CN113717137A - Preparation method of high-purity hyoscyamine - Google Patents
Preparation method of high-purity hyoscyamine Download PDFInfo
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- CN113717137A CN113717137A CN202111035221.9A CN202111035221A CN113717137A CN 113717137 A CN113717137 A CN 113717137A CN 202111035221 A CN202111035221 A CN 202111035221A CN 113717137 A CN113717137 A CN 113717137A
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- RKUNBYITZUJHSG-UHFFFAOYSA-N Hyosciamin-hydrochlorid Natural products CN1C(C2)CCC1CC2OC(=O)C(CO)C1=CC=CC=C1 RKUNBYITZUJHSG-UHFFFAOYSA-N 0.000 title claims abstract description 28
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims abstract description 70
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/04—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring
- C07D311/06—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2
- C07D311/08—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring
- C07D311/16—Benzo[b]pyrans, not hydrogenated in the carbocyclic ring with oxygen or sulfur atoms directly attached in position 2 not hydrogenated in the hetero ring substituted in position 7
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
The invention discloses a preparation method of high-purity hyoscyamine, which comprises the steps of crushing wolfberry fruits, degreasing by using a subcritical technology, and carrying out reflux extraction on a degreased product by using an ethanol solution to obtain a wolfberry crude extract; separating and enriching the crude extract of the Chinese wolfberry by using macroporous adsorption resin, and eluting by using ethanol to obtain a scopoletin crude extract; dissolving the crude scopoletin extract with water, layering by using an organic solvent, and concentrating and drying the upper layer to obtain a crude scopoletin product; recrystallizing the crude scopoletin product with chromatographic pure methanol, and drying the concentrated solution to obtain the high-purity scopoletin. The hyoscyamine extracted by the method has high purity which is more than 98.5 percent, and good uniformity and stability, and can be used as a standard sample of the hyoscyamine.
Description
Technical Field
The invention relates to a preparation method of high-purity hyoscyamine, belonging to the technical field of extraction of active ingredients.
Background
Hyoscyamine (Scopoletin) is a coumarin compound, has effects of inhibiting human prostate cell proliferation and inducing apoptosis, inhibiting vascular endothelial cell regeneration, resisting inflammation and depression, and has high content in fructus Lycii up to 2-8%. Hyoscyamine, CAS: 92-61-5, molecular formula: c10H8O4Molecular weight: 192.17, chemical nomenclature: 2H-1-Benzopyran-2-one,7-hydroxy-6-methoxy, property: pale yellow needle-like or prism-like crystals, melting point: 204 ℃ and solubility: dissolving in hot ethanol, hot methanol or hot glacial acetic acid, slightly dissolving in cold ethanol, cold methanol or water, and slightly dissolving in chloroform. Chemical structural formula:
the current research considers that various physiological activities of the medlar have certain relation with the hyoscyamine. Therefore, more and more researchers use scopoletin as one of the main indicators of the activity of lycium barbarum to measure the quality of lycium barbarum. At present, the domestic market lacks standard and unified hyoscyamine standard samples, and the quality of related products is not uniform, so that the development of the hyoscyamine standard samples is urgently needed, the quality of the Chinese wolfberry extract taking hyoscyamine as a main component is standardized, and the quality of functional foods, health-care foods and cosmetics taking the hyoscyamine as a raw material is ensured.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a preparation method of high-purity scopoletin aiming at the defects in the prior art, the extracted scopoletin has high purity which is more than 98.5 percent, and good uniformity and stability, and can be used as a standard sample of scopoletin.
In order to achieve the purpose, the invention adopts the following technical scheme:
a preparation method of high-purity hyoscyamine comprises the following steps:
(1) pulverizing fructus Lycii, defatting with subcritical technology, reflux extracting the defatted product with ethanol solution to obtain fructus Lycii crude extract;
(2) separating and enriching the crude extract of the Chinese wolfberry by using macroporous adsorption resin, and eluting by using ethanol to obtain a scopoletin crude extract;
(3) dissolving the crude scopoletin extract with water, layering by using an organic solvent, and concentrating and drying the upper layer to obtain a crude scopoletin product;
(4) recrystallizing the crude scopoletin product with chromatographic pure methanol, and drying the concentrated solution to obtain the high-purity scopoletin.
