Method for preparing high-purity gallic acid from polygonatum odoratum

Abstract

The invention relates to a method for preparing high-purity gallic acid from polygonatum odoratum, and relates to the technical field of extraction, separation and purification of traditional Chinese medicine components. Comprising the following steps: (1) preparing a polygonatum odoratum crude extract; (2) preparing an extract rich in gallic acid; (3) purifying the extract; and (4) preparing the high-purity gallic acid. The gallic acid is extracted and separated by taking the overground part of the polygonatum odoratum as a raw material for the first time, and the gallic acid with high content (content gt; 2.1%), high purity (purity gt; the gallic acid monomer compound provides a new way for comprehensive development and deep utilization of radix polygonati officinalis traditional Chinese medicine resources. The preparation method disclosed by the invention is quick and efficient in operation, relatively low in environmental pollution, cheap and easily available in raw materials, easy in mass production and wide in application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of extraction, separation and purification of traditional Chinese medicine components, and in particular to a method for preparing high-purity gallic acid from polygonatum odoratum. Background Art

[0002] Polygonatum odoratum (Polygonatum odoratum Rhizome), also known as bell vegetable, ground tube, yellow chicken vegetable root, tail ginseng, and weirui, is widely distributed in Central Asia and southwestern China. Studies have shown that the main components of Polygonatum odoratum are polysaccharides, flavonoids, steroidal saponins, etc., which have biological activities such as hypoglycemic, immunomodulatory, anti-tumor, antioxidant, and anti-fatigue. The dried rhizome of Polygonatum odoratum is a traditional Chinese medicine with a sweet taste, neutral nature, and a slightly cold nature. It has the effects of promoting fluid production and moistening the lungs, nourishing yin and nourishing the stomach, and tonifying the kidneys.

[0003] At present, the research on Polygonatum odoratum at home and abroad is mostly limited to the biological activity, medicinal components, physiology, ecology and other aspects of the underground rhizomes, and there is almost no relevant research on the aboveground parts of Polygonatum odoratum. As a perennial herb, the aboveground parts have not been effectively utilized. Polygonatum odoratum is a plant with medicinal and edible properties, rich medicinal value, and has broad development and application prospects. The effective utilization of the aboveground parts of Polygonatum odoratum will improve the comprehensive utilization value of the plant.

[0004] Gallic acid, also known as 3,4,5-trihydroxybenzoic acid, is commonly known as gallic acid or gallic acid. It has multiple physiological activities such as antioxidant, anti-inflammatory, and antibacterial, and is widely used in food, feed, agriculture, chemical industry, and other fields. Gallic acid is widely available and often exists in a bound state in plant materials rich in tannins. Strong acids or strong bases are often required as catalysts during the extraction process. It usually exists in a free state in fruits, but the content is relatively low. At present, there are no reports on the preparation of gallic acid from Polygonatum odoratum. In view of this, the present invention provides a method for preparing high-purity gallic acid from Polygonatum odoratum. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing high-purity gallic acid from polygonatum odoratum, and the purpose is to extract high-purity gallic acid from the above-ground parts of polygonatum odoratum.

[0006] The technical solution of the present invention to solve the above technical problems is as follows:

[0007] A method for preparing high-purity gallic acid from Polygonatum odoratum comprises the following steps:

[0008] (1) Preparing a crude extract of Polygonatum odoratum: extracting the above-ground part of Polygonatum odoratum with a polar solvent to obtain an extract, and concentrating the extract to obtain a crude extract of Polygonatum odoratum;

[0009] (2) preparing an extract rich in gallic acid: dissolving the crude extract of Polygonatum odoratum in water, extracting with ethyl acetate, collecting the aqueous solution after extraction, and concentrating the aqueous solution after extraction to obtain an extract rich in gallic acid;

[0010] (3) purifying the extract: dissolving the gallic acid-rich extract in water, subjecting it to macroporous resin column chromatography, eluting it with pure water, collecting the eluate, and concentrating it until floccules precipitate to obtain a purified extract;

[0011] Cooling the purified extract to crystallize, and collecting crystals;

[0012] (4) Preparation of high-purity gallic acid:

[0013] The crystals are dissolved in trifluoroacetic acid aqueous solution and concentrated to obtain a concentrated aqueous solution. The concentrated aqueous solution is subjected to gel column chromatography, first eluted with pure water, the eluate is discarded, and then eluted with methanol aqueous solution. The methanol aqueous solution eluate is collected and concentrated to obtain gallic acid.

