CN110724044A - Preparation method of arylturmerone reference substance - Google Patents
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Abstract
The invention provides a preparation method of an aromacrone reference substance, which takes turmeric as a raw material, the turmeric is crushed into turmeric powder with the average grain diameter less than or equal to 10 mu m, n-pentane-ethanol mixed liquor is firstly used for extracting to obtain an extract I, then the extract I is extracted by isopropyl ether-butyl acetate-ethyl acetate-water mixed liquor to obtain an extract II, and finally the isopropyl ether-butyl acetate mixed liquor is used as an eluent to carry out silica gel column chromatography separation on the extract II, so that the high-purity aromacrone reference substance can be obtained at high yield.
Description
Technical Field
The invention relates to the technical field of natural product extraction and separation, in particular to a preparation method of an aromatic turmerone reference substance.
Background
The turmeric is a traditional Chinese medicine in China, rhizomes mainly contain volatile oil and curcumin components, and in addition, resins, saccharides, sterols, fatty acids, polypeptides and the like, and according to literature reports, dozens of volatile oil components are found in the traditional Chinese medicine turmeric, including: borneol, camphor, terpineol, curcumene, arylturmerone, turmerone, phellandrene and the like.
Wherein, the arylturmerone belongs to terpene compounds, is one of important components of turmeric, and has wide prospects in aspects of clinical development and treatment of metabolic comprehensive diseases such as leukemia, malignant lymphoma, bacterial inflammation, fungal inflammation, diabetes, obesity and the like, birth control and the like.
The reference substance is a standard substance used for identifying, checking, measuring content and correcting performance of a checking instrument, is a reference for determining authenticity of a medicine in medicine inspection, and is an essential tool for controlling medicine quality, so that research and development of a preparation method of the ar-turmerone reference substance are very important.
Patent CN104478686B discloses a preparation method of a turmerone reference substance in turmeric volatile oil, which is characterized in that turmeric volatile oil is used as a raw material, and is subjected to pressure normal phase silica gel column rough separation, then pressure normal phase silica gel column fine separation and finally high performance liquid chromatography separation to obtain a turmerone reference substance with the purity of 99.95%. However, the preparation method has low yield of the arylturmerone, and in addition, the turmeric volatile oil is taken as the raw material, so that on one hand, the raw material cost is high, and on the other hand, a part of the arylturmerone is probably lost in the preparation process of the turmeric volatile oil, so that the preparation method of the patent is not suitable for industrial production as a whole.
Disclosure of Invention
The invention aims to provide a preparation method of an arylturmerone reference substance, which takes turmeric as a raw material, has low cost and high product yield and purity and is suitable for industrial production.
In order to achieve the purpose, the invention is realized by the following scheme:
a method for preparing ar-turmerone reference substance comprises the following steps:
(1) firstly, crushing turmeric to prepare turmeric powder, and then, mixing the turmeric powder with a mixture of the following components in a volume ratio of 1: 2-3, extracting with n-pentane-ethanol mixed solution to obtain an extract I;
(2) then adding the extract I into a mixture with a volume ratio of 1: 8-10: 5-8: 6-9 parts of isopropyl ether-butyl acetate-ethyl acetate-water mixed solution, fully and uniformly oscillating, extracting, taking an organic phase, and concentrating under reduced pressure to obtain an extract II;
(3) then, by using the volume ratio of 1: 0.2-0.3 of isopropyl ether-butyl acetate mixed solution is used as an eluent, the extract II is subjected to silica gel column chromatography separation, and the obtained eluent is subjected to rotary evaporation and drying to obtain the zingiberone reference substance;
wherein the preparation method of the turmeric powder in the step (1) comprises the following steps: firstly, mechanically crushing turmeric to prepare coarse powder with the particle size of less than or equal to 5mm, then pouring the coarse powder into deionized water with the weight of 5-8 times, ultrasonically oscillating for 30-40 minutes, grinding the coarse powder by a colloid mill for 0.5-1 hour, then quickly transferring the coarse powder into a vacuum freeze dryer, reducing the temperature to-40 to-50 ℃, then vacuumizing to 15-20 Pa, raising the temperature to 20-30 ℃, and continuously treating for 7-8 hours under the current vacuum degree condition to obtain medium powder; finally, micronizing the medium powder by using a superfine pulverizer to obtain turmeric powder with the average particle size of less than or equal to 10 microns.