In the above technical solution, the operation steps of step (1) are: pulverizing fructus Lycii, sieving to obtain fructus Lycii powder, adding ethanol solution into the fructus Lycii powder, stirring, soaking to obtain fructus Lycii mixed solution, defatting with subcritical butane, adding ethanol solution into defatted product, and reflux extracting to obtain fructus Lycii crude extract;
in the technical scheme, in the step (1), the medlar is dried mature fruit of Ningxia medlar (Lycium barbarum L.), the dried mature fruit is crushed and sieved by a 50-mesh sieve to obtain medlar powder, the medlar powder is added into 70% ethanol with the weight being 10 times of that of the medlar powder, the mixture is heated to 60 ℃, and the mixture is stirred and soaked for 1 hour at the temperature to obtain medlar mixed solution.
In the above technical solution, in the step (1), the subcritical butane is degreased under the following operating conditions: the extraction temperature is 45-55 deg.C, the extraction pressure is 0.3-0.6MPa, the extraction time is 35-45min, and the fructus Lycii residue obtained after extraction is defatted product.
In the technical scheme, in the step (1), the degreased product is added into 95% ethanol with the weight of 8-12 times, and the extract obtained by heating to 50-60 ℃ and reflux-extracting for 3-5h is the medlar crude extract (the yield is 23-27%, and the extraction rate is 78-82%).
In the above technical solution, the specific operation of step (2) is: adsorbing fructus Lycii crude extract with macroporous adsorbent resin, eluting with 20%, 50%, 70%, and 95% ethanol sequentially after adsorption, collecting 50% ethanol eluate, spray drying to water content of product lower than 10% to obtain hyoscyamine crude extract (containing 13-18% flavone extract).
In the above technical scheme, in the step (2), the macroporous adsorbent resin is any one of HPD400A, LSA10, DA201-CIII and D101; the mass ratio of the medlar crude extract to the macroporous adsorption resin is 1: 50, adsorbing by using an upper column, wherein the adsorption flow rate is 1-3 BV/h, the elution flow rate of the eluent is 2-4 BV/h, and the using amount of the eluent is 2-5 BV.
In the above technical solution, the operation steps of step (3) are: dissolving the crude scopoletin extract in pure water, adding n-butanol, fully shaking, placing into a separating funnel, standing for 8h for layering, collecting the upper n-butanol phase and the lower water phase, rotary evaporating the upper n-butanol phase to remove the solvent, concentrating and recovering to obtain a concentrated solution, and volatilizing the concentrated solution in a water bath to obtain a crude scopoletin product (containing 28-30% of flavone extract).
In the technical scheme, in the step (3), when the scopoletin crude extract is dissolved in pure water, 60g of the scopoletin crude extract is dissolved in 3000mL of pure water, and is dissolved thoroughly by ultrasonic for 20min, and 3000mL of n-butanol is added for layering after the scopoletin crude extract is dissolved thoroughly.
In the above technical solution, in the step (3), the solvent is removed by rotary evaporation, and the concentrated solution is recovered by concentration, wherein the operating conditions are as follows: the vacuum degree is-0.9-0.1 mpa, the temperature is 50-60 ℃, a cooling water circulation system is arranged in the evaporation process, the temperature of condensed water is below-10 ℃, preferably-20-10 ℃, part of solvent obtained by evaporation is taken and added with acid potassium dichromate solution, if the solution turns to grey green, the solution is not evaporated, and if the solution does not change color, the evaporation is proved to be finished.
In the above technical solution, the operation steps of step (4) are: dissolving the crude scopoletin product with a proper amount of methanol, mixing with silica gel, filling the mixture into a column by a wet method, and mixing with dichloromethane: and eluting with mixed solution of methanol at sequentially increased ratio, collecting eluate in stages, collecting eluate from the second column volume, mixing the eluates from the second to the fourth column volumes, vacuum drying under reduced pressure, and volatilizing solvent to obtain a large amount of yellowish needle crystals, which are high-purity hyoscyamine.
In the technical scheme, in the step (4), the crude scopoletin product is dissolved by methanol to obtain scopoletin solution with the concentration of 140-160mg/mL, and 45g of silica gel and 10mL of scopoletin solution are taken to be packed into a column by a wet method.
