CN110437053B - Method for extracting and separating eupatorium adenophorum ketone compounds from eupatorium adenophorum - Google Patents

Abstract

The invention discloses a method for extracting and separating zelanone compounds from Euratopseria japonica. The steps are as follows: air-drying and pulverizing medicinal materials, supercritical CO ₂ extraction, molecular distillation separation, industrial chromatography purification, and finally obtaining 9-carbonyl-10 ,11-dehydrozelenone and 9-carbonyl-10Hβ-zelenone, the obtained products have high purity and high transfer rate, the whole process is reasonable and feasible, the process is simple, the solvent is single and easy to recover, and it is suitable for large-scale industrial production, Has a strong practical value.

Description

A kind of method for extracting and separating zelanone compounds from Euratopsia

technical field

The present invention relates to the technical field of extraction and separation of active components of natural products, in particular to a method for extracting and separating zelanone compounds from Eurasian zelanus.

Background technique

Eupatorium adenophorum Spreng is a perennial sub-shrub-like herb of the genus Asteraceae. As a malignant weed, it is distributed in more than 30 countries in the world and poses a serious threat to the environment. It has the characteristics of strong adaptability, fast transmission speed and strong reproductive ability. It repels the decline of native plants through allelopathic action to form a single dominant population. It was introduced to Yunnan in my country in the 1940s and was listed as one of the first foreign invasions in my country. species. It is now widely distributed in southwest China, and has become the most serious invasive alien weed in my country. For many years, the control effect of Euratopseria is not ideal, and its comprehensive utilization provides a new way to control invasive alien species.

Studies have shown that the zelanone compounds such as 9-carbonyl-10,11-dehydrozeranone (euptoxA), 9-carbonyl-10Hβ zelanone (9-Oxoageraphorone), etc. Ranone substances have good anti-tumor, anti-virus, bacteriostatic, insecticidal and other biological activities, and have great potential for the development of botanical pesticides and disease treatment drugs. At present, the research on the extraction and purification process of zelanone compounds in A. chinensis is still in the laboratory stage. It is necessary to purify the extracted part of zelanone by repeated column chromatography, which is complicated in operation, complicated in solvent and large in amount. , low product purity, low yield and other problems. The inability to obtain high-purity samples quickly and in large quantities is the main factor restricting the further development of zelanones.

The Chinese invention patent announcement number is CN103274935B, and the name of the invention is "A method for extracting zelanone compounds in A. chinensis". , extract with methanol or water by ultrasonic vibration, and concentrate under reduced pressure to obtain the extract; the third step, dissolve the extract in methanol, dilute with water, extract multiple times with ethyl acetate, combine the extracts, and concentrate; the fourth step, separate by silica gel column chromatography , the eluent is a mixture of dichloromethane, dichloromethane and ethyl acetate in turn; the fifth step, the macroporous resin is decolorized, and the eluent is a mixture of methanol, water and chloroform; the sixth step, silica gel again Column chromatography, the eluents were dichloromethane and ethyl acetate. Although this technical solution can extract 2-deoxy-2-acetoxy-9-carbonyl zelenone, 9-carbonyl-10Hβ zelenone, 9-carbonyl-10Hα zelenone, 9-carbonyl-10,11 de- Hydrozelanone, but the extraction process steps are complicated, and the toxic and dangerous solvents such as dichloromethane and chloroform are used in the process, and the eluents used in silica gel column chromatography separation and macroporous resin decolorization are complicated.

The Chinese invention patent publication number is: CN103351290A, and the name of the invention is "The Extraction and Purification Method of Two Allelopathic Active Secondary Substances of Echinacea". The following technical scheme is disclosed: the first step is to soak in distilled water to obtain an aqueous extract; , n-hexane extraction; the third step, two glass silica gel column chromatography separation, the eluent is a mixture of ethyl acetate and chloroform, TLC silica gel plate detection, qualitative collection of zerandione and hydroxyzeranone . Although there are not many steps in this technical solution, chloroform is used as a toxic and dangerous solvent, and zerandione and hydroxyzelanone are finally separated, not 9-carbonyl-10,11-dehydrozelan Ketone, 9-Carbonyl-10Hβ Zelenone.

