and obtaining compounds 1-6 with the following structures, respectively: ##STR00013##

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 illustrates fractions F4 and F5 eluted by pure methanol.

[0028] FIG. 2 illustrates HPLC chromatogram of the fraction F4 from seabuckthorn pomace.

[0029] FIG. 3 illustrates HPLC chromatogram of the fraction F4-2 from seabuckthorn pomace.

[0030] FIG. 4 illustrates chromatogram under comparative condition 1.

[0031] FIG. 5 illustrates chromatogram under comparative condition 2.

DETAILED DESCRIPTION

[0032] The present invention is further explained below in combination with specific embodiments, but the embodiments do not limit the present invention in any form.

Example 1

[0033] Extraction of Triterpenoid Acids from Seabuckthorn Pomace

[0034] The dried seabuckthorn pomace was crushed, added with 95% of ethanol at the ratio of material to liquid of 1:10 kg/L, heated and refluxed at a temperature of 70° C. for 1.5 h, 3 times, and filtered. The filtrate was pooled and concentrated by rotary evaporation at 60° C. to remove the solvent in order to obtain an ethanol extract from seabuckthorn pomace, which was frozen at −20° C.

[0035] Enrichment of Triterpene Acids from Seabuckthorn Pomace

[0036] Primary treatment of triterpenoid acids from seabuckthorn pomace: 1.0 kg of seabuckthorn extract was weighed, added with 2.0 L of distilled water, and heated in a water bath at 60° C. until the extract was dissolved, and stands still for precipitation, and filtered and separated to obtain a filter residue, which is dried and pulverized to obtain a crude extract of triterpene acids.

[0037] Preparation of triterpenic acids by ODS under a reduced pressure: 6.20 g of the dried crude extract was weighted, added with an appropriate amount of methanol to prepare a suspension, mixed with the dry ODS filler in a mass ratio of 1:1.5 (g/g) and then dried.

[0038] 3-5 BV of 20% methanol in water (v/v), 60% methanol in water (v/v), 80% methanol in water (v/v), and pure methanol was used successively for gradient elution. The fractions eluted with 20%, 60%, and 80% of methanol were named as F1, F2, and F3 successively. The fraction eluted with methanol was divided into two bands (see FIG. 1), the first yellow band was named as F4 and the second red band was named as F5.

[0039] (3) 0.1 g of F4 dry powder was formulated into a solution with a concentration of 50 mg/mL in methanol, centrifuged to obtain a supernatant. The supernatant was filtered with a filter membrane of 0.45 μm.

[0040] Chromatographic column: Xaqua C18 (250×21.2 mm, 5 μm); mobile phase: a methanol-water solution (85:15, v/v), isocratic elution; elution time: 80 min; flow rate: 16 mL/min; detection wavelength: 210 nm.

[0041] According to peak times of compounds, the peak time of fraction F4-1 is 0-20 min, the peak time of fraction F4-2 is 29-50 min, and the peak time of fraction F4-3 is 60-70 min. The collected fractions were centrifugated, concentrated and dried for the next step of separation.

[0042] The prepared spectrum was shown in FIG. 2.

[0043] (4) The fraction F4-2 was purified and separated by the following method:

[0044] Column: Kromasil C18 (250×21.2 mm, 5 μm); an elution program: A-water, B-acetonitrile, 65%-72% B at 0-17.5 min, 72%-73% B at 17.5-20 min, 73%-77% B at 20-40 min, 77%-100% B at 40-60 min; elution time: 50 min; flow rate: 16 mL/min; detection wavelength: 210 nm.

[0045] The prepared chromatogram of the fraction F4-2 was shown in FIG. 3. There were 7 distinct chromatographic peaks in total, and the main compounds can achieve baseline separation. By this method, a total of 6 monomer compounds of Fr-1, Fr-2, Fr-3, Fr-4, Fr-5 and Fr-6 were obtained.

[0046] Compound 1: Maslinic acid, a white amorphous powder, chemical formula: C.sub.30H.sub.48O.sub.4.