[0014] Among them, the structure of gallic acid is as follows:

[0015] Principle of the preparation method of gallic acid of the present invention:

[0016] The present invention has discovered for the first time in experimental research that the aerial parts of Polygonatum odoratum contain abundant free gallic acid. Through the extraction method and purification means of the present invention, high-purity gallic acid can be obtained quickly and effectively, and the extraction and purification process is environmentally friendly, overcoming the shortcomings of existing industrial production of gallic acid. The details are as follows:

[0017] Step (1) of the present invention utilizes the principle of like dissolves like and adopts a polar solvent, such as methanol or ethanol solution, to better extract gallic acid from the aerial parts of Polygonatum odoratum; wherein the aerial parts of Polygonatum odoratum are fresh stems and leaves and / or dried stems and leaves; in addition to the target compound, the crude extract of Polygonatum odoratum obtained in step (1) mainly contains polysaccharides and flavonoids as impurities; step (2) can remove part of the flavonoid compounds in the aqueous solution by extraction with ethyl acetate; the macroporous resin chromatography in step (3) can adsorb most of the flavonoids and a small amount of small polar compounds, and can achieve further purification of the target compound; gallic acid is easy to crystallize and precipitate at low temperature, and the saturated solution state of gallic acid is more conducive to the precipitation of gallic acid crystals; gallic acid can stably exist in a weakly acidic solution, and the weakly acidic solution is conducive to removing other impurities; the gel column chromatography in step (4) can adsorb gallic acid, and the pure water elution can remove residual large polar compounds such as polysaccharides. In conclusion, the present invention can obtain gallic acid with high content (content>2.1%) and high purity (purity>93%) through the above operation, which provides a new approach for the comprehensive development and in-depth utilization of Polygonatum odoratum Chinese medicinal resources.

[0018] The beneficial effects of the present invention are:

[0019] (1) The present invention is the first to extract and separate gallic acid from the aerial parts of Polygonatum odoratum as raw materials, and can obtain high-content (content>2.1%) and high-purity (purity>93%) gallic acid monomer compounds, providing a new approach for the comprehensive development and in-depth utilization of Polygonatum odoratum Chinese medicinal resources.

[0020] (2) The preparation method of the present invention is quick and efficient to operate, has little pollution to the environment, uses cheap and readily available raw materials, is easy to mass produce, and has broad application prospects.

[0021] Based on the above technical solution, the present invention can also be improved as follows.

[0022] Furthermore, the polar solvent in step (1) comprises an alcohol aqueous solution with a volume fraction of 60% to 80%;

[0023] Or / and, the extraction in step (1) comprises at least one of leaching, heating extraction and ultrasonic extraction.

[0024] The beneficial effect of adopting the above further scheme is that the above extraction method is more conducive to the extraction of gallic acid and improves the extraction rate.

[0025] Furthermore, the alcohol in the alcohol aqueous solution includes at least one of methanol and ethanol.

[0026] Furthermore, the specific steps of the extraction are: after the aerial parts of the polygonatum odoratum are chopped, an alcohol aqueous solution with a volume fraction of 60%-80% is added in an amount of 8-20 times the weight of the aerial parts of the polygonatum odoratum, and extraction is performed at room temperature, the extraction time for each time is 2d-3d, the extraction is repeated 2-4 times, and the extraction solution of each time is combined to obtain an extract;

[0027] Or / and, the specific steps of the heating extraction are: after the aerial parts of the polygonatum odoratum are chopped, an alcohol aqueous solution with a volume fraction of 60%-80% is added in an amount of 8-15 times the weight of the aerial parts of the polygonatum odoratum, and extraction is performed at a temperature of 35°C-40°C, the extraction time for each time is 3h-7h, the extraction is repeated 3-5 times, and the heated extracts are combined each time to obtain an extract;