Preferably, before the turmeric is crushed, the turmeric is subjected to impurity removal, water washing and drying in the shade, and the drying in the shade is further preferably carried out for 2-3 days.
Preferably, the mechanical pulverization is any one or more selected from jaw pulverization, cone pulverization, roll pulverization, hammer pulverization or impact pulverization.
Preferably, the temperature of the materials is controlled to be 20-25 ℃ during grinding treatment of the rubber mill.
Preferably, the ultrafine grinding is carried out by an ultrafine grinder, and more preferably, a jet mill is used, and the working conditions are as follows: the air flow pressure is 1000kPa, the feeding speed is 200r/min, the grading frequency is 35Hz, and the crushing time is 60-80 minutes.
Preferably, in the step (1), the mass-to-volume ratio of the turmeric powder to the n-pentane-ethanol mixed solution is 1 g: 6-9 mL.
Preferably, the specific method for extracting in step (1) is as follows: extracting under reflux for 2-3 hours, repeatedly extracting for 2-3 times, filtering, combining filtrates, and removing the solvent under reduced pressure to obtain an extract I; in each extraction, the solid-liquid mass ratio is 1: 5 to 8.
Preferably, in the step (2), the mass volume ratio of the extract I to the isopropyl ether-butyl acetate-ethyl acetate-water mixed solution is 1 g: 6-9 mL.
Preferably, in the step (3), a 100-200 mesh silica gel column is adopted for chromatographic separation, and the flow rate of passing through the column is 0.5-0.8 mL/min.
The invention has the beneficial effects that:
according to the method, turmeric is used as a raw material, the turmeric is crushed into turmeric powder with the average grain diameter of less than or equal to 10 microns, an n-pentane-ethanol mixed solution is used for extracting to obtain an extract I, an isopropyl ether-butyl acetate-ethyl acetate-water mixed solution is used for extracting to obtain an extract II, and finally the isopropyl ether-butyl acetate mixed solution is used as an eluent to carry out silica gel column chromatography separation on the extract II, so that a high-purity turmerone reference substance can be obtained at a high yield.
When preparing the turmeric powder, firstly, mechanically crushing the turmeric to prepare coarse powder with the grain diameter of less than or equal to 5mm, then mixing the coarse powder with water, grinding the mixture by using a colloid mill, freezing and drying the mixture in vacuum to obtain medium powder, and finally, carrying out superfine grinding on the medium powder by using a superfine grinder to obtain the turmeric powder with the average grain diameter of less than or equal to 10 mu m. The turmeric powder obtained by the method has small average particle size, is beneficial to subsequent full extraction, and greatly improves the extraction yield.
The method mainly comprises two times of extraction and one time of silica gel column chromatography separation to obtain the final artocarpin contrast product, wherein the selection of an extraction reagent and an eluent is very critical. The method comprises the following steps of (1) extracting turmerone which is a terpene compound and belongs to a fat-soluble component, selecting an extraction reagent and an eluent for two-time extraction and one-time silica gel column chromatography separation, and carrying out a series of screening and exploration by an applicant; the second extraction is to extract by using isopropyl ether-butyl acetate-ethyl acetate-water mixed liquor with a specific proportion to obtain an extract II, wherein the extract II contains arylturmerone and fat-soluble components with similar polarity; the silica gel column chromatography separation adopts isopropyl ether-butyl acetate mixed liquor with a specific ratio as an eluent to realize complete separation and obtain the high-purity ar-turmerone reference substance.
