A method for simultaneously preparing deoxynojirimycin, chlorogenic acid and morin from mulberry bark.

Abstract

The present invention relates to a method for simultaneously preparing deoxynojirimycin, chlorogenic acid and morin from mulberry bark, so as to solve the technical problem that the existing extraction of a single component from mulberry bark leads to waste of effective components. The improved method comprises: 1. obtaining a concentrated extract of mulberry bark; 2. preparing a deoxynojirimycin extract, a chlorogenic acid extract and a morin extract; 3. removing impurities and purifying the deoxynojirimycin extract, the chlorogenic acid extract and the morin extract respectively to obtain a deoxynojirimycin product, a chlorogenic acid product and a morin product.

Description

Technical Field

[0001] The invention relates to a method for simultaneously extracting deoxynojirimycin, chlorogenic acid and morin, and in particular to a method for simultaneously preparing deoxynojirimycin, chlorogenic acid and morin from mulberry bark. Background Art

[0002] Mulberry bark is the dried root bark of the mulberry plant of the Moraceae family. It has aliases such as mulberry root white bark, mulberry root bark, mulberry bark, and white mulberry bark. It is mainly used for lung heat and cough due to asthma, mainly due to lung heat. The antitussive effect is relatively weak or not obvious. It mainly calms the lung breath and has liver coldness, with a slight tendency to nourish yin. Therefore, it is the most suitable medicine for excessive lung heat and initial injury to lung yin. It is generally used in pediatrics to clear the lungs and relieve asthma.

[0003] Morus alba contains chemical components such as deoxynojirimycin, morin, rutin, resveratrol, scopoletin, chlorogenic acid, etc. Deoxynojirimycin (1-DNJ) is a potent α-glucosidase inhibitor, which can effectively inhibit the conversion of human sugar and reduce the sugar content in the blood; chlorogenic acid, also known as "green acid", "coffee tannic acid", and "coffee tannic acid", is a phenylpropanoid compound produced by plants through the shikimic acid pathway during aerobic respiration. It is a derivative of caffeoylquinic acid, has a weak sour taste and is astringent. Its solubility in water is 4% at 25°C, and it is more soluble in hot water, soluble in ethanol and acetone, slightly soluble in ethyl acetate, insoluble in chloroform, ether and carbon disulfide, and has strong antioxidant capacity. It also has the functions of anti-HIV, anti-tumor cells, antibacterial, improving central excitability, choleretic, anti-teratogenic, anti-allergic and regulating the activity of cytochrome P450 ligase.

[0004] Morin is a light yellow pigment extracted from the bark of mulberry trees, orange trees and other mulberry plants and many Chinese herbal medicines. Its chemical structure is characterized by an oxygen-containing heterocyclic ring connecting two aromatic rings. It is usually yellow or grayish yellow needle-shaped crystals. It is easily oxidized to brown when left in the air for a long time. Its melting point is 285℃~290℃ (decomposition) and it is slightly soluble in water. It can inhibit enzyme activity and has antioxidant, anti-pain, antibacterial, anti-inflammatory, anti-atherosclerotic, blood sugar lowering and anti-stress effects.

[0005] Currently, mulberry bark is mainly used to extract deoxynojirimycin, while the extraction of chlorogenic acid and mulberry pigment in mulberry bark is less common. Most reports are on the detection of chlorogenic acid and mulberry pigment content in mulberry bark. The single extraction and purification of deoxynojirimycin, chlorogenic acid and mulberry pigment leads to the waste of the other two components in the raw material, resulting in underutilization of the raw material. Summary of the invention

[0006] In order to solve the technical problem that the existing extraction of a single component from Morus alba bark leads to waste of effective components, the present invention provides a method for simultaneously preparing deoxynojirimycin, chlorogenic acid and morin from Morus alba bark.

[0007] The technical solution provided by the present invention is:

[0008] A method for simultaneously preparing deoxynojirimycin, chlorogenic acid and morin from mulberry bark is characterized in that it comprises the following steps:

[0009] S1, taking dried mulberry bark raw material and crushing it, adding alcohol extraction solution according to the ratio of mulberry bark raw material mass: alcohol extraction solution volume = 1g: (8-10)mL, heating and extracting to obtain mulberry bark extract, concentrating the mulberry bark extract until there is no alcohol, and obtaining mulberry bark concentrated extract;

[0010] S2. Add 1 to 3 times the volume of pure water to the concentrated extract of Morus alba to dilute it, so as to obtain a pre-chromatographic solution of Morus alba;

[0011] Take 1 / 2 to 2 / 3 times the mass of the mulberry bark raw material of the macroporous adsorption resin to prepare a macroporous adsorption resin chromatography column;

[0012] Adding the pre-prepared chromatography liquid of Morus alba bark to the macroporous adsorption resin chromatography column, collecting the adsorption effluent; eluting with pure water until no deoxynojirimycin is present in the eluate, combining the adsorption effluent and the pure water eluate to obtain the deoxynojirimycin extract;

[0013] Continue to elute with 15-25% ethanol until the eluate does not contain chlorogenic acid, and collect the 15-25% ethanol eluate as the chlorogenic acid extract;

[0014] Continue to use 70-90% ethanol to elute until the eluate does not contain morin, and collect the 70-90% ethanol eluate as morin extract;

[0015] S3. Deoxynojirimycin extract, chlorogenic acid extract and mulberry pigment extract are purified to obtain deoxynojirimycin product, chlorogenic acid product and mulberry pigment product respectively.

[0016] Furthermore, in step S3, the deoxynojirimycin extract is purified by: purifying the deoxynojirimycin extract 1-3 times by cation exchange resin chromatography column purification method to obtain a deoxynojirimycin concentrated extract; adding 1-3% by mass of activated carbon of the mass of the deoxynojirimycin concentrated extract, stirring and decolorizing at 65-75°C for 40-60min, filtering, and obtaining a decolorized liquid, drying it at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 65°C-75°C to obtain a deoxynojirimycin product.