[0028] Or / and, the specific steps of the ultrasonic extraction are: after chopping the aerial part of Polygonatum odoratum, adding 15-20 times the weight of the aerial part of Polygonatum odoratum with a volume fraction of 60%-80% alcohol aqueous solution, using a power of 80W-100W and a frequency of 30kHz-80kHz for ultrasonic extraction, each extraction time is 30min-45min, repeating the extraction 3-5 times, combining the ultrasonic extracts each time to obtain an extract.

[0029] The beneficial effects of adopting the above further scheme are: adopting the leaching method is conducive to the extraction of gallic acid and maintaining the stability of the compound; adopting the heating extraction method is more conducive to the extraction of gallic acid, and low-temperature heating avoids the destruction of the gallic acid structure; adopting the ultrasonic extraction method is more conducive to the rapid and effective extraction of gallic acid.

[0030] Furthermore, the length of the aerial part of Polygonatum odoratum after being chopped is 1cm-3cm.

[0031] The beneficial effect of adopting the above further scheme is that adopting the above length is more conducive to the extraction of gallic acid.

[0032] Furthermore, in step (2), dissolving the crude extract of Polygonatum odoratum in water is specifically performed by adding water equivalent to 1-2 times the weight of the crude extract of Polygonatum odoratum to dissolve it; and in step (2), extracting with ethyl acetate is specifically performed by extracting with ethyl acetate, wherein the volume ratio of ethyl acetate to the crude extract of Polygonatum odoratum after adding water is (3-5):5, and the number of extractions with ethyl acetate is 2-4 times.

[0033] The beneficial effect of adopting the above further scheme is that most of the medium polar flavonoid compounds can be removed by multiple extractions.

[0034] Furthermore, in step (3), dissolving the extract rich in gallic acid in water is specifically performed by adding water equivalent to 1-3 times the weight of the extract rich in gallic acid to dissolve it;

[0035] Or / and, the macroporous resin column layer in step (3) is DIAION HP-20, and the specification of DIAION HP-20 is 11 cm×100 cm;

[0036] Or / and, the step (3) of eluting with pure water specifically comprises: eluting with 3-4 times the column volume of pure water;

[0037] Or / and, the parameters of the cooling crystallization in step (3) are: temperature of 4±0.5°C and time of more than 48 hours.

[0038] The beneficial effects of adopting the above further scheme are: the use of the DIAION HP-20 chromatography column can better adsorb the remaining flavonoids in the crude extract, so that the gallic acid can be further purified; the use of the above-mentioned DIAION HP-20 specifications can adsorb flavonoids to a greater extent, and achieve efficient separation of gallic acid and flavonoids.

[0039] Furthermore, in step 3, the temperature of the cooling crystallization is 4°C.

[0040] The beneficial effect of adopting the above further scheme is that the above temperature can quickly and effectively precipitate gallic acid crystals.

[0041] Furthermore, the step (4) wherein the crystalline substance is dissolved in a trifluoroacetic acid aqueous solution comprises: adding a trifluoroacetic acid aqueous solution equivalent to 1-2 times the weight of the crystalline substance to dissolve the crystalline substance; the trifluoroacetic acid aqueous solution in the step (4) is a trifluoroacetic acid aqueous solution with a volume fraction of 0.1%;

[0042] Or / and, in step (4), the gel column is Sephadex-LH 20, the specification of the Sephadex-LH 20 column is 6 cm×80 cm, and the filler particle size is 100 μm-163 μm;

[0043] Or / and, the step (4) of eluting with pure water specifically comprises: eluting with 2-3 times the column volume of pure water;

[0044] Or / and, the methanol aqueous solution in step (4) is a methanol aqueous solution with a volume fraction of 40%-50%.

[0045] The beneficial effect of adopting the above further scheme is that by adopting gel column chromatography, large polar impurities such as polysaccharides remaining in the solution can be removed, and the purity of gallic acid can be further improved.