In the above technical scheme, in the step (4), the eluent is a mixed solution in which the volume ratios of dichloromethane to methanol are respectively 1:1, 1:3 and 1:5 are sequentially increased; the flow rate of each eluent is 1-2BV/h, and the dosage is 1-3 BV.
In the above technical solution, in the step (4), the reduced pressure vacuum drying is performed under the following operating conditions: the vacuum degree is 2-8mp, and the temperature is-25 to-10 ℃.
The hyoscyamine extracted by the method has high purity which is more than 98.5 percent, good uniformity and stability, can be used as a standard sample of the hyoscyamine, standardizes the quality of the medlar extract taking the hyoscyamine as a main component, and ensures the quality of functional food, health-care food and cosmetics which take the hyoscyamine as a raw material.
Drawings
FIG. 1-1 is a thin layer chromatogram of system I for detection of scopoletin obtained in example 1;
FIGS. 1-2 are thin-layer chromatograms of system II for detection of scopoletin from example 1;
FIGS. 1-3 are thin-layer chromatograms of system III for detection of scopoletin obtained in example 1;
FIG. 2-1 is an HPLC chromatogram of scopoletin obtained in example 1 under a first elution condition;
FIG. 2-2 is a detection of the hyoscyamine HPLC profile obtained in example 1 under the second elution condition;
2-3 is a blank spectrogram under the second elution condition;
FIG. 3-1 is an enlarged chromatogram of scopoletin obtained in detection example 1 at a detection wavelength of 228 nm;
FIG. 3-2 is an enlarged chromatogram of scopoletin obtained in detection example 1 at a detection wavelength of 295 nm;
FIGS. 3 to 3 are enlarged chromatograms of scopoletin obtained in detection example 1 at a detection wavelength of 345 nm;
FIG. 4-1 is Hypersil ODS 2C18HPLC chromatogram of scopoletin obtained in example 1 was detected by a column (4.6 mm. times.250 mm,5 μm);
FIG. 4-2 is a Sinochrom ODS-BP C18HPLC chromatogram of scopoletin obtained in example 1 was detected by a column (4.6 mm. times.250 mm,5 μm);
FIG. 5 is a total ion flow diagram of detection of scopoletin in positive ion mode obtained in example 1 by HPLC-MS;
FIG. 6-1 is a TG curve (N) of scopoletin obtained in example 12Atmosphere);
FIG. 6-2 shows the TG curve (O) of scopoletin obtained in example 12Atmosphere);
FIG. 7-1 is a UV spectrum of scopoletin obtained in example 1;
FIG. 7-2 is an infrared spectrum of scopoletin obtained in example 1;
FIGS. 7-3 are high-resolution mass spectrograms of scopoletin obtained in example 1.
Detailed Description
The following detailed description of the embodiments of the present invention is provided, but the present invention is not limited to the following descriptions:
the invention will now be illustrated with reference to specific examples:
example 1: preparation method of high-purity hyoscyamine
The method comprises the following steps:
(1) taking dried mature fruit of fructus Lycii as fructus Lycii of Ningxia (Lycium barbarum L.) as raw material, pulverizing, sieving with 50 mesh sieve to obtain fructus Lycii powder, adding fructus Lycii powder into 10 times of 70% ethanol, heating to 60 deg.C, stirring at this temperature, and soaking for 1 hr to obtain fructus Lycii mixed solution; degreasing the medlar mixed solution by using subcritical butane, wherein the operation conditions are as follows: extracting at about 50 deg.C under about 0.4MPa for 43min to obtain fructus Lycii residue as defatted product; adding the degreased product into 9 times of 95% ethanol, heating to about 52 deg.C, reflux-extracting for 3.5 hr to obtain extractive solution of fructus Lycii crude extract (yield of 25% and extraction rate of 80%).
(2) Adsorbing the medlar crude extract by using LSA10 macroporous adsorption resin, wherein the mass ratio of the medlar crude extract to the macroporous adsorption resin is 1: adsorbing with 50 column at adsorption flow rate of 1BV/h, eluting with 2.54BV/h eluent at 4 BV; eluting with 20%, 50%, 70%, and 95% ethanol sequentially after adsorption, collecting the liquid eluted with 50% ethanol, and spray drying until the water content of the product is less than 10% to obtain hyoscyamine crude extract (containing 15% flavone extract).
