CN114573446B - Method for preparing protocatechuic acid from phellinus linteus medicinal material - Google Patents

Abstract

The invention provides a method for preparing protocatechuic acid from phellinus linteus medicinal material, which comprises the steps of extracting the phellinus linteus medicinal material with pure water, adopting a two-dimensional orthogonal liquid chromatography technology, enriching pure water resistant C8YE silica gel filler as a separation material to obtain crude protocatechuic acid in one dimension, and preparing and purifying the crude protocatechuic acid in two dimensions by using polar copolymerization C18HC bonded silica gel filler to obtain a protocatechuic acid product with the purity of more than 95 percent. The preparation process of the invention has stable process, high yield, high automation degree and easy operation, and can meet the requirement of large-scale production.

Description

A method for preparing protocatechuic acid from phellinus medicinal material

technical field

The method relates to the technical field of separation and purification, in particular to a method for preparing protocatechuic acid from Phellinus phellifolia. Further, the present invention is a method for preparing high-purity protocatechuic acid from the Phellinus phylloxera medicinal material by using two-dimensional liquid chromatography technology, and the purity can reach more than 95%.

Background technique

Phellinus is a precious perennial large medicinal fungus, known as "forest gold", which is recorded in "Compendium of Materia Medica". Belonging to the Basidiomycota Phyllomycetes, Polyporaceae Acupunculus genus, there are 40 species distributed in my country. It mainly parasitizes on the trunks of mulberry, willow, birch, poplar and oak, among which the wild mulberry Phellinus is the most precious.

Phellinus Phellinus, as a traditional fungal traditional Chinese medicine in my country, is currently internationally recognized as the most effective medicinal fungus in the field of biological cancer treatment. It contains chemical components of various structural types, including polysaccharides, flavonoids, triterpenoids, aromatic acids, amino acids, etc.; modern pharmacological experiments also show that Phellinus has anti-cancer, immune regulation, anti-obesity, anti-oxidation, and anti-inflammatory properties. Various pharmacological activities.

In recent years, searching for dual-target drugs with neurotrophic-like effects and enhancing the effects of neurotrophic factors in natural compounds has become a research hotspot. According to research, protocatechuic acid has various functions such as anti-cancer, anti-oxidation, anti-inflammation, and hypoglycemia, and is a healthy and safe beneficial small molecule substance.

Protocatechuic acid (3,4-dihydroxybenzoic acid), the property is a white powder, the structural formula is as follows:

There are multiple -OH in the molecular formula of protocatechuic acid, which has a certain binding effect with the silanol groups on the surface of silica gel, which is the main reason for the serious tailing and difficult separation during the preparation of the substance. In order to solve this problem, the patent The one-dimensional and two-dimensional separation fillers used are respectively treated with multi-point mild bonding, end-sealing technology and polar copolymerization technology to treat the surface of the filler, which greatly improves the tailing problem in the preparation process;

Protocatechuic acid has various pharmacological activities such as anti-platelet aggregation, reducing myocardial oxygen consumption, antibacterial, analgesic, etc., and is an important natural active substance. In recent years, studies have found that it has many new pharmacological effects such as anti-oxidation, anti-cancer and mediating tumor cell apoptosis; it has a certain protective effect on nerve cells in ischemic and hypoxic neurons; it can significantly relieve interleukin 1β Inflammatory response to chondrocytes; can maintain the phenotype of damaged chondrocytes and promote proliferation; maintain chondrocyte phenotype and promote cell proliferation, etc.

Protocatechuic acid has strong antioxidant capacity, has obvious inhibitory effect on Staphylococcus aureus, streptococcus, etc., and has the effect of astringent and promoting wound healing. It can be used to treat inflammation, fever and cancer.

There are few reports on the preparation of protocatechuic acid from Phellinus chinensis, and the spectrum-effect relationship is not clear. Therefore, the preparation of this compound is of great significance for the basic research and pharmacological activity of Phellinus chinensis;

In view of the above technical background and development prospects, the present invention is proposed.

