ISOMERIZATION FEATURE-BASED METHOD FOR PURIFYING PUNICALAGIN

Abstract

An isomerization feature-based method for purifying and preparing punicalagin is provided, wherein pomegranate peel extract is used as a raw material. The isomerization feature-based method avoids impurities contained in the punicalagin based on structural characteristics, for example, the punicalagin in the pomegranate peel extract has two mutually convertible isomers. In the isomerization feature-based method, a pilot-scale preparative liquid chromatography is used to obtain a large amount of the punicalagin having a purity higher than 98% from a complex pomegranate peel extract. The isomerization feature-based method is simple, an obtained punicalagin has high purities and a preparation is in a massive scale. The isomerization feature-based has a strong reference value for purification and preparation of compounds with isomerization features.

Claims

1. An isomerization feature-based method for purifying punicalagin, comprising the following steps of: a) taking a reversed-phase chromatographic column, wherein a mobile phase is consisted of methanol and 0.01%-5% formic acid water in a volume ratio of 1-5:9-5, a flow rate is controlled at 50-300 mL/min, and a UV detection wavelength is 220-280 nm and 350-380 nm, and the reversed-phase chromatographic column is balanced for 3-30 min to obtain a balanced reversed-phase chromatographic column; b) weighing a pomegranate peel extract, dissolving the pomegranate peel extract in water, taking a supernatant after centrifuging an extract solution and loading the supernatant into the balanced reversed-phase chromatographic column, collecting two components around two chromatographic peaks around α-punicalagin and β-punicalagin respectively, then concentrating the two components at 30° C.-50° C. respectively to obtain two concentrated components; c) reloading the two concentrated components at a position of the α-punicalagin obtained in step b) into the reversed-phase chromatographic column under identical conditions as step a), and collecting the two components of the two chromatographic peaks around the α-punicalagin and the β-punicalagin respectively, wherein a first component of the two components collected around a chromatographic peak of the β-punicalagin is concentrated by a rotary evaporator at a temperature of 30° C.-50° C. and lyophilized to obtain g powder of the punicalagin with a purity of higher than 98% after detected by a high-performance liquid chromatography; concentrating a second component of the two components collected from a chromatographic peak of the α-punicalagin by the rotary evaporator at a temperature of 30° C.-50° C., and circularly loading the two concentrated components under an identical chromatographic condition as step a) to prepare high-purity punicalagin; and/or, reloading the two concentrated components collected from the chromatographic peak of the β-punicalagin obtained in step b) into the reversed-phase chromatographic column under identical conditions as step a), and collecting the two components from the two chromatographic peaks around the α-punicalagin and the β-punicalagin respectively, wherein the second component of the two components collected from the chromatographic peak of the α-punicalagin is a qualified product; concentrating the first component of the two components collected from the chromatographic peak of the β-punicalagin by the rotary evaporator at the temperature of 30° C.-50° C., reloading the two concentrated components into the reversed-phase chromatographic column of step a), collecting the two components from the two chromatographic peaks of the α-punicalagin and the β-punicalagin respectively, wherein the second component of the two components collected from the chromatographic peak of the α-punicalagin is the qualified product; combining the second component collected from the chromatographic peak of the α-punicalagin and the first component collected from the chromatographic peak of the β-punicalagin in step c) to obtain combined components, concentrating the combined components by the rotary evaporator at the temperature of 30° C.-50° C. to obtain concentrated combined components, lyophilizing the concentrated combined components to obtain a pale yellow powder of the punicalagin with a purity of higher than 98% after detected by the high-performance liquid chromatography; concentrating the first component collected from the chromatographic peak of the β-punicalagin at the temperature of 30° C.-50° C., and circularly loading the two concentrated components under the identical chromatographic condition as step a) to prepare the high-purity punicalagin.

2. The isomerization feature-based method according to claim 1, wherein in step b), 0.1-10 g of the pomegranate peel extract is weighed and dissolved in 10-50 mL of water.

3. The isomerization feature-based method according to claim 1, wherein in step b), the extract solution is centrifuged at a rotation speed of 5000-10000 revolutions per minute for 3-10 minutes.

4. The isomerization feature-based method according to claim 1, wherein a concentration is performed to reduce a volume to 10-50 mL.

5. The isomerization feature-based method according to claim 1, wherein a concentration is performed using the rotary evaporator.