[0017] Further, in step S3, the deoxynojirimycin extract is purified by a cation exchange resin chromatography column purification method as follows:

[0018] Take cation exchange resin in an amount of 1 / 3 to 1 / 2 times the mass of Morus alba raw material to prepare a cation exchange resin chromatography column; add deoxynojirimycin extract to the cation exchange resin chromatography column, and elute 2-3BV with pure water; continue to use ammonia water with a volume fraction of 1% to 2% to elute until there is no deoxynojirimycin in the eluate, concentrate the collected ammonia water eluate to a specific gravity of 1.10-1.15 at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 55°C to 65°C, add pure water to continue concentrating to neutrality, and complete a purification.

[0019] Further, in step S3, the chlorogenic acid extract is purified by contamination removal as follows: the chlorogenic acid extract is concentrated to a specific gravity of 1.13-1.18, 4-6 times the volume of ethyl acetate is added, the extraction is stirred at 25-28°C for 30-40 min and the liquid is separated, the extraction is performed 3-5 times, the ethyl acetate phase is combined, and the ethyl acetate phase is concentrated to 1 / 10-1 / 8 of the total volume of the ethyl acetate phase, and the crystallization is allowed to stand at 25-28°C for 20-26h, and the crystals are rinsed with ethanol with a volume fraction of 95-98%, and the chlorogenic acid product is obtained by drying at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 65°C-75°C.

[0020] Further, in step S3, the mulberry pigment extract is purified by: concentrating the mulberry pigment extract to be free of alcohol, adding 1.5-2.5 times of pure water by volume to obtain a mulberry pigment diluted extract; according to the volume ratio, the mulberry pigment diluted extract: the extractant = 1: (3-5), stirring and extracting at 25-28 ° C for 20-30 minutes and separating the liquids, extracting 3-5 times, combining the extractant phase, and concentrating to 1 / 8-1 / 6 of the total volume of the extractant phase to obtain a mulberry pigment extract;

[0021] The mulberry pigment extract is added into a polyamide resin chromatography column, wherein the mass of the polyamide resin in the polyamide resin chromatography column is 1 / 6-1 / 5 of the mass of the mulberry bark raw material; ethanol solutions with volume fractions of 30%, 60% and 80% are used for gradient elution, and the eluent is detected by high performance liquid chromatography, the eluent containing mulberry pigment is collected and concentrated to 1 / 12-1 / 10 of the total volume of the eluent, and the eluent is placed at a temperature of 25-28° C. for crystallization for 20-26 hours, the crystals are filtered, and the crystals are rinsed with ethanol with a volume fraction of 95-98%, and vacuum dried at a temperature of 55° C. and a vacuum pressure of -0.09Mpa--0.07Mpa to obtain a mulberry pigment product.

[0022] Furthermore, in step S3, the extractant is ethyl acetate or dichloromethane or n-butanol or n-hexane or petroleum ether or cyclohexane; or any one of ethyl acetate, dichloromethane, n-butanol is combined with any one of n-hexane, petroleum ether, and cyclohexane.

[0023] Further, in step S1, the alcohol extraction solution is methanol or ethanol, and the volume fractions of methanol and ethanol are both 70-80%;

[0024] The extraction conditions are as follows: extracting at 70-80°C for 2-4 times, each time for 1.5-2.5 hours, and then combining to obtain the mulberry bark extract.

[0025] Furthermore, in step S2, the macroporous adsorption resin is LX-38 resin, LX-18 resin, LX-15 resin or AB-8 resin.

[0026] Beneficial effects of the present invention:

[0027] 1. The present invention proposes a method for simultaneously preparing deoxynojirimycin, chlorogenic acid and morin from mulberry bark, using ethanol to extract the raw materials, and then applying macroporous adsorption resin, according to the properties of deoxynojirimycin, chlorogenic acid and morin, eluting with water, 15-25% ethanol, and 70-90% ethanol in turn, and collecting water-soluble eluent, low alcohol-soluble eluent, and high alcohol-soluble eluent respectively. Through tracking detection, it is found that the components of the three eluents are all eluted, achieving a good separation effect.

[0028] 2. The present invention extracts and purifies deoxynojirimycin, chlorogenic acid and morin in the white mulberry bark by a combined extraction and purification method, and finally obtains three products, which saves a lot of process in the extraction and purification steps, that is, saves manpower, physical and financial resources, greatly reduces the cost, and makes the white mulberry bark raw material and the waste generated when extracting a certain component can be fully utilized. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The present invention is a flow chart of an embodiment of a method for simultaneously preparing deoxynojirimycin, chlorogenic acid and morin from Morus alba bark;

[0030] Figure 2 This is a schematic diagram of the HPLC detection results of deoxynojirimycin in Example 1 of the present invention;

[0031] Figure 3 This is a schematic diagram of the HPLC detection results of chlorogenic acid in Example 1 of the present invention;

[0032] Figure 4 This is a schematic diagram of the HPLC detection results of morin in Example 1 of the present invention. DETAILED DESCRIPTION

[0033] The following is further described in conjunction with specific embodiments.

[0034] Example 1

[0035] See also Figure 1100 kg of dry mulberry bark raw material was crushed, 1000 L of 75% ethanol by volume was added, and the mixture was extracted three times at 70°C for 2 hours each time. The mulberry bark extract was combined to obtain the mulberry bark extract. The mulberry bark extract was concentrated at -0.08Mpa--0.06Mpa and 75°C until it was alcohol-free to obtain a mulberry bark concentrated extract. According to the volume ratio, 2 times the amount of pure water was added to the mulberry bark concentrated extract to dilute it to obtain a mulberry bark pre-chromatographic solution.