Contents of the invention

The invention provides a method for preparing protocatechuic acid by using two-dimensional liquid chromatography with Phellinus phellifolia as a raw material, and the operation steps are as follows:

1. As a preference, use the Phellinus medicinal material to extract with pure water, the ratio of solid to liquid is 1:15-30, soak for 10-24 hours, heat and boil water for 1-2 hours, extract 2-3 times, and combine to obtain the water extract;

2. As a preference, adopt membrane filtration technology to carry out hollow fiber membrane filtration to the water extract, and the precision of the hollow fiber membrane filter element is 10,000-800,000 molecular weight to obtain the hollow fiber membrane permeate;

富集获得原儿茶酸粗品。3. As a preference, pure water-resistant C8YE silica gel filler is used, which has a good enrichment effect on protocatechuic acid. It can directly enrich the water phase permeate of the hollow fiber membrane in a large volume. The filler has a particle size of 30 ~60μm, the pore size is

Enrichment to obtain crude protocatechuic acid.

4. In order to realize the purpose of one-dimensionally preparing and enriching the crude product of protocatechuic acid, the present invention adopts the following technical solutions:

The elution condition used in the preparation of one-dimensional liquid chromatography is a mixture of acetone, acetonitrile, isopropanol (organic phase) and acidic water (water phase), and the water phase is one or two of acetic acid or phosphoric acid, the ratio is 0.1～ 0.5% (v/v), according to organic phase 5-25% isocratic, 5-25% linear gradient or step gradient, flow rate 0.1-0.2 times packing volume/min, temperature 25-45°C, sample loading 0.5- 3.0% (the ratio of the solid mass of the sample solution to the packing mass), the detector is a DAD ultraviolet detector, the elution time is 20-60min, the fractions are collected according to the chromatographic peak curve of the detector, and all fractions are analyzed by liquid phase to confirm the original Theophyllic acid fractions, combined and concentrated to 20-80mg/mL, are one-dimensional preparation of crude protocatechuic acid;

制备获得纯度>95％原儿茶酸；5. As a preference, polar copolymer C18HC bonded silica gel filler is used for two-dimensional preparation, the particle size of the filler is 10-40 μm, and the pore size is

Preparation of protocatechuic acid with a purity >95%;

6. In order to realize the purpose of two-dimensional preparation of protocatechuic acid, the present invention adopts the following technical solutions:

The two-dimensional liquid chromatography preparation adopts the elution condition of mixing acetone, acetonitrile, isopropanol (organic phase) and formic acid water (water phase), according to the organic phase 5-25% isocratic, 5-25% gradient or step isocratic , the water phase is formic acid, the proportion is 0.1-1.2% (v/v), the flow rate is 0.2-0.3 times the filler volume/min, the temperature is 25-45°C, and the sample loading is 0.2-2.0% (ratio of sample mass to filler mass) , the detector is a DAD ultraviolet detector, and the elution time is 30 to 80 minutes. The components of protocatechuic acid are collected according to the chromatographic peak curve of the detector, and the obtained fraction is vacuum-dried to obtain protocatechu with a purity of >95%. acid.

The present invention has the following advantages:

Compared with the prior art, the present invention has the following characteristics:

1. The one-dimensional separation packing of Phellinus compound adopts a pure water-resistant synthesis process, which can load a large volume of pure water phase samples, and can avoid the problem of curling and loss of retention of the bonded phase of ordinary C18 bonded packings. Two-dimensional The separation packing adopts the polar copolymer C18HC bonded silica gel packing, which is used for the orthogonal separation of the one-dimensional Phellinus compound protocatechuic acid, which is complementary to the one-dimensional separation packing;

2. High purity of protocatechuic acid: due to the technical method used in the present invention, the protocatechuic acid contained in the medicinal materials is directly prepared without heating and acid-base changes, and the protocatechuic acid itself exists in the medicinal materials The state is prepared without hydrolyzate doping, so the obtained protocatechuic acid has a high purity of more than 95%.

3. The method for preparing protocatechuic acid involved in the present invention has a high degree of automation of the instrument, is easy to operate, and has a high yield, which can meet the needs of large-scale production.

Description of drawings

Fig. 1 is the one-dimensional preparation spectrogram of protocatechuic acid of embodiment 1;

Fig. 2 is the two-dimensional preparation spectrogram of protocatechuic acid of embodiment 1;

Fig. 3 is the protocatechuic acid liquid phase analysis spectrogram of embodiment 1.

Detailed ways

Now in conjunction with examples, the present invention will be further described. It should be pointed out that for those skilled in the art, some improvements and modifications can be made without departing from the technical principle of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention. The examples are only for illustrating the present invention, and the present invention is not limited thereto.