6. A method for purifying punicalagin according to claim 1, comprising following steps of: a) taking a pilot-scale reverse-phase chromatographic column, wherein the mobile phase is consisted of the methanol and 0.1% formic acid water in a volume ratio of 12:88, the flow rate is controlled at 180 mL/min, the temperature is set at room temperature, and the UV detection wavelength is 254 nm and 366 nm, the pilot-scale reverse-phase chromatographic column is balanced for 15 min and ready for use to obtain a balanced pilot-scale reverse-phase chromatographic column; b) weighing 6 g of the pomegranate peel extract, dissolving the 6 g of the pomegranate peel extract in 20 mL of water, taking the supernatant after centrifuging the extract solution at 10,000 rpm for 5 minutes and loading the supernatant into the balanced pilot-scale reversed-phase chromatographic column through a six-way valve, collecting the two components from the two chromatographic peaks of the α-punicalagin and the β-punicalagin, then concentrating the two components to a volume of 20 mL with a rotary evaporator at 50° C. respectively to obtain two concentrated components; c) reloading the second component of the two concentrated components collected from the chromatographic peak of the α-punicalagin obtained in step b) into the pilot-scale reversed-phase chromatographic column under the identical conditions as step a), and collecting the two components from the two chromatographic peaks around the α-punicalagin and the β-punicalagin respectively, wherein the first component collected from the chromatographic peak of the β-punicalagin is the qualified product; concentrating the qualified product with the rotary evaporator at a temperature of 39° C. to obtain a concentrated product and lyophilizing the concentrated product to obtain 137 mg of the pale yellow powder of the punicalagin with a purity higher than 98% after detected by the high-performance liquid chromatography; concentrating the second component collected from the chromatographic peak of α-punicalagin to a volume of 20 mL with the rotary evaporator at a temperature of 50° C., and circularly loading the two concentrated components under the identical chromatographic condition as step a) to prepare the high-purity punicalagin; or, reloading the two concentrated components collected from the chromatographic peak of β-punicalagin obtained in step b) into the pilot-scale reversed-phase chromatographic column under the identical conditions as step a), and collecting the two components from the two chromatographic peaks of the α-punicalagin and the β-punicalagin respectively, wherein the second component collected from the chromatographic peak of the α-punicalagin is the qualified product; concentrating the second component collected by cutting at a position of the β-punicalagin to the volume of 20 mL with the rotary evaporator at the temperature of 50° C., reloading the two concentrated components into the pilot-scale reversed-phase chromatographic column of step a), collecting the two components from the two chromatographic peaks of the α-punicalagin and the β-punicalagin respectively, wherein the second component collected from the chromatographic peak of the α-punicalagin is the qualified product; combining the second component collected from the chromatographic peak of the α-punicalagin, and the first component collected from the chromatographic peak of the β-punicalagin in step c) to obtain the combined components, concentrating the combined components at the temperature of 39° C., lyophilizing the two concentrated components to obtain 280 mg of the pale yellow powder of the punicalagin with a purity of higher than 98% after detected by the high-performance liquid chromatography; concentrating the first component collected from the chromatographic peak of the β-punicalagin to the volume of 20 mL with the rotary evaporator at the temperature of 50° C., and circularly loading the two concentrated components under the identical chromatographic condition as step a) to prepare the high-purity punicalagin.

7. The isomerization feature-based method according to claim 4, wherein the concentration is performed using the rotary evaporator.

8. The method according to claim 6, wherein in step b), 0.1-10 g of the pomegranate peel extract is weighed and dissolved in 10-50 mL of water.

9. The method according to claim 6, wherein in step b), the extract solution is centrifuged at a rotation speed of 5000-10000 revolutions per minute for 3-10 minutes.

10. The method according to claim 6, wherein a concentration is performed to reduce the volume to 10-50 mL.

11. The method according to claim 6, wherein a concentration is performed using the rotary evaporator.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 is a pilot-scale reversed-phase chromatogram of the pomegranate peel extract involved in the present invention;

[0028] FIG. 2 is a preparative chromatogram of the α-punicalagin component 1 involved in the present invention after being concentrated and reloaded onto the pilot-scale reversed-phase chromatographic column;

[0029] FIG. 3 is a liquid chromatographic identification diagram of the β-punicalagin component 3 obtained in the present invention;

[0030] FIG. 4 is a preparative chromatogram of the β-punicalagin component 4 involved in the present invention after being concentrated and reloaded onto the pilot-scale reversed-phase chromatographic column;

[0031] FIG. 5 is a liquid chromatographic identification diagram of the combined α-punicalagin component 5 and α-punicalagin component 7 obtained in the present invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0032] The present invention will be further described in detail below in conjunction with specific embodiments.

Example 1

[0033] a. A pilot-scale reversed-phase chromatographic column was taken, wherein the column had a size of 250 mm (column length)×80 mm (inner diameter); its filler had a particle size of 10 μm; the mobile phase was consisted of methanol and 0.1% formic acid water in a volume ratio of 12:88; the flow rate was kept at 180 mL/min; the temperature was set at room temperature, and the UV detection wavelength was 254 nm and 366 nm, the column was equilibrated for 15 min, and ready for use.

