阅读说明：本技术 一种郁金的微提取方法 (Micro-extraction method of radix curcumae ) 是由 曹君 陈嫣 于 2020-07-24 设计创作，主要内容包括：本发明涉及天然药物的分离富集领域,尤其是涉及一种郁金的微提取方法,包括以下步骤：(1)将郁金经粉碎后过筛得到郁金粉末；(2)将葫芦脲研磨后烘干得到葫芦脲粉末；(3)将葫芦脲粉末与郁金粉末混合研磨后得到混合粉末；(4)在混合粉末中加入提取溶剂萃取；(5)离心、过滤取上清后用HPLC进样分析。本发明采用葫芦脲作为吸附剂用于基质固相分散萃取郁金的有效成分,吸附能力强,选择性好,提取效率高,并且无毒不污染环境；并且可以同时分析并提取郁金中多种有效成分,快速便捷,整个提取过程操作简易,一步完成预提取和纯化,无论是样品提取还是进样分析都比较快速,大大节省了时间和劳动成本。(The invention relates to the field of separation and enrichment of natural medicines, in particular to a micro-extraction method of radix curcumae, which comprises the following steps: (1) pulverizing radix Curcumae, and sieving to obtain radix Curcumae powder; (2) grinding cucurbituril and drying to obtain cucurbituril powder; (3) mixing and grinding cucurbituril powder and curcuma aromatica powder to obtain mixed powder; (4) adding an extraction solvent into the mixed powder for extraction; (5) centrifuging, filtering, collecting supernatant, and analyzing by HPLC sample injection. The cucurbituril is used as an adsorbent for matrix solid-phase dispersion extraction of effective components of the curcuma aromatica, and the cucurbituril has the advantages of strong adsorption capacity, good selectivity, high extraction efficiency, no toxicity and no environmental pollution; and can simultaneously analyze and extract multiple effective components in the curcuma aromatica, is quick and convenient, has simple and easy operation in the whole extraction process, completes the pre-extraction and purification in one step, has rapider sample extraction and sample injection analysis, and greatly saves the time and labor cost.)

1. A micro-extraction method of radix curcumae is characterized by comprising the following steps:

(1) pulverizing radix Curcumae, and sieving to obtain radix Curcumae powder;

(2) grinding cucurbituril and drying to obtain cucurbituril powder;

(3) mixing and grinding the cucurbituril powder and the curcuma aromatica powder according to the mass ratio of 30-80:70 for 40-160s to obtain mixed powder;

(4) adding an extraction solvent into the mixed powder for extraction;

(5) centrifuging, filtering, collecting supernatant, and analyzing by HPLC sample injection.

2. The method of claim 1, wherein the solvent is one or more of methanol, ethanol, triton, sodium dodecyl sulfate, dodecyl trimethyl ammonium chloride, 3-sulfopropyl dodecyl dimethyl ammonium, 3-sulfopropyl tetradecyl dimethyl ammonium, and 3-sulfopropyl hexadecyl dimethyl ammonium.

3. The method of claim 2, wherein the solvent is 3-sulfopropylhexadecyldimethylammonium.

4. The method of claim 3, wherein the concentration of the extraction solvent is 50-250 mM.

5. The method as claimed in claim 4, wherein the concentration of the extraction solvent is 150-250 mM.

6. The method for micro-extracting curcuma aromatica according to claim 1, wherein the mass ratio of the cucurbituril powder to the curcuma aromatica powder in the step (3) is 60-80: 70.

7. The method as claimed in claim 1 or 6, wherein the grinding time in step (3) is 100-140 s.

8. The method for micro-extracting curcuma aromatica according to claim 1, wherein the extraction mode in step (4) is violent vortex extraction.

9. The method for micro-extracting radix curcumae according to claim 1, wherein in the step (1), the radix curcumae is sieved by a 20-60 mesh sieve after being crushed.

10. The method as claimed in claim 1, wherein in the step (5), the centrifugation is 10000-.

Technical Field

The invention relates to the field of separation and enrichment of natural medicines, in particular to a micro-extraction method of radix curcumae.