Drawings
FIG. 1 is a carbon spectrum of the product obtained in example 1;
FIG. 2 is a structural diagram of ar-turmerone;
FIG. 3 shows hydrogen and carbon spectra data of ar-turmerone.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The traditional Chinese medicine turmeric of the invention is purchased from Bozhou city Fanghai pharmaceutical industry Co.
Example 1
A method for preparing ar-turmerone reference substance comprises the following steps:
(1) firstly, crushing turmeric to prepare turmeric powder, and then carrying out the following steps of: extracting with 2 mL of n-pentane-ethanol mixed solution to obtain an extract I;
(2) then adding the extract I into a mixture with the volume ratio of 1 mL: 8mL of: 5mL of: in 6mL of isopropyl ether-butyl acetate-ethyl acetate-water mixed solution, fully and uniformly oscillating, extracting, taking an organic phase, and concentrating under reduced pressure to obtain an extract II;
(3) then, using a volume ratio of 1 mL: 0.2 mL of isopropyl ether-butyl acetate mixed solution is used as an eluent, the extract II is subjected to silica gel column chromatography separation, and the obtained eluent is subjected to rotary evaporation and drying to obtain the artocarpin contrast product;
wherein the preparation method of the turmeric powder in the step (1) comprises the following steps: firstly, mechanically crushing turmeric to prepare coarse powder with the particle size of less than or equal to 5mm, then pouring the coarse powder into deionized water with the weight of 5 times of the weight of the coarse powder, ultrasonically oscillating for 30 minutes, grinding the coarse powder by a colloid mill for 0.5 hour, then quickly transferring the coarse powder into a vacuum freeze dryer, reducing the temperature to-40 ℃, then vacuumizing the vacuum freeze dryer to 15Pa, then increasing the temperature to 20 ℃, and continuously processing the coarse powder for 7 hours under the current vacuum degree condition to obtain medium powder; finally, micronizing the medium powder by using a superfine pulverizer to obtain turmeric powder with the average particle size of less than or equal to 10 microns.
Before the crushing treatment, the turmeric is subjected to impurity removal, water washing and shade drying treatment, wherein the shade drying time is 2 days. Jaw crushing is adopted for mechanical crushing. And controlling the material temperature to be 20-25 ℃ during grinding treatment of the rubber grinding machine. Carrying out superfine grinding by using a superfine grinder, specifically adopting a jet mill, wherein the working conditions are as follows: the air pressure was 1000kPa, the feed rate was 200r/min, the classification frequency was 35Hz, and the pulverizing time was 60 minutes.
In the step (1), the mass-to-volume ratio of the turmeric powder to the n-pentane-ethanol mixed solution is 1 g: 6 mL. The specific method for extracting in the step (1) is as follows: extracting under reflux for 2 hr, repeating the extraction for 2 times, filtering, mixing filtrates, and removing solvent under reduced pressure to obtain extract I; in each extraction, the solid-liquid mass ratio is 1 g: 5g of the total weight.
In the step (2), the mass-volume ratio of the extract I to the isopropyl ether-butyl acetate-ethyl acetate-water mixed solution is 1 g: 6 mL.
In the step (3), a 100-mesh silica gel column is adopted for chromatographic separation, and the flow rate of column passing is 0.5 mL/min.
And (3) carrying out structure confirmation on the obtained arylturmerone reference substance:
EI-MSm/z：216（M+，6）, 201（M+-Me，7）,132（11）, 119（44）, 91（25）, 83（100）, 77（14）, 55（70）；
the Bruker nuclear magnetic resonance spectrometer ANCE 400 uses chloroform as an internal standard, a carbon spectrum is shown in figure 1, and hydrogen spectrum and carbon spectrum data corresponding to a structure are shown in figures 2 and 3.