[0034] b. 6 g of pomegranate peel extract was weighed, wherein the content of punicalagin was about 32%. The extract was dissolved in 20 mL of water and centrifuged at 10,000 rpm for 5 minutes. The supernatant was collected and loaded into the equilibrated pilot-scale reversed-phase chromatographic column through a six-way valve, as shown in FIG. 1. The component named as component 1 in the time range from 10 min to 15 min i.e. α-punicalagin was collected in a component tank and concentrated to a volume of 20 mL with a rotary evaporator at 50° C. respectively.

[0035] c. The component 1 was reloaded into the balanced pilot-scale reversed-phase chromatographic column according to step a. Its chromatogram is shown in FIG. 2. The components named as component 2 in the time range from 12.5 min to 20 min (around α-punicalagin) and component 3 in the time range from 25 to 35 minutes (around s-punicalagin) were collected in different component tanks respectively. The component 3 was concentrated with a rotary evaporator at a temperature of 39° C., lyophilized to obtain pale yellow powder. Such powder was weighed 137 mg in total and showed a purity of higher than 98% after detected by high-performance liquid chromatography, as shown in FIG. 3. The component 2 was concentrated to a volume of 20 mL with a rotary evaporator at a temperature of 50° C., and circularly loaded under the same chromatographic condition as step a to prepare high-purity punicalagin.

Example 2

[0036] a. A pilot-scale reversed-phase chromatographic column was taken, wherein the column had a size of 250 mm (column length)×80 mm (inner diameter); its filler had a particle size of 10 μm; the mobile phase was consisted of methanol and 0.1% formic acid water in a volume ratio of 12:88; the flow rate was kept at 180 mL/min; the temperature was set at room temperature, and the UV detection wavelength was 254 nm and 366 nm, the column was equilibrated for 15 min, and ready for use.

[0037] b. 6 g of pomegranate peel extract was weighed, wherein the content of punicalagin was about 32%. The extract was dissolved in 20 mL of water and centrifuged at 10,000 rpm for 5 minutes.

[0038] The supernatant was collected and loaded into the balanced pilot-scale reversed-phase chromatographic column through a six-way valve, as shown in FIG. 1. The component named as component 4 around β-punicalagin (ranging from 17 min to 30 min) was collected in a component tank and concentrated to a volume of 20 mL with a rotary evaporator at 50° C. respectively.

[0039] c. The component 4 was reloaded into the balanced pilot-scale reversed-phase chromatographic column according to step a. Its chromatogram is shown in FIG. 4. The components named as component 5 in the time range from 12.5 min to 20 min (around α-punicalagin) and component 6 in the time range from 25 to 35 minutes (around β-punicalagin) were collected in different component tanks respectively. The n component 5 was the qualified product.

[0040] The component 6 was concentrated to a volume of 20 mL with a rotary evaporator at a temperature of 50° C., and reloaded into the balanced pilot-scale reversed-phase chromatographic column according to step a. Its chromatogram is also shown in FIG. 4. The components named as component 7 in the time range from 12.5 min to 20 min (around α-punicalagin) and component 8 in the time range from 25 to 35 minutes (around β-punicalagin) were collected in different component tanks respectively. The α- component 7 was the qualified product. It was combined with the component 5, and concentrated with a rotary evaporator at a temperature of 39° C., then lyophilized to obtain pale yellow powder. Such powder was weighed 280 mg in total and showed a purity of higher than 98% after detected by high-performance liquid chromatography, as shown in FIG. 5. The component 8 was concentrated to a volume of 20 mL with a rotary evaporator at a temperature of 50° C., and circularly loaded under the same chromatographic condition as step a to prepare high-purity punicalagin.

[0041] Besides methanol-0.1% formic acid water mixture, the mobile phase of the reversed-phase chromatographic column used in the Examples can also be acetonitrile-0.1% formic acid water mixture, tetrahydrofuran-0.1% formic acid water mixture, methanol-acetonitrile-0.1% formic acid water mixture, methanol-tetrahydrofuran-0.1% formic acid water mixture, acetonitrile-tetrahydrofuran-0.1% formic acid water mixture, methanol-acetonitrile-tetrahydrofuran-0.1% formic acid water mixture. The content of 0.1% formic acid water should be controlled between 50% and 95% in the mobile phase.

[0042] In addition to the reversed-phase C18 chromatographic column, other reversed-phase chromatographic columns including reversed-phase C8 column, reversed-phase C4 column, reversed-phase C30 column, reversed-phase C2 column, reversed-phase cyano column, reversed-phase polystyrene column can also be used as the reversed-phase chromatographic columns of the Examples.

[0043] Besides the column length of 250 mm, the inner diameter of 80 mm, and the filler particle size of 10 μm, the reversed-phase chromatographic column involved in the Examples, can also has a column length of 50 mm-1000 mm, an inner diameter of 2.1 mm-2000 mm, and a filler particle size of 1.2 μm-500 μm.