Example 1

Weigh 100g of Phellinus medicinal material, add 3L of aqueous solution, soak for 24 hours, heat and boil water for 8 hours, collect the water extract, add 3L of water solution and repeatedly add water and boil to collect the water extract, repeat the extraction once, and combine to obtain The aqueous extract was obtained to obtain an aqueous extract of the Phellinus medicinal material with a concentration of 1.8 mg/mL, which was filtered through a hollow fiber membrane (the precision of the membrane core was 100,000 molecular weight).

质量1200g，华谱新创科技有限公司)，流动相采用(A)乙腈(有机相)-(B)0.1％乙酸水(水相，v/v)，梯度洗脱方式：纯水洗脱10min，乙腈(流动相A)体积浓度由10％经60min提高到20％乙腈(其余为流动相B)梯度洗脱60min，采用DAD紫外检测器选择254nm吸收波长，制备温度25℃，上样体积为3.3L/针，载样量0.5％(样品溶液固体质量与填料质量之比)，流动相流速为300mL/min，按照检测器色谱峰曲线收集馏分，对所有馏分进行液相分析，确认原儿茶酸馏分，合并后浓缩至30mg/mL，得到一维制备原儿茶酸粗品，纯度36.51％，收率86.9％。One-dimensional preparation conditions, chromatographic conditions using C8YE filler (preparation column size: 100 × 250mm, particle size 30μm, pore size

Mass 1200g, Huapu Xinchuang Technology Co., Ltd.), the mobile phase is (A) acetonitrile (organic phase) - (B) 0.1% acetic acid water (water phase, v/v), gradient elution method: pure water elution 10min , acetonitrile (mobile phase A) volume concentration is increased to 20% acetonitrile (all the other are mobile phase B) gradient elution 60min by 10% through 60min, adopts DAD ultraviolet detector to select 254nm absorption wavelength, preparation temperature 25 ℃, loading sample volume is 3.3L/needle, sample load 0.5% (ratio of solid mass of sample solution to packing mass), flow rate of mobile phase is 300mL/min, collect fractions according to detector chromatographic peak curve, perform liquid phase analysis on all fractions, confirm original The tea acid fractions were combined and concentrated to 30 mg/mL to obtain a crude product of protocatechuic acid prepared in one dimension with a purity of 36.51% and a yield of 86.9%.

质量300g，华谱新创科技有限公司)，流动相选择(A)丙酮(有机相)-(B)含0.1％甲酸水(水相，质量浓度)，采用体积浓度10％丙酮(流动相(A)等度洗脱(其余为流动相B)，采用DAD紫外检测器选择320nm吸收波长，制备温度为25℃，进样量为20mL/针，流动相流速为60mL/min，按照检测器色谱峰曲线对原儿茶酸的馏分进行收集(按色谱峰收集)，旋转蒸发至干，得到原儿茶酸化合物(产物经核磁和高分辨质谱结构解析，确定其为化合物原儿茶酸)，经液相色谱分析，纯度为95.55％，收率93.2％。Two-dimensional preparation conditions, chromatographic conditions using reversed-phase C18HC bonded silica gel packing (preparative column specification: 50×150mm, particle size 10μm, pore size

Quality 300g, Huapu Xinchuang Science and Technology Co., Ltd.), mobile phase selection (A) acetone (organic phase)-(B) contains 0.1% formic acid water (water phase, mass concentration), adopts volume concentration 10% acetone (mobile phase ( A) Isocratic elution (the rest is mobile phase B), using a DAD ultraviolet detector to select 320nm absorption wavelength, the preparation temperature is 25°C, the injection volume is 20mL/needle, the mobile phase flow rate is 60mL/min, according to the detector chromatogram The fractions of protocatechuic acid were collected according to the peak curve (collected according to the chromatographic peak), and evaporated to dryness by rotary evaporation to obtain the protocatechuic acid compound (the product was determined to be the compound protocatechuic acid by NMR and high-resolution mass spectrometry structure analysis), After analysis by liquid chromatography, the purity was 95.55%, and the yield was 93.2%.