[0046] Furthermore, the concentration in steps (1) to (4) is reduced pressure and low temperature concentration; the parameters of the reduced pressure and low temperature concentration are: pressure of 40 KPa-90 KPa, temperature of ≤45°C.

[0047] The beneficial effect of adopting the above further scheme is that gallic acid is easily decomposed at high temperature, and the possibility of gallic acid decomposition can be reduced by adopting the above parameters of reduced pressure and low temperature concentration.

[0048] Furthermore, the aerial parts of Polygonatum odoratum in step (1) are fresh stems and leaves of Polygonatum odoratum and / or dried stems and leaves of Polygonatum odoratum. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The gallic acid obtained in Example 1 of the present invention 1 H NMR spectrum;

[0050] Figure 2 The gallic acid obtained in Example 1 of the present invention 13 C NMR spectrum. DETAILED DESCRIPTION

[0051] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0052] Description of the sources of the materials and reagents used:

[0053] DIAION HP-20 was purchased from Mitsubishi Chemical Corporation, Japan.

[0054] Example 1

[0055] The present embodiment relates to a method for preparing high-purity gallic acid from Polygonatum odoratum, comprising the following steps:

[0056] (1) Preparation of Polygonatum odoratum crude extract:

[0057] Take 2.5kg of dried aerial parts of Polygonatum odoratum (samples were collected in Ziyuan County, Guilin in September), wash, chop into 2-3cm, add 70% methanol aqueous solution with a volume percentage of 15 times the weight of the raw material, soak and extract at room temperature for 2 days, filter for the first time, and obtain the first filtrate and the first filter residue; add 60% methanol aqueous solution with a volume percentage of 12 times the weight of the raw material to the first filter residue, soak and extract at room temperature for 2 days, filter for the second time, and obtain the second filtrate and the second filter residue; add 70% methanol aqueous solution with a volume percentage of 15 times the weight of the raw material to the second filter residue, soak and extract at room temperature for 2 days, filter for the third time, and obtain the third filtrate. Combine the first filtrate, the second filtrate and the third filtrate, and concentrate under reduced pressure at 50KPa and a temperature of 45°C to an extract to obtain a crude extract of Polygonatum odoratum;

[0058] (2) Preparation of extracts rich in gallic acid:

[0059] Dissolve the crude extract of Polygonatum odoratum obtained in step (1) in water, pour the solution into a separatory funnel, add ethyl acetate in a ratio of 1:1 to the separatory funnel for extraction, collect the aqueous solution, and concentrate at 50 KPa and 45° C. to remove the organic solvent; repeat the extraction 3 times to obtain an extract rich in gallic acid;

[0060] (3) Purification of extracts:

[0061] DIAION HP-20 macroporous resin was loaded into a column with a size of 11 cm × 100 cm. The column was first activated and balanced with 3 column volumes of ethanol, and then 3 column volumes of deionized water were added to replace the ethanol.

[0062] The gallic acid-rich extract obtained in step (2) was dissolved in water, eluted with 4 column volumes of pure water at a rate of 15 mL / min through a DIAION HP-20 column, and the eluate was collected and concentrated under reduced pressure at 80 KPa and 45° C. until a small amount of floccules precipitated from the solution;

[0063] (4) Further purification of the extract:

[0064] The solution obtained in step (3) is placed in an environment of 4° C. and allowed to stand for 48 hours, a large amount of crystals are precipitated, and the first crystals are collected by suction filtration; the mother liquor is collected, and the solution is concentrated under reduced pressure at 80 KPa and a temperature of 45° C. until a small amount of floccules are precipitated from the solution, and the solution is placed in an environment of 4° C. and allowed to stand for 24 hours, crystals are precipitated, and the second crystals are collected by suction filtration; the first crystals and the second crystals are combined to obtain a crystalline substance;

[0065] (5) Preparation of high-purity gallic acid:

[0066] The crystals obtained in step 4 are dissolved in a 0.1% by volume trifluoroacetic acid aqueous solution, and concentrated under reduced pressure at 80 KPa and 45° C. to obtain an aqueous solution;

[0067] Take Sephadex LH-20 (purchased from GE Company, USA) and pressurize it into a column. The column size is 6cm×80cm and the filler particle size is 100μm-163μm. First, use 3 column volumes of methanol to activate and balance the column, and then add 3 column volumes of deionized water to replace the methanol.

