Method for efficiently preparing pentagalloylglucose reference substance from mango kernels on large scale

Abstract

The invention provides a method for efficiently preparing a pentagalloylglucose chemical reference substance from mango kernels on a large scale. According to the method, the pentagalloylglucose chemical reference substance with the purity of 98% or above can be obtained by adopting two steps of polyamide column chromatography enrichment and separation and high performance liquid chromatography refining for mango kernel alcohol extract extracted by conventional heating reflux. The process steps are simple, the preparation efficiency of the pentagalloylglucose chemical reference substance is high, the preparation amount is large, and the method is suitable for industrial mass production.

Description

1. Technical Field

[0001] The present invention relates to a method for efficiently and massively preparing pentagalloylglucose chemical reference substance from mango kernels. The invention belongs to the field of modernization of traditional Chinese medicine. The pentagalloylglucose compound has the following structural formula:

[0002] 2. Background Technology

[0003] Pentagalloylglucose is widely present in many Chinese medicinal materials such as mango kernel, white peony root, red peony root, gallnut, peony bark, scutellaria, aloe, cassia seed, herba eucommiae, litchi kernel, chrysanthemum, safflower, eucommia bark and rhubarb. It has multiple pharmacological activities such as anti-tumor, anti-inflammatory, hypoglycemic, antioxidant, antimutagenic, anticonvulsant, antiallergic, antiviral, antibacterial and inhibition of kidney stone formation (Pharmaceutical Research, 2009, 26(9): 2066-2080). It is an important indicator for quality control of Chinese medicine. However, due to the lack of reference substances, the quality control of pentagalloylglucose is limited to paper publication and is rarely used in practice. Therefore, it is of great significance to provide a method for preparing pentagalloylglucose chemical reference substances with simple preparation process and high efficiency and scale.

[0004] Li Xiao et al. used white peony root as the research object and prepared pentagalloylglucose reference substance from it (patent CN10312510B). However, since the content of pentagalloylglucose in white peony root is very low, only 0.18% (Journal of Beijing University of Chinese Medicine, 2009, 32(2): 122-124), the yield of pentagalloylglucose reference substance is very low. In addition, the separation process is complicated and the preparation amount is small because the preparation method undergoes two macroporous resin enrichment and desorption steps and then two gel column chromatography steps.

[0005] Mango is a tropical evergreen tree of the genus Mangifera in the Anacardiaceae family. It is widely distributed. More than 70 countries in the world produce mangoes, with an annual output of nearly 2.0×107t, of which my country's annual output is 1.0×10 6 t. Mangoes are eaten fresh or processed to produce a large number of pits, and the kernel in the pit accounts for about 20% of the total weight of mangoes, but the mango kernel is often discarded and not effectively utilized. Mango kernel has the effect of tonifying the kidney and removing kidney cold, and is used for kidney deficiency, kidney cold, low back pain, hernia and orchitis. Studies in recent years have shown that mango kernel has antibacterial and antidiarrheal, antioxidant and anti-inflammatory pharmacological effects.

[0006] Mango kernel is a traditional Chinese medicine with significant pharmacological effects and clinical efficacy and broad development prospects, but mango kernel is often treated as waste and has not been effectively utilized. Pentagalloylglucose is the main chemical component of mango kernel, and its content in mango kernel is as high as 4.63-9.42% (Chinese Journal of Hospital Pharmacy, 2018, 38(13): 1372-1376). Therefore, preparing pentagalloylglucose reference substance from mango kernel not only has a high yield, but also can turn waste into treasure. Therefore, the present invention aims to provide a method for efficiently and massively preparing pentagalloylglucose chemical reference substance from mango kernel. The process steps of the present invention are simple, the preparation efficiency of pentagalloylglucose chemical reference substance is high, and the preparation amount is large. III. Summary of the invention

[0007] The present invention aims to provide a preparation process for efficiently and massively preparing pentagalloylglucose chemical reference substance from mango kernels. The process steps of the present invention are simple, the preparation efficiency of pentagalloylglucose chemical reference substance is high, and the preparation amount is large.