(3) Dissolving 60g of crude scopoletin extract in 3000mL of pure water, dissolving the crude scopoletin extract thoroughly by ultrasonic dissolution for 20min, adding 3000mL of n-butyl alcohol, fully oscillating, placing the mixture into a separating funnel, standing for 8h for layering, collecting the upper n-butyl alcohol phase, performing rotary evaporation to remove the solvent, concentrating and recovering to obtain a concentrated solution, and volatilizing the concentrated solution in a water bath to obtain crude scopoletin (containing 29.5% of flavone extract). The operating conditions for the trans-evaporation were: the vacuum degree is-0.9 mpa, the temperature is about 52 ℃, a cooling water circulation system is arranged in the evaporation process, the temperature of condensed water is-20 ℃, part of solvent obtained by evaporation is taken and added into the acid potassium dichromate solution, if the solution turns into grey green, the solution is not evaporated, and if the solution does not change color, the evaporation is finished.
(4) Dissolving the crude scopoletin product with a proper amount of methanol to obtain scopoletin solution with the concentration of 140-160mg/mL, and performing wet column packing on 45g of silica gel and 10mL of scopoletin solution by using dichloromethane: eluting with mixed solution of methanol at sequentially increased ratio (1:1, 1:3, 1:5) at flow rate of 2BV/h and dosage of 2 BV; collecting eluate by stages, collecting from the second column volume, mixing the eluates of the second to the fourth column volumes, vacuum drying under reduced pressure (vacuum degree of 8mp, temperature of-15 deg.C) until the solvent is evaporated to dryness to obtain a large amount of yellowish needle crystals, which are high-purity hyoscyamine.
Verification test I: thin layer chromatography purity analysis of the high purity scopoletin obtained in example 1 using 3 different developing reagents
The detection is carried out by adopting 3 development systems, and the sample application amount of five thin-layer plate silica gel 254G plates is respectively designed to be 20 mug (point 1), 40 mug (point 2), 60 mug (point 3), 80 mug (point 4) and 100 mug (point 5).
1. Deployment system
1.1 System I
Thin-layer plate: HPTLC precast slab of Yangtze river friend silica gel development Co., Ltd
Developing agent: petroleum ether: ethyl acetate (1:1, v/v)
Color developing agent: i is2
The color development method comprises the following steps: iodine steam fumigation
Rf value: 0.34
And (4) conclusion: no impurity spots appeared at each amount, as shown in FIGS. 1-1.
1.2 System II
Thin-layer plate: HPTLC precast slab of Yangtze river friend silica gel development Co., Ltd
Developing agent: petroleum ether: acetone (3:2, v/v)
Color developing agent: i is2
The color development method comprises the following steps: iodine steam fumigation
Rf value: 0.38
And (4) conclusion: no impurity spots appeared at each amount, as shown in FIGS. 1-2.
1.3 System III
Thin-layer plate: HPTLC precast slab of Yangtze river friend silica gel development Co., Ltd
Developing agent: dichloromethane: methanol (50:1, v/v)
Color developing agent: i is2
The color development method comprises the following steps: iodine steam fumigation
Rf value: 0.40
And (4) conclusion: no impurity spots appeared at each amount, as shown in FIGS. 1-3.
2. Conclusion
The results of thin layer chromatography detection by using 3 different developing agents show that only one obvious color spot is seen in the sample application amount range of 20-100 mu g of scopoletin sample, no impurity spot is seen, and the purity of the sample is high.
And (5) verification test II: purity analysis of the high purity scopoletin obtained in example 1 by HPLC
1. Purity analysis under different elution conditions
1.1 elution conditions one
Analysis conditions were as follows: a chromatographic column: hypersil ODS 2C18Chromatography column (4.6 mm. times.250 mm,5 μm); mobile phase: 0min 0.1% TFA water methanol 80: 20; 14min 0.1% TFA water methanol 60: 40; 16-22 min 0.1% TFA water and methanol is 30: 70; 25min 0.1% TFA water methanol 80: 20; flow rate: 1 mL/min; column temperature: 30 ℃; detection time: 25 min; detection wavelength: 345 nm. After the solvent chromatographic peak is subtracted, the sample chromatographic peak is quantified by an area normalization method, and the purity of the scopoletin is 99.32 percent through integration. The HPLC measurement chart is shown in FIG. 2-1.