Background

Radix Curcumae is dry root tuber of Curcuma wenyujin Y.H.Chen et C.Ling, Curcuma longa L.Chen et C.Ling, Curcuma kwangsiensis C.Ling or Curcuma zedoaria C.Ling of Zingiberaceae, and is mainly distributed in Zhejiang, Sichuan, Guangdong, Guangxi, Taiwan, Jiangxi, etc., where it mostly grows in wet sunny slopes or fields. The curcuma aromatica is a common Chinese herbal medicine and is widely applied to the treatment of qi stagnation and blood stasis, jaundice and cholelithiasis in medicine. Radix Curcumae contains curdione, isocurcumenol, curodene, germacrone, and furanodiene as active ingredients. These active ingredients have been proved to be effective in promoting regeneration of liver cells, sterilizing, resisting viruses, relieving inflammation and pain, inhibiting regeneration of cancer cells, etc. The extraction of the active ingredients from the turmeric root tuber samples is also significant because of the strong pharmacological action of the turmeric root tuber.

To date, various detection methods have been reported in many published documents, such as gas chromatography-mass spectrometry (GC-MS), Nuclear Magnetic Resonance (NMR), Capillary Electrophoresis (CE), and High Performance Liquid Chromatography (HPLC), in combination with traditional extraction techniques such as Ultrasonic Extraction (UE), Pressurized Liquid Extraction (PLE), water distillation extraction, and decoction extraction. For example, the method for extracting the alpinetin from the curcuma root with the publication number of CN104193715A comprises the following steps: pulverizing radix Curcumae, adding water and biological enzyme with the same volume, performing enzymolysis for 3-5 hr, adding saturated lime water solution, performing ultrasonic extraction for 2-3 times, adjusting pH to 5-6, adding into macroporous resin, adsorbing, gradient eluting with ethanol solution, concentrating eluate, standing for crystallization, dissolving crystal with alkaline water, adding appropriate amount of ethanol, filtering, adjusting pH to 1-3, standing for crystallization, crystallizing with ethanol solution, and drying.

The method uses a large amount of toxic organic solvent, and has long extraction time and low extraction efficiency. The water distillation and decoction methods are complicated to operate, require high extraction temperatures, and take a long time. Moreover, the extraction method can only extract one active substance at a time, and has low extraction efficiency.

Disclosure of Invention

The invention provides a micro-extraction method of radix curcumae, which aims to overcome the defects that the traditional UE method and PLE method use a large amount of toxic organic solvents, have long extraction time and low extraction efficiency, and the water distillation method and decoction method have complex operation, need higher extraction temperature, have long time consumption and can only extract one active substance.

In order to achieve the purpose, the invention adopts the following technical scheme:

a micro-extraction method of radix curcumae comprises the following steps:

(1) pulverizing radix Curcumae, and sieving to obtain radix Curcumae powder;

(2) grinding cucurbituril and drying to obtain cucurbituril powder;

(3) mixing and grinding the cucurbituril powder and the curcuma aromatica powder according to the mass ratio of 30-80:70 for 40-160s to obtain mixed powder;

(4) adding an extraction solvent into the mixed powder for extraction;

(5) centrifuging, filtering, collecting supernatant, and analyzing by HPLC sample injection.

The cucurbituril is used as an adsorbent for matrix solid phase dispersion extraction of effective components of the radix curcumae, and the cucurbituril is provided with a hydrophobic inner cavity and two hydrophilic ports formed by polar carbonyl groups, so that the cucurbituril can be highly selectively reacted with the effective components in the radix curcumae through interaction of hydrophobicity, hydrogen bonds and ion dipoles to form a stable inclusion compound, thereby extracting the effective components in the radix curcumae, separating the effective components through a subsequent extraction process, and then carrying out sample injection and analysis of HPLC. The whole process is simple and convenient to operate, various active ingredients can be pre-extracted and purified in one step, sample extraction and sample introduction analysis are rapid, and time and labor cost are greatly saved.

The method has wide application range, and volatile active ingredients can be detected by using the method regardless of radix curcumae or other traditional Chinese medicinal materials, namely the invention provides a novel environment-friendly technology which skillfully combines the extraction technology of the zwitterionic surfactant with the adsorption characteristic of cucurbituril and can quickly and efficiently extract the volatile active ingredients in natural products.

Preferably, the extraction solvent is one or more of methanol, ethanol, triton, sodium dodecyl sulfate, dodecyl trimethyl ammonium chloride, 3-sulfopropyl dodecyl dimethyl ammonium, 3-sulfopropyl tetradecyl dimethyl ammonium and 3-sulfopropyl hexadecyl dimethyl ammonium.

Preferably, the extraction solvent is 3-sulfopropylhexadecyldimethylammonium.