[0036] Take 50kg of LX-38 macroporous adsorption resin to prepare a macroporous adsorption resin chromatography column; add the pre-prepared chromatography liquid of Morus alba bark to the macroporous adsorption resin chromatography column, and collect the adsorption effluent; elute with pure water until there is no deoxynojirimycin in the eluent, and combine the adsorption effluent and the pure water eluent to form a deoxynojirimycin extract; continue to elute with 20% ethanol until the eluent does not contain chlorogenic acid, and collect the 20% ethanol eluent as a chlorogenic acid extract; continue to elute with 80% ethanol until the eluent does not contain mulberry pigment, and collect the 80% ethanol eluent as a mulberry pigment extract.

[0037] Preparation of deoxynojirimycin product: 40 kg of 001*7 cation exchange resin is taken to prepare a cation exchange resin chromatography column; the deoxynojirimycin extract is added to the cation exchange resin chromatography column, and 2-3 BV is eluted with pure water; 1% volume fraction of ammonia water is continued to be used for elution until there is no deoxynojirimycin in the eluent, and the collected ammonia water eluent is concentrated to a specific gravity of 1.10 at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 60°C. -1.15, add pure water and continue to concentrate to neutrality, complete one purification, repeat purification once, and obtain a concentrated extract of deoxynojirimycin; add 2% of the mass of the concentrated extract of deoxynojirimycin activated carbon, stir and decolorize at 70°C for 40 minutes, filter, and obtain a decolorized solution, and dry it at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 70°C to obtain 384.7g of deoxynojirimycin powder, with a yield of 0.384%; see Figure 2 , detected by HPLC: the content of deoxynojirimycin is 62.51%. The specific test results are shown in the following table:

[0038] Element Retention time / min Peak area / AU*s Peak height / AU content / % 1-DNJ 9.639 2465703 192056 62.51

[0039] Preparation of chlorogenic acid product: The chlorogenic acid extract was concentrated to a specific gravity of 1.13-1.18, 5 times the volume of ethyl acetate was added, and the mixture was stirred and extracted at 25°C for 30 minutes and separated, and the extraction was performed 4 times. The ethyl acetate phase was combined and concentrated to 1 / 8 of the total volume of the ethyl acetate phase. At 25°C, the mixture was allowed to stand for crystallization for 24 hours, and the crystals were rinsed with 95% ethanol by volume, and dried at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 70°C to obtain 128.3g of chlorogenic acid powder with a yield of 0.128%; see Figure 3 , detected by HPLC: the chlorogenic acid content is 28.17%, and the specific test results are shown in the following table:

[0040] Element Retention time / min Peak area / AU*s Peak height / AU content / % Chlorogenic acid 17.905 2486005 103200 28.17

[0041] Preparation of mulberry pigment product: after concentrating the mulberry pigment extract to be free of alcohol, add 2 times of pure water by volume to obtain a mulberry pigment diluted extract; according to the volume ratio, the mulberry pigment diluted extract: ethyl acetate = 1:3, stir and extract at 26°C for 30 minutes and separate the liquids, extract 4 times, combine the ethyl acetate phases, and concentrate to 1 / 8 of the total volume of the ethyl acetate phase to obtain a mulberry pigment extract; add the mulberry pigment extract to a polyamide resin chromatography column, and the mass of the polyamide resin in the polyamide resin chromatography column is 18 kg; Gradient elution was performed using ethanol solutions with a volume fraction of 30%, 60%, and 80%, and the eluent was detected by high performance liquid chromatography. The eluent containing morin was collected and concentrated to 1 / 12 of the total volume of the eluent, and crystallized at a temperature of 25°C for 24 hours. The crystals were filtered and rinsed with ethanol with a volume fraction of 95%, and vacuum dried at a temperature of 65°C and a vacuum pressure of -0.09Mpa--0.07Mpa to obtain 98.3g of morin powder with a yield of 0.098%; see Figure 4 , detected by HPLC: the content of mulberry pigment is 24.38%, and the specific test results are shown in the following table:

[0042] Element Retention time / min Peak area / AU*s Peak height / AU content / % Morin 8.909 636420 54327 24.38

[0043] Example 2

[0044] 100 kg of dry mulberry bark raw material was crushed, 800 L of 80% ethanol by volume was added, and extraction was performed at 70°C for 3 times, each time for 2 hours. The mulberry bark extract was combined to obtain the mulberry bark extract. The mulberry bark extract was concentrated at -0.08Mpa--0.06Mpa and 75°C until it was alcohol-free to obtain a mulberry bark concentrated extract. According to the volume ratio, 2 times the amount of pure water was added to the mulberry bark concentrated extract to dilute it to obtain a mulberry bark pre-chromatographic solution.

[0045] Take 60kg of LX-18 macroporous adsorption resin to prepare a macroporous adsorption resin chromatography column; add the pre-prepared chromatography liquid of Morus alba bark to the macroporous adsorption resin chromatography column, and collect the adsorption effluent; elute with pure water until there is no deoxynojirimycin in the eluent, and combine the adsorption effluent and the pure water eluent to obtain a deoxynojirimycin extract; continue to elute with 25% ethanol until the eluent does not contain chlorogenic acid, and collect the 25% ethanol eluent as a chlorogenic acid extract; continue to elute with 70% ethanol until the eluent does not contain mulberry pigment, and collect the 70% ethanol eluent as a mulberry pigment extract.