3.3.1.2 elution conditions two
Analysis conditions were as follows: a chromatographic column: hypersil ODS 2C18Chromatography column (4.6 mm. times.250 mm,5 μm); mobile phase: 0min 0.1% TFA water acetonitrile methanol 80:10: 10; 14min 0.1% TFA water acetonitrile methanol 60:20: 20; 16-22 min, 0.1% TFA water, acetonitrile and methanol, wherein the ratio is 30:40: 30; 25min 0.1% TFA water acetonitrile methanol 80:10: 10; flow rate: 1 mL/min; column temperature: 30 deg.C(ii) a Detection time: 25 min; detection wavelength: 345 nm. After the solvent chromatographic peak is subtracted, the sample chromatographic peak is quantified by an area normalization method, and the purity of the scopoletin is 99.34 percent through integration. The elution conditions were selected and the subsequent homogeneity, stability and quantification tests were performed on the scopoletin standards. The scopoletin HPLC chromatogram is shown in fig. 2-2, and the blank chromatogram is shown in fig. 2-3.
2. Purity analysis at different detection wavelengths
Analysis conditions were as follows: a chromatographic column: hypersil ODS 2C18Chromatography column (4.6 mm. times.250 mm,5 μm); mobile phase: 0min 0.1% TFA water acetonitrile methanol 80:10: 10; 14min 0.1% TFA water acetonitrile methanol 60:20: 20; 16-22 min, 0.1% TFA water, acetonitrile and methanol, wherein the ratio is 30:40: 30; 25min 0.1% TFA water acetonitrile methanol 80:10: 10; flow rate: 1 mL/min; column temperature: 30 ℃; detection time: 25 min; detection wavelength: 200-400 nm. Except for the solvent peak, no other impurity peaks appear at other wavelengths. Amplifying chromatograms under the conditions of 228nm, 295nm and 345nm, wherein the purity and amplification chromatograms are shown in the following table:
TABLE 1 results for different detection wavelengths
| Detection wavelength (nm) | Purity value (%) | Enlarged chromatogram |
| ·228· | ·99.49· | FIG. 3-1. fig |
| ·295· | ·99.37· | FIG. 3-2 |
| ·345· | ·99.34· | FIG. 3-3 |
3. Purity analysis under different chromatographic column conditions
Using two different C18Chromatography column, the purity of the scopoletin standard sample was determined under the same elution conditions.
3.1Hypersil ODS2 C18Chromatographic column (4.6mm X250 mm,5 μm)
Mobile phase: 0min 0.1% TFA water acetonitrile methanol 80:10: 10; 14min 0.1% TFA water acetonitrile methanol 60:20: 20; 16-22 min, 0.1% TFA water, acetonitrile and methanol, wherein the ratio is 30:40: 30; 25min 0.1% TFA water acetonitrile methanol 80:10: 10; flow rate: 1 mL/min; column temperature: 30 ℃; detection time: 25 min; detection wavelength: 345 nm. After the solvent chromatographic peak is subtracted, the sample chromatographic peak is quantified by an area normalization method, and the purity of the scopoletin is 99.34 percent through integration. The HPLC profile is shown in FIG. 4-1.
3.2SinoChrom ODS-BP C18Chromatographic column (4.6mm X250 mm,5 μm)
Mobile phase: 0min 0.1% TFA water acetonitrile methanol 80:10: 10; 14min 0.1% TFA water acetonitrile methanol 60:20: 20; 16-22 min, 0.1% TFA water, acetonitrile and methanol, wherein the ratio is 30:40: 30; 25min 0.1% TFA water acetonitrile methanol 80:10: 10; flow rate: 1 mL/min; column temperature: 30 ℃; detection time: 25 min; detection wavelength: 345 nm. After the solvent chromatographic peak is deducted, the sample chromatographic peak is quantified by an area normalization method, and the purity of the scopoletin is 99.37 percent through integration. The HPLC profile is shown in FIG. 4-2.
4. Conclusion
And (3) quantifying the chromatographic peaks of the sample by an area normalization method under different elution conditions, different detection wavelengths and different chromatographic column conditions, wherein the purity of the scopoletin sample is more than 98.5% through integration.