Therefore, it is necessary to develop a new technology to obtain 9-carbonyl-10,11-dehydrozelenone and 9-carbonyl-10Hβ-zelenone.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a kind of method for extracting and separating zelanone compounds from E. japonica, and solve the problem that the method for extracting and purifying zelanone compounds in the prior art is complicated in process, complicated in solvent, large in dosage, low in product purity, and in yield. problems such as low rates.

In order to achieve the above object, the present invention adopts the following technical solutions:

(1) Treatment of medicinal materials: After collecting the leaves of Rhizoma Rhizopus chinensis, it was air-dried in a cool place, and pulverized with a pulverizer to obtain Rhododendron chinensis powder.

(2) supercritical CO ₂ extraction: add the powder prepared in step (1) to the extraction kettle of the supercritical CO ₂ extraction device, and the extraction device is respectively heated and pressurized to the extraction cycle condition, and the cyclic extraction is started, and the constant temperature and pressure are maintained. To the required extraction time, adjust the temperature and pressure of the analysis kettle to obtain the Echinacea japonica extract;

(3) molecular distillation separation: adding the extract of Step (2) gained Euratopsis to the molecular distillation device constant temperature tank, be warming up to the required distillation temperature, adjust the vacuum degree to the required pressure, and control the E. Accelerate, start distillation, collect zelenone-enriched fractions;

(4) Industrial chromatographic purification: Dissolve the enriched fractions of zelanone obtained in step (3), filter and apply industrial chromatography, elute with organic solvent, collect 9-carbonyl-10,11-dehydrozelanone and 9- The carbonyl-10Hβ-zelenone eluate was concentrated under reduced pressure and dried to obtain 9-carbonyl-10,11-dehydrozelenone and 9-carbonyl-10Hβzelenone products. The zelanone-enriched fraction obtained in the mobile phase dissolving step (3) can be used. Of course, those skilled in the art can also configure a suitable solvent by themselves according to the technical content disclosed in the present invention and common knowledge in the field.

Further, in the step (1), the leaves of Rhododendron chinensis are pulverized to 60-120 mesh powder, preferably to 80 mesh.

Further, in the step (2), the extraction pressure is 25-35MPa, the extraction temperature is 45-65°C, and the extraction time is 0.5-1.5h. Preferably, the extraction pressure is 30MPa, the extraction temperature is 55°C, and the extraction time is 55°C. is 1.0h.

Further, in the step (2), the pressure of the analysis kettle I is 12 to 21 MPa, and the temperature is 45 to 55 ° C, preferably: the pressure of the analysis kettle I is 18 MPa, and the temperature is 50 ° C. The pressure of the analysis kettle II was 6MPa and the temperature was 45°C.

Further, in the step (3), the feed flow rate of E. chinensis extract is 0.5 drops/sec～4 drops/sec, the distillation temperature is 120～200°C, the vacuum degree is 10～40Pa, and the speed of the scraping film is 200～300r/min, condensing temperature is 0～50 ℃; preferably: feed flow rate is 2 drops/second, distillation temperature is 150 ℃, vacuum degree is 20Pa, film scraping speed is 250r/min, condensing temperature is 25 ℃ .

Further, in the step (4), the filler of the process chromatography is one or more of UniPS10, UniPMM10, UniPSA 10, UniPSN 10 and UniSil 10.

Further, in the step (4), the organic solvent used for elution is ethanol, methanol or acetonitrile, preferably methanol.

Further, in the step (4), the volume fraction of the organic solvent used for elution is 50%-80%, preferably 70%.

Further, in the step (4), during elution, the organic solvent is eluted in stages at a flow rate of 6-15 mL/min, preferably 10 mL/min.

Further, in the step (4), under the condition that the detection wavelength is 230 nm, the elution peaks of 9-carbonyl-10,11-dehydrozelenone and 9-carbonyl-10Hβ-zelenone are collected; concentrated under reduced pressure The temperature was 55°C.

The beneficial effects of the invention are as follows: taking supercritical CO ₂ extraction-molecular distillation separation-industrial chromatographic purification as the core complete set of technology to extract and separate zelanone compounds from E. The process route of separating and purifying zelanone compounds, the product obtained by this process has high purity and high transfer rate, the whole process is reasonable and feasible, the process is simple, the solvent is single and easy to recover, and it is easy to be industrialized in large quantities, and has strong practicality. value.