[0046] Preparation of deoxynojirimycin product: 50 kg of 001*7 cation exchange resin is used to prepare a cation exchange resin chromatography column; the deoxynojirimycin extract is added to the cation exchange resin chromatography column, and 2-3 BV is eluted with pure water; 1.5% volume fraction of ammonia water is continued to be used for elution until there is no deoxynojirimycin in the eluate, and the collected ammonia water eluate is concentrated to a specific gravity of 1.10-1.15 at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 60°C, and pure water is added to continue to concentrate. The mixture was reduced to neutrality, and purification was completed once, and the purification was repeated once to obtain a concentrated extract of deoxynojirimycin; 767 activated carbon with an amount of 2% by mass of the concentrated extract of deoxynojirimycin was added, and the mixture was stirred for decolorization at 70°C for 40 minutes, and the decolorized liquid was filtered to obtain a decolorized liquid, which was dried at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 70°C to obtain 379.4g of deoxynojirimycin powder with a yield of 0.379%; HPLC detection showed that the content of deoxynojirimycin was 65.26%.

[0047] Preparation of chlorogenic acid product: Concentrate the chlorogenic acid extract to a specific gravity of 1.13-1.18, add 4 times the volume of ethyl acetate, stir and extract at 25°C for 30 minutes and separate the liquids, extract 4 times, combine the ethyl acetate phases, concentrate to 1 / 10 of the total volume of the ethyl acetate phase, stand and crystallize at 25°C for 24 hours, rinse the crystals with 95% by volume ethanol, and dry at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 70°C to obtain 122.4g of chlorogenic acid powder with a yield of 0.122%; HPLC detection: the chlorogenic acid content is 28.63%.

[0048] Preparation of mulberry pigment products: After the mulberry pigment extract is concentrated to be alcohol-free, 2 times of pure water is added by volume to obtain a mulberry pigment diluted extract; according to the volume ratio, the mulberry pigment diluted extract: dichloromethane = 1:4, stirred and extracted at 26°C for 30 minutes and separated, extracted 4 times, the dichloromethane phase is combined, and concentrated to 1 / 6 of the total volume of the dichloromethane phase to obtain a mulberry pigment extract; the mulberry pigment extract is added to a polyamide resin chromatography column, and the mass of the polyamide resin in the polyamide resin chromatography column is 20kg; gradient elution is performed using ethanol solutions with volume fractions of 30%, 60%, and 80%, and the eluent is detected by high performance liquid chromatography, the eluent containing mulberry pigment is collected and concentrated to 1 / 10 of the total volume of the eluent, and crystallized at a temperature of 25°C for 24 hours, the crystals are filtered, and the crystals are rinsed with ethanol with a volume fraction of 95%, and the temperature is 65°C and the vacuum pressure is -0.09Mpa-

[0049] After vacuum drying at -0.07 MPa, 97.1 g of mulberry pigment powder was obtained with a yield of 0.097%. HPLC detection showed that the mulberry pigment content was 26.41%.

[0050] Example 3

[0051] 100 kg of dry mulberry bark raw material was crushed, 1000 L of 70% by volume ethanol was added, and extraction was performed at 80°C for 3 times, each time for 1.5 h. The mulberry bark extract was combined to obtain the mulberry bark extract. The mulberry bark extract was concentrated at -0.08Mpa--0.06Mpa and 75°C until it was alcohol-free to obtain a mulberry bark concentrated extract. According to the volume ratio, 2 times the amount of pure water was added to the mulberry bark concentrated extract to dilute it to obtain a mulberry bark pre-chromatographic solution.

[0052] Take 50kg of LX-15 macroporous adsorption resin to prepare a macroporous adsorption resin chromatography column; add the pre-prepared chromatography liquid of Morus alba bark to the macroporous adsorption resin chromatography column, and collect the adsorption effluent; elute with pure water until there is no deoxynojirimycin in the eluent, and combine the adsorption effluent and the pure water eluent to obtain a deoxynojirimycin extract; continue to elute with 20% ethanol until the eluent does not contain chlorogenic acid, and collect the 20% ethanol eluent as a chlorogenic acid extract; continue to elute with 80% ethanol until the eluent does not contain mulberry pigment, and collect the 80% ethanol eluent as a mulberry pigment extract.

[0053] Preparation of deoxynojirimycin product: 35 kg of 001*7 cation exchange resin is used to prepare a cation exchange resin chromatography column; the deoxynojirimycin extract is added to the cation exchange resin chromatography column, and 2-3 BV is eluted with pure water; 2% volume fraction of ammonia water is continued to be used for elution until there is no deoxynojirimycin in the eluate, and the collected ammonia water eluate is concentrated to a specific gravity of 1.10-1.15 at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 60°C, and pure water is added to continue to concentrate. The mixture was reduced to neutrality, and purification was completed once, and the purification was repeated once to obtain a concentrated extract of deoxynojirimycin; 320 activated carbon with a mass of 1% of the mass of the concentrated extract of deoxynojirimycin was added, and the mixture was stirred for decolorization at 70°C for 40 minutes, and the decolorized liquid was filtered to obtain a decolorized liquid, which was dried at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 70°C to obtain 380.5g of deoxynojirimycin powder with a yield of 0.38%; HPLC detection showed that the content of deoxynojirimycin was 64.13%.

[0054] Preparation of chlorogenic acid product: Concentrate the chlorogenic acid extract to a specific gravity of 1.13-1.18, add 6 times the volume of ethyl acetate, stir and extract at 25°C for 30 minutes and separate the liquids, extract 3 times, combine the ethyl acetate phases, concentrate to 1 / 9 of the total volume of the ethyl acetate phase, let stand at 25°C for 24 hours to crystallize, rinse the crystals with 96% volume fraction of ethanol, dry at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 70°C to obtain 129.2g of chlorogenic acid powder with a yield of 0.129%; HPLC detection: the chlorogenic acid content is 27.82%.