And (3) verification test III: purity analysis of the high purity scopoletin obtained in example 1 by HPLC-MS
Analysis conditions were as follows: a chromatographic column: sinochrom ODS-BP C18Chromatography column (4.6 mm. times.250 mm,5 μm); mobile phase: 0min 0.1% formic acid acetonitrile 80:10:10, 14min 0.1% formic acid acetonitrile 60:20:20, 16-18 min 0.1% formic acid acetonitrile 30:40: 30; flow rate: 1.0 mL/min; column temperature: 30 ℃; operating time: and (4) 18 min. The MS conditions were as follows: gas Temp: 225 ℃; drying Gas: 5L/min; nebulizer: 20 psig; shear Gas Temp: 400 ℃; shear Gas Flow: 12L/min; scan: 150to 2000 m/z. As shown in fig. 5, no significant impurity peak was found in the total ion flow pattern of the positive ions.
And (4) verification test four: thermogravimetric analysis of the high purity scopoletin obtained in example 1
In N2Under the atmosphere condition, the temperature is within the range of 25-105 ℃, and the TG curve is a horizontal line (figure 6-1), which shows that the sample has no adsorbed water and crystal water and has good thermal stability. When the temperature exceeded 205 ℃, the sample began to decompose. At O2Under the atmosphere condition, the weight loss of the sample is 99.41-99.84% when the sample is heated to the temperature of 900 ℃ (figure 6-2) (parallel analysis is carried out for 3 times), and the sample is free from obvious inorganic impurities.
And (5) verification test five: the structure of the high purity scopoletin obtained in example 1 was confirmed
The ultraviolet spectrum of high purity scopoletin obtained in example 1 is shown in fig. 7-1, and scopoletin uv (meoh) × max (log ∈) ═ 345(4.15), which is consistent with the characteristic of scopoletin ultraviolet absorption.
The infrared spectrum of the high purity scopoletin obtained in example 1 is shown in FIG. 7-2: 3338cm-1: -stretching vibrations of OH; 1703cm-1: stretching vibration of O-C-; 1628, 1608, 1566cm-1: stretching vibration of the multi-conjugated double bonds; 1291cm-1: stretching vibration of asymmetric C-O-C; 1018cm-1: symmetric C-O-C stretching vibration; 820, 745cm-1: aromatic C-H out-of-plane bending vibration; 714cm-1: ring C ═ C bending vibration. The compound has functional groups of-OH, O ═ C-, C-O-C-, aromatic ring conjugated double bonds, and completely conforms to the structure of scopoletin.
Obtained in example 1The high resolution mass spectrum of high purity scopoletin is shown in FIGS. 7-3: hyoscyamine plus sodium ion [ C ]10H8O4+Na]+The theoretical accurate mass number of the product is 215.0320, the accurate mass number of the hyoscyamine and sodium ion measured by the high-resolution mass spectrum is 215.0320, the data of the product and the data are completely consistent, and the basic requirements of the high-resolution mass spectrum are met.
The above examples are only for illustrating the technical concept and features of the present invention, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (10)
1. A preparation method of high-purity hyoscyamine is characterized by comprising the following steps:
(1) pulverizing fructus Lycii, defatting with subcritical technology, reflux extracting the defatted product with ethanol solution to obtain fructus Lycii crude extract;
(2) separating and enriching the crude extract of the Chinese wolfberry by using macroporous adsorption resin, and eluting by using ethanol to obtain a scopoletin crude extract;
(3) dissolving the crude scopoletin extract with water, layering by using an organic solvent, and concentrating and drying the upper layer to obtain a crude scopoletin product;
(4) recrystallizing the crude scopoletin product with chromatographic pure methanol, and drying the concentrated solution to obtain the high-purity scopoletin.
2. The method according to claim 1, wherein the step (1) comprises the steps of: pulverizing fructus Lycii, sieving to obtain fructus Lycii powder, adding ethanol solution into the fructus Lycii powder, stirring, soaking to obtain fructus Lycii mixed solution, defatting with subcritical butane, adding ethanol solution into defatted product, and reflux-extracting to obtain fructus Lycii crude extract.
3. The method according to claim 2, wherein the fructus Lycii is dried mature fruit of Lycium barbarum L, and is obtained by pulverizing and sieving with 50 mesh sieve to obtain fructus Lycii powder, adding 10 times weight of 70% ethanol, heating to 60 deg.C, and soaking at this temperature under stirring for 1 hr to obtain fructus Lycii mixed solution.