Example 2
A method for preparing ar-turmerone reference substance comprises the following steps:
(1) firstly, crushing turmeric to prepare turmeric powder, and then carrying out the following steps of: extracting with 3mL of n-pentane-ethanol mixed solution to obtain an extract I;
(2) then adding the extract I into a mixture with the volume ratio of 1 mL: 10 mL of: 8mL of: fully and uniformly oscillating 9mL of isopropyl ether-butyl acetate-ethyl acetate-water mixed solution, extracting, taking an organic phase, and concentrating under reduced pressure to obtain an extract II;
(3) then, using a volume ratio of 1 mL: 0.3 mL of isopropyl ether-butyl acetate mixed solution is used as an eluent, the extract II is subjected to silica gel column chromatography separation, and the obtained eluent is subjected to rotary evaporation and drying to obtain the artocarpin contrast product;
wherein the preparation method of the turmeric powder in the step (1) comprises the following steps: firstly, mechanically crushing turmeric to prepare coarse powder with the particle size of less than or equal to 5mm, then pouring the coarse powder into deionized water with the weight of 8 times, ultrasonically oscillating for 40 minutes, grinding the coarse powder by a colloid mill for 1 hour, then quickly transferring the coarse powder into a vacuum freeze dryer, reducing the temperature to minus 50 ℃, then vacuumizing to 20Pa, raising the temperature to 30 ℃, and continuously processing the coarse powder for 8 hours under the current vacuum degree condition to obtain medium powder; finally, micronizing the medium powder by using a superfine pulverizer to obtain turmeric powder with the average particle size of less than or equal to 10 microns.
Before the crushing treatment, the turmeric is subjected to impurity removal, water washing and shade drying treatment, wherein the shade drying time is 3 days. The mechanical pulverization adopts cone pulverization. The material temperature is controlled at 25 ℃ during the grinding treatment of the rubber mill. Carrying out superfine grinding by using a superfine grinder, specifically adopting a jet mill, wherein the working conditions are as follows: the air pressure was 1000kPa, the feed rate was 200r/min, the classification frequency was 35Hz, and the pulverizing time was 80 minutes.
In the step (1), the mass-to-volume ratio of the turmeric powder to the n-pentane-ethanol mixed solution is 1 g: 9 mL. The specific method for extracting in the step (1) is as follows: extracting under reflux for 3 hr, repeating the extraction for 3 times, filtering, mixing filtrates, and removing solvent under reduced pressure to obtain extract I; in each extraction, the solid-liquid mass ratio is 1 g: 8 g.
In the step (2), the mass-volume ratio of the extract I to the isopropyl ether-butyl acetate-ethyl acetate-water mixed solution is 1 g: 9 mL.
In the step (3), a 200-mesh silica gel column is adopted for chromatographic separation, and the flow rate of column passing is 0.8 mL/min.
Example 3
A method for preparing ar-turmerone reference substance comprises the following steps:
(1) firstly, crushing turmeric to prepare turmeric powder, and then carrying out the following steps of: extracting with 2 mL of n-pentane-ethanol mixed solution to obtain an extract I;
(2) then adding the extract I into a mixture with the volume ratio of 1 mL: 10 mL of: 5mL of: fully and uniformly oscillating 9mL of isopropyl ether-butyl acetate-ethyl acetate-water mixed solution, extracting, taking an organic phase, and concentrating under reduced pressure to obtain an extract II;
(3) then, using a volume ratio of 1 mL: 0.2 mL of isopropyl ether-butyl acetate mixed solution is used as an eluent, the extract II is subjected to silica gel column chromatography separation, and the obtained eluent is subjected to rotary evaporation and drying to obtain the artocarpin contrast product;
wherein the preparation method of the turmeric powder in the step (1) comprises the following steps: firstly, mechanically crushing turmeric to prepare coarse powder with the particle size of less than or equal to 5mm, then pouring the coarse powder into deionized water with the weight of 8 times, ultrasonically oscillating for 30 minutes, grinding the coarse powder by a colloid mill for 1 hour, then quickly transferring the coarse powder into a vacuum freeze dryer, reducing the temperature to-40 ℃, then vacuumizing to 20Pa, raising the temperature to 20 ℃, and continuously treating the coarse powder for 8 hours under the current vacuum degree condition to obtain medium powder; finally, micronizing the medium powder by using a superfine pulverizer to obtain turmeric powder with the average particle size of less than or equal to 10 microns.
