CN114573446A - Method for preparing protocatechuic acid from phellinus igniarius medicinal material - Google Patents

Abstract

The invention provides a method for preparing protocatechuic acid from a phellinus igniarius medicinal material, which is characterized in that the phellinus igniarius medicinal material is extracted by pure water, a two-dimensional orthogonal liquid chromatography technology is adopted, pure water resistant C8YE silica gel filler is used as a separation material in one dimension for enrichment to obtain a protocatechuic acid crude product, and polar copolymerization C18HC bonded silica gel filler is used in two dimensions for preparation and purification to obtain the protocatechuic acid product with the purity of more than 95%. The preparation process has the advantages of stable process, high yield, high automation degree and easy operation, and can meet the requirement of large-scale production.

Description

A kind of method for preparing protocatechuic acid from Phellinus linteus

technical field

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

Background technique

Phellinus linteus is a precious perennial large medicinal fungus, known as "forest gold", which is recorded in "Compendium of Materia Medica". It belongs to the Basidiomycota Laminaria, Nonphyllum, Polyporaceae, and there are forty species distributed in my country. It is mainly parasitic on the trunks of mulberry, willow, birch poplar, etc. Among them, the wild mulberry Phellinus is the most precious.

Phellinus linteus, as a traditional fungal Chinese medicine in my country, is currently internationally recognized as the most efficient 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 linteus has anti-cancer, immune regulation, anti-obesity, antioxidant and anti-inflammatory properties. Various pharmacological activities.

In recent years, the search 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 anti-cancer, antioxidant, anti-inflammatory, hypoglycemic and other effects, and is a healthy and safe beneficial small molecule substance.

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

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

Protocatechuic acid has many pharmacological activities such as anti-platelet aggregation, reducing myocardial oxygen consumption, bacteriostasis and analgesia, 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 neuroprotective effect on ischemic and hypoxic neurons; it can significantly relieve interleukin-1β. Inflammatory response to chondrocytes; can maintain the phenotype and pro-proliferation of damaged chondrocytes; 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, and can be used to treat inflammation, fever and cancer.

The extraction and preparation of protocatechuic acid from Phellinus linteus is rarely reported, and the spectrum-effect relationship is not clear. Therefore, the preparation of this compound is of great significance for the research on the material basis and pharmacological activity of Phellinus linteus.

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

SUMMARY OF THE INVENTION

The invention provides a method for preparing protocatechuic acid by taking Phellinus linteus as raw material and adopting two-dimensional liquid chromatography. The operation steps are as follows:

1. As preferably, using Phellinus linteus medicinal materials to extract with pure water, the ratio of material to liquid is 1:15～30, soaking for 10～24 hours, heating and boiling water for 1～2 hours, extracting 2～3 times, and combining to obtain an aqueous extract;

2. As a preference, the water extract is filtered with a hollow fiber membrane using membrane filtration technology, and the accuracy of the hollow fiber membrane filter element is 10,000-800,000 molecular weight to obtain the permeate of the hollow fiber membrane;

富集获得原儿茶酸粗品。3. As a preference, the use of pure water-resistant C8YE silica gel fillers has a good enrichment effect on protocatechuic acid, and the water phase permeate of the hollow fiber membrane can be directly loaded and enriched in large volumes. ~60μm, the pore size is

After enrichment, crude protocatechuic acid was obtained.

4. in order to realize the purpose of one-dimensional preparation and enrichment of protocatechuic acid crude product, the present invention adopts the following technical scheme:

The elution conditions used in the preparation of one-dimensional liquid chromatography are acetone, acetonitrile, isopropanol (organic phase) mixed with acid water (aqueous phase), and the aqueous phase is one or two of acetic acid or phosphoric acid, and the ratio is 0.1～ 0.5% (v/v), according to the organic phase 5-25% isocratic, 5-25% linear gradient or step gradient, the flow rate is 0.1-0.2 times the filler volume/min, the temperature is 25-45 ℃, the sample loading is 0.5- 3.0% (the ratio of the solid mass of the sample solution to the mass of the filler), the detector is a DAD UV detector, the elution time is 20-60 min, 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 The tea acid fractions are combined and concentrated to 20-80 mg/mL to prepare crude protocatechuic acid in one dimension;

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

Prepared to obtain >95% protocatechuic acid;

6. in order to realize the purpose of two-dimensional preparation of protocatechuic acid, the present invention has adopted the following technical scheme:

Two-dimensional liquid chromatography preparation adopts the elution conditions of acetone, acetonitrile, isopropanol (organic phase) and formic acid water (aqueous phase) mixing, according to the organic phase 5-25% isocratic, 5-25% gradient or step isocratic , the water phase is formic acid, the ratio is 0.1-1.2% (v/v), the flow rate is 0.2-0.3 times the volume of filler/min, the temperature is 25-45°C, and the sample loading is 0.2-2.0% (the ratio of sample mass to filler mass) , the detector is DAD ultraviolet detector, the elution time is 30-80min, the components of protocatechuic acid are collected according to the chromatographic peak curve of the detector, and the obtained fractions are vacuum-dried to obtain protocatechu with purity>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 linteus uses a synthetic process that is resistant to pure water, which can be used for large-volume sample loading of pure water phase samples, which can avoid the curling of the bonded phase and the loss of retention of ordinary C18 bonded packing. The problem of two-dimensional The separation packing adopts polar copolymerized C18HC bonded silica gel packing, which can orthogonally separate the one-dimensional Phellinus compound protocatechuic acid, which is complementary to the one-dimensional separation packing;