[0047] .sup.1H-NMR data (600 MHz, MeOD, δ, ppm): 5.28 (t, J=3.30 Hz, H-12); 3.95 (dt, J1=10.32 Hz, J2=4.36 and 4.32 Hz, H-2); 3.50 (d, J=9.36 Hz, H-18); 3.25 (dd, J1=13.68 Hz, J2=4.24 Hz, H-3); 3.10 (dd, J1=7.58 Hz, J2=11.64 Hz, H-2); 2.85 (td, J1=14.10 Hz, J2=4.14 Hz, H-18); 2.30 (s, H-3); 1.17 (s, Me-27); 1.08 (s, Me-23); 0.94 (s, Me-25); 0.92 (s, Me-30); 0.90 (s, Me-29); 0.86 (s, Me-24); 0.84 (s, Me-26). .sup.13C-NMR data (600 MHz, MeOD, δ, ppm): 181.8 (C-28); 145.3 (C-13); 123.5 (C-12); 84.5 (C-3); 69.5 (C-2); 56.7 (C-5) 48.1 (C-9); 48.5 (C-1); 47.2 (C-17); 47.6 (C-19); 42.7 (C-14); 42.9 (C-18); 40.5 (C-8); 40.6 (C-4); 39.3 (C-10); 34.9 (C-21); 33.8 (C-22); 33.9 (C-29); 33.6 (C-7); 31.6 (C-20); 29.3 (C-23); 28.8 (C-15); 26.4 (C-27); 24.1 (C-11); 24.0 (C-30); 24.6 (C-16); 19.6 (C-6); 17.5 (C-26); 17.1 (C-25); 17.7 (C-24). The compound was determined to be maslinic acid by comparing with the literature data and the standard substance.

##STR00005##

[0048] Compound 2: corosolic acid, a white amorphous powder, chemical formula: C.sub.30H.sub.48O.sub.4.

[0049] .sup.1H-NMR data (600 MHz, MeOD, δ, ppm): 5.28 (t, J=3.30 Hz, H-12); 3.61 (1H, m), 3.30 (dd, J1=7.58 Hz, J2=11.64 Hz, H-2); 2.92 (1H, d, J=9.5 Hz), 2.20 (d, J1=14.10 Hz, J2=4.14 Hz, H-18); 2.00 (s, H-3), 1.62 (m, H-3), 1.38 (1H, m), 1.10 (s, Me-27), 1.03 (s, Me-23), 0.98 (s, Me-25), 0.88 (s, Me-30), 0.85 (s, Me-29), 0.81 (s, Me-24), 0.76 (s, Me-26); .sup.13C-NMR data (600 MHz, MeOD, δ, ppm): δC 181.6 (C-28), 139.8 (C-13), 126.7 (C-12), 84.5 (C-3), 69.5 (C-2), 55.8 (C-5), 54.4 (C-18), 49.5 (C-17), 48.3 (C-1, C-9), 40.5 (C-4), 56.7 (C-5), 40.8 (C-19), 40.4 (C-20), 43.3 (C-8, C-14), 39.2 (C-10), 34.2 (C-7), 38.1 (C-22), 29.3 (C-15), 31.8 (C-21), 29.2 (C-23), 24.5 (C-27), 17.5 (C-29), 24.1 (C-11), 25.3 (C-16), 19.5 (C-6), 21.6 (C-30), 17.8 (C-26), 17.6 (C-25), 17.2 (C-24). The compound was determined to be corosolic acid by comparing with the literature data and the standard substance.

##STR00006##

[0050] Compound 3: 2-O-trans-p-coumaroyl maslinic acid, a white amorphous powder, chemical formula: C39145406.