The method adopts a novel green extraction system, the long-chain zwitterionic surfactant, especially 3-sulfopropyl hexadecyl dimethyl ammonium, is used as an extraction solvent for extracting effective components of the radix curcumae, and because the electrostatic interaction and the hydrogen bond between the 3-sulfopropyl hexadecyl dimethyl ammonium and a target molecule are much stronger than the intermolecular interaction between the target molecule and an adsorbent, the extraction efficiency is even more superior than that of organic solvents such as methanol, and the extraction method is almost nontoxic, avoids the use of toxic reagents such as methanol, and greatly improves the safety and the environmental friendliness of experiments.

Preferably, the concentration of the extraction solvent is 50-250 mM.

Preferably, the concentration of the extraction solvent is 150-250 mM.

The 3-sulfopropylhexadecyldimethylammonium elution solvent can suitably resist the comparatively slight interaction between the adsorbent and the target molecule due to the strong hydrogen bond and electrostatic interaction between the analyte and the aqueous 3-sulfopropylhexadecyldimethylammonium solution, and thus the extraction efficiency can be improved by suitably increasing the concentration of 3-sulfopropylhexadecyldimethylammonium, whereas when the concentration of the aqueous 3-sulfopropylhexadecyldimethylammonium solution exceeds 250mM, the viscosity of the elution solvent increases, so that it becomes more difficult to transfer the target molecule from the adsorbent into the solvent, the extraction recovery rate of the analyte starts to decrease, and thus the concentration of the extraction solvent is preferably 150-.

Preferably, the mass ratio of the cucurbituril powder to the curcuma aromatica powder in the step (3) is 60-80: 70.

With the increase of the using amount of the dispersing agent, the electrostatic interaction and the hydrogen bond interaction between the cucurbituril and the sample are enhanced, so that the extraction efficiency of the effective components in the curcuma aromatica can be improved by the cucurbituril with higher quality; however, excessive adsorbent can show excessive adsorption capacity to the effective components in the curcuma aromatica, so that the extraction solvent is difficult to resist strong intermolecular force, the effective components in the curcuma aromatica cannot be completely eluted, and therefore, the mass ratio of the cucurbituril powder to the curcuma aromatica powder is selected to be 60-80: 70.

Preferably, the grinding time in the step (3) is 100-140 s.

As the grinding time is longer, the contact area between the cucurbituril powder and the curcuma aromatica powder can be increased, so that the effective components in the curcuma aromatica can be better adsorbed by the cucurbituril, and the extraction efficiency of the analyte is improved, but after the adsorption rates of the cucurbituril and the curcuma aromatica reach the peak value, the extraction efficiency cannot be obviously influenced by prolonging the grinding time again, and the labor cost and the energy consumption caused by the extraction efficiency cannot be increased, so that the grinding time of 100-140 is selected.

Preferably, the extraction mode in the step (4) is violent vortex extraction. The violent turbine extraction mode is simple and quick, the extraction efficiency is high, and the matrix interference can be well removed; and as the cucurbituril and the effective components of the radix curcumae are adsorbed together, the violent turbine extraction mode can be favorable for separating the effective components of the radix curcumae adsorbed in the cucurbituril from the cucurbituril, and the extraction efficiency is improved.

Preferably, in the step (1), the curcuma aromatica is ground and sieved by a sieve with 20-60 meshes.

Preferably, in the step (5), the centrifugation conditions are 10000-.

In conclusion, the invention has the following beneficial effects: (1) cucurbituril is used as an adsorbent for matrix solid phase dispersion extraction of effective components of the curcuma aromatica, and the cucurbituril has strong adsorption capacity, good selectivity, high extraction efficiency, no toxicity and no environmental pollution; (2) the method can simultaneously analyze and extract various effective components in the curcuma aromatica, is rapid and convenient, has simple and easy operation in the whole extraction process, completes the pre-extraction and purification in one step, is rapid in both sample extraction and sample injection analysis, and greatly saves time and labor cost.

Drawings

Fig. 1 is a structural diagram of five active ingredients of curcuma aromatica, which are respectively as follows: a, curdione; b, furan diene; c, curcumene furan dienone; d, germacrone; e, isocurcumenol.

FIG. 2 is a flow chart of matrix solid phase dispersion extraction.

Fig. 3 is a line graph of the extraction effect of different grinding times, wherein 1,2, 3, 4 and 5 are respectively as follows: 1. curdione; 2. isocurcumenol; 3. curcumenol dienone; 4. germacrone; 5. a furanodiene.