[0068] Slowly add the water solution of Polygonatum odoratum to the sample, elute with 2 times the column volume of deionized water at a rate of 5 mL / min, and discard the eluent. Then elute with 50% methanol aqueous solution, collect the eluent of 50% methanol aqueous solution, concentrate it under reduced pressure at 80 KPa and 45°C to extract, and vacuum dry it at 45°C for 10 hours to obtain high-purity gallic acid.

[0069] Gallic acid structure identification:

[0070] 1 H NMR (500MHz, CD3OD) δ: 7.05 (2H, s, H-2,6); 1 H NMR spectrum Figure 1 shown.

[0071] 13 C NMR (125MHz, CD3OD) δ: 168.0 (C-7), 144.3 (C-3,5), 137.4 (C-4), 120.1 (C-1), 108.6 (C-2,6); 13 C NMR spectrum Figure 2 shown.

[0072] 1 H NMR and 13 The C NMR spectral data were consistent with the reference (Reference: Wang Jingyi, Yu Jundong, Chen Yue, et al. Study on the chemical composition of raspberry [J]. Chinese Herbal Medicine, 2022, 53, (13): 3897-3903), so this compound was identified as gallic acid.

[0073] Example 2

[0074] The present embodiment relates to a method for preparing high-purity gallic acid from Polygonatum odoratum, comprising the following steps:

[0075] (1) Preparation of Polygonatum odoratum crude extract:

[0076] Take 1kg of dried above-ground parts of Polygonatum odoratum, wash, chop into 1-2cm, add 60% methanol aqueous solution with a volume fraction of 10 times the weight of the raw material, ultrasonically extract for 30min, power 100W, frequency 40kHz, filter for the first time, obtain the first filtrate and the first filter residue; add 70% methanol aqueous solution with a volume fraction of 10 times the weight of the raw material to the first filter residue, use power 80W, frequency 40kHz, ultrasonically extract for 45min, filter for the second time, obtain the second filtrate and the second filter residue; add 60% methanol aqueous solution with a volume fraction of 12 times the weight of the raw material to the second filter residue, use power 80W, frequency 40kHz, ultrasonically extract for 45min, filter for the third time, obtain the third filtrate. Combine the first filtrate, the second filtrate and the third filtrate, concentrate to extract at 65KPa, temperature 45℃ under reduced pressure and low temperature to obtain Polygonatum odoratum crude extract.

[0077] (2) Preparation of extracts rich in gallic acid:

[0078] The crude extract of Polygonatum odoratum obtained in step (1) is dissolved in water to form a solution, the solution is placed in a separatory funnel, ethyl acetate is added to the separatory funnel in a ratio of 5:4 for extraction, the aqueous solution is collected, and the organic solvent is removed by low-temperature concentration under reduced pressure; the extraction is repeated twice to obtain an extract rich in gallic acid;

[0079] (3) Purification of extracts:

[0080] DIAION HP-20 macroporous resin was loaded into a column with a size of 11 cm × 100 cm. The column was first activated and balanced with 3 column volumes of ethanol, and then 3 column volumes of deionized water were added to replace the ethanol.

[0081] The gallic acid-rich extract obtained in step (2) was dissolved in water, eluted with 4 column volumes of pure water at a rate of 15 mL / min through a DIAION HP-20 column, and the eluate was collected and concentrated under reduced pressure at 80 KPa and 45° C. until a small amount of floccules precipitated from the solution;

[0082] (4) Further purification of the extract

[0083] The solution obtained in step (3) is placed in an environment of 4° C. and allowed to stand for 48 hours, a large amount of crystals are precipitated, and the first crystals are collected by suction filtration; the mother liquor is collected, and the solution is concentrated under reduced pressure at 80 KPa and a temperature of 45° C. until a small amount of floccules are precipitated from the solution, and the solution is placed in an environment of 4° C. and allowed to stand for 24 hours, crystals are precipitated, and the second crystals are collected by suction filtration; the first crystals and the second crystals are combined to obtain a crystalline substance;