[0055] Preparation of mulberry pigment products: After the mulberry pigment extract is concentrated to be alcohol-free, 2 times of pure water is added by volume to obtain a mulberry pigment diluted extract; according to the volume ratio, the mulberry pigment diluted extract: n-butanol = 1:5, stirred and extracted at 25°C for 30 minutes and separated, extracted 3 times, the n-butanol phase is combined, and concentrated to 1 / 6 of the total volume of the n-butanol phase to obtain a mulberry pigment extract; the mulberry pigment extract is added to a polyamide resin chromatography column, and the mass of the polyamide resin in the polyamide resin chromatography column is 19kg; gradient elution is performed using ethanol solutions with volume fractions of 30%, 60%, and 80%, and the eluent is detected by high performance liquid chromatography, the eluent containing mulberry pigment is collected and concentrated to 1 / 11 of the total volume of the eluent, and crystallized at a temperature of 25°C for 24 hours, the crystals are filtered, and the crystals are rinsed with ethanol with a volume fraction of 95%, and the temperature is 65°C and the vacuum pressure is -0.09Mpa-

[0056] After vacuum drying at -0.07 MPa, 98.6 g of mulberry pigment powder was obtained with a yield of 0.098%. HPLC detection showed that the mulberry pigment content was 25.15%.

[0057] Example 4

[0058] 30 kg of dry mulberry bark raw material was crushed, 270 L of 75% by volume methanol was added, and extraction was performed twice at 75° C., each time for 2.5 h. The mulberry bark extract was combined to obtain the mulberry bark extract. The mulberry bark extract was concentrated at -0.08 MPa--0.06 MPa and a temperature of 75° C. until it was alcohol-free to obtain a mulberry bark concentrated extract. According to the volume ratio, 1 times the amount of pure water was added to the mulberry bark concentrated extract for dilution to obtain a mulberry bark pre-chromatographic solution.

[0059] Take 20kg of AB-8 macroporous adsorption resin to prepare a macroporous adsorption resin chromatography column; add the pre-prepared chromatography liquid of Morus alba bark to the macroporous adsorption resin chromatography column, and collect the adsorption effluent; elute with pure water until there is no deoxynojirimycin in the eluent, and combine the adsorption effluent and the pure water eluent to obtain a deoxynojirimycin extract; continue to elute with 15% ethanol until the eluent does not contain chlorogenic acid, and collect the 15% ethanol eluent as a chlorogenic acid extract; continue to elute with 90% ethanol until the eluent does not contain mulberry pigment, and collect the 90% ethanol eluent as a mulberry pigment extract.

[0060] Preparation of deoxynojirimycin product: 15 kg of 001*7 cation exchange resin is taken to prepare a cation exchange resin chromatography column; the deoxynojirimycin extract is added to the cation exchange resin chromatography column, and 2-3 BV is eluted with pure water; 2% volume fraction of ammonia water is continued to be used for elution until there is no deoxynojirimycin in the eluate, and the collected ammonia water eluate is concentrated to a specific gravity of 1.10-1.15 at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 55°C, and pure water is added to continue to concentrate to neutrality, completing one purification, repeating the purification twice to obtain a concentrated extract of deoxynojirimycin; adding 3% of the mass of the concentrated extract of deoxynojirimycin to 320 activated carbon, stirring and decolorizing at 65°C for 50 minutes, filtering, and obtaining a decolorized liquid, drying it at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 65°C to obtain 117.00g of deoxynojirimycin powder, with a yield of 0.39%; HPLC detection: the content of deoxynojirimycin is 63.86%.

[0061] Preparation of chlorogenic acid product: Concentrate the chlorogenic acid extract to a specific gravity of 1.13-1.18, add 5 times the volume of ethyl acetate, stir and extract at 27°C for 40 minutes and separate the liquids, extract 3 times, combine the ethyl acetate phases, concentrate to 1 / 9 of the total volume of the ethyl acetate phase, let stand at 28°C for 20 hours to crystallize, rinse the crystals with 98% by volume ethanol, and dry at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 65°C to obtain 37.50g of chlorogenic acid powder with a yield of 0.125%; HPLC detection: the chlorogenic acid content is 28.86%.

[0062] Preparation of mulberry pigment product: after concentrating the mulberry pigment extract to be alcohol-free, add 1.5 times of pure water by volume to obtain a mulberry pigment diluted extract; according to the volume ratio, the mulberry pigment diluted extract: petroleum ether = 1:4, stir and extract for 20 minutes at 28°C and separate the liquids, extract 3 times, combine the petroleum ether phase, and concentrate to 1 / 6 of the total volume of the petroleum ether phase to obtain a mulberry pigment extract; add the mulberry pigment extract to a polyamide resin chromatography column, the mass of the polyamide resin in the polyamide resin chromatography column is 5kg; use ethanol solutions with volume fractions of 30%, 60%, and 80% for gradient elution, and use high performance liquid chromatography to detect the eluent, collect the eluent containing mulberry pigment and concentrate it to 1 / 12 of the total volume of the eluent, place it at a temperature of 28°C for crystallization for 20 hours, filter the crystals by suction, rinse the crystals with ethanol with a volume fraction of 96%, and perform chromatography at a temperature of 50°C and a vacuum pressure of -0.09Mpa-

[0063] After vacuum drying at -0.07 MPa, 30.30 g of mulberry pigment powder was obtained with a yield of 0.101%. HPLC detection showed that the mulberry pigment content was 27.08%.

[0064] Example 5

[0065] 30 kg of dry mulberry bark raw material was crushed, 240 L of 80% methanol by volume was added, and extraction was performed at 75° C. for 4 times, each time for 2 hours. The mulberry bark extract was combined to obtain the mulberry bark extract. The mulberry bark extract was concentrated at -0.08 MPa--0.06 MPa and a temperature of 75° C. until it was alcohol-free to obtain a mulberry bark concentrated extract. According to the volume ratio, 3 times the amount of pure water was added to the mulberry bark concentrated extract for dilution to obtain a mulberry bark pre-chromatographic solution.