[0008] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0009] For the mango kernel alcohol extract extracted by conventional heating and reflux, two steps of polyamide column chromatography enrichment and separation and high performance liquid phase preparative chromatography purification are used to obtain a pentagalloylglucose chemical reference substance with a purity of more than 98%. The specific steps are as follows:

[0010] (1) Enrichment and separation by polyamide column chromatography: The alcohol extract prepared from mango kernel by heating reflux method is enriched by polyamide column chromatography, and then eluted with water, 10% ethanol aqueous solution, 30% ethanol aqueous solution, and 50-70% ethanol aqueous solution in sequence. The polyamide thin layer chromatography developing solvent is a 50:50 methanol aqueous solution, and the color developer is a 0.5% ferric chloride aqueous solution. The 50%-70% ethanol aqueous solution portion is collected, and the solvent is recovered under reduced pressure to obtain a crude pentagalloylglucose product.

[0011] (2) Reverse phase high performance liquid preparative chromatography purification: The crude product is dissolved in a methanol aqueous solution with a volume concentration of 40-60%, and filtered with a microporous filter membrane to obtain a test sample. The test sample is purified and separated by reverse phase high performance preparative liquid chromatography, the chromatographic column is filled with C18, the column length is 20-22 cm, and the diameter is 5-7.5 cm. The mobile phase is a methanol aqueous solution with a volume concentration of 45-55%. The pH of the methanol aqueous solution is adjusted to 3 with formic acid, the injection volume is 1-5 mL, the flow rate is 80-180 mL / min, and the ultraviolet detector is monitored at 273 nm. The main chromatographic peak fractions in the preparative chromatogram are collected, and the chromatographic peak fractions are dried under reduced pressure to obtain a white powdery pentagalloyl glucose reference substance with a purity greater than 98%.

[0012] The present invention separates and prepares pentagalloyl glucose chemical reference substance from mango kernel, which has the following advantages and progress:

[0013] 1. Mango kernels are often used as waste. The present invention prepares pentagalloylglucose chemical reference substance from mango kernels, which can turn waste into treasure.

[0014] 2. The process of the present invention is simple. The mango kernel alcohol extract is separated by polyamide column chromatography and purified by high performance liquid phase preparative chromatography.

[0015] The steps can obtain the pentagalloylglucose chemical reference substance with a purity of more than 98%.

[0016] 3. The yield of the pentagalloylglucose chemical reference substance of the present invention is relatively high, reaching about 2.0%.

[0017] 4. The present invention has a large preparation volume and can prepare a 10-gram reference substance through one separation and purification. 4. Specific implementation methods

[0018] The present invention is now described in further detail with reference to the examples, which are intended to illustrate the present invention but not to limit the present invention.

[0019] Example 1

[0020] (1) Polyamide column chromatography enrichment and separation: The mango kernel alcohol extract was enriched and separated by polyamide column chromatography, eluted with water, 10% ethanol aqueous solution, 30% ethanol aqueous solution, and 50% ethanol aqueous solution in sequence, and identified by polyamide thin layer chromatography (developing agent is 50:50 methanol aqueous solution, color developer is 0.5% ferric chloride aqueous solution), and the 50% ethanol aqueous solution fraction was collected. The solvent was recovered under reduced pressure to obtain a crude pentagalloylglucose product.

[0021] (2) Reverse phase high performance liquid preparative chromatography purification: The crude product was dissolved in a methanol aqueous solution with a volume concentration of 40%, and filtered with a microporous filter membrane to obtain the test sample. The test sample was purified and separated by reverse phase high performance preparative liquid chromatography, the chromatographic column was filled with C18, the column length was 20 cm, the diameter was 5 cm, the volume concentration of 55% methanol aqueous solution was used as the mobile phase, the pH of the methanol aqueous solution was adjusted to 3 with formic acid, the injection volume was 5 mL, the flow rate was 100 mL / min, the ultraviolet detector was monitored at 273 nm, the main chromatographic peak fractions in the preparative chromatogram were collected, and the chromatographic peak fractions were dried under reduced pressure to obtain a white powdery pentagalloyl glucose reference substance with a purity greater than 98%.