[0084] (5) Preparation of high-purity gallic acid

[0085] The crystals obtained in step (4) are dissolved in a 0.1% by volume trifluoroacetic acid aqueous solution, and concentrated under reduced pressure at 80 KPa and 45° C. to obtain an aqueous solution;

[0086] Take Sephadex LH-20 and pack it into a column with a size of 6 cm × 80 cm and a filler particle size of 100 μm-163 μm. First, activate and balance the column with 3 column volumes of methanol, and then add 3 column volumes of deionized water to replace the methanol.

[0087] Slowly drop the Polygonatum odoratum aqueous solution into the sample, elute with 2 times the column volume of deionized water at a rate of 6 mL / min, and discard the eluent; then elute with 40% methanol aqueous solution, collect the eluent portion of 40% methanol aqueous solution, concentrate it under reduced pressure at 80 KPa and 45°C to an extract, and vacuum dry it at 45°C for 10 hours to obtain high-purity gallic acid.

[0088] Gallic acid structure identification:

[0089] 1 H NMR (500MHz, CD3OD) δ: 7.05 (2H, s, H-2,6); 1 H NMR spectrum Figure 1 shown.

[0090] 13 C NMR (125MHz, CD3OD) δ: 168.0 (C-7), 144.3 (C-3,5), 137.4 (C-4), 120.1 (C-1), 108.6 (C-2,6); 13 C NMR spectrum Figure 2 shown.

[0091] Example 3

[0092] The present embodiment relates to a method for preparing high-purity gallic acid from Polygonatum odoratum, comprising the following steps:

[0093] (1) Preparation of Polygonatum odoratum crude extract:

[0094] Take 2kg of dried above-ground parts of Polygonatum odoratum, wash, chop into 1-2cm, add 60% methanol aqueous solution with a volume fraction of 10 times the weight of the raw material, heat and extract at 38℃ for 5h, filter for the first time, obtain the first filtrate and the first filter residue; add 70% methanol aqueous solution with a volume fraction of 8 times the weight of the raw material to the first filter residue, heat and extract at 40℃ for 5h, filter for the second time, obtain the second filtrate and the second filter residue; add 60% methanol aqueous solution with a volume fraction of 12 times the weight of the raw material to the second filter residue, heat and extract at 36℃ for 7h, filter for the third time, obtain the third filtrate. Combine the first filtrate, the second filtrate and the third filtrate, concentrate at 65KPa, 45℃ under reduced pressure to extract, and obtain the crude extract of Polygonatum odoratum.

[0095] (2) Preparation of extracts rich in gallic acid:

[0096] The crude extract of Polygonatum odoratum obtained in step (1) is dissolved in water to form a solution, the solution is placed in a separatory funnel, ethyl acetate is added in a ratio of 1:1 in the separatory funnel for extraction, the aqueous solution is collected, and the organic solvent is removed by low-temperature concentration under reduced pressure; the extraction is repeated twice to obtain an extract rich in gallic acid;

[0097] (3) Purification of extracts:

[0098] DIAION HP-20 macroporous resin was loaded into a column with a size of 11 cm × 100 cm. The column was first activated and balanced with 3 column volumes of ethanol, and then 3 column volumes of deionized water were added to replace the ethanol.

[0099] The gallic acid-rich extract obtained in step (2) was dissolved in water, eluted with 4 column volumes of pure water at a rate of 10 mL / min through a DIAION HP-20 column, and the eluate was collected and concentrated under reduced pressure at 80 KPa and 45° C. until a small amount of floccules precipitated from the solution;

[0100] (4) Further purification of the extract

[0101] The solution obtained in step (3) is placed in an environment of 4° C. and allowed to stand for 48 hours, a large amount of crystals are precipitated, and the first crystals are collected by suction filtration; the mother liquor is collected, and the solution is concentrated under reduced pressure at 80 KPa and a temperature of 45° C. until a small amount of floccules are precipitated from the solution, and the solution is placed in an environment of 4° C. and allowed to stand for 24 hours, crystals are precipitated, and the second crystals are collected by suction filtration; the first crystals and the second crystals are combined to obtain a crystalline substance;