[0066] Take 18 kg of LX-18 macroporous adsorption resin to prepare a macroporous adsorption resin chromatography column; add the pre-prepared chromatography liquid of Morus alba bark to the macroporous adsorption resin chromatography column, and collect the adsorption effluent; elute with pure water until there is no deoxynojirimycin in the eluent, and combine the adsorption effluent and the pure water eluent to obtain a deoxynojirimycin extract; continue to elute with 18% ethanol until the eluent does not contain chlorogenic acid, and collect the 18% ethanol eluent as a chlorogenic acid extract; continue to elute with 85% ethanol until the eluent does not contain mulberry pigment, and collect the 85% ethanol eluent as a mulberry pigment extract.

[0067] Preparation of deoxynojirimycin product: 10 kg of 001*7 cation exchange resin is taken to prepare a cation exchange resin chromatography column; the deoxynojirimycin extract is added to the cation exchange resin chromatography column, and 2-3 BV is eluted with pure water; 1.5% volume fraction of ammonia water is continued to be used for elution until there is no deoxynojirimycin in the eluate, and the collected ammonia water eluate is concentrated to a specific gravity of 1.10-1.15 at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 65°C, and pure water is added to continue to concentrate. The mixture was reduced to neutrality, and purification was completed once, and the purification was repeated twice to obtain a concentrated extract of deoxynojirimycin; by mass, 2% of the mass of the concentrated extract of deoxynojirimycin was added to Senhuan activated carbon, and the mixture was stirred and decolorized at 75°C for 60 minutes, and filtered to obtain a decolorized liquid, which was dried at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 75°C to obtain 115.80g of deoxynojirimycin powder with a yield of 0.386%; HPLC detection showed that the content of deoxynojirimycin was 64.17%.

[0068] Preparation of chlorogenic acid product: Concentrate the chlorogenic acid extract to a specific gravity of 1.13-1.18, add 5 times the volume of ethyl acetate, stir and extract at 26°C for 35 minutes and separate the liquids, extract 5 times, combine the ethyl acetate phases, concentrate to 1 / 8 of the total volume of the ethyl acetate phase, stand and crystallize at 27°C for 26 hours, rinse the crystals with 98% by volume ethanol, dry at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 75°C to obtain 36.30g of chlorogenic acid powder with a yield of 0.121%; HPLC detection: the chlorogenic acid content is 28.52%.

[0069] Preparation of mulberry pigment product: after concentrating the mulberry pigment extract to be free of alcohol, add 1.5 times of pure water by volume to obtain a mulberry pigment diluted extract; according to the volume ratio, the mulberry pigment diluted extract: the extractant = 1:5, stir and extract for 25 minutes at 27°C and separate the liquids, extract 5 times, the extractant is a mixed solution of ethyl acetate and cyclohexane, combine the extractant phases, concentrate to 1 / 7 of the total volume of the extractant phases, and obtain a mulberry pigment extract; add the mulberry pigment extract to a polyamide resin chromatography column, the mass of the polyamide resin in the polyamide resin chromatography column is 5kg; adopt volume Gradient elution was performed with 30%, 60% and 80% ethanol solutions, and the eluent was detected by high performance liquid chromatography. The eluent containing mulberry pigment was collected and concentrated to 1 / 11 of the total volume of the eluent, and crystallized at 26°C for 22 hours. The crystals were filtered and rinsed with 98% ethanol by volume. The crystals were dried in vacuo at 70°C and a vacuum pressure of -0.09Mpa to 0.07Mpa to obtain 29.40g mulberry pigment powder with a yield of 0.098%. HPLC detection showed that the mulberry pigment content was 26.48%.

[0070] Example 6

[0071] 60 kg of dry mulberry bark raw material was crushed, 600 L of 75% by volume methanol was added, and extraction was performed at 80°C for 3 times, each time for 1.5 h. The mulberry bark extract was combined to obtain the mulberry bark extract. The mulberry bark extract was concentrated at -0.08Mpa--0.06Mpa and 75°C until it was alcohol-free to obtain a mulberry bark concentrated extract. According to the volume ratio, 3 times the amount of pure water was added to the mulberry bark concentrated extract to dilute it to obtain a mulberry bark pre-chromatographic solution.

[0072] Take 30kg of AB-8 macroporous adsorption resin to prepare a macroporous adsorption resin chromatography column; add the pre-prepared chromatography liquid of Morus alba bark to the macroporous adsorption resin chromatography column, and collect the adsorption effluent; elute with pure water until there is no deoxynojirimycin in the eluent, and combine the adsorption effluent and the pure water eluent to obtain a deoxynojirimycin extract; continue to elute with 24% ethanol until the eluent does not contain chlorogenic acid, and collect the 24% ethanol eluent as a chlorogenic acid extract; continue to elute with 75% ethanol until the eluent does not contain mulberry pigment, and collect the 75% ethanol eluent as a mulberry pigment extract.

[0073] Preparation of deoxynojirimycin product: 20 kg of 001*7 cation exchange resin is taken to prepare a cation exchange resin chromatography column; the deoxynojirimycin extract is added to the cation exchange resin chromatography column, and 2-3 BV is eluted with pure water; 1.5% volume fraction of ammonia water is continued to be used for elution until there is no deoxynojirimycin in the eluate, and the collected ammonia water eluate is concentrated to a specific gravity of 1.10-1.15 at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 55°C, and pure water is added to continue to concentrate. The mixture was reduced to neutrality, and purification was completed once, and the purification was repeated once to obtain a concentrated extract of deoxynojirimycin; 320 activated carbon with an amount of 2% by mass of the concentrated extract of deoxynojirimycin was added, and the mixture was stirred and decolorized at 75°C for 55 minutes, and the decolorized liquid was filtered to obtain a decolorized liquid, which was dried at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 65°C to obtain 225.60g of deoxynojirimycin powder with a yield of 0.376%; HPLC detection showed that the content of deoxynojirimycin was 62.78%.