[0022] Example 2

[0023] (1) Polyamide column chromatography enrichment and separation: The mango kernel alcohol extract was separated by polyamide column chromatography, eluted with water, 10% ethanol aqueous solution, 30% ethanol aqueous solution, and 70% ethanol aqueous solution in sequence, and identified by polyamide thin layer chromatography, and the 60% ethanol aqueous solution fraction was collected. The solvent was recovered under reduced pressure to obtain a crude pentagalloylglucose product.

[0024] (2) Reverse phase high performance liquid chromatography purification: The crude product was dissolved in a methanol aqueous solution with a volume concentration of 60%, and filtered with a microporous filter membrane to obtain the test sample. The test sample was purified and separated by reverse phase high performance preparative liquid chromatography, the chromatographic column was filled with C18, the column length was 22 cm, the diameter was 6 cm, the volume concentration of methanol aqueous solution was 45% as the mobile phase, the pH of the methanol aqueous solution was adjusted to 3 with formic acid, the injection volume was 3 mL, the flow rate was 150 mL / min, the ultraviolet detector was monitored at 273 nm, the main chromatographic peak fractions in the preparative chromatogram were collected, and the chromatographic peak fractions were dried under reduced pressure to obtain a white powdery pentagalloyl glucose reference substance with a purity greater than 98%.

[0025] Example 3

[0026] (1) Polyamide column chromatography enrichment and separation: The mango kernel alcohol extract was separated by polyamide column chromatography, eluted with water, 10% ethanol aqueous solution, 30% ethanol aqueous solution, and 70% ethanol aqueous solution in sequence, and identified by polyamide thin layer chromatography, and the 70% ethanol aqueous solution fraction was collected. The solvent was recovered under reduced pressure to obtain a crude pentagalloylglucose product.

[0027] (2) Reverse phase high performance liquid chromatography purification: The crude product was dissolved in a methanol aqueous solution with a volume concentration of 50%, and filtered with a microporous filter membrane to obtain the test sample. The test sample was purified and separated by reverse phase high performance preparative liquid chromatography, the chromatographic column was filled with C18, the column length was 22 cm, the diameter was 7 cm, the volume concentration of 50% methanol aqueous solution was used as the mobile phase, the pH of the methanol aqueous solution was adjusted to 3 with formic acid, the injection volume was 4 mL, the flow rate was 180 mL / min, the ultraviolet detector was monitored at 273 nm, the main chromatographic peak fractions in the preparative chromatogram were collected, and the chromatographic peak fractions were dried under reduced pressure to obtain a white powdery pentagalloyl glucose reference substance with a purity greater than 98%.

Claims

1. A method for efficiently and massively preparing pentagalloylglucose chemical reference substance from mango kernels, characterized in that Follow these steps: (1) Polyamide column chromatography enrichment and separation: The alcohol extract prepared from mango kernel by conventional heating reflux method is enriched by polyamide column chromatography, and then eluted with water, 10% ethanol aqueous solution, 30% ethanol aqueous solution, and 50-70% ethanol aqueous solution in sequence, the polyamide thin layer chromatography detection developing solvent is 50:50 methanol aqueous solution, the color developing agent is 0.5% ferric chloride aqueous solution, the 50-70% ethanol aqueous solution portion is collected, and the solvent is recovered under reduced pressure to obtain a crude pentagalloylglucose product; The mango kernel is the dried fruit kernel of Mangifera indica L., a plant of the Anacardiaceae family; (2) Reverse phase high performance liquid chromatography purification: The crude product was dissolved in a methanol aqueous solution with a volume concentration of 40-60%, and filtered through a microporous filter membrane to obtain a test product; The test sample is purified and separated by reverse phase high performance preparative liquid chromatography, the chromatographic column is filled with C18, the column length is 20-22 cm, the diameter is 5-7.5 cm, a methanol aqueous solution with a volume concentration of 45-55% is used as the mobile phase, the pH of the methanol aqueous solution is adjusted to 3 with formic acid, the injection volume is 1-5 mL, the flow rate is 80-180 mL / min, the ultraviolet detector is monitored at 273 nm, and the main chromatographic peak fractions in the preparation chromatogram are collected. The chromatographic peak fractions are dried under reduced pressure to obtain a white powdery pentagalloyl glucose reference substance with a purity greater than 98%.

Images