Before the crushing treatment, the turmeric is subjected to impurity removal, water washing and shade drying treatment, wherein the shade drying time is 2 days. The mechanical pulverization adopts roller type pulverization. The material temperature is controlled at 25 ℃ during the grinding treatment of the rubber mill. Carrying out superfine grinding by using a superfine grinder, specifically adopting a jet mill, wherein the working conditions are as follows: the air pressure was 1000kPa, the feed rate was 200r/min, the classification frequency was 35Hz, and the pulverizing time was 60 minutes.
In the step (1), the mass-to-volume ratio of the turmeric powder to the n-pentane-ethanol mixed solution is 1 g: 9 mL. The specific method for extracting in the step (1) is as follows: reflux extracting for 2 hr, repeating the extraction for 3 times, filtering, mixing filtrates, and removing solvent under reduced pressure to obtain extract I; in each extraction, the solid-liquid mass ratio is 1 g: 5g of the total weight.
In the step (2), the mass-volume ratio of the extract I to the isopropyl ether-butyl acetate-ethyl acetate-water mixed solution is 1 g: 9 mL.
In the step (3), a 100-mesh silica gel column is adopted for chromatographic separation, and the flow rate of column passing is 0.8 mL/min.
Example 4
A method for preparing ar-turmerone reference substance comprises the following steps:
(1) firstly, crushing turmeric to prepare turmeric powder, and then carrying out the following steps of: extracting with 3mL of n-pentane-ethanol mixed solution to obtain an extract I;
(2) then adding the extract I into a mixture with the volume ratio of 1 mL: 8mL of: 8mL of: in 6mL of isopropyl ether-butyl acetate-ethyl acetate-water mixed solution, fully and uniformly oscillating, extracting, taking an organic phase, and concentrating under reduced pressure to obtain an extract II;
(3) then, using a volume ratio of 1 mL: 0.3 mL of isopropyl ether-butyl acetate mixed solution is used as an eluent, the extract II is subjected to silica gel column chromatography separation, and the obtained eluent is subjected to rotary evaporation and drying to obtain the artocarpin contrast product;
wherein the preparation method of the turmeric powder in the step (1) comprises the following steps: firstly, mechanically crushing turmeric to prepare coarse powder with the particle size of less than or equal to 5mm, then pouring the coarse powder into deionized water with the weight of 5 times of the weight of the coarse powder, oscillating the coarse powder for 40 minutes by ultrasonic waves, grinding the coarse powder by a colloid mill for 0.5 hour, then quickly transferring the coarse powder into a vacuum freeze dryer, reducing the temperature to minus 50 ℃, then vacuumizing the vacuum freeze dryer to 15Pa, then increasing the temperature to 30 ℃, and continuously processing the coarse powder for 7 hours under the current vacuum degree condition to obtain medium powder; finally, micronizing the medium powder by using a superfine pulverizer to obtain turmeric powder with the average particle size of less than or equal to 10 microns.
Before the crushing treatment, the turmeric is subjected to impurity removal, water washing and shade drying treatment, wherein the shade drying time is 3 days. The mechanical pulverization adopts hammer type pulverization. The material temperature is controlled at 20 ℃ during the grinding treatment of the rubber mill. Carrying out superfine grinding by using a superfine grinder, specifically adopting a jet mill, wherein the working conditions are as follows: the air pressure was 1000kPa, the feed rate was 200r/min, the classification frequency was 35Hz, and the pulverizing time was 80 minutes.