4. The method of claim 2, wherein the subcritical butane is degreased under the following operating conditions: extracting at 45-55 deg.C under 0.3-0.6MPa for 35-45min to obtain fructus Lycii residue as defatted product; the degreased product is added into 95% ethanol with the weight of 8-12 times, and the extract obtained by heating to 50-60 ℃ and reflux extraction for 3-5h is the crude extract of the medlar.
5. The preparation method according to claim 1, wherein the specific operation of step (2) is: adsorbing the fructus Lycii crude extract with macroporous adsorbent resin, eluting with 20%, 50%, 70%, and 95% ethanol sequentially after adsorption, collecting 50% ethanol eluate, spray drying to water content of product lower than 10% to obtain hyoscyami crude extract.
6. The preparation method according to claim 5, wherein in the step (2), the macroporous adsorbent resin is any one of HPD400A, LSA10, DA201-CIII and D101; the mass ratio of the medlar crude extract to the macroporous adsorption resin is 1: 50, adsorbing by using an upper column, wherein the adsorption flow rate is 1-3 BV/h, the elution flow rate of the eluent is 2-4 BV/h, and the using amount of the eluent is 2-5 BV.
7. The method according to claim 1, wherein the step (3) is performed by: dissolving the crude scopoletin extract in pure water, adding n-butanol, fully oscillating, placing into a separating funnel, standing for 8h for layering, collecting the upper n-butanol phase and the lower water phase, rotary evaporating the upper n-butanol phase to remove the solvent, concentrating and recovering to obtain a concentrated solution, and volatilizing the concentrated solution in a water bath to obtain the crude scopoletin.
8. The preparation method according to claim 7, wherein when the crude scopoletin extract is dissolved in pure water, 60g of the crude scopoletin extract is dissolved in 3000mL of pure water, and dissolved thoroughly by ultrasonic dissolution for 20min, and then 3000mL of n-butanol is added for layering after complete dissolution; the rotary evaporation is used for removing the solvent, the concentrated solution is obtained by concentration and recovery, and the operation conditions are as follows: the vacuum degree is-0.9-0.1 mpa, the temperature is 50-60 ℃, a cooling water circulation system is arranged in the evaporation process, the temperature of condensed water is below-10 ℃, part of solvent obtained by evaporation is added into the acid potassium dichromate solution, if the solution turns to grey green, the evaporation is not finished, and if the solution does not change color, the evaporation is finished.
9. The method according to claim 1, wherein the step (4) is performed by: dissolving the crude scopoletin product with a proper amount of methanol, mixing with silica gel, filling the mixture into a column by a wet method, and mixing with dichloromethane: and eluting with mixed solution of methanol at sequentially increased ratio, collecting eluate in stages, collecting eluate from the second column volume, mixing the eluates from the second to the fourth column volumes, vacuum drying under reduced pressure, and volatilizing solvent to obtain a large amount of yellowish needle crystals, which are high-purity hyoscyamine.
10. The method according to claim 9, wherein the crude scopoletin is dissolved in methanol to obtain a scopoletin solution with a concentration of 140-160mg/mL, and 45g of silica gel and 10mL of scopoletin solution are loaded on the column by a wet method; the eluent is a mixed solution of dichloromethane and methanol with the volume ratio of 1:1, 1:3 and 1:5 which are increased in sequence; the flow rate of each eluent is 1-2BV/h, and the dosage is 1-3 BV; the vacuum drying under reduced pressure comprises the following operating conditions: the vacuum degree is 2-8mp, and the temperature is-25 to-10 ℃.
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| CN102617674A (en) * | 2012-03-15 | 2012-08-01 | 中国科学院西北高原生物研究所 | Preparation method of scopolin monomer in anisodus tanguticus root |
| CN112898258A (en) * | 2021-03-05 | 2021-06-04 | 中国科学院兰州化学物理研究所 | Separation preparation method of high-purity hyoscyamine |
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| CN102617674A (en) * | 2012-03-15 | 2012-08-01 | 中国科学院西北高原生物研究所 | Preparation method of scopolin monomer in anisodus tanguticus root |
| CN112898258A (en) * | 2021-03-05 | 2021-06-04 | 中国科学院兰州化学物理研究所 | Separation preparation method of high-purity hyoscyamine |
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