Description of drawings

The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

Fig. 1 is the molecular structure diagram of 9-carbonyl-10,11-dehydrozelanone;

Fig. 2 is the molecular structure diagram of 9-carbonyl-10Hβ zelanone;

Fig. 3 is the high performance liquid chromatogram of molecular distillation fraction in the embodiment of the present invention 1, and chromatographic peak 1 and peak 2 are respectively 9-carbonyl-10,11-dehydrozelanone and 9-carbonyl-10Hβ-zelanone;

4 is a chromatogram (230 nm) of an industrial chromatogram in Example 1 of the present invention, and the chromatographic peaks 1 and 2 are 9-carbonyl-10,11-dehydrozelenone and 9-carbonyl-10Hβ-zelenone, respectively.

Fig. 5 is the high performance liquid chromatogram of the product after industrial chromatographic purification in the embodiment of the present invention 1, and chromatographic peak 1 is 9-carbonyl-10,11-dehydrozelanone;

Fig. 6 is the high-performance liquid chromatogram of the product after purification by industrial chromatography in Example 1 of the present invention, and peak 2 is 9-carbonyl-10Hβ zeranone.

Detailed ways

The present invention will be described in detail below with reference to specific embodiments, and the illustrative embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

Example 1

(1) Treatment of medicinal materials: After collecting the leaves of Eurasian chinensis, air-dry them in a cool place, and pulverize them to 80 mesh powder with a pulverizer;

(2) supercritical CO ₂ extraction: take by weighing 1.0Kg of the powder prepared in step (1), put it in a supercritical CO ₂ extraction device extraction kettle, and the extraction kettle is boosted to 30MPa, and heated to 55°C; 18MPa, heated to 50°C; pressure of analysis kettle II was increased to 6MPa, heated to 45°C, cyclic extraction was started, constant temperature and pressure were maintained, extraction was performed for 1.0h, after extraction was completed, the extract of analysis kettle II was collected to obtain 65.2g of extract;

(3) Molecular distillation separation: take 60.0 g of the extract obtained in step (2), add it to a constant temperature tank of a molecular distillation device, heat up to 150 ° C, adjust the vacuum to 20 Pa, wipe the film rotation speed 250 r/min, and condense temperature 25 ° C. The distillation process was started at a speed of 2 drops/second, and 40.8 g of zelanone fraction was collected;

(4) Industrial chromatographic purification: take 10.0 g of the zelenone-enriched fraction obtained in step (3), add 70% methanol to dissolve, filter and apply to industrial chromatography, use UniSil 10 as the filler, 70% methanol as the eluent, and use 10 mL of The elution was carried out at a flow rate of /min in stages, and at a wavelength of 230 nm, the elution peaks of 9-carbonyl-10,11-dehydrozelenone and 9-carbonyl-10Hβ-zelenone were collected, concentrated under reduced pressure at 55°C, and dried. 7.43 g of 9-carbonyl-10,11-dehydrozelenone product and 1.51 g of 9-carbonyl-10Hβ zelenone product were obtained.

The experimental results are shown in Figures 3-6. Compound 1 is: 9-carbonyl-10,11-dehydrozelanone (euptoxA); compound 2 is: 9-carbonyl-10Hβ-zelanone (9-Oxoageraphorone).

Example 2

(1) Treatment of medicinal materials: After collecting the leaves of Eurasian chinensis, air-dry them in a cool place, and pulverize them to 60 mesh powder with a pulverizer;

( ₂ ) supercritical CO extraction: take by weighing 1.0Kg of the powder prepared in step (1), put it in a supercritical _CO extraction device extraction kettle, the extraction kettle is boosted to 35MPa, and heated to 45°C; the analytical kettle 1 is boosted to 21MPa, heat up to 45°C; increase the pressure of analytical kettle II to 6MPa, heat up to 45°C, start cyclic extraction, maintain constant temperature and pressure, extract for 0.5h, collect the extract of analytical kettle II after the extraction is completed, and obtain 65.9g of extract;