[0102] (5) Preparation of high-purity gallic acid

[0103] The crystals obtained in step (4) are dissolved in a 0.1% by volume trifluoroacetic acid aqueous solution, and concentrated under reduced pressure at 70 KPa and 45° C. to obtain an aqueous solution;

[0104] Take Sephadex LH-20 and pack it into a column with a size of 6 cm × 80 cm and a filler particle size of 100 μm-163 μm. First, activate and balance the column with 3 column volumes of methanol, and then add 3 column volumes of deionized water to replace the methanol.

[0105] Slowly drop the Polygonatum odoratum aqueous solution into the sample, elute with 2 times the column volume of deionized water at a rate of 6 mL / min, and discard the eluent; then elute with 40% methanol aqueous solution, collect the eluent portion of 40% methanol aqueous solution, concentrate it under reduced pressure at 80 KPa and 45°C to an extract, and vacuum dry it at 45°C for 10 hours to obtain high-purity gallic acid.

[0106] Gallic acid structure identification:

[0107] 1 H NMR (500MHz, CD3OD) δ: 7.05 (2H, s, H-2,6); 1 H NMR spectrum Figure 1 shown.

[0108] 13 C NMR (125MHz, CD3OD) δ: 168.0 (C-7), 144.3 (C-3,5), 137.4 (C-4), 120.1 (C-1), 108.6 (C-2,6); 13 C NMR spectrum Figure 2 shown.

[0109] In summary, the present invention has found for the first time in experimental research that the aerial parts of Polygonatum odoratum contain rich free gallic acid. Through the extraction method and purification means of the present invention, high-purity gallic acid can be obtained quickly and effectively, and the extraction and purification process is environmentally friendly, overcoming the shortcomings of existing industrial production of gallic acid. The preparation method of the present invention is fast and efficient in operation, has less environmental pollution, and has cheap and readily available raw materials, is easy to mass produce, and has broad application prospects.

[0110] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A method for preparing high-purity gallic acid from Polygonatum odoratum, characterized in that: The steps include: (1) Preparing a crude extract of Polygonatum odoratum: extracting the above-ground part of Polygonatum odoratum with a polar solvent to obtain an extract, and concentrating the extract to obtain a crude extract of Polygonatum odoratum; (2) preparing an extract rich in gallic acid: dissolving the crude extract of Polygonatum odoratum in water, extracting with ethyl acetate, collecting the aqueous solution after extraction, and concentrating the aqueous solution after extraction to obtain an extract rich in gallic acid; (3) purifying the extract: dissolving the gallic acid-rich extract in water, subjecting it to macroporous resin column chromatography, eluting it with pure water, collecting the eluate, and concentrating it until floccules precipitate to obtain a purified extract; Cooling the purified extract to crystallize, and collecting crystals; (4) Preparation of high-purity gallic acid: The crystals are dissolved in a trifluoroacetic acid aqueous solution and concentrated to obtain a concentrated aqueous solution. The concentrated aqueous solution is subjected to gel column chromatography, first eluted with pure water and then with a methanol aqueous solution, the methanol aqueous solution eluate is collected and concentrated to obtain gallic acid.

2. A method for preparing high-purity gallic acid from Polygonatum odoratum according to claim 1, characterized in that: The polar solvent in step (1) comprises an alcohol aqueous solution with a volume fraction of 60% to 80%; Or / and, the extraction in step (1) comprises at least one of leaching, heating extraction and ultrasonic extraction.

3. A method for preparing high-purity gallic acid from Polygonatum odoratum according to claim 2, characterized in that: The alcohol in the alcohol aqueous solution includes at least one of methanol and ethanol.