[0074] Preparation of chlorogenic acid product: Concentrate the chlorogenic acid extract to a specific gravity of 1.13-1.18, add 4 times the volume of ethyl acetate, stir and extract at 28°C for 35 minutes and separate the liquids, extract 45 times, combine the ethyl acetate phases, concentrate to 1 / 8 of the total volume of the ethyl acetate phase, stand and crystallize at 26°C for 22 hours, rinse the crystals with 97% volume fraction of ethanol, dry at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 75°C, and obtain 76.20g of chlorogenic acid powder with a yield of 0.127%; HPLC detection: the chlorogenic acid content is 27.90%.

[0075] Preparation of mulberry pigment product: after concentrating the mulberry pigment extract to be alcohol-free, add 2.5 times of pure water by volume to obtain a mulberry pigment diluted extract; according to the volume ratio, the mulberry pigment diluted extract: the extractant = 1:3, stir and extract for 22 minutes at 25°C and separate the liquids, extract 5 times, the extractant is a mixture of n-butanol and n-hexane, combine the extractant phases, concentrate to 1 / 7 of the total volume of the extractant phases, and obtain a mulberry pigment extract; add the mulberry pigment extract to a polyamide resin chromatography column, the mass of the polyamide resin in the polyamide resin chromatography column is 12kg; adopt a volume Gradient elution was performed with 30%, 60% and 80% ethanol solutions, and the eluent was detected by high performance liquid chromatography. The eluent containing mulberry pigment was collected and concentrated to 1 / 10 of the total volume of the eluent, and crystallized at 26°C for 26 hours. The crystals were filtered and rinsed with 95% ethanol by volume. The crystals were dried in vacuo at 70°C and a vacuum pressure of -0.09Mpa to 0.07Mpa to obtain 58.80g mulberry pigment powder with a yield of 0.098%. HPLC detection showed that the mulberry pigment content was 25.31%.

[0076] Example 7

[0077] 60 kg of dry mulberry bark raw material was crushed, 600 L of 70% by volume methanol was added, and extraction was performed at 70°C for 4 times, each time for 2.5 hours. The mulberry bark extract was combined to obtain the mulberry bark extract. The mulberry bark extract was concentrated at -0.08Mpa--0.06Mpa and 75°C until it was alcohol-free to obtain a mulberry bark concentrated extract. According to the volume ratio, 1 times the amount of pure water was added to the mulberry bark concentrated extract for dilution to obtain a mulberry bark pre-chromatographic solution.

[0078] Take 40kg of LX-38 macroporous adsorption resin to prepare a macroporous adsorption resin chromatography column; add the pre-prepared chromatography liquid of Morus alba bark to the macroporous adsorption resin chromatography column, and collect the adsorption effluent; elute with pure water until there is no deoxynojirimycin in the eluent, and combine the adsorption effluent and the pure water eluent to obtain a deoxynojirimycin extract; continue to elute with 25% ethanol until the eluent does not contain chlorogenic acid, and collect the 25% ethanol eluent as a chlorogenic acid extract; continue to elute with 80% ethanol until the eluent does not contain mulberry pigment, and collect the 80% ethanol eluent as a mulberry pigment extract.

[0079] Preparation of deoxynojirimycin product: 25 kg of 001*7 cation exchange resin is taken to prepare a cation exchange resin chromatography column; the deoxynojirimycin extract is added to the cation exchange resin chromatography column, and 2-3 BV is eluted with pure water; 1% volume fraction of ammonia water is continued to be used for elution until there is no deoxynojirimycin in the eluate, and the collected ammonia water eluate is concentrated to a specific gravity of 1.10-1.15 at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 60°C, and pure water is added to continue to concentrate to neutrality, completing one purification, repeating the purification once to obtain a concentrated extract of deoxynojirimycin; adding 320 activated carbon with a mass of 1% of the mass of the concentrated extract of deoxynojirimycin, stirring and decolorizing at 70°C for 45 minutes, filtering, and obtaining a decolorized liquid, drying it at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 70°C to obtain 235.20g of deoxynojirimycin powder, with a yield of 0.392%; HPLC detection: the content of deoxynojirimycin is 64.96%.

[0080] Preparation of chlorogenic acid product: Concentrate the chlorogenic acid extract to a specific gravity of 1.13-1.18, add 6 times the volume of ethyl acetate, stir and extract at 25°C for 35 minutes and separate the liquids, extract 4 times, combine the ethyl acetate phases, concentrate to 1 / 10 of the total volume of the ethyl acetate phase, stand and crystallize at 25°C for 25 hours, rinse the crystals with 95% by volume ethanol, and dry at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 65°C to obtain 75.60g of chlorogenic acid powder with a yield of 0.126%; HPLC detection: the chlorogenic acid content is 28.95%.

[0081] Preparation of mulberry pigment product: after concentrating the mulberry pigment extract to be free of alcohol, add 2.5 times of pure water by volume to obtain a mulberry pigment diluted extract; according to the volume ratio, the mulberry pigment diluted extract: extractant = 1:4, stir and extract for 28 minutes at 26°C and separate the liquids, extract 4 times, the extractant is a mixture of dichloromethane and petroleum ether, combine the extractant phases, concentrate to 1 / 8 of the total volume of the extractant phases, and obtain a mulberry pigment extract; add the mulberry pigment extract to a polyamide resin chromatography column, the mass of the polyamide resin in the polyamide resin chromatography column is 12kg; use Gradient elution was performed with ethanol solutions with volume fractions of 30%, 60% and 80%, and the eluent was detected by high performance liquid chromatography. The eluent containing mulberry pigment was collected and concentrated to 1 / 10 of the total volume of the eluent, and crystallized at a temperature of 25°C for 26 hours. The crystals were filtered and rinsed with ethanol with a volume fraction of 97%, and vacuum dried at a temperature of 60°C and a vacuum pressure of -0.09Mpa--0.07Mpa to obtain 61.80g of mulberry pigment powder with a yield of 0.103%. HPLC detection showed that the mulberry pigment content was 26.88%.