(3) Molecular distillation separation: take 60.0 g of the extract obtained in step (2), add it to a constant temperature tank of a molecular distillation device, heat up to 200 ° C, adjust the vacuum to 40 Pa, wipe the film rotation speed 300 r/min, and condensate temperature 0 ° C. The distillation process was started at a speed of 4 drops/second, and 41.2 g of zelanone fraction was collected;

(4) Industrial chromatographic purification: take 10.0 g of the zelenone enriched fraction obtained in step (3), add 50% acetonitrile to dissolve, filter and apply to industrial chromatography, use UniPS10 as the filler, 50% acetonitrile as the eluent, take 15 mL/ The elution was carried out at a flow rate of 1 min, and at a wavelength of 230 nm, the elution peaks of 9-carbonyl-10,11-dehydrozelenone and 9-carbonyl-10Hβ-zelenone were collected, concentrated under reduced pressure at 55 °C, and dried to obtain 7.25 g of 9-carbonyl-10,11-dehydrozelenone product and 1.43 g of 9-carbonyl-10Hβ zelenone product.

Example 3

(1) Treatment of medicinal materials: After collecting the leaves of Rhododendron chinensis, air-dry them in a cool place, and pulverize them to 120 mesh powder with a pulverizer.

(2) supercritical CO ₂ extraction: take by weighing 1.0Kg of the powder prepared in step (1), put it in a supercritical CO ₂ extraction device extraction kettle, the extraction kettle is boosted to 25MPa, and heated to 65°C; 18MPa, the temperature was raised to 55°C; the pressure of the analysis kettle II was increased to 6MPa, the temperature was raised to 45°C, the cyclic extraction was started, the constant temperature and pressure were maintained, and the extraction was performed for 1.0 h. After the extraction was completed, the extract of the analysis kettle II was collected to obtain 64.7g of extract;

(3) Molecular distillation separation: take 60.0 g of the extract obtained in step (2), add it to a constant temperature tank of a molecular distillation device, heat up to 120 ° C, adjust the vacuum degree to 10 Pa, wipe the film rotation speed 200 r/min, and condense temperature 25 ° C. The distillation process was started at a rate of 0.5 drops/second, and 40.9 g of zelanone fraction was collected;

(4) Industrial chromatographic purification: take 10.0 g of the zelenone-enriched fraction obtained in step (3), add 80% ethanol to dissolve, filter and apply to industrial chromatography, use UniPSN 10 as the filler, 80% ethanol as the eluent, and use 6 mL of The elution was carried out at a flow rate of /min in stages, and at a wavelength of 230 nm, the elution peaks of 9-carbonyl-10,11-dehydrozelenone and 9-carbonyl-10Hβ-zelenone were collected, concentrated under reduced pressure at 55°C, and dried. 7.33 g of 9-carbonyl-10,11-dehydrozelenone product and 1.47 g of 9-carbonyl-10Hβ zelenone product were obtained.

Example 4

The zelanone samples of each step in Example 1, Example 2, and Example 3 were detected by liquid chromatography, and the peak area was recorded according to the detection result of high performance liquid chromatography. The peak area of the active ingredient in the reference standard solution was the same as the The linear relationship between the concentrations was used to calculate the content and transfer rate of zelanone compounds in each step. Compound 1 is: 9-carbonyl-10,11-dehydrozeranone (euptoxA); compound 2 is: 9-carbonyl-10Hβ zelanone (9-Oxoageraphorone). Details are as follows:

Table 1 Results of supercritical _CO extraction of zeranone compounds

Table 2 Molecular distillation enrichment results

Table 3 Industrial chromatographic separation results

Table 4 Industrial chromatographic separation results

In addition, in order to verify that different extraction methods of crushed medicinal materials will obtain different technical effects, three different extraction experiments were performed as follows:

The first: the supercritical _CO extraction of the embodiment of the present invention 1;

The second method: steam distillation method, the process is as follows: take 320 g of dried leaves (coarse powder) of Rhododendron chinensis, add an appropriate amount of water, and after distillation for 6 hours, collect the pale yellow volatile oil, and store it in a refrigerator at 4°C in a dark place and sealed;

The third type: solvent ultrasonic extraction method, the process is as follows: Accurately weigh 2.0 g of dried leaves of Eurasian japonica, add 40 mL of methanol reagent, ultrasonically extract at room temperature for 30 min, centrifuge the extracts and combine them, dilute to a 50 mL volumetric flask, and refrigerate at 4°C Store in an airtight seal away from light;

Comparing the technical effects of the above three extraction methods, as shown in Table 5 below, it can be clearly concluded that the technical solution of the present invention has great advantages.