4. A method for preparing high-purity gallic acid from Polygonatum odoratum according to claim 2, characterized in that: The specific steps of the extraction are: chopping the aerial parts of the polygonatum odoratum, adding 8-20 times the weight of the aerial parts of the polygonatum odoratum and a volume fraction of 60%-80% alcohol aqueous solution, extracting at room temperature, each extraction time is 2d-3d, repeating the extraction 2-4 times, combining each extraction solution to obtain an extract; Or / and, the specific steps of the heating extraction are: after the aerial parts of the polygonatum odoratum are chopped, an alcohol aqueous solution with a volume fraction of 60%-80% is added in an amount of 8-15 times the weight of the aerial parts of the polygonatum odoratum, and extraction is performed at a temperature of 35°C-40°C, the extraction time for each time is 3h-7h, the extraction is repeated 3-5 times, and the heated extracts are combined each time to obtain an extract; Or / and, the specific steps of the ultrasonic extraction are: after chopping the aerial part of Polygonatum odoratum, adding 15-20 times the weight of the aerial part of Polygonatum odoratum with a volume fraction of 60%-80% alcohol aqueous solution, using a power of 80W-100W and a frequency of 30kHz-80kHz for ultrasonic extraction, each extraction time is 30min-45min, repeating the extraction 3-5 times, combining the ultrasonic extracts each time to obtain an extract.

5. A method for preparing high-purity gallic acid from Polygonatum odoratum according to claim 4, characterized in that: The length of the aerial part of Polygonatum odoratum after being chopped is 1cm-3cm.

6. The method for preparing high-purity gallic acid from Polygonatum odoratum according to claim 1, characterized in that: The step (2) of dissolving the crude extract of Polygonatum odoratum in water specifically comprises: adding water equivalent to 1-2 times the weight of the crude extract of Polygonatum odoratum to dissolve it; the step (2) of extracting with ethyl acetate specifically comprises: extracting with ethyl acetate, wherein the volume ratio of ethyl acetate to the crude extract of Polygonatum odoratum after adding water is (3-5):5, and the number of times of ethyl acetate extraction is 2-4 times.

7. The method for preparing high-purity gallic acid from Polygonatum odoratum according to claim 1, characterized in that: In step (3), dissolving the extract rich in gallic acid in water comprises: adding water equivalent to 1-3 times the weight of the extract rich in gallic acid to dissolve; Or / and, the macroporous resin column layer in step (3) is DIAION HP-20, and the specification of DIAION HP-20 is 11 cm×100 cm; Or / and, the step (3) of eluting with pure water specifically comprises: eluting with 3-4 times the column volume of pure water; Or / and, the parameters of the cooling crystallization in step (3) are: temperature of 4±0.5°C and time of more than 48 hours.

8. The method for preparing high-purity gallic acid from Polygonatum odoratum according to claim 1, characterized in that: The step (4) wherein the crystals are dissolved in a trifluoroacetic acid aqueous solution comprises: adding a trifluoroacetic acid aqueous solution equivalent to 1-2 times the weight of the crystals to dissolve the crystals; the trifluoroacetic acid aqueous solution in the step (4) is a trifluoroacetic acid aqueous solution with a volume fraction of 0.1%; Or / and, in step (4), the gel column is Sephadex-LH 20, the specification of the Sephadex-LH 20 column is 6 cm×80 cm, and the filler particle size is 100 μm-163 μm; Or / and, the step (4) of eluting with pure water specifically comprises: eluting with 2-3 times the column volume of pure water; Or / and, the methanol aqueous solution in step (4) is a methanol aqueous solution with a volume fraction of 40%-50%.

9. The method for preparing high-purity gallic acid from Polygonatum odoratum according to claim 1, characterized in that: The concentration in steps (1) to (4) is reduced pressure and low temperature concentration; the parameters of the reduced pressure and low temperature concentration are: pressure of 40KPa-90KPa, temperature of ≤45°C.

10. The method for preparing high-purity gallic acid from Polygonatum odoratum according to any one of claims 1 to 9, characterized in that: The above-ground parts of Polygonatum odoratum in step (1) are fresh stems and leaves of Polygonatum odoratum and / or dried stems and leaves of Polygonatum odoratum.

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