In the step (1), the mass-to-volume ratio of the turmeric powder to the n-pentane-ethanol mixed solution is 1 g: 6 mL. The specific method for extracting in the step (1) is as follows: extracting under reflux for 3 hr, repeating the extraction for 2 times, filtering, mixing filtrates, and removing solvent under reduced pressure to obtain extract I; in each extraction, the solid-liquid mass ratio is 1 g: 8 g.
In the step (2), the mass-volume ratio of the extract I to the isopropyl ether-butyl acetate-ethyl acetate-water mixed solution is 1 g: 6 mL.
In the step (3), a 200-mesh silica gel column is adopted for chromatographic separation, and the flow rate of column passing is 0.5 mL/min.
Example 5
A method for preparing ar-turmerone reference substance comprises the following steps:
(1) firstly, crushing turmeric to prepare turmeric powder, and then carrying out the following steps of: 2.5 mL of n-pentane-ethanol mixed solution is extracted to obtain an extract I;
(2) then adding the extract I into a mixture with the volume ratio of 1 mL: 9mL of: 7 mL of: in 8mL of isopropyl ether-butyl acetate-ethyl acetate-water mixed solution, fully and uniformly oscillating, extracting, taking an organic phase, and concentrating under reduced pressure to obtain an extract II;
(3) then, using a volume ratio of 1 mL: 0.25 mL of isopropyl ether-butyl acetate mixed solution is used as an eluent, the extract II is subjected to silica gel column chromatography separation, and the obtained eluent is subjected to rotary evaporation and drying to obtain the artocarpin contrast product;
wherein the preparation method of the turmeric powder in the step (1) comprises the following steps: firstly, mechanically crushing turmeric to prepare coarse powder with the particle size of less than or equal to 5mm, then pouring the coarse powder into deionized water with the weight of 7 times, ultrasonically oscillating for 35 minutes, grinding the coarse powder by a colloid mill for 1 hour, then quickly transferring the coarse powder into a vacuum freeze dryer, reducing the temperature to-45 ℃, then vacuumizing to 18Pa, increasing the temperature to 25 ℃, and continuously treating the coarse powder for 7 hours under the current vacuum degree condition to obtain medium powder; finally, micronizing the medium powder by using a superfine pulverizer to obtain turmeric powder with the average particle size of less than or equal to 10 microns.
Before the crushing treatment, the turmeric is subjected to impurity removal, water washing and shade drying treatment, wherein the shade drying time is 3 days. The mechanical crushing adopts impact crushing. The material temperature is controlled at 22 ℃ during the grinding treatment of the rubber mill. Carrying out superfine grinding by using a superfine grinder, specifically adopting a jet mill, wherein the working conditions are as follows: the air pressure was 1000kPa, the feed rate was 200r/min, the classification frequency was 35Hz, and the pulverizing time was 70 minutes.
In the step (1), the mass-to-volume ratio of the turmeric powder to the n-pentane-ethanol mixed solution is 1 g: 8 mL. The specific method for extracting in the step (1) is as follows: reflux extracting for 2 hr, repeating the extraction for 3 times, filtering, mixing filtrates, and removing solvent under reduced pressure to obtain extract I; in each extraction, the solid-liquid mass ratio is 1 g: 6 g.
In the step (2), the mass-volume ratio of the extract I to the isopropyl ether-butyl acetate-ethyl acetate-water mixed solution is 1 g: 8 mL.
In the step (3), a 200-mesh silica gel column is adopted for chromatographic separation, and the flow rate of column passing is 0.6 mL/min.