Fig. 4 is a line graph of the extraction effect of different adsorbent sample ratios, wherein 1,2, 3, 4 and 5 are respectively: 1. curdione; 2. isocurcumenol; 3. curcumenol dienone; 4. germacrone; 5. a furanodiene.

FIG. 5 is a bar graph showing the extraction effect of different types of elution solvents, wherein A-F are respectively corresponding to methanol, ethanol, dodecyltrimethylammonium chloride, 3-sulfopropyldodecyldimethylammonium, 3-sulfopropyltetradecyldimethylammonium and 3-sulfopropylhexadecyldimethylammonium, and five peaks in each segment of the label are, from left to right, curdione, isocurcumenol, curcumenone, germacrone and furanodiene.

FIG. 6 is a line graph showing the extraction effect of different concentrations of elution solvents, wherein 1,2, 3, 4 and 5 are respectively: 1. curdione; 2. isocurcumenol; 3. curcumenol dienone; 4. germacrone; 5. a furanodiene.

Fig. 7 is a chromatogram of effective components extracted from curcuma aromatica of the present invention and a control chromatogram of 10 μ g/mL standard mixture, wherein fig. 7A is a chromatogram of effective components extracted from curcuma aromatica of the present invention, fig. 7B is a control chromatogram of 10 μ g/mL standard mixture, and fig. 1,2, 3, 4, and 5 are respectively: 1. curdione; 2. isocurcumenol; 3. curcumenol dienone; 4. germacrone; 5. a furanodiene.

Detailed Description

The invention is further described with reference to the following detailed description and accompanying drawings.

General examples

A micro-extraction method of radix curcumae comprises the following steps:

(1) drying radix Curcumae, pulverizing into powder, and filtering with 20-60 mesh sieve;

(2) grinding the absorbent cucurbituril white particles into powder, and putting the powder into an oven for drying;

(3) accurately weighing 50mg of radix curcumae powder, transferring the powder into an agate mortar, then accurately weighing 30-80mg of cucurbituril powder, adding the powder into the mortar, slightly grinding the powder for 40-160s by using a pestle to obtain uniformly distributed mixed powder, and transferring the mixture into a 2ml centrifuge tube after grinding;

(4) adding 1mL of extraction solvent with the concentration of 50-250mM, and carrying out violent vortex extraction for 2 minutes;

(5) centrifuge the tube at 10000-.

A chromatographic column: eclipse C18 column (50X 2.1mm, 1.8 μm). The mobile phase A, B is water and acetonitrile respectively, the flow rate is 0.4mL/min, the flow gradient is 0-3min, and the flow rate is 60-70% B; 3-6min, 70-100% B; 6-7min, 100-60% B. The column temperature was 25 ℃, the sample size was 2.0 μ L, and the detection wavelength was 214 nm.

The invention relates to a standard mixed control solution, which is prepared by the following steps: precisely weighing appropriate amount of curdione, isocurcumenol, curcumenol, germacrone, and furanodiene, placing into 1.5mL centrifuge tube, adding appropriate amount of water, ultrasonic vortex dissolving to obtain 1mg/mL standard mother solution, and diluting with ultrapure water to obtain standard control solution with desired concentration before use.

Precisely weighing proper amount of extraction solvent including triton, sodium dodecyl sulfate, dodecyl trimethyl ammonium chloride, 3-sulfopropyl dodecyl dimethyl ammonium, 3-sulfopropyl tetradecyl dimethyl ammonium and 3-sulfopropyl hexadecyl dimethyl ammonium, ultrasonic dissolving with proper amount of water to obtain 500mM mother liquor, and diluting with ultrapure water to desired concentration before use.

The conditions for HPLC sample injection, preparation of the standard mixed control solution, and preparation of the extraction solvent are not specifically described, and are used in the following examples.

The structure diagram of five active ingredients of the extracted curcuma aromatica is shown in figure 1, and the active substances extracted by the method are mainly ketone substances and also part of furan compounds, which have different pharmacological or other beneficial effects. The flow chart of the matrix solid-phase dispersion extraction is shown in figure 2, the method can simultaneously analyze and extract various effective components in the curcuma aromatica, is rapid and convenient, has simple and easy operation in the whole extraction process, completes the pre-extraction and purification in one step, is rapid in both sample extraction and sample injection analysis, and greatly saves time and labor cost.