Claims

1. A method for simultaneously preparing deoxynojirimycin, chlorogenic acid and morin from Morus alba bark, characterized in that: The following steps are involved: S1, taking dried mulberry bark raw material and crushing it, adding alcohol extraction solution according to the ratio of mulberry bark raw material mass: alcohol extraction solution volume = 1g: (8-10)mL, heating and extracting to obtain mulberry bark extract, concentrating the mulberry bark extract until there is no alcohol, and obtaining mulberry bark concentrated extract; S2. Add 1 to 3 times the volume of pure water to the concentrated extract of Morus alba to dilute it, so as to obtain a pre-chromatographic solution of Morus alba; Take a macroporous adsorption resin in an amount of 1 / 2 to 2 / 3 times the mass of the mulberry bark raw material to prepare a macroporous adsorption resin chromatography column; add the mulberry bark pre-prepared chromatography liquid into the macroporous adsorption resin chromatography column, and collect the adsorption effluent; The deoxynojirimycin is eluted with pure water until there is no deoxynojirimycin in the eluent, and the adsorption effluent and the pure water eluent are combined to form a deoxynojirimycin extract; Continue to elute with 15-25% ethanol until the eluate does not contain chlorogenic acid, and collect the 15-25% ethanol eluate as the chlorogenic acid extract; Continue to use 70-90% ethanol to elute until the eluate does not contain morin, and collect the 70-90% ethanol eluate as morin extract; S3, respectively removing impurities and purifying the deoxynojirimycin extract, the chlorogenic acid extract and the mori pigment extract to obtain the deoxynojirimycin product, the chlorogenic acid product and the mori pigment product; In step S3, the deoxynojirimycin extract is purified by: purifying the deoxynojirimycin extract 1-3 times by cation exchange resin chromatography column purification method to obtain a concentrated deoxynojirimycin extract; adding 1-3% by mass of activated carbon of the mass of the concentrated deoxynojirimycin extract, stirring and decolorizing at 65-75° C. for 40-60 minutes, filtering, and obtaining a decolorized solution, drying it at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 65° C.-75° C. to obtain a deoxynojirimycin product; In step S3, the chlorogenic acid extract is purified by: concentrating the chlorogenic acid extract to a specific gravity of 1.13-1.18, adding 4-6 times the volume of ethyl acetate, stirring and extracting at 25-28°C for 30-40min and separating the liquids, extracting 3-5 times, combining the ethyl acetate phases, and concentrating to 1 / 10-1 / 8 of the total volume of the ethyl acetate phase, standing and crystallizing at 25-28°C for 20-26h, rinsing the crystals with ethanol with a volume fraction of 95-98%, and drying at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 65°C-75°C to obtain a chlorogenic acid product; In step S3, the mulberry pigment extract is purified by: concentrating the mulberry pigment extract to be free of alcohol, adding 1.5-2.5 times of pure water by volume to obtain a mulberry pigment diluted extract; according to the volume ratio, the mulberry pigment diluted extract: the extractant = 1: (3-5), stirring and extracting at 25-28° C. for 20-30 minutes and separating the liquids, extracting 3-5 times, combining the extractant phases, and concentrating to 1 / 8-1 / 6 of the total volume of the extractant phase to obtain a mulberry pigment extract; The mulberry pigment extract is added to a polyamide resin chromatography column, wherein the mass of the polyamide resin in the polyamide resin chromatography column is 1 / 6-1 / 5 of the mass of the mulberry bark raw material; gradient elution is performed using ethanol solutions with volume fractions of 30%, 60%, and 80%, and the eluent is detected by high performance liquid chromatography, the eluent containing mulberry pigment is collected and concentrated to 1 / 12-1 / 10 of the total volume of the eluent, and crystallized at a temperature of 25-28° C. for 20-26 hours, the crystals are filtered, and the crystals are rinsed with ethanol with a volume fraction of 95-98%, and vacuum dried at a temperature of 55° C. and a vacuum pressure of -0.09Mpa--0.07Mpa to obtain a mulberry pigment product; In step S3, the deoxynojirimycin extract is purified by a cation exchange resin chromatography column purification method, specifically: Take cation exchange resin in an amount of 1 / 3 to 1 / 2 times the mass of Morus alba raw material to prepare a cation exchange resin chromatography column; add deoxynojirimycin extract to the cation exchange resin chromatography column, and elute 2-3BV with pure water; continue to use ammonia water with a volume fraction of 1% to 2% to elute until there is no deoxynojirimycin in the eluate, concentrate the collected ammonia water eluate to a specific gravity of 1.10-1.15 at a vacuum degree of -0.08Mpa--0.06Mpa and a temperature of 55°C to 65°C, add pure water to continue concentrating to neutrality, and complete a purification.

2. The method for simultaneously preparing deoxynojirimycin, chlorogenic acid and morin from Morus alba bark according to claim 1, characterized in that: In step S3, the extractant is ethyl acetate or dichloromethane or n-butanol or n-hexane or petroleum ether or cyclohexane; or any one of ethyl acetate, dichloromethane, n-butanol is combined with any one of n-hexane, petroleum ether, and cyclohexane.

3. The method for simultaneously preparing deoxynojirimycin, chlorogenic acid and morin from Morus alba bark according to claim 2, characterized in that: In step S1, the alcohol extraction solution is methanol or ethanol, and the volume fractions of methanol and ethanol are both 70-80%; The extraction conditions are as follows: extracting at 70-80°C for 2-4 times, each time for 1.5-2.5 hours, and then combining to obtain the mulberry bark extract.

4. The method for simultaneously preparing deoxynojirimycin, chlorogenic acid and morin from Morus alba bark according to claim 3, characterized in that: In step S2, the macroporous adsorption resin is LX-38 resin, LX-18 resin, LX-15 resin or AB-8 resin.

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