Table 5 Comparison of technical effects of three extraction methods

From the above comparison, it can be known that only with the complete technology of supercritical CO ₂ extraction-molecular distillation separation-industrial chromatographic purification as the core process can a better technical effect be obtained, and the conventional technology can not obtain the technical effect of the present invention by replacing the operation therein. of.

The technical solutions provided by the embodiments of the present invention have been introduced in detail above. The principles and implementations of the embodiments of the present invention are described in this paper by using specific examples. The descriptions of the above embodiments are only applicable to help understand the embodiments of the present invention. At the same time, for those of ordinary skill in the art, according to the embodiments of the present invention, there will be changes in the specific implementation and application scope. To sum up, the content of this specification should not be construed as a limitation of the present invention.

Claims (3)

1. a method for extracting and separating zelan ketone compounds from Euratopseria, is characterized in that: Proceed as follows: (1) Treatment of medicinal materials: after collecting the leaves of Eurasiania japonica, it is air-dried in a cool place, and pulverized with a pulverizer to obtain E. japonica powder; (2) supercritical CO ₂ extraction: add the powder prepared in step (1) to the extraction kettle of the supercritical CO ₂ extraction device, and the extraction device is respectively heated and pressurized to the extraction cycle condition, and the cyclic extraction is started, and the constant temperature and pressure are maintained. To the required extraction time, adjust the temperature and pressure of the analysis kettle to obtain the Echinocarpus chinensis extract, the extraction pressure is 25 ~ 35MPa, the extraction temperature is 45 ~ 65 ° C, the extraction time is 0.5 ~ 1.5h, and the pressure of the analysis kettle I is 12 ～21MPa, the temperature is 45～55℃; the pressure of the analytical kettle II is 6MPa, and the temperature is 45℃; (3) molecular distillation separation: adding the extract of Step (2) gained Euratopsis to the molecular distillation device constant temperature tank, be warming up to the required distillation temperature, adjust the vacuum degree to the required pressure, and control the E. Accelerate, start the distillation, collect the zelanone enriched fractions, the feed flow rate of the Zelan japonica extract is 0.5 drops/sec~4 drops/sec, the distillation temperature is 120~200°C, the vacuum degree is 10~40Pa, and the film is scraped. The rotating speed is 200～300r/min, and the condensing temperature is 0～50℃; (4) Industrial chromatographic purification: dissolve the zelanone enriched fraction obtained in step (3), filter and apply industrial chromatography, and the filler of the process chromatography is one or both of UniPS10, UniPMM10, UniPSA 10, UniPSN 10 and UniSil10 Above, eluted with organic solvent, collected 9-carbonyl-10,11-dehydrozelenone and 9-carbonyl-10Hβ-zelenone eluent, concentrated under reduced pressure and dried to obtain 9-carbonyl-10,11- Dehydrozeranone and 9-carbonyl-10Hβzelenone products, the organic solvent used for elution is ethanol, methanol or acetonitrile, the volume fraction of organic solvent is 50%-80%, and the flow rate of organic solvent during elution is 6 Elution was performed in fractions of ~15 mL/min. 2. a kind of method of extracting and separating zelanone compounds from Eurasian zelanus according to claim 1, is characterized in that: In the step (1), the leaves of Rhododendron chinensis are pulverized to 60-120 mesh powder. 3. a kind of method of extracting and separating zelanone compounds from Eurasian purpurea according to claim 1, is characterized in that: In the step (4), under the condition that the detection wavelength is 230 nm, the elution peaks of 9-carbonyl-10,11-dehydrozelenone and 9-carbonyl-10Hβ-zelenone are collected; The temperature for concentration under reduced pressure was 55°C.

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