Example 2

Weigh Phellinus 100g, dissolve in 2L aqueous solution, soak for 30 hours, heat and boil for 6 hours, collect the water extract, add 2L of aqueous solution, add water and boil again, collect the water extract, repeat the extraction twice, combine The water extract was obtained, and the Phellinus medicinal material water extract solution was obtained, the concentration of which was 1.2 mg/mL, the hollow fiber membrane was filtered, the precision of the hollow fiber membrane core was 500000 molecular weight, and the permeate was concentrated to 40 mg/mL.

质量1200g，华谱新创科技有限公司)，流动相采用(A)乙腈(有机相)-(B)0.1％磷酸水(水相，v/v)，台阶等度洗脱方式：纯水洗脱20min，乙腈(流动相A)体积浓度20％等度洗脱(其余为流动相B)60min，采用DAD紫外检测器选择254nm吸收波长，制备温度25℃，进样量为300mL/针，载样量0.75％(样品溶液固体质量与填料质量之比)，流动相流速为300mL/min，按照检测器色谱峰曲线收集馏分，对所有馏分进行液相分析，确认原儿茶酸馏分，合并后浓缩至40mg/mL，得到一维制备原儿茶酸粗品，纯度46.82％，收率90.1％。One-dimensional preparation conditions, chromatographic conditions using C8YE packing (preparative column specifications: 100 × 250mm, particle size 60μm, pore size

Mass 1200g, Huapu Innovation Technology Co., Ltd.), the mobile phase is (A) acetonitrile (organic phase) - (B) 0.1% phosphoric acid water (water phase, v/v), step isocratic elution method: pure water washing Remove for 20min, acetonitrile (mobile phase A) volume concentration 20% isocratic elution (the rest is mobile phase B) for 60min, use DAD ultraviolet detector to select 254nm absorption wavelength, preparation temperature 25 ℃, injection volume is 300mL/needle, load Sample volume 0.75% (ratio of sample solution solid mass to filler mass), mobile phase flow rate is 300mL/min, collect fractions according to detector chromatographic peak curve, carry out liquid phase analysis to all fractions, confirm protocatechuic acid fraction, after merging Concentrate to 40 mg/mL to obtain the crude product of protocatechuic acid prepared in one dimension with a purity of 46.82% and a yield of 90.1%.

质量300g，华谱新创科技有限公司)，流动相选择(A)丙酮(有机相)-(B)0.5％磷酸水(水相，质量浓度)，采用5％丙酮(流动相A)60min提高至25％丙酮(其余为流动相B)梯度洗脱，采用DAD紫外检测器选择320nm吸收波长，制备温度为18℃，进样量为25mL/针，流动相流速为60mL/min，按照检测器色谱峰曲线对原儿茶酸的馏分进行收集(按色谱峰收集)，旋转蒸发至干，得到原儿茶酸化合物(产物经核磁和高分辨质谱结构解析，确定其为化合物原儿茶酸)，经液相色谱分析，纯度为96.68％，收率93.8％。Two-dimensional preparation conditions, chromatographic conditions using reversed-phase C18HC bonded silica gel packing (preparative column specification: 50×150mm, particle size 10μm, pore size

Quality 300g, Hua Pu Xinchuang Science and Technology Co., Ltd.), mobile phase selection (A) acetone (organic phase)-(B) 0.5% phosphoric acid water (water phase, mass concentration), adopt 5% acetone (mobile phase A) 60min to improve Gradient elution to 25% acetone (the rest is mobile phase B), using a DAD ultraviolet detector to select 320nm absorption wavelength, the preparation temperature is 18°C, the injection volume is 25mL/needle, the mobile phase flow rate is 60mL/min, according to the detector The chromatographic peak curve collects the fractions of protocatechuic acid (collected according to the chromatographic peak), and rotary evaporates to dryness to obtain the protocatechuic acid compound (the product is determined to be the compound protocatechuic acid by NMR and high-resolution mass spectrometry structure analysis) , analyzed by liquid chromatography, the purity was 96.68%, and the yield was 93.8%.

Comparative example 1

The difference from Example 1 is that non-group-modified silica gel (100-200 mesh) is used as the separation material, and other conditions are the same as in Example 1, and the crude product of protocatechuic acid cannot be enriched, so two-dimensional preparation and separation cannot be carried out. Purification, high-purity protocatechuic acid cannot be obtained.

Comparative example 2

The difference from Example 1 is that ordinary octadecylsilane bonded silica gel is used as the one-dimensional separation filler, and the rest of the preparation conditions are the same. The crude product of protocatechuic acid is prepared one-dimensionally. The purity of liquid phase analysis is 42.58%, and the yield is 44.9%. .