2. The purity of protocatechuic acid is high: due to the technical method used in the present invention, the protocatechuic acid contained in the medicinal material is directly prepared without heating and changes in acid and alkali, and the protocatechuic acid exists in the medicinal material itself. It is prepared in the state of , without hydrolysis product doping, so the obtained protocatechuic acid has high purity, reaching more than 95%.

3. The method for preparing protocatechuic acid involved in the present invention has the advantages of high degree of instrument automation, simple operation and high yield, and 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 protocatechuic acid two-dimensional preparation spectrogram of embodiment 1;

FIG. 3 is a liquid phase analysis spectrum of protocatechuic acid in Example 1. FIG.

Detailed ways

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

Example 1

Weigh 100g of Phellinus linteus medicinal materials, add 3L aqueous solution, soak for 24 hours, heat and boil for 8 hours, collect the water extract, add 3L aqueous solution to the solid material again, add water and boil again to collect the water extract, repeat the extraction once, and combine to obtain The aqueous extract is obtained to obtain an aqueous extract solution of Phellinus linteus with a concentration of 1.8 mg/mL, which is filtered by a hollow fiber membrane (membrane core precision is 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 use C8YE packing (preparative column specifications: 100 × 250 mm, particle size 30 μm, pore size

Mass 1200g, Huapu Xinchuang Technology Co., Ltd.), mobile phase adopts (A) acetonitrile (organic phase)-(B) 0.1% acetic acid water (aqueous phase, v/v), gradient elution mode: pure water elution 10min , the volume concentration of acetonitrile (mobile phase A) was increased from 10% to 20% acetonitrile (the rest were mobile phase B) gradient elution for 60min over 60min, the DAD ultraviolet detector was used to select the absorption wavelength of 254nm, the preparation temperature was 25°C, and the sample volume was 3.3L/needle, the sample load is 0.5% (the ratio of the solid mass of the sample solution to the mass of the filler), the flow rate of the mobile phase is 300mL/min, the fractions are collected according to the detector chromatographic peak curve, and all fractions are analyzed by liquid phase to confirm the original The tea acid fractions were combined and concentrated to 30 mg/mL to obtain one-dimensionally prepared crude protocatechuic acid 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 use reverse-phase C18HC bonded silica gel packing (preparative column size: 50 × 150 mm, particle size 10 μm, pore size

Mass 300g, Huapu Xinchuang Technology Co., Ltd.), mobile phase selection (A) acetone (organic phase)-(B) water containing 0.1% formic acid (aqueous phase, mass concentration), using volume concentration 10% acetone (mobile phase ( A) Isocratic elution (the rest are mobile phase B), use DAD ultraviolet detector to select 320nm absorption wavelength, preparation temperature is 25°C, injection volume is 20mL/needle, mobile phase flow rate is 60mL/min, according to the detector chromatography The peak curve is to collect the fractions of protocatechuic acid (collected according to the chromatographic peaks), and rotary evaporate 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 structural analysis), Through liquid chromatography analysis, the purity was 95.55%, and the yield was 93.2%.

Example 2

Weigh 100g of Phellinus linteus, dissolve in 2L aqueous solution, soak for 30 hours, heat and boil for 6 hours, collect the water extract, add 2L aqueous solution to the solid material again, add water and boil again to collect the water extract, repeat the extraction twice, and combine An aqueous extract is obtained to obtain an aqueous extraction solution of Phellinus linteus with a concentration of 1.2 mg/mL, filtered through a hollow fiber membrane, the core precision of the hollow fiber membrane is 500,000 molecular weight, and the permeate is 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 use C8YE packing (preparative column specifications: 100 × 250 mm, particle size 60 μm, pore size

Mass 1200g, Huapu Xinchuang Technology Co., Ltd.), mobile phase adopts (A) acetonitrile (organic phase)-(B) 0.1% phosphoric acid water (aqueous phase, v/v), step isocratic elution method: pure water washing Remove 20min, acetonitrile (mobile phase A) volume concentration 20% isocratic elution (the rest are mobile phase B) 60min, use DAD ultraviolet detector to select 254nm absorption wavelength, preparation temperature 25 ℃, injection volume is 300mL/needle, load The sample volume is 0.75% (the ratio of the solid mass of the sample solution to the mass of the filler), the flow rate of the mobile phase is 300 mL/min, and the fractions are collected according to the chromatographic peak curve of the detector. All fractions are analyzed by liquid phase to confirm the protocatechuic acid fraction. It was concentrated to 40 mg/mL to obtain one-dimensionally prepared crude protocatechuic acid 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 size: 50 × 150 mm, particle size 10 μm, pore size