Comparative example 1
A method for preparing ar-turmerone reference substance comprises the following steps:
(1) firstly, crushing turmeric to prepare turmeric powder, and then carrying out the following steps of: extracting with 2 mL of n-pentane-ethanol mixed solution to obtain an extract I;
(2) then adding the extract I into a mixture with the volume ratio of 1 mL: 8mL of: 5mL of: in 6mL of isopropyl ether-butyl acetate-ethyl acetate-water mixed solution, fully and uniformly oscillating, extracting, taking an organic phase, and concentrating under reduced pressure to obtain an extract II;
(3) then, using a volume ratio of 1 mL: 0.2 mL of isopropyl ether-butyl acetate mixed solution is used as an eluent, the extract II is subjected to silica gel column chromatography separation, and the obtained eluent is subjected to rotary evaporation and drying to obtain the artocarpin contrast product;
wherein the preparation method of the turmeric powder in the step (1) comprises the following steps: pulverizing Curcuma rhizome mechanically to obtain Curcuma rhizome powder with particle size of less than or equal to 5 mm. Before the crushing treatment, the turmeric is subjected to impurity removal, water washing and shade drying treatment, wherein the shade drying time is 2 days. Jaw crushing is adopted for mechanical crushing.
In the step (1), the mass-to-volume ratio of the turmeric powder to the n-pentane-ethanol mixed solution is 1 g: 6 mL. The specific method for extracting in the step (1) is as follows: extracting under reflux for 2 hr, repeating the extraction for 2 times, filtering, mixing filtrates, and removing solvent under reduced pressure to obtain extract I; in each extraction, the solid-liquid mass ratio is 1 g: 5g of the total weight.
In the step (2), the mass-volume ratio of the extract I to the isopropyl ether-butyl acetate-ethyl acetate-water mixed solution is 1 g: 6 mL.
In the step (3), a 100-mesh silica gel column is adopted for chromatographic separation, and the flow rate of column passing is 0.5 mL/min.
Comparative examples 2 to 11
The composition of the extraction solvent or eluent was adjusted as in example 1, and is shown in Table 1.
TABLE 1 adjustment of the ratio of extraction solvent or eluent in comparative examples 2 to 11
Note: "- -" represents the same as in example 1.
Comparative example 12
The same procedure as in example 1 was repeated except that n-hexane was used instead of n-pentane and a n-hexane-ethanol mixture of the same ratio was used instead of the n-pentane-ethanol mixture.
Comparative example 13
Replace butyl acetate with ethyl acetate, using 1 mL: 13mL of: the extraction of step (2) was performed using 6mL of a mixture of isopropyl ether, ethyl acetate and water, and the same procedure as in example 1 was repeated.
Comparative example 14
The same procedure as in example 1 was repeated except that ethyl acetate was used instead of butyl acetate and isopropyl ether-butyl acetate was used instead of isopropyl ether-butyl acetate.
Test examples
The extraction yield of the arylturmerone reference substances (the quality of the obtained arylturmerone reference substance/the quality of turmeric) of examples 1 to 5 and comparative examples 1 to 14 was counted, and the purity was calculated by using a high performance liquid area normalization method, and the results are shown in table 2.