[0051] .sup.1H NMR (600 MHz, MeOD, δ, ppm): 7.65 (1H, d, J=16.1, H-7′), 7.45 (1H, d, J=8.4, H-2′, 6′), 6.88 (1H, d, J=8.4, H-3′, 5′), 6.73 (1H, d, J=16.1, H-8′), 5.26 (1H, m, H-12), 4.58 (1H, d, J=9.8, H-3), 3.79 (1H, m, H-3), 2.87 (1H, dd, J=14.0, 4.2, H-18), 1.30 (3H, s, H-27), 1.18 (3H, s, H-25), 1.03 (3H, s, H-24), 0.95 (3H, s, H-30), 0.91 (3H, s, H-29), 0.88 (3H, s, H-23), 0.83 (3H, s, H-26); .sup.13C NMR (600 MHz, MeOD, δ, ppm): 181.9 (C-28), 169.8 (C-9′), 159.3 (C-4′), 145.1 (C-7′), 144.0 (C-13), 133.1 (C-2′, 6′), 127.5 (C-1′), 122.5 (C-12), 116.8 (C-3′, 5′), 115.1 (C-8′), 84.6 (C-3), 66.9 (C-2), 55.8 (C-5), 51.1 (C-9), 49.2 (C-1), 48.4 (C-17), 47.2 (C-19), 47.2 (C-14), 46.8 (C-18), 42.3 (C-4), 42.2 (C-8), 34.5 (C-10), 33.3 (C-21), 32.5 (C-7), 31.2 (C-22), 30.1 (C-29), 29.9 (C-20), 29.4 (C-23), 29.0 (C-15), 26.6 (C-27), 24.8 (C-11), 24.2 (C-16), 24.1 (C-30), 19.6 (C-6), 18.5 (C-24), 17.9 (C-26), 17.2 (C-25). The compound was determined to be 2-O-trans-p-coumaroyl maslinic acid by comparing with the literature data and the standard substance.

##STR00007##

[0052] Compound 4: 2-O-trans-p-coumaroyl corosolic acid, a white amorphous powder, chemical formula: C39145406.

[0053] .sup.1H NMR (600 MHz, MeOD, δ, ppm): 7.64 (1H, d, J=16.1, H-7′), 7.46 (1H, d, J=8.4, H-2′, 6′), 6.87 (1H, d, J=8.4, H-3′, 5′), 6.72 (1H, d, J=16.1, H-8′), 5.24 (1H, m, H-12), 4.59 (1H, d, J=9.8, H-3), 3.80 (1H, m, H-3), 2.23 (1H, d, J=11.9, H-18), 1.14 (3H, s, H-27), 1.05 (3H, s, H-25), 0.97 (3H, d, J=6.3, H-30), 0.95 (3H, s, H-24), 0.89 (3H, s, H-23), 0.88 (3H, d, J=6.3, H-29), 0.83 (3H, s, H-26); .sup.13C NMR (600 MHz, MeOD, δ, ppm): 181.9 (C-28), 169.8 (C-9′), 159.3 (C-4′), 145.1 (C-7′), 144.0 (C-13), 133.1 (C-2′, 6′), 127.5 (C-1′), 122.5 (C-12), 116.8 (C-3′, 5′), 115.1 (C-8′), 84.6 (C-3), 66.9 (C-2), 55.8 (C-5), 51.1 (C-9), 49.2 (C-1), 48.4 (C-17), 47.2 (C-19), 47.2 (C-14), 46.8 (C-18), 42.3 (C-4), 42.2 (C-8), 34.5 (C-10), 33.3 (C-21), 32.5 (C-7), 31.2 (C-22), 30.1 (C-29), 29.9 (C-20), 29.4 (C-23), 29.0 (C-15), 26.6 (C-27), 24.8 (C-11), 24.2 (C-16), 21.7 (C-30), 19.6 (C-6), 18.6 (C-24), 17.9 (C-26), 17.8 (C-29), 17.4 (C-25). The compound was determined to be 2-O-trans-p-coumaroyl corosolic acid by comparing with the literature data and the standard substance.

##STR00008##

[0054] Compound 5: 3-O-trans-p-coumaroyl maslinic acid, a white amorphous powder, chemical formula: C39145406.