Mass 300g, Huapu Xinchuang Technology Co., Ltd.), mobile phase selection (A) acetone (organic phase)-(B) 0.5% phosphoric acid water (aqueous phase, mass concentration), using 5% acetone (mobile phase A) 60min to improve To 25% acetone (the rest are mobile phase B) gradient elution, use DAD UV detector to select 320nm absorption wavelength, preparation temperature is 18 ℃, injection volume is 25mL/needle, mobile phase flow rate is 60mL/min, according to the detector Chromatographic peak curve The fractions of protocatechuic acid were collected (collected according to the chromatographic peaks), and rotary evaporated to dryness to obtain the protocatechuic acid compound (the product was determined to be the compound protocatechuic acid by NMR and high-resolution mass spectrometry structural analysis) , by liquid chromatography analysis, the purity is 96.68%, and the yield is 93.8%.

Comparative Example 1

The difference from Example 1 is that the non-group-modified silica gel (100-200 mesh) is used as the separation material, and other conditions are the same as those in Example 1, and the crude protocatechuic acid cannot be obtained by enrichment, so two-dimensional preparation and separation cannot be performed. After purification, high-purity protocatechuic acid could not be obtained.

Comparative Example 2

The difference from Example 1 is that the one-dimensional separation packing adopts ordinary octadecylsilane-bonded silica gel, and the other preparation conditions are the same, and the crude protocatechuic acid is one-dimensionally prepared. .

The two-dimensional separation packing adopts ordinary octadecylsilane-bonded silica gel, and the other preparation conditions are the same. According to the 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, the steps are as follows: (1) carrying out pure water boiling extraction with Phellinus linteus medicinal material to obtain Phellinus linteus water extract; (2) carrying out hollow fiber membrane filtration with Phellinus linteus water extract to obtain membrane permeate; (3) Use pure water-resistant C8YE silica gel filler to carry out one-dimensional enrichment of the membrane permeate to obtain crude protocatechuic acid. The chromatographic filler used is C8YE filler, and the elution conditions are one of acetone, acetonitrile, and isopropanol. Or both are organic phase and acid water is water phase as mobile phase, water phase is one or both of acetic acid or phosphoric acid, and the ratio is 0.01～3.0% (v/v); according to organic phase 1～30%, etc. degree, 1-30% linear gradient or step gradient, flow rate is 0.01-1.0 times the volume of filler/min, temperature is 15-55°C, sample loading is 0.2-6.0% (ratio of solid mass of sample solution to mass of filler), detector It is a DAD UV detector, the elution time is 40-300min, the fractions are collected according to the chromatographic peak curve of the detector, and all fractions are analyzed by liquid phase to confirm the protocatechuic acid fraction. Preparation of crude protocatechuic acid; (4) Two-dimensional preparation of crude protocatechuic acid by using polar copolymerized C18HC-bonded silica gel filler to obtain high-purity protocatechuic acid product, and the elution condition is that the elution condition is one of acetone, acetonitrile, and isopropanol or Two kinds are organic phase and acid water is water phase as mobile phase, and the water phase is one or both of acetic acid or phosphoric acid, and the ratio is 0.05～3.0% (v/v); according to the organic phase 1～50% isocratic , 1～50% linear gradient or step gradient, flow rate is 0.02～1.2 times of packing volume/min, temperature is 15～55 ℃, sample load is 0.1～4% (ratio of solid mass of sample solution to packing mass), the detector is DAD ultraviolet detector, the elution time is 15-200min, the components of protocatechuic acid are collected according to the chromatographic peak curve of the detector, and the obtained fractions are vacuum dried to obtain protocatechuic acid products with a purity of >95%. 2. The method according to claim 1, characterized in that, using pure water to extract Phellinus linteus medicinal materials, the ratio of material to liquid is 1:20～1:150 (m/v), soaking for 2～72 hours; heating and boiling for extraction For 1 to 20 hours, collect the water extract, and repeat the extraction of the solid material by adding water to boil again according to the material-to-liquid ratio of 1:20 to 1:150 (m/v). Extraction. 3 . The method according to claim 1 , wherein the water extract is filtered with a hollow fiber membrane by using a membrane filtration technology, and the precision of the hollow fiber membrane filter element is 5,000-1,000,000 molecular weight to obtain the water-phase permeate of the hollow fiber membrane. 4 . 4. The method according to claim 1, wherein the one-dimensional preparation adopts pure water-resistant C8YE silica gel filler, which has a very good enrichment effect on protocatechuic acid, and the hollow fiber membrane water phase permeate can be carried out. The large volume is directly loaded and enriched, the particle size of the filler is 5-200 μm, and the pore size is

After enrichment, crude protocatechuic acid was obtained. 5. The method according to claim 1, wherein the two-dimensional preparation adopts the C18HC silica gel filler of polar copolymerization bonding technology as the chromatographic filler, which can well retain the compound protocatechuic acid, and is compatible with the one-dimensional C18HC silica gel filler. The separation has good orthogonality, and impurities such as small molecular organics can be separated and removed to obtain a high-purity compound protocatechuic acid.

Prepared to obtain a protocatechuic acid product with a purity of >95%.

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