Ordinary octadecylsilane bonded silica gel is used as the filler for two-dimensional separation, and the rest of the preparation conditions are the same. According to liquid chromatography analysis, the purity of protocatechuic acid is 79.48%, and the yield is 73.8%.

Claims (5)

1. A method for preparing protocatechuic acid from Phellinus linteus medicinal material comprises the following steps:

(1) Boiling and extracting Phellinus linteus medicinal material with pure water to obtain Phellinus linteus water extract;

(2) Filtering the Phellinus linteus water extract with hollow fiber membrane to obtain membrane permeate;

(3) Carrying out one-dimensional enrichment on membrane permeate liquid by using pure water-resistant C8YE silica gel filler to obtain crude protocatechuic acid, wherein the used chromatographic filler is C8YE filler, the elution condition is that acetonitrile is an organic phase, acid water is an aqueous phase, the aqueous phase is one or two of acetic acid and phosphoric acid, and the ratio is 0.01-3.0%, v/v; according to the isocratic of 1-30 percent of the organic phase, the linear gradient or the step gradient of 1-30 percent, the flow rate of 0.01-1.0 times of the filler volume/min, the temperature of 15-55 ℃ and the sample loading amount of 0.2-6.0 percent expressed by the ratio of the solid mass of the sample solution to the filler mass, the detector is a DAD ultraviolet detector, the elution time is 40-300 min, fractions are collected according to the chromatographic peak curve of the detector, liquid phase analysis is carried out on all the fractions, the protocatechuic acid fractions are confirmed, and the mixture is concentrated to 10-300 mg/mL to prepare a protocatechuic acid crude product in one dimension; (4) Preparing a protocatechuic acid crude product in two dimensions by using a polar copolymerization C18HC bonded silica gel filler to obtain a high-purity protocatechuic acid product, wherein the elution condition is that acetone is an organic phase, acid water is an aqueous phase and is a mobile phase, the aqueous phase is formic acid or phosphoric acid, and the ratio of the aqueous phase to the formic acid or phosphoric acid is 0.05-3.0%, and v/v; according to the isocratic of 1-50% of the organic phase, the linear gradient or the step gradient of 1-50%, the flow rate of 0.02-1.2 times of the filler volume/min, the temperature of 15-55 ℃, the sample loading amount expressed by the ratio of the solid mass of the sample solution to the filler mass of 0.1-4%, the detector is a DAD ultraviolet detector, the elution time is 15-200 min, the components of protocatechuic acid are collected according to the chromatographic peak curve of the detector, and the obtained fraction is dried in vacuum, so that the protocatechuic acid product with the purity of more than 95% can be obtained.

2. The method of claim 1, wherein the Phellinus linteus medicinal material is extracted by pure water at a feed liquid ratio of 1:20-1:150, m/v, and soaked for 2-72 hours; heating and boiling for extraction for 1-20 hours, collecting water extract, repeatedly adding water for boiling and extracting solid materials according to the feed-liquid ratio of 1:20-1:150 and m/v, collecting water extract, repeatedly extracting for 1-6 times, and combining to obtain the water extract.

3. The method of claim 1, wherein the water extract is subjected to hollow fiber membrane filtration by a membrane filtration technology, and the hollow fiber membrane filter core has a molecular weight of 5000-1000000, so as to obtain the hollow fiber membrane water phase permeate.

4. The method of claim 1, wherein pure water resistant C8YE silica gel filler is adopted for one-dimensional preparation, so that the method has a good enrichment effect on protocatechuic acid, the hollow fiber membrane aqueous phase permeate can be subjected to large-volume direct loading enrichment, the filler has a particle size of 5-200 mm, the pore diameter is 50-300 a, and crude protocatechuic acid is obtained through enrichment.

5. The method of claim 1, wherein the two-dimensional preparation adopts chromatographic packing as C18HC silica gel packing of polar copolymerization bonding technology, which can well retain protocatechuic acid, has good orthogonality with one-dimensional separation, and can separate and remove small molecular organic impurities, thereby obtaining high-purity protocatechuic acid, wherein the used chromatographic packing has a particle size of 5-200 mm and a pore diameter of 50-300A, and the protocatechuic acid product with a purity of >95% is prepared.

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