[0055] .sup.1H-NMR (600 MHz, MeOD, δ, ppm) δ: 7.63 (1H, d, J=15.9 Hz, H-7′), 7.47 (2H, d, J=8.2 Hz, H-2′, 6′), 6.90 (2H, d, J=8.4 Hz, H-3′, 5′), 6.38 (1H, d, J=15.8 Hz, H-8′), 5.25 (1H, br s, H-12), 4.63 (1H, d, J=9.8 Hz, H-3), 3.84 (1H, m, H-2); .sup.13C-NMR (600 MHz, MeOD, δ, ppm) δ: 180.7 (C-28), 168.3 (C-39), 161.7 (C-34), 145.3 (C-37), 145.1 (C-13), 131.0 (C-32, 36), 126.6 (C-31), 122.6 (C-12), 117.2 (C-33, 35), 116.5 (C-38), 85.4 (C-3), 66.7 (C-2), 55.9 (C-5), 50.8 (C-9), 49.1 (C-1), 47.8 (C-19), 47.7 (C-17), 47.6 (C-14), 47.4 (C-18), 47.3 (C-8), 47.1 (C-4), 38.8 (C-10), 34.6 (C-21), 33.6 (C-22, 29), 33.4 (C-7), 31. 3 (C-20), 29.3 (C-23), 28. 6 (C-15), 26.6 (C-27), 24.3 (C-16), 24.1 (C-30), 24.0 (C-11), 19.0 (C-6), 18.6 (C-26), 17.8 (C-25), 17.1 (C-24). The compound was determined to be 3-O-trans-p-coumaroyl maslinic acid by comparing with the literature data and the standard substance.

##STR00009##

[0056] Compound 6: 3β-O-trans-p-coumaroyloxy-2a-hydroxyurs-12-en-28-oic acid, a white amorphous powder, chemical formula: C.sub.39H.sub.54O.sub.6.

[0057] .sup.1H-NMR data (600 MHz, MeOD, δ, ppm): 7.64 (2H, d, J=8.6 Hz, H-2′, 6′), 7.61 (1H, d, J=16.8 Hz, H-7′), 6.91 (2H, d, J=8.6 Hz, H-3′, 5′), 6.38 (1H, d, J=16.8 Hz, H-8′), 5.23 (1H, t, J=4.6 Hz, H-12), 4.63 (1H, d, J=10.2 Hz, H-3), 3.94 (1H, m, H-2), 3.3 (1H, m, H-18), 1.15 (3H, s, H-27), 1.06 (3H, s, H-25), 0.96 (6H, s, H-24, 30), 0.95 (3H, s, H-29), 0.89 (3H, s, H-23), 0.82 (3H, s, H-26); .sup.13C-NMR (600 MHz, MeOD, δ, ppm): 180.3 (C-28), 167.5 (C-9′), 160.8 (C-4′), 144.0 (C-7′), 139.7 (C-13), 134.1 (C-2′, 6′), 127.0 (C-1′), 125.7 (C-12), 117.4 (C-8′), 116.2 (C-3′, 5′), 85.3 (C-3), 66.6 (C-2), 55.9 (C-5), 53.8 (C-18), 50.6 (C-1), 49.0 (C-17), 47.9 (C-9), 47.7 (C-14), 47.6 (C-21), 47.4 (C-8), 47.3 (C-4), 39.8 (C-19, 20), 38.6 (C-10), 37.8 (C-22), 33.7 (C-7), 29.4 (C-23), 29.0 (C-15), 25.2 (C-16), 24.3 (C-27), 24.0 (C-11), 21.8 (C-30), 19.0 (C-6), 18. 6 (C-26), 17.9 (C-29), 17.8 (C-25), 17.2 (C-24). The compound was determined to be 3β-O-trans-p-coumaroyloxy-2a-hydroxyurs-12-en-28-oic acid by comparing with the literature data.

##STR00010##

[0058] The inventor's experiments found that the separation of the compounds in the fraction F4-2 was relatively difficult. After many selections, 7 main chromatographic peaks were finally separated through specific chromatographic conditions, and 6 main compounds among them were identified. The following conditions cannot be used for better separation of the compounds in the fraction F4-2.

[0059] Comparative condition 1: Column: Xaqua C18 (250×4.6 mm, 5 μm); mobile phase: A-water, B-acetonitrile, gradient elution: 40%-100% B at 0-50 min; analysis time: 50 min; flow rate: 1 mL/min; detection wavelength: 210 nm; column temperature: 25° C. The chromatogram was shown in FIG. 4.

[0060] Comparative condition 2: Column: Xcharge C18 (250×4.6 mm, 5 μm); mobile phase: A-water, B-acetonitrile, a gradient elution: 0-50 min 40%-100% B; analysis time: 50 min; flow rate: 1 mL/min; detection wavelength: 210 nm; column temperature: 25° C. The chromatogram was shown in FIG. 5.