TABLE 2 extraction yield and purity results
| Extraction yield (mg/kg) | Purity (%) | |
| Example 1 | 3880 | ≥99.99 |
| Example 2 | 3885 | ≥99.99 |
| Example 3 | 3900 | ≥99.99 |
| Example 4 | 3899 | ≥99.99 |
| Example 5 | 3911 | ≥99.99 |
| Comparative example 1 | 2031 | ≥99.99 |
| Comparative example 2 | 3277 | 90.33 |
| Comparative example 3 | 3018 | 90.12 |
| Comparative example 4 | 3459 | 92.88 |
| Comparative example 5 | 3332 | 92.17 |
| Comparative example 6 | 3488 | 92.25 |
| Comparative example 7 | 3318 | 92.58 |
| Comparative example 8 | 3469 | 92.17 |
| Comparative example 9 | 3385 | 92.12 |
| Comparative example 10 | 3715 | 95.38 |
| Comparative example 11 | 3589 | 94.85 |
| Comparative example 12 | 3011 | 83.67 |
| Comparative example 13 | 3233 | 85.22 |
| Comparative example 14 | 3187 | 87.91 |
As shown in Table 2, the preparation methods of examples 1 to 5 have high extraction yield and high purity of the obtained product. Comparative example 1 turmeric powder with a particle size of not more than 5mm is directly prepared by mechanical crushing, and the extraction yield is obviously reduced because the subsequent extraction is not sufficient due to large particle size of the turmeric powder; the composition of the extraction solvent or the eluent is respectively adjusted in the comparative examples 2 to 11, and the individual reagents in the comparative examples 12 to 14 are replaced, so that the purity of the obtained product is obviously reduced, and the combination of the extraction solvent and the eluent in a specific ratio can obtain a good extraction effect.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (9)
1. The preparation method of the arylturmerone reference substance is characterized by comprising the following steps:
(1) firstly, crushing turmeric to prepare turmeric powder, and then, mixing the turmeric powder with a mixture of the following components in a volume ratio of 1: 2-3, extracting with n-pentane-ethanol mixed solution to obtain an extract I;
(2) then adding the extract I into a mixture with a volume ratio of 1: 8-10: 5-8: 6-9 parts of isopropyl ether-butyl acetate-ethyl acetate-water mixed solution, fully and uniformly oscillating, extracting, taking an organic phase, and concentrating under reduced pressure to obtain an extract II;
(3) then, by using the volume ratio of 1: 0.2-0.3 of isopropyl ether-butyl acetate mixed solution is used as an eluent, the extract II is subjected to silica gel column chromatography separation, and the obtained eluent is subjected to rotary evaporation and drying to obtain the zingiberone reference substance;
wherein the preparation method of the turmeric powder in the step (1) comprises the following steps: firstly, mechanically crushing turmeric to prepare coarse powder with the particle size of less than or equal to 5mm, then pouring the coarse powder into deionized water with the weight of 5-8 times, ultrasonically oscillating for 30-40 minutes, grinding the coarse powder by a colloid mill for 0.5-1 hour, then quickly transferring the coarse powder into a vacuum freeze dryer, reducing the temperature to-40 to-50 ℃, then vacuumizing to 15-20 Pa, raising the temperature to 20-30 ℃, and continuously treating for 7-8 hours under the current vacuum degree condition to obtain medium powder; finally, micronizing the medium powder by using a superfine pulverizer to obtain turmeric powder with the average particle size of less than or equal to 10 microns.
2. The method according to claim 1, wherein the turmeric is subjected to impurity removal, water washing and drying in the shade before the pulverization treatment.
3. The method according to claim 1, wherein the mechanical pulverization is any one or more selected from jaw pulverization, cone pulverization, roll pulverization, hammer pulverization, and impact pulverization.
4. The preparation method according to claim 1, wherein the temperature of the material is controlled to be 20-25 ℃ during the grinding treatment of the colloid mill.
5. The process according to claim 1, wherein the micronizer is used for micronization, preferably a jet mill, under the following operating conditions: the air flow pressure is 1000kPa, the feeding speed is 200r/min, the grading frequency is 35Hz, and the crushing time is 60-80 minutes.
6. The method according to claim 1, wherein in the step (1), the ratio of the turmeric powder to the n-pentane-ethanol mixture is about 1 g: 6-9 mL.
7. The preparation method according to claim 1, wherein the specific method of extraction in step (1) is: extracting under reflux for 2-3 hours, repeatedly extracting for 2-3 times, filtering, combining filtrates, and removing the solvent under reduced pressure to obtain an extract I; in each extraction, the solid-liquid mass ratio is about 1: 5 to 8.
8. The preparation method according to claim 1, wherein in the step (2), the mass-to-volume ratio of the extract I to the isopropyl ether-butyl acetate-ethyl acetate-water mixture is 1 g: 6-9 mL.
9. The preparation method according to claim 1, wherein in the step (3), the silica gel column with 100-200 meshes is adopted for chromatographic separation, and the flow rate of the silica gel column passing through the column is 0.5-0.8 mL/min.
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